Gene Therapy & Molecular Biology Volume 1 Issue A - PART 1 by Niko Boulikas

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Gene Therapy & Molecular Biology FROM BASIC MECHANISMS TO CLINICAL APPLICATIONS

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GENE THERAPY AND MOLECULAR BIOLOGY: FROM BASIC MECHANISMS TO CLINICAL APPLICATIONS !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!

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Table of Contents Volume 1, March 1998 832 pages, color figures

Boulikas T (1998) Status of gene therapy in 1997: molecular mechanisms, disease targets, and clinical applications. Gene Ther Mol Biol Vol 1, 1-172. Martin F and Boulikas T (1998) The challenge of liposomes in gene therapy. Gene Ther Mol Biol Vol 1, 173-214. Soares MK, Goins WH, Glorioso JC and Fink DJ (1998) Gene transfer to the nervous system using HSV vectors. Gene Ther Mol Biol Vol 1, 215-229. Hofmann C, Lehnert W, and Strauss M (1998) The baculovirus vector system for gene delivery into hepatocytes. Gene Ther Mol Biol Vol 1, 231-239. Lee CG L, Vieira WD, Pastan I, and Gottesman MM (1998) Delivery systems for the M D R 1 gene. Gene Ther Mol Biol Vol 1, 241-251. Ramesh R, Marrogi AJ, and Freeman SM (1998) Tumor killing using the HSV-tk suicide gene. Gene Ther Mol Biol Vol 1, 253-263. Bohn MC and Choi-Lundberg DL (1998) Neurotrophic factor gene therapy for neurodegenerative diseases. Gene Ther Mol Biol Vol 1, 265-277. Connelly S and Kaleko M (1998) Hemophilia A: current treatment and future gene therapy. Ther Mol Biol Vol 1, 279-292. Hoeben RC (1998) Gene therapy for haemophilia. Gene Ther Mol Biol Vol 1, 293-300. Chao J and Chao L (1998) Kallikrein gene therapy in hypertension, cardiovascular and renal diseases. Gene Ther Mol Biol Vol 1, 301-308. Buschle M, Schmidt W, Berger M, Schaffner G, Kurzbauer R, Killisch I, Tiedemann J-K, Trska B, Kirlappos H, Mechtler K, Schilcher F, Gabler C, and Birnstiel ML (1998) Chemically defined, cell-free cancer vaccines: use of tumor antigen-derived peptides or polyepitope proteins for vaccination. Gene Ther Mol Biol Vol 1, 309-321. Xiong S, Gerloni M and Zanetti M (1998) Somatic transgenesis by immunoglobulin genes. Ther Mol Biol Vol 1, 323-332. White-Scharf ME, Banerjee P, Sachs DH, and LeGuern C (1998) Applications of gene therapy in transplantation. Gene Ther Mol Biol Vol 1, 333-344. Law P, Goodwin T, Fang Q, Vastagh G, Jordan T, Jackson T, Kenny S, Duggirala V, Larkin C, Chase N, Phillips W, Williams G, Neel M, Krahn T, and Holcomb R (1998) Myoblast transfer as a platform technology of gene therapy. Gene Ther Mol Biol Vol 1, 345-363. Webster MK and Donoghue DJ (1998) Constitutive activation of fibroblast growth factor receptors in human developmental syndromes. Gene Ther Mol Biol Vol 1, 365-379. Georgiev GP, Kiselev SL and Lukanidin EM (1998) Genes involved in the control of tumor progression and their possible use for gene therapy. Gene Ther Mol Biol Vol 1, 381-398. Noteborn MHM, Danen-van Oorschot AAAM and van der Eb AJ (1998) The Apoptin " gene of chicken anemia virus in the induction of apoptosis in human tumorigenic cells and in gene therapy of cancer. Gene Ther Mol Biol Vol 1, 399-406. Voeks DJ, Clawson GA, and Norris JS (1998) Tissue-specific triple ribozyme vectors for prostate cancer gene therapy. Gene Ther Mol Biol Vol 1, 407-418. Fujita S, Hamada M, Jigami Y, Kise H, and Taira K (1998) A novel system for selection of intracellularly active ribozymes using the gene for dihydrofolate reductase (DHFR) as a selective marker in Escherichia coli. Gene Ther Mol Biol Vol 1, 419-434. Kuwabara T, Warashina M, Nakayama A, Hamada M, Amontov S, Takasuka Y, Komeiji Y, and Taira K (1998) Comparison of the specificities and catalytic activities of conventional hammerhead ribozymes, Joyce's DNA enzymes, and novel dimeric minizymes with respect to the cleavage of BCR-ABL chimeric L6 (b2a2) mRNA. Gene Ther Mol Biol Vol 1, 435-449. Yamamoto R, Murakami K, Taira K, and Kumar PKR (1998) Isolation and characterization of an RNA that binds with high affinity to Tat protein of HIV-1 from a completely random p o o l o f R N A . Gene Ther Mol Biol Vol 1, 451-466. HĂŠlĂ¨ne C, Garestier T, Giovannangeli C, and Sun J-S (1998) Sequence-specific control of gene expression by antigene and clamp oligonucleotides. Gene Ther Mol Biol Vol 1, 467-474.

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van Holde K, Leuba SH and Zlatanova J (1998) Physical approaches to the study of chromatin fibers. Gene Ther Mol Biol Vol 1, 475-482. Bresnick EH, Versaw WK, Lam LT, Forsberg EC, and Eisenman HC (1998) Activation at a distance: involvement of nucleoprotein complexes that remodel chromatin. Gene Ther Mol Biol Vol 1, 483-494. Iborra F, Pombo A and Jackson DA (1998) Dedicated sites of gene expression in the nuclei of mammalian cells. Gene Ther Mol Biol Vol 1, 495-508. Davie JR, Samuel S, Spencer V, Bajno L, Sun J-M, Chen HY, and Holth LT (1998) Nuclear matrix: application to diagnosis of cancer and role in transcription and modulation of chromatin structure. Gene Ther Mol Biol Vol 1, 509-528. Harel A, Goldberg M, Ulitzur N and Gruenbaum Y (1998) Structural organization and biological roles of the nuclear lamina. Gene Ther Mol Biol Vol 1, 529-542. Will K and Deppert W (1998) Analysis of mutant p53 for MAR-DNA binding: determining the dominant-oncogenic function of mutant p53. Gene Ther Mol Biol Vol 1, 543-549. Bode J, Bartsch J, Boulikas T, Iber M, Mielke C, SchĂźbeler D, Seibler J, and Benham C (1998) Transcription-promoting genomic sites in mammalia: their elucidation and architectural principles. Gene Ther Mol Biol Vol 1, 551-580. Tsutsui K (1998) Synthetic concatemers as artificial MAR: importance of a particular configuration of short AT-tracts for protein recognition. Gene Ther Mol Biol Vol 1, 581-590. Verbovaia LV and Razin SV (1998) Replicon map of the human dystrophin gene: asymmetric replicons and putative replication barriers. Gene Ther Mol Biol Vol 1, 591-598. Tsutsumi K-i and Zhao Y (1998) Initiation of DNA replication at the rat aldolase B locus.â&#x20AC;&#x201D;An overlapping set of DNA elements regulates transcription and replication? Gene Ther Mol Biol Vol 1, 599-608. Resnick MA, Kouprina N, and Larionov V (1998) TARgeting the human genome to make gene isolation easy. Gene Ther Mol Biol Vol 1, 609-612. White RJ (1998) Control of growth and proliferation by the retinoblastoma protein. Gene Ther Mol Biol Vol 1, 613-628. Zachos G and Spandidos DA (1998) Transcriptional regulation of the H-ras1 proto-oncogene by DNA binding proteins: mechanisms and implications in human tumorigenesis. Gene Ther Mol Biol Vol 1, 629-639. Kiyama R (1998) Periodicity of DNA bend sites in eukaryotic genomes. Gene Ther Mol Biol Vol 1, 641-647. Szyf M (1998) DNA methyltransferase: a downstream effector of oncogenic programs; implications for therapy. Gene Ther Mol Biol Vol 1, 649-660. Zardo G, Marenzi S and Caiafa P (1998) Correlation between DNA methylation and poly(ADPribosyl)ation processes. Gene Ther Mol Biol Vol 1, 661-679. Quesada P (1998) Poly (ADP-ribosyl)ation as one of the molecular events that accompany mammalian spermatogenesis. Gene Ther Mol Biol Vol 1, 681-699. Haucke V (1998) Membrane biogenesis: from mechanism to disease. Gene Ther Mol Biol Vol 1, 701-706. Eckstein JW (1998) Cdc25 protein phosphatase: regulation and its role in cancer. Gene Ther Mol Biol Vol 1, 707-711. Boulikas T (1998) Nucleocytoplasmic trafficking: implications for the nuclear import of plasmid DNA during gene therapy. Gene Ther Mol Biol Vol 1, 713-740. Satyanarayana C and Horst M (1998) The ATP-driven protein translocation-motor of mitochondria. Gene Ther Mol Biol Vol 1, 741-748.

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Gene Therapy and Molecular Biology Vol 1, page 1 Gene Ther Mol Biol Vol 1, 1-172. March, 1998.

Status of gene therapy in 1997: molecular mechanisms, disease targets, and clinical applications Teni Boulikas Institute of Molecular Medical Sciences, 460 Page Mill Road, Palo Alto, California 94306 and Regulon Inc., 249 Matadero Avenue, Palo Alto, CA 94306 __________________________________________________________________________________________________ Correspondence: Teni Boulikas, Regulon Inc., 249 Matadero Avenue, Palo Alto, CA 94306, Tel (650) 813-9264, Fax: (650) 424-9594, E-mail: Boulikas@Worldnet.att.net Key words: gene therapy, gene transfer, clinical trials, cancer, immunotherapy, p53, adenovirus, retrovirus, adeno-associated virus, HIV-1, HSV-1, EBV, AIDS, tumor vaccines, IFN-!, TNF-", VEGF, retinoblastoma, purine nucleoside phosphorylase, HSVtk, E1A, E1B, Cre, LoxP, recombination, HIV vectors, liposomes, fusogenic peptides, plasmovirus, transcription factor, TIL, IL2, IL-3, IL-7, IL-12, GM-CSF, prostate cancer, p21, p16, apoptosis, Bcl-2, Bax, Bcl-xs, E2F, bystander effect, MDR1, IGF-I, antisense, triplex DNA, Parkinsonâ&#x20AC;&#x2122;s disease, lysosomal storage disease, hemophilia, cystic fibrosis, CFTR, rheumatoid arthritis, hypertension, familial hypercholesterolemia, LDL, angiopoietin, restenosis, angiogenesis, TGF-#, arterial injury, atherosclerosis, ADA deficiency, obesity, leptin.

Summary Gene therapy is a newly emerging field of biomedical research aimed at introducing therapeutically important genes into somatic cells of patients; a new and revolutionary era in molecular medicine has begun. Diseases already shown to be amenable to therapy with gene transfer in clinical trials include cancer (melanoma, breast, lymphoma, head & neck, ovarian, colon, prostate, brain, chronic myelogenous leukemia, non-small cell lung, lung adenocarcinoma, colorectal, neuroblastoma, glioma, glioblastoma, astrocytoma, and others), AIDS, cystic fibrosis, adenosine deaminase deficiency, cardiovascular diseases (restenosis, familial hypercholesterolemia, peripheral artery disease), Gaucher disease, 1-antitrypsin deficiency, rheumatoid arthritis and a few others. Human diseases expected to be the object of clinical trials include hemophilia A and B, Parkinsonâ&#x20AC;&#x2122;s disease, ocular diseases, xeroderma pigmentosum, high blood pressure, obesity and many others. The establishment of novel animal models for human disease, the discovery of new genes, and improvements in successful gene delivery open bright new prospects for molecular medicine. A wide variety of delivery vehicles for genes have been tested including murine retroviruses, recombinant adenoviral vectors, adeno-associated virus, HSV, EBV, HIV vectors, and baculovirus. Nonviral gene delivery methods use cationic or neutral liposomes, direct injection of plasmid DNA, and polymers. Various strategies to enhance efficiency of gene transfer have been tested such as fusogenic peptides in combination with liposomes, or polymers, to enhance the release of plasmid DNA from endosomes. Recombinant retroviruses stably integrate into the DNA and require host DNA synthesis; adenoviruses can infect nondividing cells but cause immune reactions leading to the elimination of therapeutically transduced cells. Adeno-associated virus (AAV) is not pathogenic, does not elicit immune responses but new strategies are required to obtain high AAV titers for preclinical and clinical studies. Wild-type AAVs integrate into chromosome 19 whereas recombinant AAVs are deprived of site-specific integration and may also persist episomally; HSV vectors can infect nonreplicating cells such as neuron cells, have a high payload capacity for foreign DNA but inflict cytotoxic effects. It seems that each delivery system will be developed independently of the others and that each will prove its strengths for specific applications. At present, retroviruses are most commonly used in human clinical trials followed by adenoviruses, cationic liposomes and AAV. Polymer-encapsulated syngeneic or allogeneic cells implanted into a tissue of a patient can be used to secrete therapeutic proteins; the method is in trials for

Boulikas: An overview on gene therapy amyotrophic lateral sclerosis using the ciliary neurotrophic factor gene, and can be extended to Factor VIII and IX for hemophilia, interleukin genes, dopamine-secreting cells to treat Parkinson's disease, nerve growth factor for Alzheimer's disease and other diseases. Ingenious techniques under development with great future prospects for human gene therapy, include the Cre-LoxP recombinase system to rid of undesirable viral DNA sequences used for gene transfer, use of tissuespecific promoters to express a gene in a particular cell type or use of ligands, such as peptides selected from random peptide libraries, recognizing surface molecules to direct the gene vehicle to a particular cell type, designing p53 “gene bombs” that explode into tumor cells, exploit the HIV-1 virus to engineer vectors for gene transfer, the combining of viruses with polymers or cationic lipids to improve gene transfer, the attachment of nuclear localization signal peptides to oligonucleotides to direct them to nuclei, and the invention of molecular switch systems allowing genes to be turned on or off at will. Although many human tumors are non- or weakly immunogenic, the immune system can be reinforced and instructed to eliminate cancer cells after transduction of patient’s cells ex vivo with the cytokine genes GM-CSF, IL-12, IL-2, IL-4, IL-7, IFN- , and TNF- , followed by cell vaccination of the patient (e.g. intradermally) to potentiate T-lymphocyte-mediated antitumor effects (cancer immunotherapy). DNA vaccination with genes encoding tumor antigens and immunotherapy with synthetic tumor peptide vaccines are further developments in this exciting field. The genes used for cancer gene therapy in human clinical trials include a number of tumor suppressor genes (p53, RB, BRCA1, E1A), antisense oncogenes (antisense c-fos, c - m y c , K-ras) , and suicide genes (HSV-tk, in combination with ganciclovir, cytosine deaminase in combination with 5-fluorocytosine). Important in gene therapy are also the genes of bcl-2, MDR-1, p21, p16, bax, bcl-xs, E2F, IGF-I VEGF, angiostatin, CFTR, LDL-R, TGF- , and leptin. Reports on human clinical trials using adenoviral and retroviral injections of the p53 gene have been very encouraging; future directions might go toward the use of genes involved in the control of tumor progression and metastasis. The molecular mechanisms of carcinogenesis have been largely elucidated and improvements in gene delivery methods are likely to lead to the final victory of the human race in the fight against cancer and other deadly diseases.

Abbreviations:

"1-AT, "1-antitrypsin 5FC, 5-fluorocytosine 5FU, 5-fluorouracil aa, amino acid AAV, adeno-associated virus Ad, adenovirus ADA, adenosine deaminase aFGF, acidic fibroblast growth factor AIDS, acquired immunodeficiency syndrome APCs, antigen-presenting cells bFGF, basic fibroblast growth factor bp, base pairs CAT, chloramphenicol acetyltransferase CD, cytosine deaminase CDKs, cyclin-dependent kinases CEA, carcinoembryonic antigen CF, cystic fibrosis CFTR, cystic fibrosis transmembrane regulator cfu, colony forming units CMV IE, cytomegalovirus immediateearly CMV, cytomegalovirus CNS, central nervous system CNTF, ciliary neurotrophic factor CTLs, cytotoxic T lymphocytes DBD, DNA-binding domain DSBs, double-strand DNA breaks EBV, Epstein-Barr virus EGF, epidermal growth factor EGFR, epidermal growth factor receptor FH, familial hypercholesterolemia GCV, ganciclovir GFP, green fluorescent protein

GM-CSF, granulocyte-macrophage colony stimulating factor HIV-1, human immunodeficiency virus type 1 HPV, human papillomavirus HSC, hematopoietic stem cells HSV, herpes simplex virus i.m., intramuscular i.p., intraperitoneal i.v., intravenous ICE, interleukin-1# converting enzyme IFN-!, interferon-! IGF-I, insulin-like growth factor I IGF-IR, insulin-like growth factor I receptor IL, interleukin IL-1#, interleukin-1# ITR, inverted terminal repeat LAK, lymphokine-activated killer cells LDL-R, low density lipoprotein receptor LTR, long terminal repeat mAb, monoclonal antibody MAR, matrix-attached region MeP-dR, 6-methylpurine-2’-deoxyriboside MHC, major histocompatibility complex MLV, murine leukemia virus MMTV, mouse mammary tumor virus Mo-MLV, Moloney murine leukemia virus MOI, multiplicity of infection MT, metallothionein Neo R, neomycin phosphotransferase NK, natural killer cells nt, nucleotides ODNs, oligodeoxynucleotides ORFs, open reading frames ORIs, origins of replication PAI-1, plasminogen activator inhibitor-1

PARP, poly(ADP-ribose) polymerase PBL, peripheral blood lymphocytes PCNA, proliferating cell nuclear antigen PDGF, platelet-derived growth factor PGF, placenta growth factor PKC, protein kinase C PMGT, particle-mediated gene transfer PNP, purine nucleoside phosphorylase PSA, prostate specific antigen PVR, proliferative vitreoretinopathy RA, rheumatoid arthritis RA-SF, rheumatoid arthritis synovial fibroblasts rAAV, recombinant adeno-associated virus RAC, recombinant advisory committee RB, retinoblastoma RLU, relative luciferase units RSV, Rous sarcoma virus s.c., subcutaneous SCID, severe combined immunodeficient SMC, smooth muscle cell TAD, transactivation domain TGF-#, transforming growth factor-# TIL, tumor-infiltrating lymphocyte TK, thymidine kinase TNF-", tumor necrosis factor " tPA, tissue plasminogen activator uPA, urokinase plasminogen activator VEGF, vascular endothelial growth factor VSMC, vascular smooth muscle cell VSMC, vascular smooth muscle cells VSV, vesicular stomatitis virus wt, wild-type

Gene Therapy and Molecular Biology Vol 1, page 3 modification of the protein and addition of a signal peptide (at the gene level) for secretion. All steps can be experimentally manipulated and improvements in each one can enormously enhance the level of expression and therapeutic index of a gene therapy approach. It has been proposed that the plasmid vector is unable to translocate to the nucleus unless complexed in the cytoplasm with nuclear proteins possessing nuclear localization signals (NLSs). NLSs are short karyophilic peptides on proteins destined to function in the nucleus used for binding to specific transporter molecules in the cytoplasm, mediating their passage through the pore complexes to the nucleus (see Boulikas, 1998, this volume). NLS are present on histones, transcription factors, nuclear enzymes, and a number of other nuclear proteins; nascent chains of DNA-binding polypeptides could bind to the supercoiled plasmid in the cytoplasm mediating its translocation to the nucleus. During delivery of foreign DNA in vivo vehicles may be attacked by macrophages, lymphocytes, or other components of the immune system and the vast majority will be cleared from blood, intracellular, or other body fluids before it is given the chance to reach the membrane of the cell target; the half-life of naked plasmids injected intravenously into animals is about 5 min (Lew et al, 1995). Cationic lipids, other than being very toxic, mediate efficient gene delivery passing through biological membranes; those lipid-DNA complexes surviving the immediate neutralization by serum proteins in the blood can reach the lung, heart and other tissues after vein or artery injection with one heart beat and transform endothelial vascular cells (reviewed by Boulikas, 1996d). A variety of viral vectors have been developed to exploit the characteristic properties of each group to maintain persistence and viral gene expression in infected cells. Retroviral vectors and AAV integrate into target chromosomes and the transgene they carry can be inactivated from position effects from chromatin surroundings. Vectors with persistence/integration functions may not result in high levels of gene delivery in vivo. Adenoviruses and retroviruses which are of the most frequently used vehicles for gene transfer can accommodate up to 7kb of total foreign DNA into their genome because of packaging limitations. This precludes their use for the transfer of large genomic regions. Transfer of intact yeast artificial chromosome (YAC) into transgenic mice will enable the analysis of large genes or multigenic loci such as human #-globin locus (reviewed by Peterson et al, 1997). A small portion of plasmid molecules crossing the cell membrane will escape degradation from nucleases in the lysosomes and become released to the cytoplasm; even a smaller portion of these molecules will enter nuclei; finally, after successfully reaching the nucleus, plasmids with therapeutic genes are usually degraded by nuclear enzymes and transgene expression is permanently lost after about 2-7 days from animal tissues following successful

I. Introduction Monumental progress in several fields including DNA replication, transcription factors and gene expression, repair, recombination, signal transduction, oncogenes and tumor suppressor genes, genome mapping and sequencing, and on the molecular basis of human disease are providing the foundation of a new era of biomedical research aimed at introducing therapeutically important genes into somatic cells of patients. The main targets of gene therapy are to repair or replace mutated genes, regulate gene expression and signal transduction, manipulate the immune system, or target malignant and other cells for destruction (reviewed by Anderson, 1992; Nowak, 1995; Boulikas, 1996a,b; Culver, 1996; Ross et al, 1996). Two main approaches have been pursued for gene transfer to somatic cells ( i ) direct gene delivery using murine retroviruses, adenoviruses, adeno-associated virus, HSV, EBV, liposomes, polymers, or direct plasmid injection (gene therapy in vivo); and ( i i ) ex vivo gene therapy involving removal of syngeneic cells from a specific organ or tumor of an individual, genetic correction of the defect in cell culture (ADA deficiency, LDL-R for FH) or transfer of a different gene (IL-2 to tumor infiltrating lymphocytes to potentiate the cytotoxicity to tumors, cytokine genes to tumor cells from a patient for cancer immunotherapy, multidrug resistance gene transfer to render bone marrow cells resistant to certain antineoplastic drugs), followed by reimplantation of the cells. The reimplanted cells produce the therapeutic protein. Several key factors or steps appear to be involved for the effective gene transfer to somatic cells in a patient or animal model: ( i ) the type of vehicle used for gene delivery (liposomes, adenoviruses, retroviruses, AAV, HSV, EBV, polymer, naked plasmid) which will determine not only the half-life in circulation, the biodistribution in tissues, and efficacy of delivery but also the route through the cell membrane and fate of the transgene in the nucleus; ( i i ) interaction of the gene-vehicle system with components in the serum or body fluids (plasma proteins, macrophages, immune response cells); ( i i i ) targeting to the cell type, organ, or tumor, and binding to the cell surface; ( i v ) port and mode of entrance to the cell (poration through the cell membrane, receptor-mediated endocytosis), ( v ) release from cytoplasmic compartments (endosomes, lysosomes), ( v i ) transport across the nuclear envelope (nuclear import); ( v i i ) type and potency of regulatory elements for driving the expression of the transferred gene in a particular cell type including DNA sequences that determine integration versus maintenance of a plasmid or recombinant virus/retrovirus as an extrachromosomal element; (viii) expression (transcription) of the transgene producing heterogeneous nuclear RNA (HnRNA) which is then (ix) spliced and processed in the nucleus to mature mRNA and is (x) exported to the cytoplasm to be (xi) translated into protein. Additional steps may include posttranslational

Boulikas: An overview on gene therapy gene delivery. During the peak of transgene expression (usually 7-48 h from injection) the transgene transcript can follow the normal fate of other nuclear transcripts when proper polyadenylation signals are provided; its processed mRNA will be exported to the cytoplasm and translated into the therapeutic protein. The choice of the appropriate delivery system for successful somatic gene transfer demands understanding of the drawbacks and advantages of each delivery system, such as limitations in the total length of the DNA that can be introduced, including the cDNA of the therapeutically important gene and control elements. Understanding the pathophysiology of the disease and the cell targets can give clues on the way of introducing the gene (i.v., i.p., intratumoral, s.c. injection) or direct the gene therapist to designing methods to target and secrete a therapeutic protein from a tissue which is not the normal site of production of a therapeutic protein. The type of control elements required for the anticipated tissue-specific expression of the construct, the presence of viral or other origins of replication as well as of the cDNA encoding the viral replication initiator protein for an episomal replication of the transgene, sequences that prompt integration and others that insulate the gene from the chromatin surroundings at the integration site, are also important for successful gene transfer. Cancer gene therapy and immunotherapy has been the first priority of human gene therapy protocols. New gene targets are being defined and new clinical protocols are being proposed and approved. Effective eradication of a great variety of tumors with drugs which inhibit angiogenesis has been extraordinarily successful on animal models and the method moves fast to clinical trials; transfer of anti-angiogenesis genes will be the next step. A number of anticancer genes are being tested in preclinical or clinical cancer trials including p53, RB, BRCA1, E1A, bcl-2, MDR-1, HER2, p21, p16, bax, bcl-xs, E2F, antisense IGF-I, antisense c-fos, antisense c-myc, antisense K-ras and the cytokine genes GM-CSF, IL-12, IL-2, IL-4, IL-7, IFN-! , and TNF-". A promising approach is transfer of the herpes simplex virus thymidine kinase (HSV-tk) gene (suicide gene) and systemic treatment with the prodrug ganciclovir which is converted by HSV-tk into a toxic drug killing dividing cells. Theoretically, expression of therapeutic genes preferentially in cancer cells could be achieved by regulatory elements from tumor-specific genes such as carcinoembryonic antigen. The first gene therapy products are expected to receive FDA approval by the year 2000; the market for gene therapy products is expected to exceed $45 billion by 2010. This article reviews the molecular mechanisms and recent developments for the gene therapy of cancer, HIV, ADA deficiency, Parkinson's disease, lysosomal storage disease, hemophilia A and B, "1-antitrypsin deficiency, cystic fibrosis, rheumatoid arthritis, hypertension, familial hypercholesterolemia, atherosclerosis/restenosis, wound healing, and obesity including the treatment of cancer and

heart diseases with angiogenesis inhibitors and gene transfer to the arterial wall. It is my intention to give a general overview rather to exhaust the field.

DIVISION ONE: GENE DELIVERY SYSTEMS AND GENE EXPRESSION II. Gene delivery using retroviruses A. Recombinant murine retroviruses The recombinant Moloney murine leukemia virus (Mo-MLV or MLV) has been extensively used for gene transfer. Retroviral vectors derived from Mo-MLV promote the efficient transfer of genes into a variety of cell types from many animal species; up to 8 kb of foreign DNA can be packaged in a retroviral vector. Recombinant retroviruses have been the most frequently used and promising vehicles for the delivery of therapeutic genes in human gene therapy protocols (Appendix 1). Retroviral vectors cause no detectable harm as they enter their target cells; the retroviral nucleic acid becomes integrated into chromosomal DNA, ensuring its long-term persistence and stable transmission to all future progeny of the transduced cell. The life cycle of the retrovirus is well understood and can be effectively manipulated to generate vectors that can be efficiently and safely packaged. An important contribution to their utility has been the development of retrovirus packaging cells, which allow the production of retroviral vectors in the absence of replication-competent virus. Recombinant retroviruses stably integrate into the DNA of actively dividing cells, requiring host DNA synthesis for this process (Miller et al, 1990). Although this is a disadvantage for targeting cells at G0, such as the totipotent bone marrow stem cells, it is a great advantage for targeting tumor cells in an organ without affecting the normal cells in the surroundings. This approach has been used to kill gliomas in rat brain tumors by injection of murine fibroblasts stably transduced with a retroviral vector expressing the HSV-tk gene (Culver et al, 1992; see below).

B. Retrovirus packaging cell lines The use of retroviral vectors in human gene therapy requires a packaging cell line which is incapable of producing replication-competent virus and which produces high titers of replication-deficient vector virus. The packaging cell lines have been stably transduced with viral genes and produce constantly viral proteins needed by viruses to package their genome. Wild-type virus can be produced through recombinational events between the helper virus and a retroviral vector. Methods are also available for generating cell lines which secrete a broad host range retrovirus vectors in the absence of helper virus. Retrovirus packaging cell lines containing the gag-pol genes from spleen necrosis virus and the env gene from 4

Gene Therapy and Molecular Biology Vol 1, page 5 spleen necrosis virus or from amphotropic murine leukemia virus on a separate vector have been used; retrovirus vectors were produced from these helper cell lines without any genetic interactions between the vectors and sequences in the helper cells (Dougherty et al, 1989). An ecotropic packaging cell line and an amphotropic packaging cell line, in which the viral gag and pol genes were on one plasmid and the viral env gene were on another plasmid have been constructed; both plasmids contained deletions of the packaging sequence and the 3' LTR; when the fragmented helper virus genomes were introduced into 3T3 cells they produced titers of retrovirus which were comparable to the titers produced from packaging cells containing the helper virus genome on a single plasmid (Markowitz et al, 1990). The pBabe retroviral vector constructs which transmit inserted genes at high titers and express them from the Mo-MLV LTR have been designed with one of four different dominantly acting selectable markers, allowing the growth of infected mammalian cells in the presence of G418, hygromycin B, bleomycin/phleomycin or puromycin, respectively. The packaging cell line, omega E, generated with separate gag/pol and ecotropic env expression constructs, was designed in conjunction with the pBabe vectors to reduce the risk of generation of wild type Mo-MLV via homologous recombination events (Morgenstern and Land, 1990).

VSV G protein was extended to a general transient transfection scheme for producing very high-titer VSV Genveloped pseudotypes from any Moloney murine leukemia-based retroviral vector (Yee et al, 1994). Pseudotyping of MuLV particles with VSV-G expressed transiently in cells producing MLV Gag and Pol proteins, has yielded vector preparations with a broader host range that could be concentrated by ultracentrifugation. For example, this technology allowed for efficient concentration of vector by ultracentrifugation to titers > 109 colony-forming units/ml and offers hope for potential use for gene transfer in vivo. Furthermore, these vectors could infect cells, such as hamster and fish cell lines, that are ordinarily resistant to infection with vectors containing the retroviral envelope protein (Burns et al, 1993). A human 293-derived retroviral packaging cell line was generated by Ory et al (1996) capable of producing high titers of recombinant Mo-MLV particles that have incorporated the VSV-G protein. This new packaging cell line may be used for direct in vivo gene transfer using retroviral vectors because the retroviral/VSV-G pseudotypes generated with these cells were significantly more resistant to human complement than commonly used amphotropic vectors. A human immunodeficiency virus type 1 (HIV-1)based retroviral vector containing the firefly luciferase reporter gene could be pseudotyped with a broad-host-range VSV envelope glycoprotein G; higher-efficiency gene transfer into CD34+ cells was achieved with a VSV-Gpseudotyped HIV-1 vector than with a vector packaged in an amphotropic envelope (Akkina et al, 1996). Because the VSV-G protein is toxic to cells when constitutively expressed, Yang et al (1995) have used steroid-inducible and tetracycline-modulated promoter systems to derive stable producer cell lines capable of substantial production of VSV-G pseudotyped MLV particles. Similarly, the toxic G protein of VSV could be induced in a cell line by the removal of tetracycline and the addition of estrogen; this cell line was transduced with a modified tTA transactivator gene engineered with the ligand-binding domain of the estrogen receptor to the carboxy terminus of the tTA transactivator; a single retroviral vector could transduce both the transactivator gene and the VSV-G protein gene controlled by the tTAinducible promoter into mammalian cells (Iida et al, 1996). The tetracycline-inducible system was modified by fusing the ligand binding domain of the estrogen receptor to the carboxy terminus of a tetracycline-regulated transactivator to regulate VSV-G expression in a tetracycline-dependent manner that could be modulated by #-estradiol in stable packaging cell lines (Chen et al, 1996).

C. Pseudotyped retroviral vectors The traditional retroviral vector enters the target cell by binding of a viral envelope glycoprotein to a cell membrane viral receptor. Coinfection of cells with a retrovirus and VSV (vesicular stomatitis virus) produces progeny virions containing the genome of one virus encapsidated by the envelope protein of the other (pseudotypes of viruses); this led to the development of pseudotyped retroviral vectors where the Moloney murine leukemia env gene product is replaced by the VSV-G protein able to interact with other membrane-bound receptors as well as with some components of the lipid bilayer (phosphatidylserine); because of the ubiquitous distribution of these membrane components pseudotyped particles display a very broad host range (Friedmann and Yee, 1995). Use of pseudotyped vectors has been a significant advancement for retroviral gene transfer. Pseudotypes of VSV and Mo-MLV, are released preferentially at early times after infection of MuLVproducing cells with VSV; at later times, after synthesis of M-MLV proteins has been inhibited by the VSV infection, neither Mo-MLV virions nor the VSV (Mo-MLV) pseudotypes are made. There appears to be a stringent requirement for recognition of the viral core by homologous envelope components for the production of VSV (M-MLV) pseudotypes (Witte and Baltimore, 1977). The finding that the G protein of vesicular stomatitis virus (VSV) can serve as the exclusive envelope protein component for one specific retroviral vector that expresses

D. Limitations and advancements using retroviral vectors Before the in vivo gene therapy with retroviruses becomes a successful reality a number of problems must 5

Boulikas: An overview on gene therapy be overcome. Despite the extensive use of retroviral vectors in gene therapy, there are still problems to be solved and there is an ultimate need for the development of new, improved retroviral vectors and packaging systems to fuel further advances in the field of human gene therapy. The principle limitation of retroviruses has been poor gene expression in vivo which has been overcome through the use of tissue-specific promoters. Use of internal ribosome entry sites from picornaviruses in retroviral vectors has provided stable expression of multiple gene enhancers (reviewed by Naviaux and Verma, 1992; Boris-Lawrie and Temin, 1993). Little is known about the factors that influence the efficiency of retroviral infection in vivo. Many commonly used experimental animal strains, such as mice, harbor endogenous C-type proviruses, some of which are expressed and have circulating antibodies against the viral envelope glycoproteins that cross-react with the Mo-MLV; the efficiency of retrovirus-mediated transfection in vivo using a variety of mouse strains was affected by humoral immune competence and interference between endogenous MLVs and exogenous recombinant Mo-MLV (Fassati et al, 1995). One of the drawbacks of retroviruses for their exploitation in gene therapy has been the low viral titers obtained, too low to achieve therapeutic levels of gene expression; methods for the efficient concentration from large volumes of supernatant and purification of amphotropic retrovirus particles have been developed in several laboratories. For example, Bowles et al (1996) have used concentration and further purification of virus particles by sucrose banding ultracentrifugation; animal studies have shown that viral transduction increased proportionally with titer of the retrovirus. Transduced cells producing retrovirus are tissueincompatible and are, therefore, expected to be attacked by the immune system; this will lead to the elimination of therapeutic cells from the body, a phenomenon markedly associated also with adenoviral gene transfer. A privileged exception are brain tumor cells expressing recombinant retrovirus which persist without immunologic rejection (Culver et al, 1992). Sodium butyrate treatment of murine retrovirus packaging cells producing a CFTR vector increased the production of the retrovirus vector between 40- and 1,000fold (Olsen and Sechelski, 1995). The Cre/LoxP recombinase strategy (see below) has been used to generate retroviral vectors that have the ability to excise themselves after inserting a gene into the genome, thereby avoiding problems encountered with conventional retrovirus vectors, such as recombination with helper viruses or transcriptional repression of transduced genes (Russ et al, 1996). Retroviral vectors with the Cre/LoxP technology have also been used to deliver the GM-CSF gene to K562 cell culture (Fernex et al, 1997), for the development of retroviral suicide vectors for gene therapy using the HSV-tk gene (Bergemann et al, 1995), and for the production of a high-titer producer cell

line containing a single LoxP site flanked by the viral LTRs (Vanin et al, 1997). Because retrovirus vectors are integrated into the genome, transcriptional repression of transduced genes will often take place from position effects exerted from neighboring chromatin domains; two matrix-attached regions (MARs), one at either flank of the transgene, are proposed here to insulating the gene in the retrovirus vector from chromatin effects at the integration site by creating an independent realm of chromatin structure harboring the transgene. MAR insulators have been used and can enhance up to 2,000-fold the expression of genes in transgenic animals and plants (McKnight et al, 1992; Breyne et al, 1992; Allen et al, 1993; Brooks et al, 1994; Thompson et al, 1994; Forrester et al, 1994).

E. Targeting of retrovirus to specific cell types A number of approaches have been directed to develop retroviral vectors that are able to target particular cell types; also efforts focus toward retroviral vectors that incorporate nonretroviral features and are tailored to desired needs for specific uses (reviewed by Vile and Russell, 1995; Gunzburg and Salmons, 1996). Ideally, therapeutic genes should be delivered only to the relevant cell type and/or expressed in this cell type. Viral and nonviral vectors can be targeted through ligandreceptor interactions. Retroviral targeting through proteasesubstrate interactions has also been described; epidermal growth factor (EGF) was fused to a retroviral envelope glycoprotein via a cleavable linker comprising a factor Xa protease recognition signal. Vector particles displaying the cleavable EGF domain could bind to EGF receptors on human cells but did not transfer their genes until they were cleaved by factor Xa protease (Nilson et al, 1996). A retroviral vector that infects human cells specifically through recognition of the low density lipoprotein receptor has been described by adding onto the ecotropic envelope protein of M-MLV a single-chain variable fragment derived from a monoclonal antibody recognizing the human LDLR; the chimeric envelope protein was used to construct a packaging cell line producing a retroviral vector capable of transfer of the lacZ gene to human cells expressing LDL-R (Somia et al, 1995).

F. Other retroviruses Viruses that contain RNA as their genetic material may be either negative- or positive-strand RNA viruses. The very large group of negative-strand RNA viruses includes some of the most serious and notorious pathogens subdivided into those with segmented RNA (influenza viruses, comprising eight separate segments of RNA and bunyaviruses containing three segments of single-stranded RNA, the large, L, the medium, M, and the small, S) and those with nonsegmented RNA (VSV, rabies, measles, 6

Gene Therapy and Molecular Biology Vol 1, page 7 Sendai, respiratory syncytial virus, Ebola viruses). Positive-strand RNA viruses include poliovirus. Cloned positive-strand poliovirus cDNA is infectious but neither isolated genome nor antigenome RNA of negative-strand viruses is infectious; this is because the negative-strand viral RNA is assembled with viral nucleoprotein into an RNP complex that becomes the template for the viral RNA-dependent RNA polymerase. Helper influenza virus-dependent procedures have been developed in which an influenza virus-like RNA molecule, containing a reporter gene, was mixed with disrupted virion core proteins to reconstitute RNP complexes in vitro which were then transfected into influenza virustransfected cells. Recombinant nucleocapsid and polymerase proteins for the unsegmented RNA viruses have also been used to produce infectious virus without help from an homologous virus using full-length cDNA clones of intracellularly transcribed antigenomes (rabies, VSV, measles, Sendai) (see Bridgen and Elliott, 1996 and the references cited therein). Plasmids containing full-length cDNA copies of the three RNA genome molecules of Bunyamwera bunyavirus and a negative-sense copy of the GFP gene, flanked by T7 promoter and hepatitis delta virus ribozyme sequences, were used to produce infectious virus particles without helper virus; these plasmids were used to transfect HeLa cells which expressed T7 RNA polymerase and recombinant Bunyamwera bunyavirus proteins by previous transfection with the appropriate plasmids; 24 h after infection about 1 in 1,000 HeLa cells displayed fluorescence indicative of transcription and replication of the reporter RNA (Bridgen and Elliott, 1996).

might also protect single-stranded DNA at the replication fork from nuclease attack, increase the rate of processivity of the viral DNA polymerase, and increase binding of NFI of the core origin of Ad5 (Cleat and Hay 1989). This protein has a size of 529 amino acids, is phosphorylated and apart from its role in DNA replication is also involved in transcription, recombination, transformation, and virus assembly (see Tucker et al 1994). Crystal structure at 2.6 A resolution of Ad-DBP shows that a 17 aa C-terminal domain hooks onto a second Ad-DBP molecule thus promoting its cooperativity during DNA binding; Ad DBP was proposed to act by forming a core around which single-stranded DNA winds (Tucker et al, 1994). Adenoviruses replicate episomally; they need to attach to the nuclear matrix of the host cell for their replication. Two adenoviral proteins have been found attached to the nuclear matrix and presumably mediating the anchorage of the adenovirus: ( i ) the E1a protein (11 kDa), a transcription and replication factor sufficient to immortalize primary rodent cells, which was crosslinked to 2+ matrix proteins with oxidation with o-phenanthroline/Cu (Chatterjee and Flint, 1986) and ( i i ) the adenovirus terminal protein (55 kDa) which is covalently attached to the 5' end of Ad DNA and initiates DNA replication; the adenovirus terminal protein mediated adenovirus anchorage to nuclear matrix was resistant to 1M guanidine extraction (Bodnar et al, 1989; Schaack et al, 1990; Fredman and Engler, 1993). Three types of internal matrix structures were recognized in HeLa cells infected with adenovirus 2; an amorphously dense region; granular regions representing virus capsid assembly structures; and filaments connecting these regions to one another and to the peripheral lamina (Zhonghe et al, 1987); the perinuclear matrix was also rearranged after adenovirus infection. Electron micrographs of thin sections through nuclei 3 of adenovirus-infected HeLa cells showed that the Hdeoxyuridine grains were located at the periphery as well as in the interior of nuclei. Simultaneous visualization of adenovirus transcription and replication showed that the two processes occurred in adjacent, yet distinct, foci throughout the interior and periphery of nuclei presumably in association with the nuclear matrix; DNA molecules were found to be displaced from the replication foci and to become spread in the surrounding nucleoplasm serving as templates for transcription (Pombo et al, 1994). Adenovirus infection provokes dramatic rearrangements to the nuclear matrix. A reorganization in both internal and peripheral NM was also observed in HeLa cells after infection with adenovirus 2 giving structures able to support the increased replication demands and capsid assembly of the virus (Zhonghe et al, 1987). Splicing of adenoviral HnRNA takes place on the nuclear matrix. All adenovirus 2 polyadenylated RNAs could be UV crosslinked to two host HnRNP proteins that are involved in the association of HnRNA to the matrix (Mariman et al, 1982).

III. Adenoviral gene delivery A. Adenovirus replication, transcription, and attachment to the nuclear matrix Before understanding the principle of adenoviral gene transfer, it is essential to comprehend the molecular events which are involved in the life cycle of the adenovirus. Adenoviruses posses a well-defined origin of replication which is stimulated by transcription factors NFI and NFIII (Hay, 1985; Pruijn et al, 1986). The transcription factor NF-I (also called CTF, CCAAT box-binding protein, or C/EBP) stimulates replication of adenovirus DNA in vitro (Pruijn et al, 1986; Jones et al, 1987; Santoro et al, 1988; Coenjaerts et al, 1991) by establishing cooperative interactions with Ad-DBP (Adenovirus DNA-binding protein) (Cleat and Hay, 1989). The transcription factor NFIII (also called Oct-1 or OTF-1), involved in the regulation of the histone H2B and immunoglobulin genes, can stimulate initiation of adenovirus DNA replication in vitro (O'Neil et al., 1988; Mul et al, 1990; Verrijzer et al, 1990; Coenjaerts et al, 1991). The adenovirus 5 protein Ad-DBP is a single-stranded DNA binding protein product of the viral E2A absolutely required for chain elongation during Ad5 DNA replication; other than facilitating unwinding of the DNA, Ad-DBP 7

Boulikas: An overview on gene therapy identified Bax as one of the seven 19k-interacting clones. The 50-78 amino acid domain of Bax contains a conserved region homologous to Bcl-2 which is able to interact specifically with either Bcl-2 or E1B. In p53 mutant cells expression of Bax induced apoptosis; inhibition of apoptosis by Bcl-2 may proceed via its ability to bind the death-promoting Bax protein (Han et al, 1996). The bax gene is upregulated by p53. Expression of p53 and of adenovirus E1A induce apoptosis (Debbas and White, 1993; Lowe and Rudley, 1993). A number of proteins when expressed at sufficient amounts block apoptosis; these include Bcl-2 and E1B 19 kDa protein of adenovirus (Debbas and White, 1993; Chiou et al, 1994). All four protein molecules act upstream of Bax which is a potent inducer of apoptosis: both the cellular Bcl-2 and the 19 kDa protein E1B of adenovirus are able to interact with Bax inhibiting its involvement in induction of apoptosis (Han et al, 1996; Figure 1). E1A acts upstream of p53 by increasing the half-life of p53 resulting in an accumulation of p53 molecules in the nucleus (Lowe and Ruley, 1993); increased levels of p53 are then believed to upregulate the bax gene. The transcription factor E2F was originally identified as an activator of the adenovirus E2 gene and is implicated in the regulation of DNA replication (Shirodkar et al., 1992). Following infection of cells with adenovirus, the DNA binding activity of E2F increases and as a consequence transcription of the E2 gene of adenovirus increases (Kovesdi et al., 1987). These changes in E2F are mediated by E1A protein of adenovirus. RB forms specific complexes with E2F keeping E2F in a form unable to upregulate its target regulatory sequences. E2F can form specific complexes also with cyclin A during S-phase in NIH 3T3 cells (Mudryj et al., 1991). Both types of complexes, E2F-RB and E2F-cyclin A, can be dissociated by the adenovirus E1A protein (Chellappan et al., 1991; Bagchi et al., 1990; reviewed by White, 1998 this volume) but also by phosphorylation of RB at G1/S causing release of E2F and stimulation in transcription of genes required for DNA replication (myc, DHFR). These events contribute to the uncontrolled proliferation of adenovirustransformed cells (Mudryj et al., 1990, 1991). Release of E2F from RB induced by E1A is critical for transformation of cells by E1A (for references see Hiebert et al, 1995).

Adenovirus establishes foci called replication centers within the nucleus, where adenoviral replication and transcription occur; although the rAAV genome was faintly detectable in a perinuclear distribution after successfully entering the cell, AAV was mobilized to the adenovirus replication centers when the cell was infected with adenovirus; thus AAV colocalizes with the adenovirus replication centers (Weitzman et al, 1996).

B. Adenovirus E1A and E1B proteins in apoptosis and control of the host cell cycle Viruses have developed strategies to shut down protein synthesis in the host and subdue its protein synthesizing machinery to produce progeny virus when infecting cells. In response, many cell types commit suicide after viral infection to protect the organism from further infection. Striking back, viruses have evolved mechanisms to prevent infected cells from perishing using mechanisms that inhibit apoptosis of the host cell; adenoviruses synthesize the 19 kDa E1B protein which has a domain similar to that of the cellular protein Bcl-2, the apoptosis inhibitor (Sarnow et al, 1982; van den Heuvel et al., 1990). p53 can be complexed with adenovirus E1B (Sarnow et al, 1982; van den Heuvel et al., 1990). Expression of the adenovirus E1A protein stimulates host DNA synthesis and induces apoptosis; on the contrary E1B 19 kDa associates with Bax protein and inhibits apoptosis (Figure 1). The E1A oncogene of adenovirus exerts its effect via p53 protein (Debbas and White, 1993; White, 1993). Indeed, expression of E1A increases the half-life of p53 resulting in accumulation of p53 molecules in adenovirus-infected cells leading to apoptosis. Although induction of host DNA synthesis by E1A provides a suitable environment for virus replication, the induction of apoptosis by the same protein impairs virus production since virus-infected cells are eliminated (see Han et al, 1996 for references). p53-deficient cells are transformed by E1A because of absence of the pathway for induction of apoptosis by p53 (Lowe et al, 1994). E1A represses HER-2/neu transcription and functions as a tumor suppressor gene in HER-2/neu-overexpressing cancer cells. Transfer the E1A gene into cancer cells that overexpress HER-2/neu is an interesting aspect of gene therapy (see E1A in gene therapy; Yu et al, 1995; Chang et al, 1996; Ueno NT et al, 1997; Rodriguez et al, 1997; Xing et al, 1997). The E1B oncogene products inhibit apoptosis induced by E1A expression thus preventing premature death of host cells during adenovirus infection. This gives an advantage to virus for its proliferation and E1B proteins (19 kDa and 55 kDa) are necessary for transformation of primary rodent cells by E1A. E1A alone is unable to transform primary rodent cells (White, 1993). The E1B 19K protein of adenovirus is the putative viral homolog of the cellular Bcl-2 protein; using the yeast two-hybrid system for the identification of proteins interacting with E1B, Han and coworkers (1996) have

C. Strategies of adenoviruses to enter the cell In order to enter the host cell the adenovirus first attaches with a high affinity to a cell surface receptor, whose nature still remains elusive, using the head domains of the protruding viral fibers; the fibronectin-binding integrin on the cell surface then associates with the penton base protein on the adenovirus triggering endocytosis of the virus particle via coated pits and coated vesicles (Svensson and Persson, 1984; Greber et al, 1996). The third step in adenovirus entry into the host cell includes 8

Gene Therapy and Molecular Biology Vol 1, page 9

Figure 1. Role of E1A and E1B19-kDa proteins of adenovirus in apoptosis.

Gene Therapy and Molecular Biology Vol 1, page 10 portion of the E1A coding sequence impairs viral replication (Gilardi et al, 1990; Rosenfeld et al, 1991).

penetration of the adenoviral particles by acid-catalyzed rupture of the endosomal membrane involving the penton protein and the integrins and allowing escape to the cytoplasmic compartment; a decrease in endosome pH during internalization expose hydrophobic domains of these adenoviral capsid proteins which permits these proteins to insert into the vesicle membrane in a fashion that ultimately disrupts its integrity (Seth et al, 1984). At the final step the adenoviral particle is attached to the cytoplasmic side of pore complexes and the DNA is released to the interior of pore annuli entering the nucleoplasm. These highly ordered processes are accompanied by losses or protease degradation of specific proteins on the viral particles; the fibers and some of the penton base complexes on the adenovirus surface are already lost during the process of endocytosis; a viral protease, L3/p23, located inside the capsid at 10 copies per virion, plays a key role in the stepwise dismantling and in the weakening of the capsid structure culminating with the release of the adenovirus DNA by degrading of the viral capsid protein VI (Greber et al, 1996). The mechanism of disruption of endosomes by the adenoviral particles has been exploited to augment efficiency of transfection with transferrinpolylysine-DNA complexes (see fusogenic peptides and Curiel et al, 1991; Cotten et al, 1992; Wagner et al, 1992b; Cristiano et al, 1993; Morishita et al, 1993; Harries et al, 1993; Curiel, 1994). To overcome one of the major limitations to the clinical utility of adenoviruses which is the low efficiency of gene transfer achieved in vivo, Arcasoy et al (1997) found that the presence of the polycations polybrene, protamine, DEAE-dextran, and poly-L-lysine significantly increased the transfection efficiency in cell culture using the lacZ gene; because the polyanion heparin did not significantly alter gene transfer efficiency, but completely abrogated the effects of polycations it supports the idea that the negative charges presented by membrane glycoproteins reduce the efficiency of adenovirus-mediated gene transfer, an obstacle that can be overcome by polycations.

E. Deletion of adenoviral DNA sequences for gene delivery First generation recombinant adenoviruses were rendered defective by deletion of sequences spanning the E1A and E1B genes; these adenoviruses expressed low levels of early and late viral genes responsible for activating destructive cellular immune responses. Further deletion of other essential genes and growth in new packaging cell lines or incorporation of temperature sensitive mutations which allow propagation of the virus in available packaging cell lines at permissive temperatures are strategies for improving the therapeutic efficacy of recombinant adenoviruses and for minimizing the immune response elicited to the host (Fisher et al, 1996). E1-defective, recombinant adenoviruses can be replication-enabled by the codelivery of a plasmid encoding the deleted E1 functions, a strategy now designated as â&#x20AC;&#x153;conditional replication-enablement system for adenovirusâ&#x20AC;? (CRESA); when the original replication-enabling plasmid was replaced by two separate plasmids that encoded the necessary E1A and E1B functions the E1-defective adenovirus could become conditionally replication-enabled by an RNA transcript encoding the required E1 functions. The RNA transcript of E1A enhanced the therapeutic efficacy of the E1-defective adenovirus: subcutaneous human tumor nodules containing a fraction of cells cotransduced with the replication-enabling RNA + DNA and an HSV-tk adenovirus were reduced to a greater extent than control nodules generated from the same fraction of cells cotransduced with the HSV-tk adenovirus and an irrelevant plasmid (Dion et al, 1996). A new type of recombinant adenovirus, (called deltarAd), deprived of all viral open reading frames and retaining only the essential cis elements (i.e., ITRs and contiguous packaging sequence), was propagated in 293 cells in the presence of E1-deleted helper virus (Fisher et al, 1996). This adenovirus was packaged as concatamers into virions and was used to deliver successfully the CFTR gene to human airway epithelial cells in culture derived from a cystic fibrosis patient. The new delivery system needs improvements in its production and purification to allow its evaluation and use in vivo.

D. Advantages and drawbacks of adenoviral vectors in gene delivery Adenoviruses possess a linear double-stranded genome which can be manipulated to accommodate up to 7.5 kb of DNA. Adenoviruses have the advantage of being able to infect nondividing cells. Other advantages are the rarity of recombination events between adenoviral vectors and the host chromosomes, the absence of induction of human malignancies by adenoviruses, and the relative safety of their use as vaccines (e.g. Ballay et al, 1985; Haj-Ahmad and Graham, 1986). For safety, replication-deficient, infectious adenoviruses are being used in somatic gene transfer; for example deletion in a portion of the E3 region of the virus permits encapsidation whereas deletion of a

F. Immune response to adenoviruses eliminate therapeutic cells Adenoviruses can achieve high levels of gene transfer (Haffe et al, 1992; Morsy et al, 1993; Herz and Gerard; Wilson, 1995; Kozarsky et al, 1996). However, the duration of transgene expression is limited ( i ) by clearance of the infected cells because of the cellular and humoral immune response (including those mediated by cytotoxic T lymphocytes) to adenoviral antigens (Yang Y

Gene Therapy and Molecular Biology Vol 1, page 11 et al, 1994, 1995) and ( i i ) by loss of adenoviral episomes in progeny cells (Feng et al, 1997). To circumvent this problem adenoviral/retroviral chimeric vectors were constructed where the nonintegrative adenoviral vector was able to induce target cells to function as transient retrovirus producer cells and the retroviral particles were able to transduce neighboring cells; thus the recombinant adenovirus became integrative via the intermediate generation of a retroviral producing cell (Feng et al, 1997). First generation adenovirus-mediated gene transfer of CFTR to the mouse lung resulted in the expression of viral proteins leading to the elimination of the therapeutic cells expressing CFTR by cellular immune responses and repopulation of the lung with nontransgene containing cells; second generation E1-deleted viruses, also crippled by a temperature sensitive mutation in the E2A gene, displayed substantially longer recombinant gene expression and induced a lower inflammatory response (Yang et al, 1994). In order to circumvent the elimination of adenovirustransduced cells by immune responses and for achieving persistence of transgene expression strategies to reduce the potential for viral gene expression have been developed; for example, an E4 modified adenovirus which was replication defective in cotton rats and displayed a reduced potential for viral gene expression in vivo was engineered (Armentano et al, 1997). Vectors containing a wild-type E4 region, E4 open reading frame 6, or a complete E4 deletion were compared in the lungs of BALB/c mice for persistence of CFTR or lacZ expression; expression was transient from the E1a promoter with all vectors but persisted from the CMV promoter only with a vector containing a wild-type E4 region; thus, transient expression from adenoviral vectors may result from the down-regulation of a promoter and not necessarily from immune response-related factors (Armentano et al, 1997). The elimination of therapeutically important cells from the body after recombinant adenovirus-mediated delivery seems to be a great limiting factor for the use of adenoviruses in long-term gene therapy (Dai et al, 1995). This problem can be partially circumvented by daily administration of the immunosupressant cyclosporin A prohibiting the elimination of virally-transduced cell by activated T lymphocytes (Fang et al, 1995). A different approach to suppress elimination of therapeuticallytransduced cells after intra-articular delivery of genes to treat RA is by pretreatment of the joints with the anti-T cell receptor monoclonal antibody H57, a treatment which resulted in a significant reduction in lymphocytic infiltration and a persistence of transgene expression (Sawchuk et al, 1996). The prokaryotic Cre-LoxP recombination system was adapted to generate recombinant adenoviruses with extended deletions in the viral genome in order to minimize expression of immunogenic and/or cytotoxic viral proteins. An adenovirus was produced with a 25-kb deletion that lacked E1, E2, E3, and late gene expression; this vector exhibited viral titers similar to those achieved with first-generation (E1a-deleted) vectors which was

efficient for gene transfer to cell culture but gene expression declined to undetectable levels much more rapidly than that sustained from first-generation vectors. Vectors deleted only at E1a were sustaining a better reporter gene expression because of their ability to replicate (Lieber et al, 1996). A clinical protocol proposed recently for the therapy of amyotrophic lateral sclerosis uses a semipermeable membrane to enclose the ex vivo modified xenogenic BKH cells which is implanted intrathecally to provide human ciliary neurotrophic factor; the membrane prevents immunologic rejection of the cells interposing a virus impermeable barrier between the transduced cells and the host (Deglon et al, 1996; Pochon et al, 1996); the method has been applied before for cross-species transplantation of a polymer-encapsulated dopamine-secreting cell line to treat Parkinson's disease and for the delivery of nerve growth factor in rat and primate models of the Alzheimer's disease (Kordower et al, 1994; see Pochon et al, 1996 for more references). Evidently, similar approaches could be used to protect adenovirus- and retrovirus-transduced syngeneic cells from immunologic rejection provided that the therapeutic protein is secreted. A new area of investigation is directed toward surface modification of recombinant adenoviruses to render them safer and to minimize the strong immune responses against the virus and virus-infected cells; to this end Fender et al (1997) proposed a dodecahedron made of adenovirus pentons or penton bases and having only one or two adenovirus proteins instead of the 11 contained in an adenovirus virion; the penton is a complex of two oligomeric proteins, a penton base and fiber, involved in the cell attachment, internalization, and liberation of virus from endosomes. It is certain that great improvements in adenoviral gene delivery will solve many of the current problems and permit a higher therapeutic efficacy in the near future.

G. Examples of adenoviral gene transfer Recombinant adenovirus vectors have been used: for the transfer of factor IX gene in hemophilia B dogs via vein injection (Kay et al, 1994) and in mice (Smith et al, 1993); for the transfer of genes into neurons and glia in the brain (le Gal la Salle, 1993); for the transfer of the gene of ornithine transcarmylase in deficient mouse and human hepatocytes (Morsy et al, 1993); for the transfer of the VLDL receptor gene for treatment of familial hypercholesterolaemia in the mouse model (Kozarsky et al, 1996); for the transfer of low density lipoprotein receptor gene in normal mice (Herz and Gerard, 1993); and for the ex vivo transduction of T cells from ADA-deficient patients (Blaese et al, 1995; Bordignon et al, 1995). The adenovirus major late promoter was linked to a human "1antitrypsin gene for its transfer to lung epithelia of cotton rat respiratory pathway as a model for the treatment of "1antitrypsin deficiency; both in vitro and in vivo infections

Gene Therapy and Molecular Biology Vol 1, page 12

Figure 2. Localization of a recombinant adenoviral vector carrying 6.3 kb of dystrophin cDNA by in situ PCR following intramuscular injection to immunosuppressed mdx mice. Shown are transverse cryostat sections of mdx tibialis anterior muscle. Panel A shows a strong in situ hybridization signal (an E4 adenoviral sequence was amplified and an E4 probe was used) in myonuclei of an immunosuppressed animal injected with E1, E3-deleted adenovirus at 30 days postinjection (magnification 650x). Panel B was produced without Taq polymerase during PCR as a negative control. Panel C shows an uninjected muscle processed as described in panel A showing no hybridization signal. From Zhao JE, Lochumuller H, Nalbantoglu J, Allen C, Prescott S, Massie B, Karpati G (1 9 9 7 ) Study of adenovirus-mediated dystrophin minigene transfer to skeletal muscle by combined microscopic display of adenoviral DNA and dystrophin. Hum Gene Ther 8, 1565-1573. With kind permission of the authors (George Karpati, Montreal Neurological Institute, Canada) and Mary Ann Liebert, Inc.

The maximum number of fibers containing recombinant adenovirus was maintained until 60 days in immunosuppressed mice but for only 10 days in immunocompetent animals. Thus, optimization of immunosuppression could assure successful long term dystrophin gene transfer for gene therapy of Duchenne muscular dystrophy (Zhao et al, 1997). A number of RAC-approved protocols for gene transfer to humans use recombinant adenoviruses (Appendix 1, protocols 118-157). Genes transferred to patients with recombinant adenoviruses include p53 (#130, 131, 147, 148, 152-156), RB (#140), CFTR (#118-123, 125, 128, 129), HSV-tk (126, 127, 132, 136, 139, 141, 143, 145, 146), cytosine deaminase (#134, 151), VEGF (#157), IL-2 (#135), GM-CSF (#149, 150), anti-erbB-2 single chain antibody (#133), ornithine transcarbamylase (#137), and GP100 melanoma antigen (#142).

have shown production and secretion of "1-antitrypsin by the lung cells (Rosenfeld et al, 1991). A transductional preference of adenovirus-polylysineDNA complexes and E1A/B-deleted replication-deficient adenoviruses was demonstrated for the prostate carcinoma cell lines DU145, LNCaP, and PC-3 over primary human bone marrow cells and the leukemia cell line KG-1; this finding led to a strategy to purge bone marrow of a specific subset of prostate carcinoma cells (Kim et al, 1997). Figure 2 shows the localization of a recombinant adenoviral vector carrying 6.3 kb of dystrophin cDNA, driven by the CMV promoter, by in situ PCR following intramuscular injection to immunosuppressed mdx mice. Figure 3. shows a comparison of the persistence of dystrophin expression and adenoviral genomes in immunosuppressed versus immunocompetent mdx mice. 12

Gene Therapy and Molecular Biology Vol 1, page 13

Figure 3. Comparison of the persistence of dystrophin expression and adenoviral genomes in immunosuppressed versus immunocompetent mdx mice. Shown are combined dystrophin immunostaining and in situ PCR in tibialis anterior muscles of mdx mice at 10 days (A and C) and 60 days (B and D) postinjection. In A and B, FK506 was used as an immunosuppressant, whereas in C and D no immunosuppression was employed. At 10 days there was no significant difference in adenovirus positive nuclei (arrows) fibers between the immunosuppressed and the immunocompetent groups. At 60 days, however, there was a dramatic decline in the number of positive nuclei in the immunocompetent muscle. Magnification 650X. From Zhao JE, Lochumuller H, Nalbantoglu J, Allen C, Prescott S, Massie B, Karpati G (1 9 9 7 ) Study of adenovirus-mediated dystrophin minigene transfer to skeletal muscle by combined microscopic display of adenoviral DNA and dystrophin. Hum Gene Ther 8, 1565-1573. With kind permission of the authors (George Karpati, Montreal Neurological Institute, Canada) and Mary Ann Liebert, Inc.

IV. Gene delivery with AdenoAssociated Virus (AAV)

The replication of the AAV is dependent on two copies of a 145-bp inverted terminal repeat (ITR) sequence that flanks the AAV genome which is the primary cis-acting element required for productive infection and the generation of recombinant AAV (rAAV) vectors. In the absence of helper virus, the AAV particle can penetrate cells and find its way to the cell nucleus where the linear genome is uncoated and becomes integrated at a specific site on chromosome 19q13.3; several copies of AAV may integrate in tandem arrays. Thus, the AAV establishes a latent infection; the integrated viral genome can be activated and rescued by superinfection with helper virus (either adenovirus or any type of herpes virus). Inverted repeats at the ends of the viral DNA serve for the integration appearing near the junctions with cellular DNA sequences (Bohenzky et al, 1988).

A. Replication of AAV and rAAV: the role of the inverted terminal repeats AAVs are replication-defective parvoviruses, not associated with any human disease (nonpathogenic), requiring cotransfection with a helper virus to produce infectious virus particles; they can replicate in cell culture only in the presence of coinfection with adenovirus or herpes virus. Five serotypes of distinct AAV isolates have been recovered from human and other primates. AAV infections in humans are asymptomatic acquired with other viral infections such as adenovirus or HSV infections; 8090% of adults are seropositive for antibodies against AAV (for references see Clark et al, 1995; Berns and Linden, 1995). 13

Boulikas: An overview on gene therapy CFTR expression and ITRs needs to be kept under 500 bp (Dong et al, 1996). Similar results were reported by Dong et al (1996) who have estimated that the optimal size of AAV vector is between 4.1 and 4.9 kb; the packaging efficiencies were sharply reduced above 5.2 kb and below 4.1 kb; two copies of the vector were packaged into each virion when vectors of 2.2-2.5 kb were provided.

Adenovirus establishes foci called replication centers within the nucleus, where adenoviral replication and transcription occur; AAV was colocalized with the adenovirus replication centers using in situ hybridization and immunocytochemistry; AAV may, thus, utilize adenovirus and cellular proteins for its own replication; the rAAV genome was faintly detectable in a perinuclear distribution after successfully entering the cell; however, rAAV was mobilized to replication centers when the cell was subsequently infected with adenovirus (Weitzman et al, 1996). Xiao et al (1997) have engineered the pDD-2 plasmid containing two copies of the D element, a unique sequence adjacent to the AAV nicking site, flanking a single ITR (a total of only 165 bp of AAV sequence); this modified hairpin was sufficient to sustain replication of the plasmid vector when Rep and adenovirus helper functions were supplied in trans. This plasmid has a significant prospect in gene transfer because is replicated more efficiently than infectious AAV clones; as a prelude to its replication the input circular plasmid was converted into a linear substrate by resolution of the AAV terminal repeat through a Holliday-like structure, a process most likely mediated by host factors. Linear monomer, dimer, and other highermolecular-weight replicative intermediates were generated during the replication of pDD-2, a feature characteristic of AAV replication. The replicative intermediates of this plasmid substrate were competent for AAV DNA replication, encapsidation, infection, integration, and subsequent rescue from the chromosome when superinfected with Ad and wild-type AAV (Xiao et al, 1997). The elucidation of the important role of this 165-bp ITR sequence for AAV replication and the entire life cycle invigorates the important role of inverted repeats at the origin of replication not only of viruses but also of cellular origins of replication (Boulikas, 1996e).

C. Integration of wtAAV but not of rAAV is site-specific Wild-type AAV is able to undergo targeted integration on chromosome 19 after infection in 15 out of 22 clones examined (Kotin et al, 1990, 1992). Of 51 integrations examined by fluorescence in situ hybridization (FISH) 48 (94%) were to chromosome 19 after infection of IB3-1 bronchial epithelial cells with wild-type AAV (Kearns et al, 1996). Site-specific integration has been reported for other viruses including avian leukosis virus (ALV) integrating adjacent to cellular oncogenes in tumors; however, the mechanism of ALV integration involves a process of selection of cells able to form tumors by overexpression of the oncogene due to virus integration rather than exclusive integration of the ALV at unique sites of the genome (Hayward et al, 1981). RSV also appears to be integrated at a limited number of sites (Shih et al, 1988). Adenovirus integration, a more rare event compared to the majority of episomal molecules, may also occur at a number of preferred sites (Jessberger et al, 1989). A larger number of recombinase molecules than those known today may be present in mammalian cell nuclei and promote site-specific integration and recombination events. Although the human wild-type AAV (wtAAV) is unique in its ability to target viral integration to a specific site on chromosome 19, the recombinant AAV (rAAV) vectors have lost the site-specific integration and targeting ability; furthermore, rAAVs have incapacitated ability to integrate, and can be found as episomes. When wtAAV-2 was used to infect IB3-1 bronchial epithelial cells all metaphase spreads examined by fluorescence in situ hybridization (FISH) had integrated copies and 94% of the integrations were to chromosome 19; furthermore, 36 of 56 metaphase spreads had a single copy of wtAAV integrated and 20 of 56 showed two sites within chromosome 19 (Kearns et al, 1996). On the contrary, when a recombinant AAV containing the CFTR cDNA was used to infect the same cells, examination of 67 metaphase chromosome spreads identified four integrations (only 6% of total) to different chromosomes. No integration was to chromosome 19. When these studies were repeated on the A35 epithelial cell line selected for stable CFTR expression, the episomal AAV-CFTR sequences were abundant in the low molecular weight DNA fraction (Kearns et al, 1996). Yang et al (1997) have cloned over 40 AAV and rAAV integration junctions to determine the terminal-repeat

B. Packaging capabilities of AAVs AAVs posses a 4.7 kb single-stranded DNA genome. Hermonat et al (1997) have examined the maximum amount of DNA which can be inserted into the wild-type AAV genome without compromising packaging into an infectious virus particle; the maximum effective packaging capacity of AAV, examined as increments of 100 bp ligated at map unit 96 of AAV, is approximately 900 bp larger than wild type. Thus, wtAAV therapy vectors can be generated carrying a foreign gene of 900 bp or less with the advantages of wtAAV such as the ease in which high titers of infectious virus can be generated and the ability to specifically integrate in chromosome 19. On the contrary, the payload capacity of recombinant AAV, which has been deprived of its viral genes and bears only the ITRs is in the order of 4.5-4.7 kb; this means that a cDNA up to this size can be inserted into a rAAV; for example the size of the CFTR cDNA is 4.5 kb and thus, the combined length of the promoter that drives

Gene Therapy and Molecular Biology Vol 1, page 15 sequences that mediate integration. These studies have shown that in both immortalized and normal diploid human cells, wt AAV targeted integration to chromosome 19 in head-to-tail tandem arrays; the majority of the junction sequences were involving incomplete copies of the AAV inverted terminal repeats (ITRs); inversions of genomic and/or viral DNA sequences at the wt integration site took place. The viral integration event was found to be mediated by terminal repeat hairpin structures and cellular recombination pathways. In contrast, rAAV provirus integrated on chromosome 2 and at the same locus in two independent cell lines, in both the flip and flop orientations; genomic rearrangements took place at the integration site of rAAV, mainly involving deletions and/or rearrangement-translocations. Similar data were reported by Rutledge and Russell (1997): recombinant AAV vectors were found to be integrated by nonhomologous recombination as singlecopy proviruses in HeLa cells and at random chromosomal locations; the recombination junctions were scattered throughout the vector terminal repeats with no apparent site specificity; the flanking HeLa DNA at integration sites was not homologous to AAV or to the site-specific integration locus of wild-type AAV. Furthermore, vector proviruses with nearly intact terminal repeats were excised from the genomic HeLa DNA and were amplified after infection of cells with wild-type AAV and adenovirus. The integration patterns of four recombinant AAV-2 genomes in individual clonal isolates of the human nasopharyngeal carcinoma cell line (KB) were different; the difference between the recombinant AAV-2 genomes were in the combinations of the genes for resistance to tetracycline, to neomycin, to ampicillin, with the genes for AAV replication, and the AAV capsid genes. None of the KB cell clones examined had the proviral genome covalently linked to the specific-site of integration of the wt AAV on chromosome 19 (Ponnazhagan et al, 1997a,b).

The difficulties in developing packaging cell lines for AAV relate to low levels of rep gene expression from the AAV-p5 promoter and to the propensity of Rep proteins to suppress continued growth of immortalized cell lines; expression of AAV rep under control of the LTR of the human HIV together with the development of cell populations containing rescuable AAV recombinant genomes increased 50-fold the packaging efficiency of AAV vectors (Flotte et al, 1995). After infection of cell cultures with recombinant AAV there is a decline in the percentage of cells expressing the transferred gene with time in culture. This decline was associated with ongoing losses of vector genomes (Malik et al, 1997). For example, transfer to cultures of K562 human erythroleukemia cells of a truncated rat nerve growth factor receptor (tNGFR) cDNA as a cell surface reporter under control of the LTR of the Moloney murine leukemia virus showed that about 30% of cells expressed tNGFR on the surface early after transduction at a multiplicity of infection (MOI) of 13 infectious units (IU), which declined to 3% over 1 month of culture. At an MOI of 130 IU, nearly all cells expressed tNGFR immediately and the proportion of cells expressing tNGFR declined to 62% over 2 months of culture (Malik et al, 1997). Another obstacle of rAAV vectors is the low rate of integration of rAAV into the host genome (which can be improved at high MOI). The efficiency of integration was about 2% at low MOI (1.3 IU) and increased to at about 49% at an MOI of 130 (Malik et al, 1997).

E. Advantages using AAV and improvements in AAV gene delivery AAV does not elicit an immune reaction and is a nonpathogenic virus to humans. AAV contains normally a single-stranded copy of its genome. Transduction with AAV can be enhanced in the presence of adenovirus gene products through the formation of double stranded, nonintegrated AAV genomes. AAV has been reported to have advantages over other viruses for gene transfer to hematopoietic stem cells due to their high titers and relative lack of dependence on cell cycle for target cell integration. A robust CMV/LacZ reporter gene expression in primary human CD34+CD2$ progenitor cells induced to undergo T-cell differentiation was obtained without toxicity or alteration in the pattern of T-cell differentiation. 70% to 80% of the cells isolated from either adult bone marrow or umbilical cord blood were efficiently transduced with AAV; however, the expression was transient without integration; this limits the potential use of AAV in gene therapy strategies for diseases such as AIDS (Gardner et al, 1997). Gene transduction by AAV vectors in cell culture can be stimulated over 100-fold by treatment of the target cells with agents that affect DNA metabolism, such as irradiation or topoisomerase inhibitors (Russell et al, 1995); great improvements in transduction efficiency can also be achieved in vivo: previous ! -irradiation increased

D. Drawbacks of AAV in gene therapy and their remedy Gene transfer with AAV vectors has typically been low. Difficulties in generating recombinant virions on a large scale sufficient for preclinical and clinical trials and in obtaining high-titer virus stocks after the initial transfection into producer cells is a limiting factor for the widespread usage of AAV vectors; this obstacle is expected to be overcome in the near future. The high viral titers required for preclinical and clinical studies have been achieved by a new strategy developed by Tamayose et al (1996); AAV vector particles in cell lysates could be concentrated by sulfonated cellulose column chromatography to a titer higher than 10 8 cfu/ml or 5 x 1010 particles/ml. A method for transfecting cells at extremely high efficiency with a rAAV vector and complementation plasmid while simultaneously infecting those cells with replication competent adenovirus using adenovirus-polylysine-DNA complexes has been developed by Mamounas et al (1995). 15

Boulikas: An overview on gene therapy the transduction rate in mouse liver by up to 900-fold, and the topoisomerase inhibitor etoposide increased transduction by about 20-fold after direct liver injection or after systemic delivery via tail vein injection; up to 3% of hepatocytes could be transduced after a single systemic vector injection (Koeberl et al, 1997). This is a significant advantage compared to stealth liposomes which , although concentrating in the liver, spleen and tumors can transduce Kupffer cells but not hepatocytes after systemic delivery (Martin and Boulikas, 1998, following article). A combination of the adenovirus-5 capsid protein or the Fiber protein of adenovirus with liposomes, termed adenosomes (adenovirus protein-cationic liposome complexes) improved the efficiency of gene transfer. This complex was able to mediate efficient transfer of a AAV/CMV-LacZ construct to endothelial cells (Zhou et al, 1995). Clark et al (1996) have developed a sensitive assay system to determine infectivity of AAV vectors based on the replication of input rAAV genomes rather than transgene expression which depends on the type of promoter which drives the foreign gene; this system uses a cell line that expresses AAV helper functions (rep and cap) upon induction by adenovirus infection.

degenerative model of Parkinson's disease (Mandel et al, 1997). AAV has also been used for the transduction of the mouse liver in vivo with Factor IX cDNA as a prelude to treatment of hemophiliacs (Snyder et al, 1997; Herzog et al, 1997) and for the human intratracheal instillation of CFTR cDNA into neonatal New Zealand white rabbits (Rubenstein et al, 1997), and to the lungs of rhesus macaques without eliciting inflammation (Conrad et al, 1996). AAV has also been used for the transfer of the human multidrug resistance gene (hMDR1) cDNA to NIH-3T3 cells followed by selection of successfully transfected cells based on the drug-resistant phenotype conferred by the Pglycoprotein efflux pump (see below and Lee et al, 1997, this volume); integration of MDR1 sequences into the host cell genome was demonstrated by fluorescent in situ hybridization (FISH) but also the persistence of nonintegrated AAV-MDR1 episomal plasmids (Baudard et al, 1996). Introduction of a human globin gene into murine hematopoietic bone marrow cells ex vivo with a recombinant AAV vector followed by transplantation of these cells into lethally irradiated congenic mice sustained a long-term repopulating ability: human globin gene sequences were detected in the bone marrow and spleen in primary recipient mice for at least 6 months. Kessler et al (1996) have shown that following a single intramuscular administration of a recombinant adeno-associated virus (rAAV) vector, carrying either the lacZ or the human erythropoietin gene into adult BALB/c mice leads to the local production of the foreign protein in the muscle for at least 32 weeks; furthermore, human erythropoietin was secreted and stimulated red blood cell production in the mouse for up to 40 weeks. This finding was extended by Fisher et al (1997) who arrived to the unexpected finding that intramuscular injection of highly purified recombinant AAV can sustain a high level of transgene expression in the absence of adenovirus after direct injection to the muscle in mice (Figure 4); this expands the potential of AAV for the treatment of inherited and acquired diseases. Using this approach no humoral or cellular immune responses were elicited after transfer of lacZ against the neoantigenic E. coli #-galactosidase. The rAAV genome was integrated at single sites as head-to-tail concatamers into nuclei of differentiated muscle fibers. Transfer of the lacZ gene using a highly purified preparation of AAV which was injected into the skeletal muscle of adult mice in the presence of E2a-deleted adenovirus to enhance transduction followed by direct visualization of the #-galactosidase by X-gal histochemistry revealed high transduction of muscle fibers by day 17 associated with inflammation (Fig 4a and b). Animals that received the same AAVlacZ in the absence of adenovirus demonstrated higher levels of transduction that persisted for 240 days (Fig 4c-h).

F. Examples using AAV for gene transfer AAV will infect a broad number of mammalian cell lines and has been used as a cloning vector to transduce the NeoR gene into mammalian tissue culture cells (Hermonat and Muzyczka 1984). Antisense AAV vectors have been used to inhibit HIV replication (Chatterjee et al, 1992), and to correct Fanconi's anemia in human hematopoietic cells (Walsh et al, 1994). AAVs transduce preferentially cells in S phase; topoisomerase inhibitors increase transduction efficiency (Russell et al, 1995). Using AAV, the genomic copy of a normal human #globin gene under control of the DNase l-hypersensitive site 2 (HS-2) from the locus control region was expressed in K562 human erythroleukemia cells, which normally lack the #-globin gene; following selection with G418 by virtue of the neo-resistance function which was provided in the rAAV vector, stable integration of the exogenous #globin allele was determined (Zhou et al, 1996). Similar data were reported by Einerhand et al (1995) transferring a recombinant AAV-vector containing a human #-globin gene together with the DNase1 hypersensitive sites 4, 3 and 2 of the human #-globin locus control region as an approach for the gene therapy of #-thalassemia and sickle cell anemia. The vector replicated to high titers and could efficiently transduce hematopoietic stem cells isolated from patients. In order to treat sickle cell anemia Lubovy et al (1996) have transferred lacZ with a recombinant AAV vector and stably transduced hematopoietic stem cells purified from normal and homozygous sickle cell anemia patients. AAV was able to promote delivery of functional levels of glial cell line-derived neurotrophic factor (GDNF), in a 16

Gene Therapy and Molecular Biology Vol 1, page 17

Figure 4. Purified recombinant AAV-mediated lacZ gene transfer to the muscle in adult mice sustains a high level of expression and is inflammation-free. Purified AAVlacZ (1x109 genomes in 25 µl) was injected into the tibialis anterior of 5-weekold C57BL/6 mice and tissue was harvested at days 3 (c ), 17 (d), 30 (e ), 64 (f ), and 180 (g , h ) post-injection and analyzed by Xgal histochemistry. Samples of AAVlacZ (1x10 9 genomes in 25 µl) were also supplemented with an E2a mutant adenovirus dl802 (5x10 10 A260 particles) just prior to injection and tissue was harvested at days 3 (a) and 17 (b ) post-injection. Magnification: a-g, X10; h, X5. Purified AAVlacZ (175 µl , 1x10 12 genomes/ml) was injected into the tibialis anterior of a male rhesus monkey. Biopsies were taken 14 days post-injection and frozen sections were cut and stained for #-galactosidase activity (i and j ); magnification: I, X5; j, X10. k and l : rAAV vector expressing human #-glucoronidase was injected into the tibialis anterior of 5-week-old C57BL/6 mice (1x10 9 genomes in 25 µl). 30 days post-injection the muscle was harvested and frozen sections were cut and stained for #glucoronidase. From Fisher KJ, Jooss K, Alston J, Yang Y, Haecker SE, High K, Pathak R, Raper SE, Wilson JM (1 9 9 7 ) Recombinant adeno-associated virus for muscle directed gene therapy. Nat Med 3, 306-312. Reproduced with the kind permission of the authors and Nature America, Inc.

V. Herpes Simplex Virus-1 (HSV-1) and miniviral vectors

AAV-mediated delivery of the lacZ gene by direct injection to brain tumors which were induced from human glioma cells in nude mice showed that 30-40% of the cells along the needle track expressed#-galactosidase; subsequent delivery of the HSV-tk/IL-2 genes to these tumors with AAV and administration of GCV to the animals for 6 days resulted in a 35-fold reduction in the mean volume of tumors compared with controls by a significant contribution from the bystander effect (Okada et al, 1996). A phase I clinical trial for CF is being conducted at Johns Hopkins Hospital using AAV (see Kearns et al, 1996, and protocols #165, 166 in Appendix 1).

HSV-1 has a capacity of inserting up to 30 kb of exogenous DNA which is a clear advantage over the adenovirus (up to 7.5 kb of exogenous DNA). High titer viral stocks can be prepared from HSV-1. HSV-1 also displays a wide range of host cells and can infect nonreplicating cells such as neuron cells in which the vectors can be maintained indefinitely in a latent state. However, infection with HSV-1 is cytotoxic to cells because of residual viral proteins produced by the virus. Strategies to circumvent this drawback led to the development of viral vectors with a very large capacity for insertion (almost as large as the size of the virus) which depend on defective helper virus for replication and 17

Boulikas: An overview on gene therapy packaging into infectious virions (see below). A miniviral vector can combine the advantage of cloning the gene in bacterial plasmids, the high efficiency of virus-mediated gene transfer, and the possibility to transfer large genomic DNA fragments including far upstream, downstream and intronic regulatory elements. The HSV-1 genome is a 152 kb double-stranded DNA containing three origins of replication and encoding at least 72 unique proteins; it consists of a unique long segment replicated from oriL and two repeats flanking the unique segment each replicated from oriS. Spaete and Frenkel (1982) have constructed plasmids containing the lytic viral origin of replication, foreign DNA inserts, and the terminal packaging signal sequences; in the presence of a wild-type helper virus such an amplicon was amplified into multimeric tandemly-repeated forms of the original vector by rolling-circle replication and was packaged into infectious HSV virions (Spaete and Frenkel, 1982). However, the helper virus caused death of the infected cells due to lytic replication and this system is not amenable to gene therapy. To circumvent this bottleneck two strategies have been developed leading to replication-defective helper HSV:( i ) a temperature-sensitive system permitted production of virion stocks at 31o C whereas infection of cells at 37 o C caused inactivation of the helper virus which was incapable of entering the lytic cycle and allowed delivery of the miniviral vector to the target cell without causing its death. ( i i ) In a different system, the immediately early gene IE3 was deleted from the helper virus; IE3 encodes for a protein (ICP4) essential for early and late viral gene expression and replication; the helper cell line used for packaging had a genomic insertion of the IE3 gene of HSV which was functionally expressed allowing for complementation and for lytic infection using the IE3defective HSV virus (DeLuca and Schaffer, 1987; Geller and Freese, 1990). Two types of viral vectors have been used for gene transfer to cancer cells: replication-incompetent vectors expressing a gene product that leads to the destruction of the tumor or replication-competent vectors that are inherently cytotoxic to the tumor cells. In order to combine the two modes of action Miyatake et al (1997) used a defective HSV vector that consisted of a defective particle, containing tandem repeats of the HSV-tk gene, and a replication-competent, non-neurovirulent HSV mutant as a helper virus. When glioma GL261 cells were infected with the tk-defective vector/helper virus the HSVTK activity was significantly higher than that in helper virus-infected cells which contained a single copy of HSVtk; subcutaneous injection of these cells to C57BL/6 mice inducing gliomas led to a significant decrease in tumor size after GCV treatment. An HSV-1 vector containing a 6.8-kb fragment of the rat tyrosine hydroxylase promoter (pTHlac) supported a seven- to 20-fold increase in reporter gene expression in catecholaminergic cell lines compared to noncatecholaminergic cell lines. Furthermore, 4 days after stereotactic

injection into the midbrain of adult rats and for a duration of 6 weeks, pTHlac supported a 10-fold targeting of #galactosidase expression to tyrosine hydroxylaseexpressing neurons in the substantia nigra pars compacta compared with pHSVlac; this long term expression was significant compared to that from pHSVlac which decreased approximately 30-fold between 4 days and 6 weeks after gene transfer (Song et al, 1997); this study also shows the importance of large control regions in the order of 7 kb in sustaining cell type-correct gene expression, something feasible with HSV and liposomes but nor with recombinant retrovirus, adenovirus, or AAV.

VI. HIV vectors for gene transfer Recent studies have succeeded in exploiting the deadly HIV-1 virus, after crippling some functions, as a gene delivery vehicle. An advantage of HIV vectors has been the broad range of tissues and cell types they can transduce, a property granted because lentiviral vectors are pseudotyped with vesicular stomatitis virus G glycoprotein. Human lentiviral (HIV)-based vectors can transduce non-dividing cells in vitro and deliver genes in vivo; expression of transgenes in the brain has been detected for more than six months. HIV vectors have been also used to introduce genes directly into liver and muscle; 3-4% of the total liver tissue was transduced by a single injection of 1-3 x 10 7 infectious units (I.U.) of recombinant HIV with no inflammation or recruitment of lymphocytes at the site of injection. Whereas expression of green fluorescent protein (GFP), used as a surrogate for therapeutic protein, was observed for more than 22 weeks in the liver and for over 8 weeks in the muscle using lentiviral vectors, little or no GFP could be detected in liver or muscle transduced with the Moloney murine leukemia virus (Mo-MLV), a prototypic retroviral vector (Kafri et al, 1997). The development of a stable noninfectious HIV-1 packaging cell line capable of generating high-titer HIV-1 vectors is another important step towards use of HIV vectors in gene therapy (Corbeau et al, 1996). A hybrid murine leukemia virus-based vector containing U3 and R sequences from HIV-1 in place of the MLV U3 and R regions gave single transcriptional unit retroviral vectors under the control of Tat; this vector has advantages for anti-HIV gene therapy (Cannon et al, 1996). Although replication-incompetent HIV vectors displayed a strict CD4+ T cell tropism for gene transfer, a feature important for AIDS therapy, it was thought to preclude HIV-based vectors for other gene transfer applications; a two-step gene transfer system, however, was developed to expand the host range of the HIV vector: in the first step, the CD4 gene was introduced into target cells using a replication-defective adenoviral vector; in the second step the CD4-transfected cells were incubated with HIV vectors which resulted in stable integration and HIVmediated gene transfer (Miyake et al, 1996). An HIV multiply attenuated vector in which the virulence genes env, vif, vpr, vpu, and nef were deleted 18

Gene Therapy and Molecular Biology Vol 1, page 19 was able to deliver genes in vivo into adult neurons (Zufferey et al, 1997). HIV-mediated gene transfer was used to transfer the GFP gene under control of CMV to retinal cells by injection into the subretinal space of eyes in rats; the GFP gene was efficiently expressed in both photoreceptor cells and retinal pigment epithelium; predominant expression in photoreceptor cells was achieved using the rhodopsin promoter. The transduction efficiency was high and photoreceptor cells in >80% of the area of whole retina were expressing GFP (Miyoshi et al, 1997).

DMTAP: 1,2-dimyristoyl-3-trimethylammonium propane DOGS: Dioctadecylamidoglycylspermine (Transfectam, Promega) DOPE: dioleyl phosphatidylethanolamine DOSPA: 2,3-dioleoyloxy-N-[2(sperminecarboxamido)ethyl]-N,Ndimethyl -1-propanaminium trifluoroacetate DOTAP: N-(1-(2,3-dioleoyloxy)propyl)-N,N,Ntrimethylammonium chloride DOTMA: N-[1-(2,3-dioleyloxy) propyl]-n,n,n-trimethylammonium chloride DPTAP: 1,2- dipalmitoyl-3-trimethylammonium propane DSTAP: 1,2-disteroyl-3-trimethylammonium propane Lipofectin: DOTMA:DOPE 1:1 (GIBCO BRL)

VII. Epstein-Barr virus (EBV) and baculovirus vectors

A. Immune responses and toxicity of cationic lipid-DNA complexes

EBV is an episomaly-replicating virus in synchrony with the cell cycle. EBV infects human cells causing mononucleosis; the presence of the unique latent origin of replication (oriP) in EBV allows for episomal replication of the virus in human cells without entering the lytic cycle. The presence of oriP and of the replication initiator protein EBNA1 cDNA on a vector allows episomal replication in human cells; in addition, plasmids containing only oriP can replicate episomally into cell lines expressing EBNA-1 (Sun et al, 1994; Banerjee et al, 1995). A hybrid HSV-1/EBV vector has been developed by Wang and Vos (1996), which combines ( i ) the HSV-1 lytic oriS; ( i i ) an HSV-1 packaging sequence which allows replication and packaging in the presence of defective helper virus carrying a deletion in theIE3 gene in the E5 cell line expressing the IE3 gene; ( i i i ) the latent oriP of EBV and ( i v ) the EBNA-1 cDNA allow episomal replication of the infectious vector in the E5 cell line so that viral stocks of high titer can be made. Infection of tumor-derived fibroblast and epithelial cell lines in culture and local injection of human liver tumors in nude mice was used to demonstrate 95-99% efficiency of infection and transfer of the reporter #-galactosidase gene. Genetically modified baculoviruses (Autographa californica nuclear polyhedrosis virus) were used to efficiently deliver genes into cultured hepatocytes of different origin; delivery into human hepatocytes with baculovirus vectors approached 100% efficiency in cell culture and expression levels were high when mammalian promoters were chosen. A number of drawbacks preclude their direct application in vivo; nevertheless gene transfer was feasible in ex vivo perfused human liver tissue (Sandig et al, 1996; Hofmann et al, 1998 this volume).

Cationic lipids have been widely used for gene transfer; a number of clinical trials (34 out of 220 total RACapproved protocols as of December 1997) use cationic lipids (see Table 4 in Martin and Boulikas, 1998, this volume, pages 203-206). Although many cell culture studies have been documented, systemic delivery of genes with cationic lipids in vivo has been very limited. All clinical protocols use subcutaneous, intradermal, intratumoral, and intracranial injection as well as intranasal, intrapleural, or aerosol administration but not i.v. delivery because of the toxicity of the cationic lipids and DOPE (see Table 4 in Martin and Boulikas, 1998, this volume, pages 203-206). Liposomes formulated from DOPE and cationic lipids based on diacyltrimethylammonium propane (dioleoyl-, dimyristoyl-, dipalmitoyl-, disteroyl-trimethylammonium propane or DOTAP, DMTAP, DPTAP, DSTAP, respectively) or DDAB were highly toxic when incubated in vitro with phagocytic cells (macrophages and U937 cells), but not towards non-phagocytic T lymphocytes; the rank order of toxicity was DOPE/DDAB > DOPE/DOTAP > DOPE/DMTAP > DOPE/DPTAP > DOPE/DSTAP; the toxicity was determined from the effect of the cationic liposomes on the synthesis of nitric oxide (NO) and TNF" produced by activated macrophages (Filion and Phillips, 1997). Another factor to be considered before i.v. injections are undertaken is that negatively charged serum proteins can interact and cause inactivation of cationic liposomes (Yang and Huang, 1997). Condensing agents used for plasmid delivery including polylysine, transferrinpolylysine, a fifth-generation poly(amidoamine) (PAMAM) dendrimer, poly(ethyleneimine), and several cationic lipids (DOTAP, DC-Chol/DOPE, DOGS/DOPE, and DOTMA/DOPE) were found to activate the complement system to varying extents. Strong complement activation was seen with long-chain polylysines, the dendrimer, poly(ethyleneimine), and DOGS; complement activation was considerably reduced by modifying the surface of preformed DNA complexes with polyethyleneglycol (Plank et al, 1996).

VIII. Liposomal gene delivery Abbreviations: DC-CHOL: 3# [N-(N',N'dimethylaminoethane)carbamoyl]cholesterol DDAB: dimethyldioctadecyl ammonium bromide DMRIE: N-[1-(2,3-dimyristyloxy)propyl]-N,N-dimethyl-N-(2hydroxyethyl) ammonium bromide

Boulikas: An overview on gene therapy

B. Mechanism of liposome entry to cells

When a group of four cationic lipids with identical head group but of different fatty acyl chains were tested for their transfection efficiencies (F igure 6); these included DOTAP, DMTAP, DPTAP, and DSTAP. The C14 acyl chain-lipid DMTAP had a similar transfection efficiency as DOTAP which has 18 carbon atoms in the acyl chain and one double bond (C18% 9); on the contrary, the transfection efficiencies of DPTAP (C16) was 10-100 fold lower and that of DSTAP (C18) was 100 to 1000 fold lower. Confocal microscopy of lung tissue after injection of 25 Âľg pCMV-GFP plasmid DNA complexed with DOTMA liposomes to mice (Figure 7) has shown that the type of cells that express the transgene are the endothelial cells that have typical characteristics of neighboring multiple air-sac structures (Figure 7D). A number of different organs in vivo can be targeted after liposomal delivery of genes or oligonucleotides. Intravenous injection of cationic liposome-plasmid complexes by tail vein in mice targeted mainly the lung and to a smaller extend the liver, spleen, heart, kidney and other organs (Zhu et al, 1993). Intraperitoneal injection of a plasmid-liposome complex expressing antisense K-ras RNA in nude mice inoculated i.p. with AsPC-1 pancreatic cancer cells harboring K-ras point mutations and PCR analysis indicated that the injected DNA was delivered to various organs except brain (Aoki et al, 1995). A number of factors for DOTAP:cholesterol/DNA complex preparation including the DNA:liposome ratio, mild sonication, heating, and extrusion were found to be crucial for improved systemic delivery; maximal gene expression was obtained when a homogeneous population of DNA:liposome complexes between 200 to 450 nm in size were used. Cryo-electron microscopy showed that the DNA was condensed on the interior of invaginated liposomes between two lipid bilayers in these formulations, a factor that was thought to be responsible for the high transfection efficiency in vivo and for the broad tissue distribution (Templeton et al, 1997). Steps to improve for successful liposome-mediated gene delivery to somatic cells include persistence of the plasmid in blood circulation, port of entry and transport across the cell membrane, release from endosomal compartments into the cytoplasm, nuclear import by docking through the pore complexes of the nuclear envelope, expression driven by the appropriate promoter/enhancer control elements, and persistence of the plasmid in the nucleus for long periods. A number of strategies for liposomal delivery and for enhancing the efficiency of uptake by the cells and release from endosomal compartments of plasmid or oligonucleotide DNA are reviewed in the following article (Martin and Boulikas, 1998).

Cationic lipids increase the transfection efficiency by destabilizing the biological membranes including plasma, endosomal, and lysosomal membranes; indeed, incubation of isolated lysosomes with low concentrations of DOTAP caused a striking increase in free activity of #galactosidase, and even a release of the enzyme into the medium demonstrating that lysosomal membrane is deeply destabilized by the lipid; the mechanism of destabilization was thought to involve an interaction between cationic liposomes and anionic lipids of the lysosomal membrane, allowing a fusion between the lipid bilayers; the process was less pronounced at pH 5 than at pH 7.4 and anionic amphipathic lipids were able to prevent partially this membrane destabilization (Wattiaux et al, 1997). In contrast to DOTAP and DMRIE which were 100% charged at pH 7.4, DC-CHOL was only about 50% charged as monitored by a pH-sensitive fluorophore; this difference decreases the charge on the external surfaces of the liposomes and was proposed to promote an easier dissociation of bilayers containing DC-CHOL from the plasmid DNA and an increase in release of the DNA-lipid complex into the cytosol from the endosomes (Zuidam and Barenholz, 1997).

C. Tissue targets using cationic liposomes in vivo Although cationic lipids have been used widely for the delivery of genes very few studies have used systemic i.v. injection of cationic liposome-plasmid complexes because of the toxicity of the lipid component and certainly in animal models, not humans. Administration by i.v. injection of two types of cationic lipids of similar structure, DOTMA and DOTAP, has shown that the transfection efficiency was determined mainly by the structure of the cationic lipid and the ratio of cationic lipid to DNA; the luciferase and GFP gene expression in different organs was transient, with a peak level between 4 and 24 hr, dropping to less than 1% of the peak level by day 4 (Song et al, 1997). Figure 5 shows the effect of cationic lipid:DNA ratio on transfection efficiency after i.v. tail injection. Luciferase activity was detected in all organs examined with the highest level in lung. In the absence of neutral lipid both DOTMA and DOTAP promoted a linear increase in luciferase activity in the lung with increasing lipid:DNA from 12:1 to 36:1 nmol lipid: Âľg of DNA. DOTMA was 10 times more efficient than DOTAP (10 6 versus 107 relative luciferase units (RLU) per mg protein. Cholesterol (Chol) mixed with DOTMA (1:1 molar ratio) decreased the level of gene expression in the lung whereas cholesterol did not affect the transfection efficiency of DOTAP liposomes. Inclusion of DOPE into either DOTAP or DOTMA liposomes significantly decreased the transfection efficiency by 100-fold in the lung.

Gene Therapy and Molecular Biology Vol 1, page 21

Figure 5. Effect of cationic lipid:DNA ratio on transfection efficiency after i.v. tail injection. Each mouse received 25 Âľg of pCMV-Luciferase plasmid DNA complexed with various amounts of liposomes indicated on the charts (at 1:1 ratio when two lipids were used). Luciferase activity was assayed 20 h after i.v. injection in up to 5 different tissues represented with different bar forms: the empty bar is lung, the large stripe bar is spleen, the small stripe is heart, gray bar is liver, and black bar is kidney. Four time points (12h, 24h, 36h, and 48h from i.v. injection) of luciferase activity are shown. Numbers +02 to +08 to the left of the figure indicates 102 to 108 relative luciferase units (RLU) per mg protein in the tissue. From Song YK, Liu F, Chu S, Liu D (1 9 9 7 ) Characterization of cationic liposome-mediated gene transfer in vivo by intravenous administration. Hum Gene Ther 8, 15851594 with the kind permission of the authors (Dexi Liu, University of Pittsburgh) and Mary Ann Liebert, Inc. Figure 6. Effect of fatty acyl chain composition on transfection efficiency. Luciferase activity was assayed 20 h postinjection in the lung, spleen, heart, liver, and kidney (in the order shown, see legend to previous figure for bar symbols). From Song YK, Liu F, Chu S, Liu D (1 9 9 7 ) Characterization of cationic liposomemediated gene transfer in vivo by intravenous administration. Hum Gene Ther 8, 1585-1594 with the kind permission of the authors (Dexi Liu, University of Pittsburgh) and Mary Ann Liebert, Inc.

Boulikas: An overview on gene therapy

Figure 7. Analysis of green fluorescence protein (GFP) expression in the lung using confocal microscopy. 25 µg pCMV-GFP plasmid DNA complexed with DOTMA liposomes were injected to mice and GFP expression in the lung was examined 14 h postinjection . (A): transmitted light image and (B ): fluorescence image (green) were observed at low magnification (Bar 100 µm). C and D are the images obtained at higher magnification showing the localization of GFP in endothelial cells (Bar 25 µm). E and F are the images from control animals injected with pCMV-Luc plasmid rather than GFP plasmid. From Song YK, Liu F, Chu S, Liu D (1 9 9 7 ) Characterization of cationic liposome-mediated gene transfer in vivo by intravenous administration. Hum Gene Ther 8, 1585-1594 with the kind permission of the authors (Dexi Liu, University of Pittsburgh) and Mary Ann Liebert, Inc.

Gene Therapy and Molecular Biology Vol 1, page 23 mechanism of lung uptake involved entrapment of large aggregates of oligonucleotides within pulmonary capillaries at 15 min post-injection via embolism; labeled oligonucleotide was localized primarily to phagocytic vacuoles of Kupffer cells at 24 h post-injection; nuclear uptake of oligonucleotide in vivo was not observed (Litzinger et al, 1996). Phosphorothioate oligonucleotides were found in most tissues 48 h after i.p. administration with highest concentrations in kidney and liver; complexation of the oligonucleotide with DOTMA did not affect neither the oligonucleotide uptake nor its tissue distribution in normal mice but increased the oligonucleotide cellular uptake (410 times) in LOX ascites tumors (Saijo et al, 1994). Triplex-forming ODNs were delivered to cells in culture using adenovirus-polylysine-ODN complexes designed to take advantage of the receptor mediated endocytosis of adenoviruses to transfer the ODNs to the cell nucleus; nuclear uptake peaked at 4 h and intact ODN persisted in the nucleus with a half-life of 12 h (Ebbinghaus et al, 1996).

D. Cationic lipids in oligonucleotide transfer Encapsulation of oligonucleotides into liposomes increased their therapeutic index, prevented degradation in cultured cells and in human serum and reduced toxicity to cells (Thierry and Dritschilo, 1992; Capaccioli et al, 1993; Morishita et al, 1993; Williams et al, 1996; Lewis et al, 1996); conjugation to a fusogenic peptide enhanced the biological activity of antisense oligonucleotides (Bongartz et al, 1994). However, most studies have been performed in cell culture, and very few in animals in vivo; there is still an important number of improvements needed before these approaches can move to the clinic. Zelphati and Szoka (1997) have found that complexes of fluorescently labeled oligonucleotides with DOTAP liposomes entered the cell using an endocytic pathway mainly involving uncoated vesicles; oligonucleotides redistributed from punctate cytoplasmic regions into the nucleus; this process was independent of acidification of the endosomal vesicles. The nuclear uptake of oligonucleotides depended on several factors such as charge of the particle where positively charged complexes were required for enhanced nuclear uptake; DOTAP increased over 100 fold the antisense activity of a specific antiluciferase oligonucleotide. Physicochemical studies of oligonucleotide-liposome complexes of different cationic lipid compositions indicated that either phosphatidylethanolamine or negative charges on other lipids in the cell membrane are required for efficient fusion with cationic liposome-oligonucleotide complexes to promote entry to the cell (Jaaskelainen et al, 1994). Similar results were reported by Lappalainen et al (1997); digoxigenin-labeled oligodeoxynucleotides (ODNs) complexed with the polycationic DOSPA and the monocationic DDAB (with DOPE as a helper lipid) were uptaken by CaSki cells in culture by endocytosis. The nuclear membrane was found to pose a barrier against nuclear import of ODNs which accumulated in the perinuclear area. Although DOSPA/DOPE liposomes could deliver ODNs into the cytosol, they were unable to mediate nuclear import of ODNs; on the contrary oligonucleotide-DDAB/DOPE complexes with a net positive charge were released from vesicles into the cytoplasm; it was determined that DDAB/DOPE mediated nuclear import of the oligonucleotides. DOPE-heme (ferric protoporphyrin IX) conjugates, inserted in cationic lipid particles with DOTAP, protected oligoribonucleotides from degradation in human serum and increased oligoribonucleotide uptake into 2.2.15 human hepatoma cells; the enhancing effect of heme was evident only at a net negative charge in the particles (Takle et al, 1997). Uptake of liposomes labeled with 111In and composed of DC-Chol and DOPE was primarily by liver, with some accumulation in spleen and skin and very little in the lung after i.v. tail injection; preincubation of cationic liposomes with phosphorothioate oligonucleotide induced a dramatic, yet transient, accumulation of the lipid in lung which gradually redistributed to liver. The

E. Fusogenic peptides enhance gene transfer efficiency Enveloped viruses have evolved efficient mechanisms to release their genomes from the endosomes into the cytoplasm of the host cells; specific envelope proteins of the nucleocapsid are capable of destabilizing the endosomal membrane. Therefore, inactivated viruses have been used to enhance the transfer of plasmids. Addition of adenoviral particles capable of inducing endosome lysis (Blumenthal et al, 1986), mediated by a conformational change in the adenovirus penton protein induced at the lower pH of endosomes (Seth, 1994) can increase transfection efficiency 100-1000 fold using 10 9 adenoviral particles/ml and the transferrin receptor (Curiel et al, 1991; Cotten et al, 1992; Wagner et al, 1992b; Cristiano et al, 1993; Morishita et al, 1993; Harries et al, 1993; Curiel, 1994; reviewed by Ledley, 1995). Use of fusogenic peptides from influenza virus hemagglutinin HA-2 enhanced greatly the efficiency of transferrin-polylysine-DNA complex uptake by cells; in this case the peptide was linked to polylysine and the complex was delivered by the transferrin receptor-mediated endocytosis (Wagner et al, 1992a; Plank et al, 1994). This peptide had the sequence: GLFEAIAGFIENGWEGMID GGGYC and was able to induce the release of the fluorescent dye calcein from liposomes prepared with egg yolk phosphatidylcholine which was higher at acidic pH; this peptide was also able to increase up to 10-fold the anti-HIV potency of antisense oligonucleotides, at a concentration of 0.1-1 mM, using CEM-SS lymphocytes in culture (Bongartz et al, 1994). This peptide changes conformation at the slightly more acidic environment of the endosome destabilizing and breaking the endosomal membrane (Murata et al, 1992; Bullough et al, 1994). Fusogenic peptides have been used by other investigators 23

Boulikas: An overview on gene therapy (Midoux et al, 1993; Kamata et al, 1994). It is thought that several fusogenic peptides self-assemble following their conformational change forming a transmembrane channel (Bongartz et al, 1994). Sendai virosomes were effective for delivering AAV neuropeptide Y (NPY) cDNA constructs in vivo. Injections into brain neocortex of Sendai-virosome encapsulated rAAV construct expressing NPY increased NPY-like immunoreactivity in neurons but not glia; injections into the rat hypothalamic para-ventricular nucleus increased body weight and food intake for 21 days (Wu et al, 1996). Tomita et al (1996) have found that newborn mice can sustain expression of the insulin gene delivered by Sendai virus-liposome complexes for at least 8 weeks as assayed by reverse transcriptase PCR and radioimmunoassay, compared to 2 weeks in adult animals. A 27 residue peptide vector, containing the fusion sequence of HIV gp41 and the nuclear localization sequence of SV40 T antigen was used to deliver oligonucleotides to cell nuclei very rapidly in cell culture (1h). The complexes formed strongly increased the stability of the oligonucleotide to nucleases, enhanced passage through the plasma membrane, and led to endosomal internalization (Morris et at, 1997). Certain cationic lipids are endowed with a better ability to disrupt the endosomal membrane and promote release of the plasmid to the cytoplasm, a prelude for its nuclear import. Presentation of plasmid DNA to COS cell cultures using three different lipid formulations: ( i ) vectamidine (3-tetradecylamino-N-tert-butyl-N'-tetradecylpropionamidi ne), ( i i ) DOTMA:DOPE (Lipofectin), and ( i i i ) DMRIEChol (1:1) resulted in complex entry via endocytosis for all three cationic lipids as revealed using transmission electron microscopy. However, the endosomal membrane in contact with complexes containing vectamidine or DMRIE-Chol, but not Lipofectin, often exhibited a disrupted morphology (El Ouahabi et al, 1997).

display the asialoglycoprotein receptor (Chen et al, 1994). Plasmid DNA and HMG1 were efficiently co-encapsulated in liposomes by agitation and sonication, and were cointroduced into cells by hemagglutinating virus of Japan (HVJ)-mediated membrane fusion; the presence of HMG1 enhanced 3-fold the transfection efficiency (Kato et al, 1991). The interaction of plasmid DNA with protamine sulfate followed by the addition of DOTAP cationic liposomes offered a better protection of plasmid DNA against enzymatic digestion and gave consistently higher gene expression in mice via tail vein injection compared with DOTAP/DNA complexes; 50 Âľg of luciferaseplasmid per mouse gave 20 ng luciferase protein per mg extracted tissue protein in the lung which was detected as early as 1 h after injection, peaked at 6 h and declined thereafter. Intraportal injection of protamine/DOTAP/DNA led to about a 100-fold decrease in gene expression in the lung as compared with i.v. injection; endothelial cells were the primary locus of lacZ transgene expression (Li and Huang, 1997). Protamine sulfate enhanced plasmid delivery into several different types of cells in vitro using the monovalent cationic liposomal formulations (DC-Chol and lipofectin); this effect was less pronounced with the multivalent cationic liposome formulation, lipofectamine (Sorgi et al, 1997). Spermine has been found to enhance the transfection efficiency of DNA-cationic liposome complexes in cell culture and in animal studies; this biogenic polyamine at high concentrations caused liposome fusion most likely promoted by the simultaneous interaction of one molecule of spermine (four positively charged amino groups) with the polar head groups of two or more molecules of lipids. At low concentrations (0.03-0.1 mM) it promoted anchorage of the liposome-DNA complex to the surface of cells and enhanced significantly transfection efficiency (Boulikas et al, in preparation). Because the receptor for ecotropic viruses is a transporter for basic amino acids, use of a histone as a facilitator increased the efficiency of retroviral infection (Singh and Rigby, 1996). Polybrene is the usual agent employed during retroviral infection. For supernate infections, concentrations of 5-10 Âľg/ml of protamine provided essentially the same infection efficiency as polybrene; protamine displayed low toxicity on a range of cell types and increased 7-fold the efficiency of retroviral infection (Cornetta and Anderson, 1989). The polycations polybrene, protamine, DEAE-dextran, and poly-L-lysine significantly increased the efficiency of adenovirus-mediated gene transfer in cell culture; this was thought to act by neutralizing the negative charges presented by membrane glycoproteins which reduce the efficiency of adenovirus-mediated gene transfer (Arcasoy et al, 1997).

F. Plasmid condensation with spermine, polylysine, protamine, histones enhances the transfection efficiency DNA can be presented to cells in culture as a complex with polycations such as polylysine, or basic proteins such as protamine, total histones or specific histone fractions (Fritz et al, 1996), cationized albumin, and others (Smull and Ludwig, 1962). These molecules increase the transfection efficiency. In addition to HMG1, also histone H1 and HMG17 were identified as transfection-enhancing proteins in cell culture (Zaitsev et al, 1997). Histone H2A significantly enhanced in vitro DNA transfection whereas other histones and anionic liposomes did not (Balicki and Beutler, 1997). Gene transfer through the asialoglycoprotein receptor-mediated endocytosis pathway was enhanced with the histones H1, H2a, H2b, H3, and H4 which were galactosylated with the crosslinker agent, 1ethyl-3-(3-dimethylaminopropyl)carbodiimide, conjugated to DNA and then used to transfect HepG2 cells, which

G. Targeted gene delivery 24

Gene Therapy and Molecular Biology Vol 1, page 25

H. Targeted gene delivery with peptidedisplaying phages

Targeting a specific cell type or animal tissue is an important goal of gene therapy. Many different approaches have been undertaken to achieve targeting. A recombinant adenovirus encoding an anti-erbB-2 intracellular singlechain antibody (sFv) displayed a genetic selectivity for the erbB-2-positive prostate carcinoma cell lines DU145 and LNCaP; delivery of this recombinant adenovirus resulted in cytotoxicity to the DU145 and LNCaP, but not PC-3, cell lines and reduced the clonogenic capacity of DU145 cells cultured alone or mixed with various ratios of irradiated human bone marrow. This finding led to a strategy for effectively reducing DU145 and erbB-2positive primary prostate tumor contamination in bone marrow cultures (Kim et al, 1997). Delivery of an antierbB-2 single chain (sFv) antibody gene for previously treated ovarian and extraovarian cancer patients is in clinical trials using adenoviral gene delivery (protocol #133). A luciferase expression vector (pRSVLuc) noncovalently linked to a humanized HER2 antibody (rhuMAbHER2) covalently modified with poly-L-lysine bridges was able to direct gene transfer to HER2 expressing cells in vitro (Foster and Kern, 1997). A targeting gene therapy approach for hematopoietic stem/progenitor cells has been directed to cell lines expressing the c-kit receptor; plasmid DNA containing a luciferase reporter gene was condensed with polylysine covalently linked to streptavidin (which binds biotinylated ligand) and with polylysine covalently linked to adenovirus (to achieve endosomal lysis) with the final addition of biotinylated steel factor; omission of the adenovirus endosomalytic agent from the vector resulted in the loss of gene expression (Schwarzenberger et al, 1996). Systemic administration of a c-fos antisense, regulated by mouse mammary tumor virus (MMTV) control elements in a retroviral vector, showed expression only in breast epithelium although the vector could be detected in several tissues thus supporting targeting to MMTVregulated tissues (Arteaga and Holt, 1996). Liposomes coated with polyethyleneglycol (PEG) can be efficiently targeted to tumor cells that express folate receptors (KB cells) via conjugation of folate to a PEG spacer of 25 nm in length; shorter PEG spacers were not efficient in mediating binding of the liposomes to KB cells (Lee and Low, 1995). Neri and coworkers (1997) were able to target an angiogenesis-associated oncofetal fibronectin (B-FN) isoform by affinity-matured recombinant antibody fragments. B-FN is present in vessels of neoplastic tissues during angiogenesis but is absent from mature vessels and could provide a target for diagnostic imaging and therapy of cancer. Phage display libraries were screened to isolate human antibody fragments able to recognize this isoform across species; imaging of F9 murine teratocarcinomas grafted in nude mice is shown on Figures 8 and 9.

Development of methods to display and select collections of peptides specific for binding a target provide valuable tools to identification of peptide drugs; peptides could be selected for binding biological targets including cell surface receptor molecules, DNA, antibodies, or whole cells. The technique of peptide-displaying phages has been developed for targeted gene delivery. Selection of cell surface-binding peptides, ideally specific for each type of cell in the human body, will be used for incorporation into gene delivery vehicles to achieve the long-searched tissue specificity of the vector (reviewed by Russell, 1996). Development of the random peptide library as a source of specific protein binding molecules (Devlin et al, 1990) and exposure of random peptides on the surface of phages (Cwirla et al, 1990) has been the catalyst for progress in this promising field. Libraries of random 8 to 12 amino acid peptides expressed on the N-terminus of the pIII protein of the fd phage or on the N-terminus of the pVIII major coat protein of the same phage have been selected that bind the extracellular domain of human IL-1 receptor; screening was against immobilized IL-1 receptor extracellular domain. Two families of peptides could act as antagonists blocking triggering of the IL-1 signaling pathway; because IL-1 levels become elevated in autoimmune and inflammatory disorders, these peptide antagonists of IL-1 receptor could provide novel drugs for these diseases (Yanofsky et al, 1996). Phages displaying known integrin-binding peptides have been shown to bind and enter mammalian cells (Hart et al, 1994). A peptide antagonist to thrombin receptor has been identified using phage display (Doorbar and Winter, 1994). Production of cell-targeting ligands has been achieved by cell-binding peptides specific for different cell types in culture; these peptides are selected through six rounds of binding (and amplification of phage clones) to a particular cell type from random peptide-presenting phage libraries; the selected peptides are apparently recognizing specific surface receptor molecules. For example, the 20mer peptide KTLTLEAALRNAWLREVGLK has been selected for its high affinity for PEA10 mouse fibroblast cells binding 1000 more efficiently to the cells than random peptides (Barry et al, 1996).

IX. Gene delivery with polymers, peptides and other means A. Delivery of transferrin-polylysine-DNA complexes A number of polymers have been tested and shown to enhance significantly the transfection efficiency of plasmids but also of viruses; the enhancement in transfection results from a facilitation in the interaction of plasmids with the cell surface, transport to endosomes, release to the cytoplasm, and in some cases nuclear import (reviewed by Behr, 1994). 25

Boulikas: An overview on gene therapy

F i g u r e 8 . Role of antibody valence. Targeting of fluorescently-labeled antibody fragments to the F9 murine teratocarcinoma grafted in nude mice using the monomeric scFv(CGS-1) and dimeric scFv(CGS-1)2 directed to oncofetal fibronectin; the dimeric scFv(D1.3) 2 with a binding specificity to lysozyme was used as a negative control. t: tumor; b: bladder. From Neri D, Carnemolla B, Nissim A, Leprini A, Querze G, Balza E, Pini A, Tarli L, Halin C, Neri P, Zardi L, Winter G (1 9 9 7 ) Targeting by affinity-matured recombinant antibody fragments of an angiogenesis associated fibronectin isoform. N a t B i o t e c h n o l 15, 1271-1275. Reproduced with the kind permission of the authors (Dario Neri, Inst for Mol Biology and Biophysics, Z端rich) and Nature America, Inc.

Figure 9. Role of antibody affinity. Targeting of fluorescently-labeled antibody fragments to the F9 murine teratocarcinoma grafted in nude mice using the affinitymatured scFv(CGS-2) and the lower affinity scFv(28SI) directed to the same epitope of oncofetal fibronectin; Tte dimeric scFv(D1.3)2 with a binding specificity to lysozyme was used as a negative control. t: tumor; b: bladder. From Neri D, Carnemolla B, Nissim A, Leprini A, Querze G, Balza E, Pini A, Tarli L, Halin C, Neri P, Zardi L, Winter G (1 9 9 7 ) Targeting by affinity-matured recombinant antibody fragments of an angiogenesis associated fibronectin isoform. N a t B i o t e c h n o l 15, 1271-1275. Reproduced with the kind permission of the authors (Dario Neri, Inst for Mol Biology and Biophysics, Z端rich) and Nature America, Inc.

Gene Therapy and Molecular Biology Vol 1, page 27 condensates prepared with varying stoichiometries of lipospermine or polyethylenimine in physiological solution; discrete nucleation centers of condensation were observed often surrounded by folded loops of DNA using either condensing agent; increasing the amount of lipospermine or polyethylenimine led to further aggregation through folding rather than winding of the DNA (Dunlap et al, 1997).

Curriel and coworkers (1991) have used the transferrin receptor on the surface of mammalian cells to deliver plasmid-polylysine-transferrin complexes to cells. These complexes are taken up by endosomes following receptor binding, a method which suffers from that the endocytosed DNA is trapped in the intracellular vesicle and is later largely destroyed by lysosomes; use of the capacity of the adenoviruses to disrupt endosomes as part of their entry mechanism to the cells have augmented over 1000-fold the efficiency of gene transfer. This method has been further developed in collaboration with Max Birnstiel; true chemical coupling rather than simple addition of replication-defective adenovirus particles has shown a further increase in transfection efficiency (Cotten et al, 1992; Wagner et al, 1992a,b). A monoclonal antibody against the CE7 antigen (chCE7) covalently linked to polylysine in the presence of chloroquine was able to transfect NB cells as efficiently as DOTAP, transfectam, TF-X50, or lipofectamine; furthermore, transfection was not observed in cell lines negative for the CE7 antigen (Coll et al, 1997).

C. APL PolyCat57 and other polymers APL PolyCat57 is a synthetic polyamino derivative (nonpeptide, nonlipid polymer) with a glucose backbone which was used by Goldman and coworkers (1997) for gene transfer in vivo and in vitro. A variety of human carcinoma cell lines were transfected with an efficiency superior to that of Lipofectamine. The polymer-plasmid complex was resistant to inhibition by serum allowing for efficient gene transfer in vivo. The level of the luciferase and #-galactosidase reporter gene expression after intrathecal injection, evaluated in animal models bearing stereotactically implanted D54-MG human glioma cell xenografts, was comparable to that obtained with an adenoviral vector. Liposomes composed of the cationic peptide amphiphile N,N-dihexadecyl-N" -[6-(trimethyl ammonio)hexanoyl]-L-alaninamide bromide comprising an L-alanine residue interposed between a charged head group and a double-chain segment were more effective and less toxic than lipofectin, and DOTAP for the transfection of COS-7 cells (Kato et al, 1996).

B. Polyethylenimine (PEI, ExGen500) Polyethylenimine, H2N-(CH2-CH2-NH)n-H, is an organic polymer with a potential for high cationic charge. PEI enhanced transfection efficiency in cell culture (Boussif et al, 1996). ExGen500 is a linear 22 kDa form of PEI, which was found to be more efficient than lipofectin, DOTAP and DOGS in delivering the luciferase reporter gene in both newborn and adult rabbit lungs (Ferrari et al, 1997). The PEI 800 kDa and PEI 25 kDa branched polymers have also been used to transfer marker genes to the newborn and adult mouse brain (Boussif et al, 1995; Abdallah et al, 1996). Another advantage of PEI is that it yields high transfection efficiencies with a charge ratio of DNA:PEI close to neutral; this is an advantage as particles with a net positive charge (cationic lipid-DNA complexes) interact with circulating serum proteins or anionic components of the extracellular matrix in the various tissues hindering their bio-availability (Schwartz et al, 1995). The high transfection efficiency of ExGen 500 was suggested to arise from the â&#x20AC;&#x153;proton spongeâ&#x20AC;? effect which leads to osmotic swelling of endosomes which have uptaken the DNA complexes (Ferrari et al, 1997). Different cationic compositions may result in different targeting and transfection abilities to specific organs; the branched, 25-kD polyethylenimine polymer (PEI 25k) was superior over DOTAP and DOGS (Transfectam) in the efficiency of transfection of the kidney when complexes of these cations with luciferase plasmid were injected into the left renal artery of rats; luciferase activity peaked at 2 days, was still significantly higher than controls at 7 days, but was undetectable at 14 days post-injection (Boletta et al, 1997). Scanning force microscopy allowed plasmid DNA strands to be visualized without drying in incomplete

D. Adenovirus-polymer complexes An adenovirus/DNA complex was constructed by chemically linking poly-L-lysine to the capsid of the replication-defective adenovirus dl312; this complex was then coupled with plasmid DNA via ionic interaction. This system was used to deliver the tumor suppressor protein p53 to the p53- human lung cancer cell line H1299, both in vitro and in vivo, leading to induction of apoptosis; injection of the complex carrying the p53 gene to subcutaneous tumor sites 5 days after tumor cell implantation resulted in a significant inhibition of tumorigenicity as measured by the number and size of tumors that developed 21 days after treatment (Nguyen et al, 1997a,b). Complexes of cationic polymers and cationic lipids with adenovirus increased adenovirus uptake and transgene expression in cells that were inefficiently infected by adenovirus alone; infection by both complexes was independent of adenovirus fiber and its receptor, occurred via a different cellular pathway than adenovirus alone, and enhanced gene transfer to the nasal epithelium of cystic fibrosis mice in vivo (Fasbender et al, 1997).

Boulikas: An overview on gene therapy aggregates and then immediately transfected using the gene gun; plasmid-coated gold particles are delivered to tumor cells using helium pressure with a hand-held gene delivery device overcoming the cumbersome exposure of the patient to viral antigens. PMGT with gold particles coated with human GM-CSF plasmid DNA is being used to transfect melanoma or renal carcinoma tissue from patients; tumor cells are then lethally-irradiated and patients are intradermally vaccinated to elicit anti-tumor immune responses (Mahvi et al, 1997). Gene gun-mediated DNA delivery into the epidermis overlying an established intradermal murine tumor was used to compare the antitumor effect of several transgene expression plasmids encoding the cytokines IL-2, IL-4, IL6, IL-12, IFN-! , TNF-", and GM-CSF; IL-12 was superior (see IL-12) (Rakhmilevich et al, 1997).

E. Peptides in transfer of oligonucleotides Peptide/oligonucleotide complexes containing a peptide vector and single or double stranded oligonucleotides were delivered into cultured mammalian cells in less than 1 h with relatively high efficiency (90%) at a peptide/oligonucleotide ratio of 20/1. The peptide vector, termed MPG (27 residues), contained a hydrophobic domain derived from the fusion peptide of HIV gp41 and a hydrophilic domain derived from the nuclear localization sequence of SV40 T-antigen. The complexes involved electrostatic interactions between basic peptide residues and phosphate groups from the oligos, as well as additional peptide-peptide interactions yielding oligonucleotides most likely coated with several molecules of MPG; these complexes, which strongly increased the stability of the oligonucleotide to nucleases, enhanced passage through the plasma membrane, and did lead to endosomal internalization; such complexes are promising delivery systems for oligos (Morris et at, 1997). The cationic amphipathic peptide WEAKLAKALA KALAKHLAKALAKALKACEA was synthesized by Wyman et al (1997) to display hydrophobic leucine residues on one side and hydrophilic lysine residues on the other after coiling to an amphipathic " -helix at pH 7.5; this peptide was suited for oligonucleotide nuclear delivery when complexes were prepared at a 10/1 (+/-) charge ratio and was endowed with the additional property of destabilizing membranes in cell culture.

X. Direct injection of naked plasmid DNA Naked plasmid has been injected to various tissues and has shown transfection efficiency. Muscle has been the classical tissue in a number of studies. Intramuscular (i.m.) administration of expression plasmids may directly deliver the plasmid to the cytoplasm by damaging the myofibril along the injected area. Direct i.m. injection of naked VEGF plasmid DNA was used in rabbits to optimize treatment of acute limb ischemia; after ligation of distal external iliac artery in New Zealand White rabbits, direct injection of 500 Âľg of a VEGF 165 expression vector into the ischemic thigh muscles resulted in more angiographically recognizable collateral vessels at 30 days posttransfection (Tsurumi et al, 1996, 1997). Injection, guided by intense illumination along the longitudinal axis of the mouse quadriceps muscle and parallel to the myofibers, yielded 200-fold higher levels of luciferase expression than perpendicular injection (Levy et al, 1996). Other tissues including skin, liver, brain and the gastric submucosa have been successfully transduced with reporter gene cDNA using naked plasmid delivery. Skin from transglutaminase 1 (TGase1)-deficient patients suffering with lamellar ichthyosis was regenerated on nude mice; repeated in vivo direct injections of naked DNA using a TGase1 expression plasmid showed restoration of TGase1 expression in the correct tissue location (Choate and Khavari, 1997). A naked luciferase expression vector injected intracerebrally in mice provided expression of the luciferase transgene, in both neurons and glia cells (Schwartz et al, 1996). Naked plasmid DNA in hypertonic solutions, injected intraportally in mice whose hepatic veins were transiently occluded, resulted in high levels of luciferase and #-galactosidase expression in 1% of the hepatocytes throughout the entire liver using 100 Âľg DNA (Budker et al, 1996). A single injection through the tail vein of a naked endothelium-derived nitric oxide cDNA plasmid caused a significant reduction of systemic blood pressure for 5 to 6

F. Plasmoviruses Plasmoviruses are plasmids capable of expressing all the viral genes required for generating infectious particles and packaging a defective genome; transfected as plasmids, plasmoviruses transform the transduced cells into packaging cells; the cells then release infectious replication-defective retrovirus particles of typical type C as revealed by electron microscopy, with the gag proteins correctly processed in the released particles and containing the transgene to be transferred. Released particles are capable of infecting nearby cells and to propagate the transgene in the culture, resulting in stable integration of plasmovirus proviral DNA into the host genome of infected cells. Nonintegrated plasmovirus DNA was not toxic for the cells. Plasmoviruses have been used for the propagation of the HSV-tk gene in cell culture resulting in a major improvement in therapeutic efficacy after ganciclovir treatment, when compared to that of plasmovirus constructs that cannot propagate (Morozov et al, 1997).

G. Particle-mediated gene transfer (PMGT) or gene gun The particle-mediated gene transfer (PMGT)technique, unlike retroviral transfection, does not require tumor cell proliferation in vitro for gene transfer; instead, tumor tissue can be dissociated into small tissue clumps or cell 28

Gene Therapy and Molecular Biology Vol 1, page 29 weeks in spontaneously hypertensive rats (Lin et al, 1997). In vivo delivery of a luciferase gene under control of the human cytomegalovirus immediate early gene promoter after intravenous injection (50 Âľg DNA) via the tail vein into ICR mice has shown that the DNA was degraded with a half-life of less than 5 min from the blood; plasmid DNA was differentially retained in the lung, spleen, liver, heart, kidney, bone morrow, and muscle up to 24 h postinjection; femtogram levels of plasmid were detected only in muscle at 6 months post infection (Lew et al, 1995). pCAT was rapidly degraded after incubation with mouse whole blood in vitro with a half-life of approximately 10 min and much faster after intravenous injection; i.v. injection of radioactively-labeled pCAT showed rapid elimination from the plasma due to extensive uptake by non-parenchymal cells in the liver, a process thought to be mediated via scavenger receptors on these cells (Kawabata et al, 1995). Direct injection of plasmids carrying reporter genes

into the gastric submucosa of adult rats resulted in transient expression (1-3 days and in exceptional cases for up to 21 days) in smooth muscle cells of the muscularis mucosae and the muscular layer and mesenchymal cells in the lamina propria. These studies indicate that the gastrointestinal nonepithelial tissue, a useful target for in vivo gene transfer, can be transfected with naked DNA (Takehara et al, 1996). Clinical protocols #158-161 use naked plasmid DNA. Protocol #158 proposes transferring the carcinoembryonic antigen to autologous tumor cells in patients with metastatic colorectal cancer for cancer immunotherapy (Appendix 1, page 170). Protocols 159 and 160 use an intraarterial angioplasty catheter to deliver VEGF cDNA plasmid to patients with peripheral artery disease or restenosis. Plasmid DNA coding for tumor idiotype is being used for intramuscular injection for immumotherapy of non-Hodgkinâ&#x20AC;&#x2122;s B-cell lymphoma (protocol #161). Table 1 summarizes the advantages and disadvantages of the principal gene delivery methods.

Table 1. Advantages and drawbacks of delivery systems Gene deliv. system Murine retroviral vectors Recombinant adenoviruses

AAV

HSV-1 Baculovirus EBV HIV-1

Hybrid HSV/EBV Cationic lipids

Stealth liposomes Naked plasmid DNA Gene gun

A d v a n ta g es

Drawbacks

Very safe; may achieve high efficiency of transduction; infects only dividing cells; integrates into host DNA.

Loss in expression soon after infection; low efficiency in vivo; up to 8 kb of DNA; high titers required for in vivo gene delivery; immunogenicity. Induction of immune responses that eliminates therapeutic cells; may induce unwanted infections to humans; only up to 7.5 kb of exogenous DNA can be inserted; loss of adenoviral episomes in progeny cells. Low efficiency of gene transfer; only up to 4.1 and 4.9 kb can be incorporated; wt AAV integrates on chromosome 19 but recombinant AAV integrates at different sites (e.g. chromosome 2); integration may cause inactivation of the transgene by chromatin effects. Infection with HSV-1 is cytotoxic.

Infect nondividing cells; rarity of recombination events between adenoviral vectors and the host chromosomes; high efficiency of transduction; adenovirus vectors efficiently escape from the endosome and enter the nucleus; episomaly-replicating virus. Does not stimulate inflammation or immune reaction; enters nondividing cells and does not replicate; nonpathogenic virus.

Can take up to 30 kb of exogenous DNA; high titer viral stocks; wide range; can infect nonreplicating cells Specificity for hepatocytes; high efficiency of infection Episomaly-replicating virus Transduces non-dividing cells; broad range of tissues and cell types; no inflammation; sustains expression of GFP for 8-22 weeks in muscle and liver after injection to animals High efficiency of infection (95-99% after intratumoral liver injection) High efficiency of transfection via membrane destabilization (cell membrane and endosomal); destabilize lysosomal membranes and promote release of plasmid in the cytoplasm. Non toxic, escape immune surveillance and concentrate into solid tumors by extravasation. Suited for intramuscular injection and DNA vaccination; easy to use; no viral antigens. Easy to use (plasmid-coated gold particles are delivered to tumor cells using helium pressure); rapid, suited for gene transfer to tumor specimens from patients for

Not applicable in vivo at present. wt EBV infects human cells causing mononucleosis. Start up technology, not broadly tested.

Not broadly tested. Toxic, not suited for i.v. injection; can interact with negatively charged serum proteins in vivo causing transgene inactivation; gene expression is transient; i.v. injection targets mainly the lung Not taken up by tumor cells but remain in the extracellular space. Low transfection; not widely applicable method; naked plasmid is cleared from blood rapidly. Not broadly tested.

Boulikas: An overview on gene therapy immunotherapy.

promoter (Ad-MLP) were compared for their killing efficiency in combination with GCV treatment; the rat 9L model for brain tumor and leptomeningeal metastases was used; the adenovirus containing the CMV promoter showed greater cell killing efficiency compared to the AdMLP promoter; animals with brain tumors showed significantly longer survival time and animals with leptomeningeal metastases had symptom-free periods (Vincent et al, 1997). Doll et al (1996) have compared the efficiency of expression of the #-galactosidase gene flanked by the AAV ITRs in brain tumors and primary brain cell cultures driven by four different promoters. The human CMV immediateearly enhancer/promoter was always the strongest, generally by at least one order of magnitude, compared with the SV40 early enhancer/promoter, the JC polymovirus promoter, and the chicken #-actin promoter coupled to the CMV enhancer. High level of expression was usually seen within 24 h of transgene delivery by either transfection or infection, but dropped dramatically within days; all four promoters showed the same decline in sustaining gene expression of #-galactosidase with time (Doll et al, 1996). The type of regulatory elements on plasmid vectors, including promoter, enhancer, intron, and polyadenylation signals, were systematically evaluated by Yew et al (1997) by constructing a series of plasmids. Figure 10 shows the effect of different introns (panel A) and different poly(A) signals (panel B) on CAT expression. A hybrid intron (HI) appeared to be the most effective. There was a 4-fold increase in CAT expression from the bovine growth hormone (BGH) poly(A) signal vector compared to the SV40 poly(A) signal vector.

XI. Promoters and enhancers for transgene expression A. Viral promoters After escaping serum components and immune cells, crossing the cell membrane, released from endosomes to the cytoplasm and transported through the nuclear pores to the nucleus the transgene has to accomplish two additional tasks: (i) to be efficiently transcribed and (ii) its expression to last for long periods. These two very important factors depend on the DNA regulatory elements that drive the expression of the therapeutic gene. The use of mammalian gene expression vectors has revolutionized the field of direct gene delivery. The proper choice of promoter and enhancer elements linked to the gene of interest is decisive for the successful expression of the gene in the desired tissue or cell type in gene therapy. The majority of mammalian expression vectors make use of promoter/enhancer elements from pathogenic viruses including the immediately early promoter of the human cytomegalovirus (CMV), the Rous sarcoma virus (RSV) promoter, the enhancer/origin of replication of SV40, the adenovirus type 2 major late promoter (AdMLP), as well as promoters from the mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV), herpes simplex virus (HSV), Epstein-Barr virus (EBV), and others. Many studies have compared the strength of different promoters in driving a therapeutic gene both in cell culture and in vivo. I will mention a few sample studies here. Recombinant adenoviruses carrying the HSV-tk gene under control of the human cytomegalovirus (CMV) immediate early gene promoter or the adenovirus type 2 major late

F i g u r e 1 0 . Effect of different introns (A) and polyadenylation signals (B ) on CAT expression. ELM cells were co-transfected with equimolar amounts of each plasmid using DMRIE:DOPE and CAT protein levels in cell lysates were assayed 48 h after transfection; pCMV# was used as an internal control. SVI is the SV40 19S/16S intron; HI, hybrid intron, SV40 pA, SV40 late polyadenylation signal; BGH, bovine growth hormone polyadenylation signal; #-Glo, rabbit #globin polyadenylation signal. The data are expressed as mean ÂąSD (n=3). From Yew NS, Wysokenski DM, Wang KX, Ziegler RJ, Marshall J, McNeilly D, Cherry M, Osburn W, Cheng SH (1 9 9 7 ) Optimization of plasmid vectors for high-level expression in lung epithelial cells. Hum Gene Ther 8, 575584. Reproduced with kind permission of the authors (Nelson Yew, Genzyme Corp., Framingham, MA) and Mary Ann Liebert, Inc.

Gene Therapy and Molecular Biology Vol 1, page 31 Figure 1 1 . Comparison of CAT expression from different promoters in vitro. ELM cells (solid bars) or CFT1 cells (stippled bars), a human airway epithelial cell line derived from a CF patient, were transfected as described in Figure 10. CAT ELISA assays were carried out 48 h after transfection (an average of 6 assays). CAT protein levels were normalized to pCF1-CAT (in A) or pCMVHICAT (in B). A. Expression from plasmids containing the BGH poly(A) signal. SPC, Surfactant protein C promoter; NOS, nitric oxide synthase promoter; UbB, ubiquitin B promoter; MUC1, mucin 1 promoter; IL8, interleukin 1 promoter; CE, CMV enhancer; pCAT control is a promoterless CAT plasmid. B . Expression from plasmids containing the SV40 poly(A) signal. CC10, Clara cell 10 kDa protein promoter; E1a, adenovirus E1a promoter. The data are expressed as mean ÂąSD (n=3-12). From Yew NS, Wysokenski DM, Wang KX, Ziegler RJ, Marshall J, McNeilly D, Cherry M, Osburn W, Cheng SH (1 9 9 7 ) Optimization of plasmid vectors for high-level expression in lung epithelial cells. Hum Gene Ther 8, 575-584. Reproduced with kind permission of the authors (Nelson Yew, Genzyme Corp., Framingham, MA) and Mary Ann Liebert, Inc.

Figure 11 compares the strength of different promoters from CAT constructs containing the bovine growth hormone (BGH) poly(A) signal (panel A) or the SV40 poly(A) signal (panel B) and the hybrid intron. The promoters were chosen for lung targeting. CMV yielded the highest expression in vitro. To determine whether or not incorporating two CMV enhancers could produce higher levels of CAT expression than one, a second CMV enhancer (from -118 to -522 relative to the transcription start site) was inserted 186 bp upstream of the CMV promoter and its associated enhancer; in the context of the SV40 poly(A) signal the second CMV enhancer (CE in Figure 11) increased expression 3-fold; however, when the BGH poly(A) signal was present, the second copy of CMV did not increase CAT expression (Figure 12).

Because of its wide use and the more potent effect, the CMV IE enhancer/promoter deserves some special attention. In order to understand the potent effect of the CMV promoter in the expression of foreign genes we need to understand the transcription factors (TFs) that activate this regulatory region; TFs in the transfected cell will be responsible for binding to the CMV promoter leading to the activation of the transgene. At present not all TF regulatory circuits leading to activation of CMV have been deciphered.Figure 13 shows two CMV promoters retrieved from Genbank which are being used in expression vectors. The CMV IE promoter includes the 10-bp palindromic sequence C C A T A T A T G G (Figure 13) which resembles the core motif of serum response elements and proved to bind specifically to the cellular nuclear protein serum response factor (SRF). Reporter gene constructs containing four tandem copies of these elements displayed up to 13-fold increased basal enhancer activity and 18-fold tetradecanoyl phorbol acetate responsiveness in U937 cells (Chang et al, 1993).

B. Transcription factor binding sites within the CMV promoter

Gene Therapy and Molecular Biology Vol 1, page 32

F i g u r e 1 2 . Effect of a second CMV enhancer region on CAT expression from the CMV promoter. Plasmids were transfected into ELM cells and the cells were harvested 48 h after transfection. Expression was normalized to pCMVHICAT (in B). A. CAT protein levels in cell lysates. RE, RSV LTR enhancer. B . Levels of CAT RNA. Total RNA was isolated from the transfected cells and a quantitative RNA protection assay was performed. The data are expressed as mean ÂąSD (n=3-9). From Yew NS, Wysokenski DM, Wang KX, Ziegler RJ, Marshall J, McNeilly D, Cherry M, Osburn W, Cheng SH (1 9 9 7 ) Optimization of plasmid vectors for highlevel expression in lung epithelial cells. Hum Gene Ther 8, 575-584. Reproduced with kind permission of the authors (Nelson Yew, Genzyme Corp., Framingham, MA) and Mary Ann Liebert, Inc.

This protein complex was more abundant in U-937, K562, and HeLa cell extracts than in Raji, HF, BALB/c 3T3, or HL-60 cells. A 40-fold stimulation of chloramphenicol acetyltransferase activity mediated by four tandem repeats of the SNE could be induced within 2 h (and up to 250-fold within 6 h) after addition of TPA in DNA-transfected U-937 cells, indicating that the stimulation appeared likely to be a true protein kinase Cmediated signal transduction event rather than a differentiation response (Chan et al, 1996). These studies demonstrate that different cell types are expected to sustain different levels of expression from CMV and that, for cell culture transfections, PKC transduction pathways are likely to stimulate transgene expression from CMV promoters. These findings have important implications for promoter choice in gene therapy.

Two multicopy basal enhancer motifs within the simian CMV IE enhancer, namely, 11 copies of the 16-bp cyclic AMP response element (CRE) and 3 copies of novel 17-bp serum response factor (SRF) binding sites referred to as the SNE (SRF/NF-&B-like element), as well as four classical NF-&B sites within the human CMV promoter, contributed to TPA responsiveness; the SNE sites of the simian CMV promoter contain potential overlapping core recognition binding motifs for SRF, Rel/ NF-&B, ETS, and YY1 class transcription factors but fail to respond to either serum or tumor necrosis factor "; the TPA responsiveness of both human and simian CMV elements proved to involve synergistic interactions between the core SRF binding site (C C A T A T A T G G ) and the adjacent inverted ETS binding motifs (T T C C ), which correlated directly with formation of a bound tripartite complex containing both the cellular SRF and ELK-1 proteins. 32

Gene Therapy and Molecular Biology Vol 1, page 33

7TCAATATTGGCCATTAGCCATATTATTCATTGGTTATATAGCATAAATCAATATTGGCTAT TGGCCATTGCATACGTTGTATCTATATCATAATATGTACATTTATATTGGCTCATGTCCAATA TGACCGCCATGTTGGCATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTA GTTCATAGCCCATATATGGAGTTCC GCGTTACATAACTTACGGTAAATGGCCCGCCTGGCT GACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCC AATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCA GTACATCAAGTGTATCATATGCCAAGTCCGCCCCCTAttgacgtcaaTGACGGTAAATGGCCCGC CTGGCATTATGCCCAGTACATGACCTTACGGGACTTTCCTACTTGGCAGTACATCTACGTA TTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACACCAATGGGCGTGGATAGC GGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTG GCACCAAAATCAACGGGACTTTCCAAAATGTCGTAATAACCCCGCCCCGTTGACGCAAAT GGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCGTTTAGTGAACCGTCA GATCACTAGAAGCTTTATTGCGGTAGTTTATCACAGTTAAATTGCTAACGCAGTCAGTG803 Figure 13A. The CMV promoter sequence from plasmid pRL-CMV, 4079 bp (nucleotides 7-803, Promega) (LOCUS AF025843)

37TCTGCTTATATAGACCTCCCACCGTACACGCCTACCGCCCATTTGCGTCAATGGGGC GGAGTTGTTACGACATTTTGGAAAGTCCCGTTGATTTTGGTGCCAAAACAAACTCCCATTG ACGTCAATGGGGTGGAGACTTGGAAATCCCCGTGAGTCAAACCGCTATCCACGCCCATTG ATGTACTGCCAAAACCGCATCACCATGGTAATAGCGATGACTAATACGTAGATGTACTGCCA AGTAGGAAAGTCCCATAAGGTCATGTACTGGGCATAATGCCAGGCGGGCCATTTACCGT CA TTGACGTCAATAGGGGGCGTACTTGGCATATGATACACTTGATGTACTGCCAAGTGGGCA GTTTACCGTAAATACTCCACCCATTGACGTCAATGGAAAGTCCCTATTGGCGTTACTATGG GAACATACGTCATTATTGACGTCAATGGGCGGGGGTCGTTGGGCGGTCAGCCAGGCGGGC CATTTACCGTAAGTTATGTAACGCGGAACTCCATATATGGGCTATGAACTAATGACCCCGTA ATTGATTACTATTAATAACTAGTCAATAATCAATGTCAACATGGCGGTAATGTTGGACATGA GCCAATATAAATGTACATATTATGATATGGATACAACGTATGCAATGGGCCAA695 F i g u r e 1 3 B . The CMV IE promoter (nucleotides 37-695, 658 bp) from the expression vector pCMVtkLUC+ (ACCESSION AF027129). t t g a c g t c a a is the binding site of HB16; GGGACTTTCC is the binding site for HIVEN 86A (two sites); CCATATATGG is the SRF binding site; TTCC is the ETS core motif; three CATTGACG motifs in each sequence are in bold-face (see Boulikas, 1994 for more references).

A closely related family of ubiquitous DNA binding proteins, called MDBP, binds with high affinity to two 14 base pair (bp) sites within the human cytomegalovirus immediate early gene 1 (CMV IE1) enhancer; these MDBP sites did not require cytosine methylation for optimal binding; mutation of one of the enhancer MDBP sites to prevent MDBP recognition modestly increased the function of a neighboring CREB binding site in a transient transfection assay (Zhang et al, 1995). Furthermore, the CMV promoter competed with the Egr1 promoter for transcription factors or co-factors which might be required for activation by WT1; WT1 was converted from an activator to a repressor by co-transfection of an excess of the parental CMV-based vector (Reddy et al, 1995).

A number of studies have used tissue-specific promoters and enhancers from mammalian genes in order to attain a cell type-specific expression of the transgene. The discovery of genes which are expressed at high levels in specific tumor cell types has prompted the idea of the use of their promoter or enhancer DNA sequences to express in this particular cancer cell type therapeutically important genes (Venkatesh et al, 1990; Brady et al, 1994; Dimaio et al, 1994; Osaki et al, 1994; Pang et al, 1995). Examples include the expression of the suicidal CD gene under control of the regulatory regions of the tumor marker gene carcinoembryonic antigen (Richards et al, 1995), the expression of HSV-tk gene, under control of " fetoprotein enhancer and albumin promoter, into adult liver cells in transgenic animals (Su et al, 1996), the expression of #-galactosidase in tyrosine hydroxylase-expressing neurons in the substantia nigra midbrain of adult rats using the tyrosine hydroxylase promoter (Song et al, 1997), and

C. Tissue-specific promoters in gene therapy

Boulikas: An overview on gene therapy the expression of the lacZ marker gene under control of the murine pancreatic amylase promoter in the pancreas in neonatal and adult mice (Dematteo et al, 1997). Transduction of the human LDL-R cDNA under the transcriptional control of the liver-type pyruvate kinase promoter allowed high and tissue specific expression of the gene in primary hepatocytes (Pages et al, 1996b). Fibroblasts, infected with recombinant retroviruses and selected with G418 for the expression of the vector carrying the therapeutic gene, have been used for the ex vivo treatment in animal models; when the therapeutic gene was either under control of the viral LTR or an heterologous internal promoter, expression of the transgene from the integrated retrovirus was shut off (Scharfmann et al, 1991). The use of the dihydrofolate reductase housekeeping gene promoter which is expressed in all cell types, led to sustained expression, albeit at very low levels (Scharfmann et al, 1991); it appears that the combination of a suitable enhancer and promoter for a particular cell type and the method of introduction of the transgene is crucial for sustained expression. Combination of the mouse muscle creatine kinase enhancer with the human cytomegalovirus promoter to drive the expression of the canine factor IX gene in ex vivo infected mouse primary myoblasts led to the expression of factor IX and its secretion in the blood of mice transplanted with these myoblasts for over 6 months; however, the levels of factor IX protein secreted into the plasma (10 ng/ml for 107 injected cells) were not sufficient to be of therapeutic value (Dai et al, 1992). Joki and coworkers (1995) have used the promoter of the early growth response gene 1 (EGR-1, also known as Zif/268, TIS-8, NFGI-A, or Krox-24) to confer selective expression of the luciferase gene in glioma cell lines exposed to ionizing radiation; a 10-fold higher activity in luciferase activity was found after irradiation of the cells which was detectable at 1-3 h after stimulation with 20 Gy (stereotactic radiosurgery during treatment of isolated brain metastases, arteriovenous malformations, meningiomas, craniopharyngiomas, and glioblastomas uses a single dose of 20-30 Gy). Transfection of the HSV-tk gene under control of the EGF-1 promoter rendered irradiated, but not nonirradiated, cells sensitive to GCV. Irradiation induces DNA repair, cell cycle arrest, and reinitiation of DNA synthesis in surviving cells; ! -radiation also induces higher levels of a number of proteins including p53, AP-1, NF-&B, TNF, IL-1, and EGF-1. Therefore, use of the EGF-1 promoter can activate gene expression selectively in radiation fields and could be used to drive the expression of cytotoxic genes (HSV-tk) for the killing of cancer cells. Peptides containing the three zinc fingers of Zif268 could efficiently repress activated transcription from promoter constructs prepared with Zif268 binding sites inserted at various positions with respect to the TATA box (Kim and Pabo, 1997); such strategies could find important applications in gene therapy leading to construction of artificial promoters able to activate or repress transcription of transferred genes. A potent hybrid

CAG promoter was used to drive the HSV-tk gene and showed effective eradication of pancreatic tumors in animal xenografts (Aoki et al, 1997).

D. Molecular switch systems The ability to regulate gene expression via exogenous stimuli will facilitate the study of gene functions in mammalian cells. Molecular switch systems have been devised (Wang et al, 1994) allowing the researcher to turn on or off individual genes; the switch used by Delort and Capecchi (1996) is composed of three elements: (i) the inducible UAS promoter, a synthetic promoter containing five GAL4 response elements, normally absent from mammalian genomes; ( i i ) the synthetic hybrid steroid receptor (TAXI), composed of the GAL4 DNA -binding domain, a truncated human progesterone receptor, and the acidic region from VP16 protein of HSV; the hybrid molecule activates transcription from the UAS promoter when bound to an inducer drug, and ( i i i ) the synthetic nontoxic drug inducer RU486 which is permeable to blood-brain and placental barriers; this model allows up to 100-fold induction of a gene linked to this system and can be finely tuned to lower levels of induction (Delort and Capecchi, 1996). Transient cotransfection of HeLa cells with the UASCAT and the hybrid receptor expression vector showed that the hybrid TAXI protein bound to the UAS promoter only after treatment with RU486 but not progesterone; the TAXI/UAS system was successfully used in transgenic mice to regulate the expression of a human growth hormone gene; the ex vivo approach, however, did not sustain long-term expression of the transgene. This system might allow physicians to alter the level of expression of foreign genes during somatic cell transfer in response to the clinical state of the patient (Delort and Capecchi, 1996). Iida et al (1996) have modified the tetracyclinecontrolled inducible system by the addition of the ligandbinding domain of the estrogen receptor to the carboxy terminus of the tTA transactivator; a single retroviral vector could transduce both the transactivator gene and the gene of interest controlled by the tTA-inducible promoter into mammalian cells; cell lines expressing the transactivator were established where the expression of a gene (the toxic G protein of vesicular stomatitis virus) depended on the removal of tetracycline and the addition of estrogen. A different genetic switch used consisted of the cytochrome P450 1A1 promoter driving the expression of the human apolipoprotein E (apoE) gene in transgenic mice; this switch system was induced by #naphthoflavone; the inducer could pass transplacentally and via breast milk from an injected mother to her suckling neonatal pups, giving rise to the induction of human apoE in neonate plasma and lowering the cholesterol levels in hypercholesterolemic pups (Smith et al, 1995).

Gene Therapy and Molecular Biology Vol 1, page 35

XII. DNA recombination in gene therapy

Group I introns from a variety of organisms contain long open reading frames (ORFs) that encode site-specific DNA endonucleases which promote integration of their DNA into cognate sites via homologous recombination. These endonucleases typically cleave intron-lacking DNA near the site of intron insertion (exon-exon junction) creating a staggered DSB which facilitates intron invasion (intron homing). This mechanism has been demonstrated in mitochondria, chloroplasts and nuclei of eukaryotic cells. I-CreI is a member of this class of molecules that promotes homing of the chloroplast 23S rRNA intron in Chlamydomonas reinhardtii; I-CreI contains once the LAGLI-DADG motif (whereas other members of the family contain two copies of this motif separated by 90120 amino acids); this motif is important for the endonuclease activity of the molecule. DNA cleavage by ICreI requires Mg2+ or Mn 2+ and is inhibited by monovalent cations, has an optimum for catalytic activity of 50-55oC, is stabilized by DNA and binds to 12 nt on each target strand (Wang et al, 1997).

A. Mechanisms of DNA recombination Genetic recombination, i.e., exchange of segments of DNA between two molecules of DNA, is a very frequent event. It often occurs during meiosis and also between homologous chromosomes in mitosis. Homologous recombination involving double-strand DNA breaks (DSBs), has similarities to mechanisms of repair of DSB lesions by cells. Specific recombinases have played and continue to play an important role in molecular evolution and genome shuffling; deregulation in recombination procecess is connected to chromosomal aberrations (inversions, translocations) in cancer. The double-strandbreak repair model was put forward by Szostak and collaborators (1983) to explain genetic recombination in yeast. Recent studies (reviewed by Stahl, 1996) have isolated the recombination intermediate molecules predicted by the DSB repair model; in this model, a 5â&#x20AC;&#x2122;-3â&#x20AC;&#x2122; exonuclease is responsible for the removal of segments of single strands starting bidirectionally from the DSB followed by invasion, repair synthesis and ligation to give the joint molecule which is then reduced to a pair of duplexes by a Holliday junction resolvase. The development of mature lymphocytes in mammals results from a complex combination of genetically preprogrammed events and interactions with antigens. Shared in its general mechanisms by both B (bone marrow) and T (thymus) lymphocytes this developmental program involves a series of cell migration gene rearrangements, cell-to-cell contacts, as well as positive and negative selection processes; recombination mechanisms take place at the immunoglobulin and the T cell receptor genes to generate a large number of immunoglobulin genes in different lymphocyte clones. One site-specific recombination event brings together the V and the J segments of the light chain immunoglobulin genes. In the case of the heavy chain genes, one recombination event joins a V to a D segment, sequentially followed in a time frame by the joining of the recombined V-D segment to a J segment. Recent studies have shown that the mechanism of V(D)J recombination is a two-step process involving: ( i ) site-specific DNA cleavage at the 7mer sequence and at the first nucleotide of the coding sequence, implicating the RAG-1 and RAG-2 proteins which are necessary and sufficient for this step (van Gent et al, 1996); ( i i ) joining of broken ends in a mechanism similar to the repair of double strand breaks. The murine SCID locus has provided crucial information in the elucidation of the second step in V(D)J recombination: thymocytes in SCID mice are able to catalyze joining of signal ends but display an accumulation of hairpin coding ends (Zhu et al, 1996). The murine SCID locus has been mapped to the gene encoding the catalytic subunit of DNA-dependent protein kinase (DNAPK) (Kirchgessner et al, 1995).

B. Aberrant recombinations can result in human disease Mammals carry about 1,000,000 copies of Alu sequences and 10,000 to 100,000 copies of complete and truncated versions of the L1 class of LINEs. Such sequences promote homologous recombination causing translocations of genes and have been hold responsible for a number of human disorders. Alu sequences are found in introns. One type of mutation in the LDL receptor gene responsible for familial hypercholesterolemia has incurred by Alu-Alu recombination deleting several exons and thus producing a truncated receptor molecule with loss of function (Lehrman et al, 1987). De novo insertions of an L1 element into the factor VIII gene can cause hemophilia A in humans (Kazazian et al, 1988). Foreign DNA transferred to host cells may be rejected (degraded), integrated at random sites by illegitimate recombination, integrated at homologous sites by legitimate recombination, or remain extrachromosomal and replicate autonomously. The homologous recombination between chromosomal DNA and transfected DNA sequences, an event termed "gene targeting," can be used to correct mutated genes in cultured cells. It has been known for a long time that the translocation of an active gene to the neighborhood of heterochromatin (transcriptionally inert part of the genome) results in silencing of the translocated gene, a process known as "position effect variegation", first described in Drosophila (Lewis, 1950; Wilson et al, 1990). Chromosomal translocations seem to contribute to tumorigenesis either by activating proto-oncogenes to oncogenes or by inactivating tumor suppressor genes. Mammalian chromosomes contain a number of breaksusceptible or fragile sites where breakage can be induced reproducibly by experimental manipulations. Such fragile 35

Boulikas: An overview on gene therapy sites might lie in the neighborhood of transposable elements, hypervariable minisatellites or other DNA structural peculiarities such as Z-DNA, and other hotspots of recombination (reviewed by Haluska et al., 1987). Nonrandom chromosome rearrangements, observed in a variety of human and animal tumors are associated with the enhanced expression or deregulation of cellular oncogenes. For example, the human c-myc oncogene becomes active following its translocation close to the enhancer sequence within the immunoglobulin heavy chain gene locus (Hayday et al., 1984). The chromosomal translocation (17;19) in acute lymphoblastic leukemia produces a chimeric transcription factor consisting of the amino-terminal portion of the helix-loop-helix proteins E12/E47 fused to the DNA binding and leucine zipper dimerization motifs of the liver-specific protein factor Hlf (Hepatic leukemia factor), normally not expressed in lymphoid cells (Hunger et al., 1992). In pre-B cell acute lymphoblastic leukemia (ALL) the t(1;19) translocation brings together two gene fragments encoding for transcription factors and results in the synthesis of a chimeric transcription factor composed of truncated E2A and Pbx1 (Kamps et al., 1991).

C. Exploitation of recombinases in gene therapy: the Cre/LoxP system A strategy has recently been developed which facilitates culturing of human cells derived from primary tumors. This method is based on the transient expression of T antigen of SV40 which has been shown to immortalize primary cells of human and murine origin and on the use of bacteriophage recombinase Cre which catalyzes sequence-specific recombination at the LoxP sequence inducing permanent deletion of T antigen cDNA; indeed, if two LoxP sequences were provided as direct repeats the intervening sequence could be deleted during Cre recombination and lost from cells. This is an advantage for primary cell cultures because the continuous expression of SV40 large T antigen may alter the antigenicity of the cells and induce other type of mutations not associated with the original tumor; according to this strategy large T antigen can be expressed in a time-dependent way(Li et al, 1997). Figure 14 shows the structure of the two retroviral vectors used by Li et al (1997) to facilitate culturing of primary tumor cells and Figure 15 the change in morphology as a result of Cre-Puro retrovirus-induced loss of expression of T antigen in T antigen-immortalized primary cell cultures of mouse breast cancer cells.

F i g u r e 1 4 . Structure of retroviral vectors LoxP-HyTK-large T and Cre-puro allowing expression of T antigen and Cre/LoxPcatalyzed deletion of T antigen cDNA. The 4 small arrows indicate the primers used for PCR analysis; the large arrow above the NLS-CRE box indicates the Cre recombinase mediating LoxP specific deletion. LTR are the long terminal repeats of Moloney Murine Leukemia virus; LOX is the 34 bp sequence identical to the recognition site of Cre recombinase; HyTK is the hygromycin/thymidine kinase fusion gene; NLS-CRE is the Cre recombinase gene targeted to the nucleus by a nuclear localization signal (NLS); SV40 is the SV40 promoter; Puro is a puromycin selection marker. From Li LP, Schlag PM, Blankenstein T (1 9 9 7 ) Transient expression of SV 40 large T antigen by Cre/LoxP-mediated site-specific deletion in primary human tumor cells. Hum Gene Ther 8, 1695-1700. Reproduced with the kind permission of the authors and Mary Ann Liebert, Inc.

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F i g u r e 1 5 . Large T antigen-immortalized breast cancer cells change morphology and lose expression of T antigen after infection with Cre-Puro retrovirus: after prolonged culture the cells were infected with Cre-Puro retrovirus (B , D ) or mock-infected (A, C), selected for Puromycin resistance (A, B, C, D) and resistance to ganciclovir (B, D) and analyzed by light microscopy (A, B) and staining with a large T-antibody (C, D). From Li LP, Schlag PM, Blankenstein T (1 9 9 7 ) Transient expression of SV 40 large T antigen by Cre/LoxP-mediated site-specific deletion in primary human tumor cells. Hum Gene Ther 8, 1695-1700. Reproduced with the kind permission of the authors and Mary Ann Liebert, Inc.

expression unit and molecular analyses of 30 doubly transduced subclones showed a strict correlation between Cre expression and LoxP-flanked selectable cassette excision; excision of the selectable cassette resulted in a significant increase of GM-CSF transcription driven by the retroviral promoter (Fernex et al, 1997). Novel retroviral vectors for gene transfer were developed by Bergemann et al (1995) by inserting two LoxP sites into a retroviral vector also containing the HSV-tk gene; Cre expression in cells infected with this vector was followed by BrdU selection for cells in which site-specific recombination took place. Furthermore, replacement of the enhancer/promoter elements in both LTRs by Lox sequences led to the development of retroviral suicide vectors for gene therapy. Vanin et al (1997) have used the Cre/LoxP recombinase system to generate high-titer retroviral producer cell lines;

Reversible immortalization of primary cells was achieved by Westerman and Leboulch (1996) using retrovirus-mediated transfer of an oncogene that could be subsequently excised by site-specific Cre/LoxP recombination; the FLP/FRT recombination was not efficient in primary cells. Pure populations of cells in which the oncogene was permanently excised were obtained which reverted to their preimmortalized state. Using the Cre/LoxP recombination strategy primary cells could be cultured and expanded; the method was proposed to be applicable for facilitating gene transfer to cells unresponsive to exogenous growth factors. A retroviral vector, containing both a neomycin resistance expression unit flanked by loxP sites and GMCSF cDNA, was used to transduce the human hematopoietic K-562 cell line. Superinfection of K562 cell clones with a retrovirus containing a Cre recombinase 37

Boulikas: An overview on gene therapy incorporation of LoxP sites at the flanks of a NeoR-HSVtk cassette in the proviral DNA allowed excision of these selectable markers through expression of Cre recombinase and the production of a high-titer producer cell line containing a single LoxP site flanked by the viral LTRs. Retransfection of this cell line with a plasmid containing a gene of interest flanked by LoxP sites and the Cre expression vector allowed insertional LoxP/LoxP recombination of the gene into the favorable preexisting site in the genome and the generation of a new line with a titer equivalent to that of the parental producer cell line. The Cre/LoxP recombinase strategy has been used to generate retroviral vectors with the ability to excise themselves after inserting a gene into the genome (Russ et al, 1996). Bushman and Miller (1997) fused retroviral integrase enzymes to sequence-specific DNA-binding domains and investigated target site selection by the resulting proteins. A fusion protein composed of HIV integrase linked to the DNA-binding domain of ' repressor was able to direct selective integration of retroviral cDNA in vitro into target DNA containing ' repressor binding sites. A fusion of HIV integrase to the DNA binding domain of the zinc finger protein Zif268 also directed increased integration near Zif268 recognition sites. Introduction of foreign DNA into cell nuclei with recombinase cDNA and appropriate sequences to promote recombination may promote nor only insertion of a therapeutic gene into a specific chromosomal site but also chromosomal rearrangements that could convert therapeutically transduced cells into malignant. There is a great deal of knowledge to be derived from these very promising strategies of gene therapy before they can be successfully applied to humans.

gene therapy treatment would need to be repeated twice a year, for example to a hemophilia patient or to a patient who has undergone balloon treatment after coronary heart disease and is being treated via arterial gene transfer. An approach to sustain expression of the transgene is via episomal replication of the plasmid carrying the transgene for long periods of time, maintaining the plasmid in high copy numbers, and in a form replicating in synchrony with the cell cycle; even better a plasmid can be replicated continuously independently of the cell cycle, an approach to find application in the transfection of nondividing cells by plasmids (which to date is a virtue of adenoviruses, AAV, and HIV-1 vectors; seeTable 1). A way to sustain expression of the transgene could be achieved via targeted integration into one or several different chromosomal locations and the insulation of the transgene from neighboring chromatin domains using special classes of DNA sequences able to act as insulators and maintain independent realms of gene activity (such as matrix-attached regions, MARs). In this case flanking of the foreign gene by two MAR sequences is expected to insulate it against position effect variegation and prevent inactivation of the gene at the chromatin level by chromatin condensation or other mechanisms propagated from the neighboring domains at the integration site (Boulikas, 1995b). Several studies have shown that linearization of plasmids with restriction enzymes favor highly their integration into the host's genome compared with supercoiled, covalently-closed plasmid DNA. Free ends of DNA are known to promote recombination and a number of nuclear proteins including p53, poly(ADP-ribose) polymerase, ligases I and II, Ku antigen, DNA-dependent protein kinase are known to bind to free ends of DNA, whereas other molecules such as helicases and endonucleases are known to function during repair of lesions in DNA inducing the appearance of strand breaks; especially important in this aspect are members of the RAD50-57 family of proteins involved in recombination and in repair of double-strand breaks.

XIII. Fate of the transgene in the nucleus A. How to sustain transgene expression? A major drawback in gene therapy applications is loss in gene activity within a few days from gene transfer although all previous steps were successful. In other words, the transferred gene is transiently expressed for 1-4 days and its expression thereafter declines dramatically. This is due ( i ) to the degradation of the gene in the nucleus; ( i i ) the dilution of the plasmid during replication of the cells from its inability to replicate; ( i i i ) its inactivation by position effects from chromatin surroundings after its integration into the chromosomal DNA; ( i v ) the elimination of the therapeutic cells expressing the transgene by the immune system of the organism either because of the antigenicity of the expressed protein or because of the antigenicity of viral proteins, an effect often associated with adenoviral and retroviral gene delivery. A number of strategies are being pursued to solve these problems. Sustaining the expression of a transgene into somatic cells for, lets say, 6 months would mean than a

B. Episomal plasmids for gene transfer Integration or replication of a foreign gene introduced as a plasmid into mammalian cells is a very rare event; plasmid DNA resides transiently in the nucleus as an episomal, extrachromosomal element for short periods of time after transfection of cells in culture (usually up to one or very few days) during which transcription can take place; after that the episomal DNA is degraded and lost permanently from the cells. Viral origins of replication have been introduced into the same plasmid as the reporter gene and found to increase the persistence of expression. A polyoma virus-based plasmid containing the polyoma virus origin of replication and the T antigen gene, as well as the neoR gene was maintained extrachromosomally in mouse embryonic stem (ES) cells at 10-30 copies per cell for at least 74 cell 38

Gene Therapy and Molecular Biology Vol 1, page 39 generations in the presence of G418 (Gassmann et al, 1995). Prolonged episomal persistence may be an advantage for gene therapy of nondividing cells. A limited number of studies in gene transfer have used plasmids able to replicate episomally. Most of the plasmids used contain viral origins of replication but also the gene of the replication initiator protein that after its expression in the host will interact with the origin of the plasmid to maintain a relatively high copy number of plasmids which will persist for some time. The advantage using episomal replication of plasmids is enormous in somatic human gene therapy as it can sustain expression of a transgene for a few months after a single injection of the plasmid as compared to the loss of expression after about 1-10 days (maximum at day 2) following injection of nonepisomal plasmids (Zhu et al, 1993). Thierry and coworkers (1995) have succeeded in sustaining the expression of the luciferase reporter gene in mice for up to 3 months after a single intravenous injection of a plasmid including the human papovavirus BKV early region and origin of replication, the large tumor antigen (T antigen) as the replication initiator protein, and the late viral capsid proteins in the same construct harboring the luciferase gene; this plasmid was shown to be replicated extrachromosomally for 2 weeks in the lung. Episomal replication of a hybrid HSV-1/EBV vector was achieved when the latent oriP of EBV and the EBNA-1 cDNA, which encodes for the replication initiator protein of EBV, were included in the vector (Wang and Vos, 1996). Expression of viral replication initiator proteins (e.g. T antigen) is oncogenic. Of special interest in human gene therapy is to determine human DNA sequences able to sustain the extrachromosomal replication of plasmids into permissive human cells for longer periods. Such DNA sequences known to act as origins of replication, although poorly understood, have been found in human, monkey, and other mammalian genomes and could be used to sustain the replication of the plasmid thus increasing its copy number in the cell and the time of its persistence (see page 122-123). To this end, a technology has been developed in our laboratory that permits us to isolate human origins of replication (ORIs) and to include selected ORIs together with the cDNA of the replication initiator protein responsible for activating this particular ORI, in plasmids with therapeutic genes (Boulikas et al, in preparation).

its passage through the cell membrane barrier to cytoplasmic lysosomes before entering nuclei; if DNA is cut the supercoils on the plasmid will be relaxed. Nicked DNA might be repaired and ligated in nuclei by DNA ligases and be subject to the same constrains as chromosomal DNA. Use of linear plasmids is expected to stimulate recombination during repair of double strand breaks (also would increase degradation of the plasmid in the nucleus and loss of the transgene) ultimately resulting in plasmid integration at variable chromosomal loci, determined to some extend by the nature of the free ends of DNA and the short terminal sequence of the DNA at the ends as well as the type of recombinase molecules in the cell type used. Treatment of cell cultures with sodium butyrate inducing hyperacetylation of core histones would reverse in part the relieving of the negative torsional strain by the wrapping of the plasmid around histone octamers and will provide DNA in a negatively superhelical of underwound form able to sustain transcription of the template (Schlake et al, 1994).

D. Overcoming the influences of chromosomal surroundings at plasmid integration sites Use of two MARs each flanking the reporter gene on either side is expected to form a minidomain after integration of the foreign gene into a chromosomal site. MARs potentiate the effect of promoters and enhancers when two MAR elements are placed one upstream and the other downstream from control elements but not between them. MARs will ( i ) shield reporter genes from the influences of chromosomal surroundings that most often cause inactivation of foreign genes. This effect of chromatin structure on neighboring sequences is known as position effect variegation. Indeed about 85% of the chromosomal sites are transcriptionally inactive assuming that 15% of the genomic DNA is transcribed; however, even integration of a foreign gene into an active chromatin locus may not warrantee its transcriptional activation as other parameters, such as proximity of the integration site to the natural promoter and enhancer elements of the active chromatin domain, or orientation of the integrated gene with respect to the active gene in the chromosomal DNA may determine its level of expression. ( i i ) MARs will maintain a supercoiled DNA topology within the domain thus increasing the negative supercoiling at local promoter and enhancer sites, a prerequisite for efficient transcription (see Boulikas, 1995b).

C. Considerations of chromatin structure of plasmids during gene delivery

XIV. Transfer of reporter genes A. Transfer of the -galactosidase (lacZ) reporter gene

Almost all supercoiled plasmids used in gene transfer, as produced in bacteria, are under negative supercoiling. Immediately after their import into nuclei plasmids are packaged into nucleosomes that absorb and constrain part or most likely all of the negative supercoils. This is true assuming that no cuts on the DNA are introduced during

Before a gene therapy preclinical study or even gene transfer to cells in culture begins it is essential to test the variables and pinpoint the conditions leading to the success of the operation using reporter gene transfer. LacZ, 39

Boulikas: An overview on gene therapy encoding the #-galactosidase (#-Gal) from E. coli is one of the most commonly used reporter genes. A staining procedure for this enzymatic activity can result in the generation of blue color using X-Gal as a substrate leading to the direct visualization of its activity, for example, in thin sections through animal tissues. Transfer of the reporter #-galactosidase gene to human liver tumors in nude mice was performed by Wang and Vos (1996) using a hybrid HSV-1/EBV vector which replicates episomally when the latent oriP of EBV and the EBNA-1 cDNA were included. Many mammalian tissues, especially intestine, kidney, epididymis, and lung contain endogenous #-Gal, a lysosomal enzyme participating biochemically in

glycolipid digestion. Weiss et al (1997) were able to detect mammalian #-Gal activity on histochemical preparations of mouse, rat and baboon lung tissue (Figure 16) and also to distinguish between the endogenous and bacterial #Gal activity in airway epithelial cells in the transgenic Rosa-26 mouse in based upon the differences in pH optima between the mammalian and bacterial enzymes (F igure 17). Time and temperature of exposure to X-Gal could not be used to distinguish between endogenous and exogenous #-Gal activity; thus, exposure of tissue preparation to pH 8.0-8.5, which minimized detection of the endogenous activity allowed unambiguous discrimination and was the method of choice to detect reporter#-Gal activity.

F i g u r e 1 6 . Endogenous mammalian #-Gal activity was detected in minced lung preparations from a variety of species following incubation with X-Gal in PBS. Representative fields from paraffin-embedded sections are shown. Original magnification 400X. Airway epithelium, alveolar macrophages and several unidentified cell types are stained blue and are, therefore, positive for #-Gal activity. From Weiss DJ, Liggitt D, Clark JG (1 9 9 7 ) In situ histochemical detection of #galactosidase activity in lung: assessment of X-Gal reagent in distinguishing lacZ gene expression and endogenous #galactosidase activity. Hum Gene Ther 8, 1545-1554. With the kind permission of the authors (Daniel Weiss, Fred Hutchinson Cancer Research Center) and Mary Ann Liebert, Inc.

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F i g u r e 1 7 . Endogenous mammalian #-Gal activity was not detected following incubation with X-Gal at pH>7.5 whereas bacterial #-Gal activity was detected at pH 8.0-8.5 in airway epithelial cells in the transgenic Rosa-26 mouse. Original magnification 250X. From Weiss DJ, Liggitt D, Clark JG (1 9 9 7 ) In situ histochemical detection of #-galactosidase activity in lung: assessment of X-Gal reagent in distinguishing lacZ gene expression and endogenous #-galactosidase activity. Hum Gene Ther 8, 1545-1554. With the kind permission of the authors (Daniel Weiss, Fred Hutchinson Cancer Research Center) and Mary Ann Liebert, Inc.

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Table 2. Reporter gene transfer in vivo Gene delivere d CAT and luciferase

Human disease

Vecto r

Method/Goal

Results

Reference

none

DOTM A: DOPE

Expression of reporter genes in mouse tissues after a single intravenous injection.

Zhu et al, 1993

Luciferase/ CMV construct and lacZ

none

Transfe ctam (DOGS :DOPE)

Luciferase/ CMV

none

Cation ic liposo mes AAV

To show effectiveness in gene transfer after intracranial injection of liposome-plasmid complexes into the newborn mouse. Distribution of luciferase gene expression among tissues and persistence of expression after systemic injection. Direct injection of tumors induced from human glioma cells into the brains of nude mice.

CMV promoter was most effective; expression in vascular endothelia, extravascular and parenchymal cells present in lung, spleen, and heart; lower expression in all other tissues; persisted for 9 weeks. Transient expression of luciferase in striatal parenchymal cells; lipospermine:DNA charge ratio of 2 or smaller was most effective in vivo. Use of viral origins of replication and T antigen cDNA in vector sustained expression up to 3 months primarily in the lung in mice. 30-40% of the cells along the needle track expressed #-galactosidase; administration of GCV to the HSV-tk/IL-2 treated animals for 6 days, resulted in a 35-fold reduction in the mean volume of tumors compared with controls. Protein expression was detected in myofibers for at least 32 weeks; dosedependent secretion of erythropoietin and corresponding increases in red blood cell production in mice persisted for up to 40 weeks. Successful transduction of all layers of the neuroretina as well as the retinal pigment epithelium; the efficiency of transduction of photoreceptors was significantly higher than that achieved with an equivalent adenoviral vector. Thin sections of cochleae showed intense immunohistochemical reactivity in the spiral limbus, spiral ligament, spiral ganglion cells and the organ of Corti but much weaker staining in the contralateral ear. A 6.8-kb fragment of the rat tyrosine hydroxylase promoter supported a 7- to 20fold increase in reporter gene expression in catecholaminergic tyrosine hydroxylaseexpressing neurons in the substantia nigra. Successful transduction of medulloblastoma (DAOY) cells in a nude rat model of leptomeningeal disease. Comparable gene transfer into medial and adventitial cells, with significantly higher efficiency of transduction in injured compared with normal vessels.

Thierry et al, 1995

AP staining was almost exclusively in the epithelial and smooth muscle cells in the bronchus at the region of balloon placement; staining was in ciliated cells but was also in basal cells and airway smooth muscle cells.

Halbert et al, 1997

lacZ and glioma HSV-tk/IL2

lacZ and human erythropoi etin

none

AAV

Single intramuscular injection into adult BALB/c mice.

lacZ

Retinal degeneration ; retinitis pigmentosa

AAV

Subretinal injection of recombinant AAV particles encoding lacZ.

lacZ

Inherited hearing disorders

AAV

To assess the feasibility of introducing genetic material directly into the peripheral auditory system; infusion into the cochlea of guinea pigs.

lacZ

Parkinsonâ&#x20AC;&#x2122;s disease

HSV-1

Stereotactic injection into the midbrain of adult rats.

lacZ

Leptomenin geal cancer

AAV

To test the feasibility of AAVmediated gene therapy.

lacZ

vascular disorders

AAV

human placental alkaline phosphata se (AP)

lung disease

AAV

To develop gene therapies for vascular disorders by gene transfer into isolated segments of normal and balloon-injured rat carotid arteries. To assess the ability of AAV vectors to transduce airway cells; AP gene was delivered to one lobe of the rabbit lung using a balloon catheter.

Schwartz et al, 1995

Okada et al,. 1996

Kessler et al, 1996

Ali et al, 1996

Lalwani et al, 1996

Song et al, 1997

Rosenfeld et al, 1997 Rolling et al, 1997

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a response that, in many cases, cannot be elicited in normal mice. Cells undergoing malignant transformation are believed to be eliminated from the body by white blood cells including natural killer cells (NK), lymphokine-activated killer cells (LAK), cytotoxic T lymphocytes (CTL), tumor-infiltrating lymphocytes (TIL), and activated macrophages; since established cancers in the human body may escape this potential defense mechanism of immunologic surveillance, cancer patients have been treated with IL-2 to stimulate their cellular immune mechanisms to kill cancer cells; lengthy and complete remissions, however, were at a low rate and complications were encountered by the toxicity caused by the systemic administration of IL-2 (Rosenberg, 1992). Transfection of the IL-2 gene into human melanoma cells increased cellular immune response (Uchiyama et al, 1993). This and similar approaches have established the foundation of the ex vivo cancer immunotherapy by transfer of autologous (cancer patientâ&#x20AC;&#x2122;s) cells after transduction in vitro with cytokine genes (see below). The ultimate goal is the activation of tumour-specific T lymphocytes capable of rejecting tumour cells from patients.

A phase I study involving six patients with inoperable lung cancer and an endobronchial lesion accessible by bronchoscopy was initiated to evaluate the feasibility, tolerance, and clinical effects using adenoviral delivery of the Escherichia coli lacZ marker gene driven by the RSV promoter; biopsy specimens of the tumor and surrounding mucosa in 5 patients were tested positive for #galactosidase expression (Tursz et al, 1996).

B. Transfer of the luciferase and green fluorescent protein (GFP) reporter genes A synthetic polyamino derivative was used by Goldman and coworkers (1997) to transfer the luciferase and #-galactosidase reporter genes in animal models bearing stereotactically implanted human glioma cell xenografts. The luciferase reporter gene was transferred in both newborn and adult rabbit lungs using polyethylenimine (Ferrari et al, 1997). Thierry and coworkers (1995) have succeeded in sustaining the expression of the luciferase reporter gene in mice for up to three months using episomal vectors (Table 2). GFP has been used as a reporter molecule for gene expression because it fluoresces green after blue-light excitation. However, many attempts by Hanazono et al (1997) to isolate stable retroviral producer cell clones secreting vectors containing GFP (after transfer of the neoR gene and selection in G418) have failed because stable GFP-clones were undergoing major rearrangements or other mutations which abrogated GFP expression and prevented vector production. Additional studies using reporter gene transfer are summarized on Table 2.

B. Cancer immunotherapy with tumor infiltrating lymphocytes (TILs) Ex vivo approaches in immunotherapy have been aimed at isolating T cells directly from tumors (known as tumor infiltrating lymphocytes or TILs), stimulate TILs to proliferate in cell culture with IL-2 followed by their reintroduction into the blood stream of advanced cancer patients (Rosenberg et al, 1988). The adoptive transfer of TILs was 50-100 times more potent than that of lymphokine-activated killer (LAK) cells isolated from the patient's tumors. Treatment of 20 patients with TILs after their expansion in vitro, plus IL-2, resulted in objective regression of metastatic melanomas in lungs, liver, bone, skin, and subcutaneous sites which lasted for several months (Rosenberg et al, 1988). TILs were also transfected in vitro with the bacterial neomycin-resistance gene and were reintroduced into patients with advanced cancer in order to follow their persistence in blood circulation with PCR methods (Aebersold et al, 1990). Such â&#x20AC;&#x153;gene markingâ&#x20AC;? clinical protocols using TILs are numbers 1, 3, 9. 13, 57, and 169 in Appendix 1, pages 159-172. Having shown safety in the NeoR-modified TIL protocol, the gene for tumor necrosis factor (TNF) was added to the vector for therapy of malignant melanoma in advanced cancer patients; the first patient began treatment in January 1991. TNF, however, is effective as an anticancer agent in mice at 400 mg/kg body weight, but in humans, TNF is toxic at 8 mg/Kg and so far of no proven therapeutic value at this low concentration (reviewed by Anderson, 1992). In a similar approach, the TNF gene was replaced by the gene of

DIVISION TWO: GENE THERAPY OF CANCER XV. Cancer immunotherapy and tumor vaccines A. The molecular basis of cancer immunotherapy Many human tumors are nonimmunogenic or weakly immunogenic. The immune system, evolved to rid the body of unwanted intruders, could be instructed and reinforced to eliminate cancer cells. Increasing the immunogenicity of tumors by causing local cytokine production or by increase in the expression in MHC antigen can lead to local antitumor effect (Tepper et al, 1989; Fearon et al, 1990). Indeed, immune surveillance is of the major defense mechanisms against cancer; immunosuppressed individuals are more prone to cancer and nude mice, lacking immune response, are exploited in the lab to elicit tumors after injection of tumorigenic cells,

Boulikas: An overview on gene therapy interleukin-2 (IL-2) in order to develop locally high doses of IL-2 at the tumor site by immunization with TIL cells from the patient producing systemic antitumor immunity (Rosenberg et al, 1992). TNF-", (also IL-1#, IFN-! , and vitamin D3) after binding to their transmembrane receptors stimulate the production of the second messager ceramide from sphingomyelin in the plasma membrane by activating sphingomyelinase; this results in a cascade of signal transduction events that result in down regulation of c-myc and induction of apoptosis, to terminal differentiation, or to RB-mediated cell cycle arrest (see apoptosis further below). IL-2 stimulates the differentiation of precursor lymphocytes into LAK cells and further stimulates LAK cell proliferation; LAK cells are probably produced by activation of NK cells or from activated T cells by IL-2. Administration of IL-2 plus amplified LAK cells into mice models led to marked regression of disseminated cancers and leukemia. LAK cells are able to destroy tumor cells that express only weakly histocompatibility antigens. IL2, however, has several pleiotropic effects: stimulation of B cell proliferation; activation of HLA class II antigen expression on endothelial cells, TILs, and melanoma cells; and enhanced production and release of TNF-" , and IFN-! (see Cassileth et al, 1995 and the references cited therein). However, the use of large numbers of adoptively transferred, broadly cytotoxic LAK cells in combination with IL-2 has been effective for only small subsets of cancer patients (reviewed by Wiltrout et al, 1995). TILs, which could potentially kill tumor cells, are found in many tumors but remain suppressed or anergic; this anergy may arise from the absence of lymphokines which provide signals for TIL cell activation and stimulation to proliferation although ligands may be bound to the variable region of the T cell receptor; indeed, nonimmunogenic tumors are rejected by syngeneic mice upon transfection by IL-2 or IL-4 genes; IL-2 lymphokine production by the tumor cells bypasses T helper function in the generation of an antitumor response rendering the tumor cells immunogenic; nontransfected tumors are not rejected by the animal and grow causing its death (Tepper et al, 1989; Fearon et al, 1990). Ex vivo gene therapy trials using cytokine gene transfer (see below) circumvent the problem of toxicity of IL-2 administration; for non-gene transfer therapies, white blood cells drawn from patients are fractionated, cultured, stimulated with IL-2 or other cytokines, and reintroduced in much higher numbers into the blood of the patient.

successfully transfected cells with a selectable marker such as the bacterial neomycin-resistance gene (Cassileth et al, 1995). Numerous phase I clinical trials employing either syngeneic genetically modified or allogenic tumor vaccines are in progress (see immunotherapy in Appendix 1, page 159-172). The development of tumor cells transduced with cytokine genes and their exploitation as tumor vaccines in patients with cancer is a very promising field (reviewed by Jaffee and Pardoll, 1997). Cytokine genes used for cancer immunotherapy include those of IL-2, IL-4, IL-7, IL-12, IFNs, GM-CSF, TNF-" in combination with genes encoding co-stimulatory molecules, such as B7-I. The major goal of the use of immunostimulatory cytokines is the activation of tumourspecific T lymphocytes capable of rejecting tumour cells from patients with low tumour burden or to protect patients from a recurrence of the disease. As distant metastasis is the major cause for therapeutic failures in clinical oncology, treatment of patients having a low tumor volume with immunotherapy could protect the patient from recurrence of disease. Treatment of rodent tumor models with little or no intrinsic immunogenicity with this approach resulted in regression of preexisting tumors and cure of the animals from their disease; furthermore, in some instances cured animals had retained immunological memory and resisted a second challenge with the parental tumor cells (reviewed by Gilboa, 1996; Mackensen et al, 1997). The transduction of the tumor cells of the patient with cytokine genes ex vivo and the development of tumor vaccines depends on the establishment of primary cell culture from the solid tumor. Although malignant melanomas are easy to culture, it is difficult to establish cell lines from most other primary human tumors using convenient methods; primary tumor cultures are being used ( i ) for the transduction of autologous cells from the cancer patient with cytokine genes to develop cancer vaccines after intradermal implantation to the patient; ( i i ) for characterization of tumor-specific cytotoxic T lymphocytes in order to identify specific antigens on the human primary culture; ( i i i ) for extensive phenotypic characterization of the tumor in cell culture. The Cre/LoxP system (see recombinases in gene therapy) has been used to facilitate the establishment of primary cell lines from human tumors (Li et al, 1997). Human gene therapy protocols 3 and 10 (Appendix 1) use immunization of cancer patients with autologous cancer cells transduced with the gene fortumor necrosis factor (TNF).

D. Cancer immunotherapy with the IL-2 gene

C. Cancer immunotherapy with cytokine genes

Active immunization with pancreatic tumor cells genetically engineered to secrete IL-2 were shown to inhibit pancreatic tumor growth in vivo; this was shown using a poorly immunogenic subcutaneous model of murine ductal pancreatic cancer by implanting tumor

The combination of immunotherapy with conventional treatments such as radio- and chemotherapy may be necessary to eradicate minimal residual disease. Advanced therapies involve the transfection of lymphocytes in culture with cytokine genes followed by selection of the 44

Gene Therapy and Molecular Biology Vol 1, page 45 Panc02 cells in C57BL/6 mice; whereas 90% of animals vaccinated with irradiated parental Panc02 and subsequently challenged with parental Panc02 cells developed tumors by 48 days only 40% of animals vaccinated with irradiated Panc02 cells engineered to secrete IL-2 and challenged with parental Panc02 cells developed tumors by 48 days (Clary et al, 1997). According to a RAC-approved clinical protocol the gene for human interleukin-2 (IL-2) was transduced into a cell line established from the neoplastic cells of a patient with malignant melanoma; this procedure established an IL-2-secreting cell line with integration of the IL-2 gene into genomic DNA. The IL-2-secreting cells were irradiated, in a manner sufficient to inactivate 100% of the cells but insufficient to completely inhibit IL-2 synthesis, and administered to 12 patients with metastatic malignant melanoma in a Phase I toxicity study. These cells have the capacity to induce an antimelanoma response as shown in animal studies (Das Gupta et al, 1997). A significant number of RAC-approved clinical protocols use IL-2 cDNA transfer. These include protocols 11, 16, 19, 20, 46, 48, 50, 61, 71, 102, and 135, in Appendix 1 and protocols 190, 197, 198, 200, 204, 211, 213, 215, and 219 using cationic lipids for gene transfer (Table 4 in Martin and Boulikas, 1998, this volume, pages 203-206).

cells with 10,000 cGy, which arrested tumor cell growth in vitro, did not affect the ability of the cells to secrete IL7 in the culture medium; this approach, which does not use retroviruses, could be applicable in vaccination protocols for melanoma patients (Finke et al, 1997). Transfer of the IL-7 cDNA for cancer immunotherapy is being used in a human clinical trial (protocol 70, Appendix 1).

G. Cancer immunotherapy with the IL-12 gene IL-12 gene therapy is one of the more novel and promising approaches in cancer therapy. IL-12 is a heterodimeric cytokine composed of two subunits, p40 and p35, that requires the simultaneous expression of both the p35 and p40 chain genes from the same cell for production of biologically active IL-12. Coordinate expression of the IL-12 p40 and p35 genes in several solid tumor models has been found to induce strong and specific antitumor immune responses. A variety of biological functions have been attributed to IL-12 including the induction of IFN-! and the promotion of predominantly Th1-type immune responses to antigens (Tahara et al, 1996). The local secretion of IL-12 achieved by gene transduction suppressed tumor growth and promoted the acquisition of specific antitumor immunity in mice. This was shown by intradermal inoculation of mice with NIH3T3 cells transduced with expression plasmids or a retroviral vector expressing the murine IL-12 gene admixed with murine melanoma BL-6 cells; CD4 + and CD8+ T cells, as well as NK cells, were responsible for the observed antitumor effects resulting from IL-12 paracrine secretion. Transduction of tumor cells with B7.1 gene enhanced the antitumor immune response (Tahara et al, 1996). The antitumor effect of several transgene expression plasmids encoding the cytokines IL-2, IL-4, IL-6, IL-12, IFN-! , TNF-", and GM-CSF was tested using the gene gun-mediated DNA delivery into the epidermis overlying an established intradermal murine tumor; this study showed that IL-12 gene therapy was much more effective than treatment with any other tested cytokine gene for induction of tumor regression as determined from the increased CD8+ T cell-mediated cytolytic activity in the draining lymph nodes of tumor-bearing mice; treated animals were able to eradicate not only the treated but also the untreated solid tumors at distant sites; elevated systemic levels of IFN-! , were found after IL-12 gene therapy. This approach is providing a safer alternative to IL-12 protein therapy for clinical treatment of cancers (Rakhmilevich et al, 1997). Lieu et al (1997) have evaluated three IL-12 retroviral vector designs for their level of IL-12 expression in leukemia/lymphoma cells; these retroviral vectors were based on the murine stem cell virus (MSCV) which efficiently transduces functional genes into normal hematopoietic cells. MSCVpac-mlL-12 and MIPV-mIL-12

E. Cancer immunotherapy with the IL-3 gene IL-3 was found to enhance the development of cytotoxic T lymphocytes; during antitumor response, macrophages could ingest whole tumor cells, cell fragments, or heat shock proteins complexed to antigenic peptides and then process the tumor antigens for presentation; IL-3 stimulated antigen-presenting cells (APCs), which are macrophage-like, within the tumor leading to generation of cytotoxic T lymphocytes (CTLs). This constitutes a plausible pathway for enhancement in tumor rejection by IL-3 stimulation (Pulaski et al, 1996). IL-3 signaling proceeding either via the JAK-STAT or the Ras-Raf pathways, stimulates a number of genes such as the DUB-1 encoding a deubiquitinating enzyme the overexpression of which leads to G1 arrest (Zhu et al, 1996); deubiquitination might be an additional mechanism to couple extracellular signaling to cell growth. IL-3 signaling leads to stimulation in myeloid cell proliferation.

F. Cancer immunotherapy with the IL-7 gene Primary cell cultures from 45 patients with malignant melanoma were transfected via electroporation with the gene encoding for human interleukin-7 (IL-7) resulting in the production of biologically active IL-7 without altering the expression of HLA class I and II, ICAM-1, and of a melanoma-associated antigen. Irradiation of the transfected 45

Boulikas: An overview on gene therapy contained an encephalomyocarditis virus internal ribosome entry site for internal translation of bicistronic mRNA transcripts, while MDCVpac-mIL-12 carried an expression cassette in the U3 region of the 3' LTR. The MSCVpacmIL-12 vector was more efficient and directed robust expression of both p40 and p35 IL-12 genes in several murine tumor cell lines of hematopoietic origin, including a T-cell lymphoma, a B-cell lymphoma, and a plasmacytoma/myeloma. Adenoviral delivery of the IL-12 gene was effective against breast tumors (Bramson et al, 1996) or metastatic colon carcinoma (Caruso et al, 1996) in animal models: mice bearing breast tumors, injected intratumorally with a single dose of an adenovirus expressing IL-12 showed regressions in greater than 75% of the treated tumors; this effect was accompanied with a maximum expression of IL12 within the tumor between 24 and 72 hr post-injection which lasted for 9 days and an elevation in IFN-! within the tumor; local production of IL-12 also stimulated IFN-! production in tumor-draining lymph node cells (Bramson et al, 1996). Whereas intratumoral adenoviral transfer of the HSV-tk and the murine IL-2 genes resulted in substantial hepatic tumor regression, induced an effective systemic antitumoral immunity in the host and prolonged the median survival time of the treated animals from 22 to 35 days a recombinant adenovirus expressing the murine IL-12 gene was much more effective: intratumoral administration of the IL-12 vector alone increased significantly survival time of the animals; 25% of the treated animals lived over 70 days (Caruso et al, 1996). The immunological host response to syngeneic murine mammary carcinoma cell line variants, genetically modified to express B7-1 or secrete GM-CSF and IL-12, was examined by Aruga et al (1997). The mammary adenocarcinoma MT-901 subline was weakly immunogenic by immunization/challenge experiments and induced tumor-specific T-cell responses in lymph nodes draining progressive subcutaneous tumors; however, tumor clones from this cell line expressing B7-1 or secreting GM-CSF exhibited reduced tumorigenicity and resulted in significantly enhanced T-cell reactivity to tumor-draining lymph node (TDLN) cells as compared to wild-type TDLN cells. In contrast, transduction with the IL-12 gene led to complete tumor growth inhibition. An adenovirus vector, AdIL12-B7-1, encoding the two IL-12 subunits in early region 1 (E1) and the B7-1 gene in E3 of adenovirus under control of the murine CMV promoter was used to treat mice tumors derived from a transgenic mouse mammary adenocarcinoma. A single intratumoral injection with a low dose (2.5 x107 pfu/mouse) mediated complete regression in 70% of treated animals whereas a similar dose of recombinant virus encoding IL-12 or B7-1 alone resulted in only a delay in tumor growth. Coinjection of two different viruses expressing either IL-12 or B7-1 induced complete tumor regression in only 30% of animals treated (Putzer et al, 1997).

Human peripheral blood lymphocytes (HuPBLs), injected s.c. in mixture with human lung tumor cells into severe combined immunodeficient (SCID) mice, engrafted and displayed antitumor cytotoxic activity; this antitumor + + activity was dependent upon both CD8 T cells and CD56 natural killer cells in the donor HuPBLs. IL-12 enhanced the human peripheral blood lymphocyte-mediated tumor suppression; this implies that transfer of the IL-12 gene has a prospect in this type of immunotherapy. This could be significant under the light of studies showing that PBLs isolated from a lung cancer patient also suppressed the growth of the patient's (autologous) tumor when coinjected s.c. with the tumor cells into SCID mice (Iwanuma et al, 1997). Tumor cell vaccines were transduced with IL-12 or IL-2 genes and the antitumor response induced in mice bearing lung metastases of the BALB/c colon carcinoma C51 were compared by Rodolfo et al (1996). The cells used for transduction with the IL-12 or IL-2 genes were the histologically related, and antigenically cross-reacting C26 tumor cells which were irradiated and injected s.c. Vaccination with C26/IL12 cells cured 40% of mice, while vaccination with C26/IL2 cells reduced the number of metastatic nodules without affecting survival; both cell vaccination regimens showed similar antitumor CTL activation in mice. Both treatments induced antibodies directed against tumor-associated antigens, but only sera from mice treated with C26/IL12 contained antibodies that lysed tumor cells. The better therapeutic efficacy of vaccination with C26/IL12 was found to be associated, among other factors, with an early infiltration of the metastatic lungs by activated T lymphocytes (Rodolfo et al, 1996). Transfer of the IL-12 cDNA for cancer immunotherapy is being used in human clinical trials (protocols 62, 111, 180, and 183, Appendix 1).

H. Adoptive immunotherapy with GMCSF 1. Cell culture experiments The human hematopoietic growth factor, granulocytemacrophage colony-stimulating factor (GM-CSF), is important in the management and gene therapy of a variety of malignant disorders of the human hematopoietic system. Infection of COS-1 monkey kidney cells with a recombinant AAV vector containing the GM-CSF gene resulted in the release of recombinant GM-CSF protein into the supernatant; the released GM-CSF was able to sustain the active proliferation of the GM-CSF-dependent human megakaryocytic leukemia cell line, M07e, (Luo et al, 1995). 2. Animal studies The Dunning rat R3327-MatLyLu prostate tumor model (an anaplastic androgen-dependent, nonimmunogenic tumor that metastasizes to the lymph nodes and the lung) 46

Gene Therapy and Molecular Biology Vol 1, page 47 has been used for GM-CSF therapy; IL-2- or GM-CSFsecreting human tumor cell preparations (tumor vaccines) were used for the treatment of advanced human prostate cancer in rats. All animals with subcutaneously established tumors were cured; the cancer vaccine induced immunological memory that protected the animals from subsequent tumor challenge; GM-CSF was less effective than IL-2 (Vieweg et al, 1994). Using the Dunning rat prostate carcinoma model, animals with hormone refractory prostate cancer treated with irradiated prostate cancer cells genetically engineered to secrete human GMCSF showed longer disease-free survival compared to untreated control rats. To further test the clinical feasibility of the prostate cancer cell vaccine, cancer cells from patients with stage T2 prostate cancer undergoing radical prostatectomy were successfully transduced with MFG-GM-CSF, achieving a significant human GM-CSF secretion in each of 10 consecutive cases (Sanda et al, 1994). Continuous secretion of GM-CSF and activation of macrophages may contribute to the antitumor effects of a recombinant vaccinia virus expressing the gene for murine GM-CSF injected to solid melanoma tumors twice weekly for 3 weeks; this injection regimen resulted in growth inhibition of the subcutaneous tumor and enhanced the survival of the animals (Ju et al, 1997). A recent effort has been toward potentiation of Tlymphocyte-mediated antitumor effects. T-lymphocyte response incapacitation in the murine renal cancer model could arise from an impairment of critical nuclear transcription factors. A vaccine-oriented gene therapy approach used T cells and antigen-presenting dendritic cells which were recruited through the use of antigen, chemokines and GM-CSF and further potentiated by fibroblasts expressing IL-2, IL-4, IL-7, or IL-12; the goal of this approach was to optimize MHC class I- and class II-dependent pathways for induction of T-lymphocytemediated responses to cancer in animal models (Wiltrout et al, 1995). Chen et al (1996) found that adenoviral delivery of a combination of HSV-tk, mouse IL-2, and mouse GM-CSF is much more effective for the treatment of metastatic colon carcinoma in the mouse liver than HSV-tk alone or HSV-tk combined only with IL-2; a fraction of the animals developed long-term antitumor immunity and survived for more than 4 months without tumor recurrence in the three gene combination regimen; thus, local expression of GM-CSF in the hepatic tumors and prolonged IL-2 expression were necessary to generate persistent antitumor immunity. A gene gun device was used to accelerate and introduce gold particles coated with GM-CSF cDNA plasmids into mouse and human tumor cells. Transfected and irradiated murine B16 melanoma cells produced about 100 ng/ml murine GM-CSF/million cells per 24 hr in vitro for at least 10 days. Toward development of a tumor vaccine, irradiated B16 tumor cells expressing murine GM-CSF cDNA were then injected into mice. Subsequent challenge

of these mice with nonirradiated, nontransfected B16 tumor cells showed that 58% of the animals were protected from the tumor by the prior vaccine treatment compared to only 2% of control animals inoculated with irradiated B16 cells transfected with the luciferase gene (Mahvi et al, 1996). Human tumor tissue transfected within 4 hr of surgery produced significant levels of transgenic human GM-CSF protein in vitro. Human GM-CSF was readily detectable in serum and at the injection site following subcutaneous implantation of these transfected tumor cells into nude mice (Mahvi et al, 1996). The autocrine secretion of GM-CSF by transduced tumor cells was found to serve as an effective immune adjuvant in the host response to a weakly immunogenic murine mammary carcinoma tumor: transfer of activated lymph node cells derived from mice inoculated with GMCSF-secreting (240 ng/million cells/24 hours) murine mammary carcinoma cells resulted in the prolonged survival of animals with macroscopic metastatic disease; this was not evident utilizing lymph node cells from mice inoculated with wild-type tumor (Aruga et al, 1997). 3. Clinical trials Autologous cells (sensitized T cells) geneticallymodified to secrete GM-CSF have been used for adoptive immunotherapy on humans. GM-CSF has been used for the treatment of advanced melanoma or renal cell cancers (Chang et al, 1996). The steps included retrieval of tumor from the patient for use as a vaccine; the tumor cell line was transduced with a retroviral/GM-CSF vector; cells were reintroduced into the patient (tumor vaccination). Removal of draining lymph nodes after 7-10 days and activation of lymph node cells with a monoclonal antibody directed against CD3 and expansion of the cell population with IL-2 gave anti-CD3+/IL2-activated cells which were exquisitely tumor-specific and mediated the regression of established tumors in animal models (Figure 18). According to a phase I clinical trial cancer patients are intradermally vaccinated with lethally-irradiated tumor cells that have been transfected by particle-mediated gene transfer with gold particles coated with human GM-CSF plasmid DNA; this is based on preclinical studies showing that vaccination of mice with irradiated, GM-CSF-transfected melanoma cells provided protection from subsequent challenges with non-irradiated, non-transfected tumor cells. Human tumor immunotherapy studies in course use patients' fresh specimens of melanoma or renal carcinoma; cells are dissociated, lethally-irradiated and transfected with GM-CSF plasmid DNA-coated gold particles resulting in the subsequent production of biologically active GM-CSF protein by the patientâ&#x20AC;&#x2122;s cells. Patientâ&#x20AC;&#x2122;s cells are used intradermally as a vaccine to elicit anti-tumor immune responses. Surgical excision of the vaccination sites will assess GM-CSF production and infiltration of immune effector cells; patients are being subjected to an intradermal injection in their opposite extremity of 5 million irradiated cryopreserved tumor cells taken from the patient at the time of vaccine preparation to asses immune reactions 47

Boulikas: An overview on gene therapy (DTH testing); if a positive reaction is noted on day 28 the DTH site will be surgically removed (Mahvi et al, 1997).

Three RAC-approved human gene therapy protocols use IFN-! cDNA transfer. These are protocols 36, 54, and 71 in Appendix 1.

J. Immunotherapy with synthetic tumor peptide vaccines Progress in the identification of tumor-specific antigens, that is proteins expressed at high levels by a specific tumor cell type such as prostate or breast cancer, most of which are surface glycoproteins easily recognizable by the immune system, as well as the deciphering of the mechanisms for enhancing the response of cytotoxic T cell lymphocytes have advanced the potential for developing cancer vaccines. Cancer immunotherapies based on synthetic tumor peptide vaccines have been developed. Tumor-specific + CD8 cytotoxic T lymphocytes (CTLs) recognize short peptide epitopes presented by MHC class I molecules that are expressed on the surface of cancer cells. Bone marrowderived dendritic cells, grown in vitro in media containing combinations of GM-CSF + IL-4, when pulsed with synthetic tumor peptides (which are loaded on the surface of the dendritic cells) became potent antigen-presenting cells (APCs) capable of generating a protective antitumor immune response. Injection of these cells into naive mice protected the mice against a subsequent lethal tumor challenge; in addition, treatment of mice bearing C3 sarcoma or 3LL lung carcinoma tumors with the same type of cells resulted in sustained tumor regression in over 80% of the animals (Mayordomo et al, 1995). One of the obstacles of this method has been the difficulty in obtaining sufficient numbers of APCs; + dendritic APCs have been isolated from CD34 hematopoietic progenitor cells drawn from cord blood and expanded in cell culture in the presence of GM-CSF and TNF-"; TNF-" inhibits the differentiation of dendritic cells into granulocytes. Human peripheral blood mononuclear cells or mouse bone marrow cells depleted of lymphocytes could also yield dendritic cells when cultured in the presence of GM-CSF + IL-4 (Mayordomo et al, 1995).

F i g u r e 1 8 . A clinical protocol for adoptive immunotherapy of advanced melanoma patients. Adapted from Chang et al (1996).

A number of RAC-approved human gene therapy protocols use GM-CSF cDNA transfer. These are protocols 35, 53, 63, 113, 149, 150, 162, and 181 in Appendix 1.

I. Cancer immunotherapy with the IFNgene

K. DNA vaccines

Solid tumors in nude mice have been successfully eradicated with treatment with tumor cell lines stably transfected with an IFN gene. A number of human tumor cell lines including 293, HeLa, K562, and Eskol (a malignant immunoblastic lymphoma) were infected with a rAAV carrying a synthetic type I interferon gene and the bacterial neomycin-resistant gene and geneticin-resistant cells were selected; when injected into nude mice, 293, K562, and Eskol cells failed to form tumors for a duration of up to 3 months; on the contrary, mice receiving nontransduced cells developed tumors within 7 to 10 days; in addition, treatment of an established Eskol tumor with transduced 293 cells resulted in tumor regression (Zhang et al, 1996).

Vaccines may be one of the first successful applications of foreign genes into mammalian cells under control of heterologous promoters and enhancers (Felgner and Rhodes, 1991; Thompson, 1992; Gilboa and Smith, 1994). Vaccination with DNA has been shown to be a promising approach for immunization against a variety of infectious diseases (Wang et al, 1993; Michel et al, 1995; Huygen et al, 1996; Kuhober et al, 1996). The method consists in introducing the gene of a viral or bacterial antigen which is uptaken and expressed by the hostâ&#x20AC;&#x2122;s cells to elicit an antigen-specific immune response. DNA coding for an antigen can be directly injected into muscle or skin and stimulate an immune response against the expressed antigen; the gene can either code for surface 48

Gene Therapy and Molecular Biology Vol 1, page 49 molecules, which are often used for conventional peptide vaccines, or from internal microbial proteins. During this approach the antigens are produced intracellularly where they are correctly folded and can be presented to the immune system to stimulate cytotoxic T cells; the method is safe and simple and has shown promising results on animals (reviewed by Moelling, 1997). For example, mice injected intramuscularly with an HIV-1 envelope DNA construct developed anti-HIV envelope immune responses (Wang et al, 1993); intramuscular injection of plasmid DNA expression vectors encoding the three envelope proteins of the hepatitis B virus (HBV) induced humoral responses in C57BL/6 mice specific to several antigenic determinants of the viral envelope (Michel et al, 1995). Immunization of mice with plasmid DNA constructs encoding one of the secreted components of Mycobacterium tuberculosis, antigen 85 gene induced substantial humoral and cellmediated immune responses (Huygen et al, 1996). Because immunization of cancer patients with tumor antigen proteins is a very promising approach used extensively in cancer therapy (e.g. Karanikas et al, 1997) many of these approaches could be transferred to the DNA level using the gene encoding the tumor antigen. As an extension, this method could find application using human tumor antigen genes rather than bacterial/viral antigen genes, that is genes encoding for proteins expressed in tumor but not in normal cells leading to development of tumor vaccines (Graham et al, 1996; Okamoto et al, 1997); this method mimics the infection of the cell in the host by a pathogenic virus resulting in the intracellular processing of the viral proteins and their presentation on the cell surface. Human tumor antigens are, however, weakly immunogenic compared to microbial antigens a problem connected with polymorphism in the major histocompatibility complex proteins of the host and in antigen presentation. Development of a fusigenic viral liposome vector was made possible using the HVJ (hemagglutinating virus of Japan, a Sendai virus) renowned for its cell fusion ability; plasmid DNA containing the human tumor antigen genes MAGE-1 and MAGE-3 was mixed with HMG-1 nonhistone protein (to increase nuclear import and expression of the plasmid after transfection) and was encapsulated into anionic liposomes (phosphatidylserine, phosphatidylcholine, cholesterol) followed by the addition of inactivated HVJ; intramuscular injection into mice resulted in production of MAGE-1 and -3 IgG antibodies (Okamoto et al, 1997).

imbalance and disarray in phosphorylation events and regulatory circuits of the cell cycle. As a result of transformation, tumor cells acquire a proliferation advantage compared with normal cells, most of which are quiescent in the adult organism; cancer cells acquire partial independence from regulatory signals from neighboring cells for restricted cell growth. A crucial step in cancer development is the nonelimination of pre-cancer cells by apoptosis (usually a subsequence of a mutation in the p53 gene); such cells acquire a number of unrepaired damage in their DNA, such as strand breaks, which induce chromosomal translocations and result in clonal expansion of this cell population. Tumor cells are able to survive after DNA damage, and display an increase in mutation rate; cancer cell populations are heterogenous with respect to translocations, loss of heterozygosity, point mutations and transpositions in various genes. Whenever the mutated cell acquires an advantage for rapid growth over other cells in the tumor mass, escaping cell cycle checkpoints, it may replace the original population, a phenomenon known as tumor progression; this may lead to appearance of a more malignant phenotype. As a result, tumor cells are of different genotypes and clones obtained from the same solid tumor may differ in the level of malignancy. A number of candidate genes, when become mutated or overexpressed, may lead to tumor phenotype: p53, RB, and p21 appear to be the most important. The deregulation of other genes is connected to tumor progression whereas different groups of genes are associated with tumor cell metastasis. These facts make a single gene transfer approach to tumor cell mass to inhibit its growth or change its phenotype from malignant to normal very challenging.

B. Human clinical trials The genes used for cancer gene therapy in human clinical trials include a number of tumor suppressor genes (p53, RB, BRCA1, E1A), antisense oncogenes (antisense c-fos, c-myc, K-ras), suicide genes (HSV-tk, in combination with ganciclovir, cytosine deaminase in combination with 5-fluorocytosine) which have been very effective in eradicating solid tumors in animals. Also the cytokine genes (IL-2, IL-7, IFN-! , GM-CSF) are being used for the ex vivo treatment of cancer cells isolated from human patients and are able to elicit an immunologic regression especially on immunoresponsive malignancies (melanomas, colorectal carcinomas, renal cell carcinomas) (Culver, 1996). Future directions might be toward use of genes involved in the control of tumor progression and metastasis. Discovery of new genes which are over- or under-expressed during transformation and metastasis is a promising approach for the identification of novel gene targets in cancer gene therapy (Georgiev et al, 1998, this volume). Diseases amenable to therapy with gene transfer in clinical trials (Appendix 1 and Table 4 in Martin and Boulikas, this volume) include cancer (melanoma, breast,

XVI. Gene therapy of cancer and candidate genes A. Mechanisms of carcinogenesis Whereas for inborn errors of metabolism transfer of a single gene can correct the disorder, cancer is a complex disease involving mutations in a number of protooncogenes and tumor suppressor genes as well as an 49

Boulikas: An overview on gene therapy lymphoma, head and neck, ovarian, colon, prostate, brain, chronic myelogenous leukemia, non-small cell lung, lung adenocarcinoma, colorectal, neuroblastoma, glioma, glioblastoma, astrocytoma, and others), AIDS, cystic fibrosis, adenosine deaminase deficiency, cardiovascular diseases (restenosis, familial hypercholesterolemia, peripheral artery disease), Gaucher disease, Hunter syndrome, chronic granulomatous disease, PNP deficiency, "1-antitrypsin deficiency, leukocyte adherence deficiency, partial ornithine transcarbamylase deficiency, Cubital Tunnel syndrome, Canavan disease and rheumatoid arthritis. Several RAC-approved protocols use gene marking rather than gene therapy . An important number of protocols in cancer use ex vivo immunotherapy (Appendix 1, pages 159-172 & 203-206).

plus activated ras (Finlay et al, 1989; Eliyahu et al, 1989). Both alleles of p53 need to be mutated or altered for transformation. Introduction of a null mutation by homologous recombination in murine embryonic stem cells gave mice which appeared normal but were susceptible to a variety of neoplasms by 6 months of age (Donehower et al, 1992). The tumor suppressive activity of p53 seems to involve at least six independent pathways: ( i ) induction by p53 of the p21/Waf-1/Cip-1 gene which causes growth arrest both via inhibition of cyclin-dependent kinases and via inactivation of PCNA; PCNA is the accessory molecule to DNA polymerases " and ( and its absence causes arrest of DNA synthesis at the replication fork; ( i i ) induction of the death-promoting bax gene by p53 as a mechanism which eliminates oncogenic virus-infected and transformed cells; ( i i i ) by a direct interaction of p53 with origins or replication preventing firing and initiation of DNA replication; ( i v ) via binding of p53 to a number of important molecules involved in transcription (TATA boxbinding protein or TBP, TFIIH); ( v ) by the role of p53 in DNA repair via its patrolling the genome for small insertion deletion mismatches or free ends of DNA; ( v i ) p53 is able to attract RPA, an accessory to DNA polymerases " and ( as well as TFIIH and RAD51 at the damaged DNA sites; TFIIH, RAD51, and RPA have a demonstrated role in DNA repair (F igure 19). Additional properties of p53 include the induction of Gadd45 involved in the arrest of the cell cycle and induction of Mdm2 which, after exceeding a threshold value in the cell associates with p53 to restrict its regulatory functions; thus, Mdm2 acts as a feedback loop for p53 to moderate its apoptotic and cell cycle restrictive functions (Figure 20).

XVII. Gene therapy strategies based on p53 A. p53 as a tumor suppressor protein The p53 has been a fascinating subject in cancer biology since its discovery (Lane and Crawford, 1979; Linzer and Levine, 1979). Originally assigned in the constellation of oncogenes was later shown to exert suppressive effects on cell growth (Finlay et al, 1989); indeed, the mutated p53 has many characteristics of an oncogene (Will and Deppert, 1998, this volume). Mutations in the p53 gene contribute to the emergence of the malignant phenotype (Diller et al., 1990; Baker et al., 1990). Alterations in the p53 tumor suppressor gene appear to be involved, directly or indirectly, in the majority of human malignancies (Vogelstein, 1990). For example, human lung cancer cell lines and specimens showed allelic loss for chromosome regions 3p and 17p (p53 is assigned to 17p13); these specimens displayed homozygous deletions of p53, DNA rearrangements involving the p53 gene, or expression of truncated p53 transcripts suggesting abnormal splicing, initiation, and termination arising from point or other mutations (Takahashi et al, 1989; Nigro et al, 1989). An interesting approach to unravel the molecular mechanism of action of p53 in restricting cell growth and in inducing apoptosis was the cloning of genes induced by p53 before the onset of apoptosis; this led to the identification of a group of 14 genes (out of 7,202 transcripts examined) which were markedly increased in p53-expressing cells compared with control cells many of which were predicted to encode proteins that could generate oxidative stress or respond to oxidative stress (Polyak et al, 1997). Additional studies in this line have suggested that the induction of the apoptotic pathway by p53 involves ( i ) transcriptional induction of redox-related genes; ( i i ) formation of reactive oxygen species; and ( i i i ) the oxidative degradation of mitochondrial components (Polyak et al, 1997). p53 can inhibit transformation of rat embryo fibroblasts mediated by adenovirusE1A plus activated ras and can also suppress focus formation mediated bymyc

B. Genes regulated by wild-type p53 Protein p53 appears to be a transcription factor able to recognize specific regulatory regions in a number of genes via its central DNA-binding domain; the DNA sequencespecific binding of wt p53 is regulated by the C-terminal domain of p53 and is activated by a variety of posttranslational modifications (Hupp et al, 1992). p53 is phosphorylated and is constitutively expressed at low levels in most normal tissues (Lane and Crawford, 1979; Linzer and Levine, 1979). The sequence specificity of p53 has been determined using random synthetic oligonucleotides followed by selection by wtp53 and cloning; these studies revealed the A 10 bp motif RRRCA GYYY (where R is purine and Y TT pyrimidine) as the binding and recognition site of wtp53 recognition (El-Deiry et al., 1992); two such 10 bp motifs are required for p53 binding separated by up to 13 bp of random sequence. Since the 10 bp motif is a palindrome, the binding site of p53 comprises 4 copies of the half binding sites A GYYY oriented in opposite directions, T which suggested that p53 binds either as a dimer to two cruciforms or as a tetramer with each subunit interacting with one half site. The second possibility is favored since 50

Gene Therapy and Molecular Biology Vol 1, page 51

F i g u r e 1 9 . Regulatory circuits involving p53. From Boulikas T (1 9 9 7 ) Gene therapy of prostate cancer: p53, suicidal genes, and other targets. Anticancer Res 17, 1471-1506. With the kind permission of Anticancer Research.

Boulikas: An overview on gene therapy

Figure 2 0 . A summary of the apoptotic and cell cycle restrictive activities of p53. From Boulikas T (1 9 9 7 ) Gene therapy of prostate cancer: p53, suicidal genes, and other targets. Anticancer Res 17, 1471-1506. With the kind permission of Anticancer Research.

Gene Therapy and Molecular Biology Vol 1, page 53 biophysical studies indicate that p53 exists as a tetramer in solution (Stenger et al., 1992). Increased levels of p53 upregulate the expression of specific genes including Cip-1/Waf-1/p21 (El-Deiry et al, 1993), GADD45 (Kastan et al, 1992), cyclin G (Okamoto and Beach, 1994), and mdm2 (Perry et al, 1993; Barak et al, 1993; Momand et al, 1992) which is induced by UV damage in a p53-dependent pathway (Perry et al, 1993). Gadd45 inhibits cell cycle progression (Papathanasiou et al, 1991). Mdm2 acts as a feedback loop for the biological functions of p53 apparently to moderate the G1/S arrest or apoptosis triggered by p53 following severe damage to DNA. Mdm2 protein associates with p53 causing p53 inactivation by preventing its sequence-specific binding to regulatory targets in DNA (Momand et al, 1992; Oliner et al, 1992). Elevated levels of Mdm2 mimic the effect of T antigen, E1B of adenovirus, E6 of HPV, which also inactivate p53 in a similar manner; overexpression of Mdm2 can block the induction of apoptosis by p53 (Chen et al, 1994). Additional genes up-regulated by p53 include human PCNA (Shivakumar et al, 1995), mouse muscle creatine kinase MCK (Zambetti et al, 1992), EGFR (Deb et al, 1994), the potent promoter of the death pathway Bax (Miyashita and Reed, 1995), and thrombospondin-1 (Dameron et al, 1994). Other cellular regulatory regions that interact with p53 include the RGC repeats in the ribosomal gene cluster (Farmer et al, 1992; Kern et al, 1992). The PCNA promoter is up-regulated in the presence of moderate amounts of wt p53; however, at higher levels of wt p53 the PCNA promoter is inhibited whereas tumorderived p53 mutants activate the PCNA promoter (Shivakumar et al, 1995); it has been suggested that the moderate elevation in wt p53 seen after DNA damage induces PCNA to cope with its DNA repair activities (Shivakumar et al, 1995); this inhibition in DNA replication but stimulation in repair by p53 might be accomplished by an independent pathway involving induction of p21 (El-Deiry et al, 1993) which interacts with PCNA protein auxiliary to DNA polymerase ( to inhibit the replication but not the repair functions of PCNA (Li et al, 1994). The bax gene which induces apoptosis (Figure 21) is upregulated by p53 whereas the bcl-2 gene which inhibits apoptosis in B cells is down-regulated by p53 (Miyashita et al, 1994a,b; Miyashita and Reed, 1995). Initiated cancer cells may lead to tumor development only when a dysfunction in their apoptotic pathway takes place; some of the mechanisms leading to inactivation of the apoptotic pathway in cancer cells may result from an up-regulation in the bcl-2 gene (a Bcl-2 chimeric factor is produced in leukemias as a result of a translocation) or down-regulation of the bax gene. Gene therapy for cancer could involve restoration of the apoptotic pathway in cancer cells leading to their suicidal death (see below).

F i g u r e 2 1 . Involvement of Bax and Bcl-2 proteins in apoptosis. Bax is a potent inducer of apoptosis; binding of Bcl-2 to Bax (also binding of the E1B 19 kDa protein of adenovirus to Bax) prevents Bax from its apoptotic functions. From Boulikas T (1 9 9 7 ) Gene therapy of prostate cancer: p53, suicidal genes, and other targets. Anticancer Res 17, 1471-1506. With the kind permission of Anticancer Research.

Binding sites for p53 have been found at the origin of replication of polyomavirus with an inhibitory effect on virus replication in vitro (Miller et al, 1995) and at the SV40 ORI (Bargonetti et al, 1991) as well as in putative cellular origins of replication (Kern et al, 1991). A number of genes not containing p53 response elements may be repressed by p53 (Ginsberg et al, 1991; Mercer et al, 1991; Shiio et al, 1992; Seto et al, 1992).

C. Binding of p53 to viral oncoproteins p53 was first detected in rodent cells transformed with SV40 in a complex with T antigen (Lane and Crawford, 1979; Linzer and Levine, 1987). Subsequent studies have shown that p53 can be complexed with adenovirus E1B (Sarnow et al, 1982; van den Heuvel et al., 1990) and the E6 oncoprotein of human papilloma virus (Werness et al., 1990). SV40 T antigen was unable to act as an initiator of SV40 DNA replication in vitro when complexed with wt murine p53 (Wang et al, 1989) thought to act by blocking the interaction of T antigen with DNA polymerase " (Gannon and Lane, 1987; Braithwaite et al, 1987). What appears to be important in understanding the involvement of p53 in tumorigenesis is that p53 is unable to transactivate the p53-inducible reporter genes in cells 53

Boulikas: An overview on gene therapy that express one of these viral oncoproteins (Yew and Berk, 1992). In addition, the growth suppressive effect of p53 protein may be mediated by its association with cellular proteins (Fields and Jang, 1990; Raycroft et al., 1990). Negative elements that could be required for an efficient growth shutdown leading to the reversible G0 state or to irreversible out-of-cycle conditions such as terminal differentiation, apoptosis, and senescence, may be affected by p53 (Bargonetti et al., 1991).

According to a second model, p53 can cause inhibition in DNA replication by a direct interaction with origins of replication at the DNA sequence level rather than via its interaction with replication initiator proteins. The potential role of p53 as a down-regulator of DNA replication in a DNA-binding-dependent manner has been suggested from replication assays of polyoma virus in vitro (Miller et al, 1995) and from the inhibition in nuclear DNA replication by a form of p53, truncated at its C-terminus, which is constitutively active for DNA binding in transcription incompetent extracts from Xenopus eggs (Cox et al, 1995). In the experiments of Miller and coworkers (1995) wild-type p53 suppressed DNA replication in vitro when the p53 binding site (RGC)16 from the ribosomal gene cluster was cloned on the late side of the polyomavirus (Py) core origin; when mutated p53-binding sites were used, the inhibition in Py replication was not observed. In addition, RPA (able to interact directly with p53) was unable to relieve the p53mediated repression in Py replication. Furthermore, tumorderived mutants of p53 that had lost their sequence-specific DNA-binding capacity were unable to inhibit Py replication of the construct with the wild-type oligomerized RGC sites in vitro.

D. Transcription repression by interaction of p53 with TBP Although p53 activates a number of promoters that contain p53-responsive elements, it represses transcription from many promoters that lack p53 binding sites; central to the promoter repression by p53 was thought to be its interaction with the TATA-box binding protein or TBP (Seto et al, 1992; Mack et al, 1993; Truant et al, 1993). This interaction may activate transcription when TBP interacts with a preformed p53-DNA complex or may repress transcription when p53 interacts with DNA-bound TBP (Deb et al, 1994). However, p53 acts as a repressor only in cells undergoing apoptosis and p53-mediated transcriptional repression is released by adenovirus E1B or cellular Bcl-2 (Shen and Shenk, 1994; Sabbatini et al, 1995). Both wild-type and mutant p53 interact with C/EBP on the human hsp70 promoter (Agoff, 1993), with TFIIH (Xiao et al, 1994), holo-TFIID (Chen et al, 1993; Liu et al, 1993) and the TAFII40 and TAFII60 subunits of TFIID (Thut et al, 1995).

F. Differences in biological functions between wild-type p53 and tumor-derived p53 mutants Tumor-derived mutant forms of p53 have lost their DNA sequence-specific binding capacities. For example the Trp-248 and His-273 mutants of p53 have poor DNAbinding abilities and are unable to activate transcription from constructs containing p53 binding sites (Farmer et al, 1992). Wild-type (wt) p53 tumor suppressor protein negatively regulates cell growth (Hollstein et al, 1991; Prives, 1994). Whereas the wild-type p53 acts as a tumor suppressor, several of the mutant forms display oncogenic activities (Levine, 1993; Prives and Manfredi, 1993; Deppert, 1994). Although the wt p53 has been postulated to repress growth by activating genes that repress growth (p21), many of the mutant forms have lost their DNA sequence-specific binding and transcriptional activation capacities (reviewed by Zambetti and Levine, 1993). According to one model (see Vogelstein and Kinzler, 1992), wt p53 is a positive regulator for the transcription of genes that by themselves are negative regulators of growth control and/or invasion. Indeed, p53 upregulates the genes of p21/CIP1/WAF1 (ElDeiry et al, 1993) and GADD45 (Kastan et al, 1992) whose products interact with PCNA to inhibit its association with DNA polymerase ( thus causing arrest in DNA replication (Smith et al, 1994). This feature of p53 that is central to its ability to suppress neoplastic growth is lost by mutations on p53 that result in loss of its ability to bind to DNA or to interact with other transcription protein factors.

E. Inhibition of DNA replication by wildtype p53 Several lines of evidence suggested inhibition in DNA replication by wild-type p53 but not by tumor-derived mutant forms of p53. Indeed, SV40 T antigen was unable to act as an initiator of SV40 DNA replication in vitro when complexed with p53 (Wang et al, 1989); mutant p53 was unable to cause inhibition in the initiating functions of T antigen in vitro (Friedman et al, 1990). Inhibition in DNA replication in vivo by p53 (Braithwaite et al, 1987) suggested that p53 might interact with cellular DNA replication initiator proteins or other components of the replication fork. p53 also interacts with replication protein A (RPA) implicated in DNA replication and in repair; interaction of p53 inhibits the replication functions of RPA (Dutta et al, 1993) although interaction of p53 with RPA via its acidic domains stimulates BPV-1 DNA replication in vitro (Li and Botchan, 1993). Immunolocalization of p53 (also of RB and host replication proteins) at foci of viral replication in HSVinfected cells (Wilcock and Lane, 1991) provided further evidence for a direct interaction of p53 with proteins (or DNA sequences) at the replication fork.

Gene Therapy and Molecular Biology Vol 1, page 55

H. A proposal for an efficient killing of cancer cells using p53/PAX5 expression vectors

Mutant p53 can transactivate genes that up-regulate cellular growth (Deb et al, 1992; Dittmer et al, 1993) such as PCNA (Shivakumar et al, 1995), EGFR (Deb et al, 1994), multiple drug resistance (MDR1) (Chin et al, 1992; Zastawny et al, 1993), and human HSP70 in vivo (Tsutsumi-Ishi et al, 1995). These studies support the idea for an oncogene function of the mutant p53 protein compared with the tumor suppressor function of wt p53; mutation in the p53 gene may, thus, cause gain of new functions such as transforming activation and binding to a distinct class of promoters which are not normally regulated by wt p53 (Zambetti and Levine, 1993; Tsutsumi-Ishi et al, 1995). At the same time appearance of mutations in the p53 gene result in the loss of function of the wt p53 (Zambetti and Levine, 1993). The wild-type but not mutant p53 at low levels transactivates the human PCNA promoter in a number of different cell lines; the wild-type p53-response element from the PCNA promoter functions in either orientation when placed on a heterologous synthetic promoter; thus moderate elevation of p53 can induce PCNA, enhancing the nucleotide excision repair functions of PCNA (Shivakumar et al, 1995). Whereas low levels of wild-type p53 activate the PCNA promoter, higher concentrations of wt p53 inhibit the PCNA promoter, and tumor-derived p53 mutants activate the promoter (Shivakumar et al, 1995). While the wtp53 is endowed with a 3'-to-5' exonuclease activity, associated with the central DNAbinding domain, and thought to function during repair, His replication, and recombination, the 273 mutant of p53 has lost the exonuclease activity (Mummenbrauer et al, 1996).

Introduction of a null p53 mutation by homologous recombination in murine embryonic stem cells gave mice which appeared normal but were susceptible to a variety of neoplasms by 6 months of age (Donehower et al, 1992). Relevant to the issue that p53 is dispensable for embryonic development are the studies of Stuart and coworkers (1995) suggesting that during early embryo development p53 is not expressed because of the suppression of its gene by Pax5; at later stages of development Pax5 inactivation allows p53 to be expressed and exert its control on cell growth (Figure 22). A significant factor to be considered in approaches aimed at transferring the wt p53 gene to tumor cells is the impairment of the wt p53 functions by the endogenous mutant p53 expressed in tumor cells which is able to tetramerize with wt p53; optimal results will be expected if the endogenous mutant p53 gene is inhibited concurrently with overexpression of the wt p53 gene. It has been proposed (Boulikas, 1997) that effective suppression of tumor growth with p53 vectors could be achieved by the simultaneous transfer of wt p53 plus Pax5 to cancer cells; Pax5 is a well established supressor of the p53 gene; its effect is exerted via a direct interaction of Pax5 with a control element in the first exon of the p53 gene (Stuart et al, 1995). Pax5 is an homeotic protein, controlling the formation of body structures during development; Pax5 is expressed in early embryo stages to keep the levels of p53 low and allow rapid proliferation of embryonic tissues. Simultaneous transfer to solid tumors of a PAX5 and p53 genes in the same expression vector but with the wt p53 mutagenized at 2-3 nucleotides to abort the PAX5 suppressive site was proposed as a strategy to effectively suppress tumor cell proliferation (Boulikas, 1997).

G. Involvement of p53 in repair and control of the cell cycle p53 controls the level of expression of the p21 gene, encoding a protein that inhibits the activity of cyclindependent kinases (CDKs); CDK activity is essential for the phosphorylation of RB at the G1/S checkpoint of the cell cycle resulting in the release of E2F transcription factor from RB-E2F complexes and in the up-regulation by the released E2F of genes required for DNA synthesis. p21 levels are reduced considerably in tumor cells that have lost the p53 protein or contain a nonfunctional mutated form of p53 (El-Deiry et al, 1993). In addition, the p21 inhibitor of cyclin-dependent kinases associates with PCNA thus blocking its ability to activate DNA polymerase (; this could give rise to the abnormal control in DNA replication or to the loss of coordination between DNA replication and cell cycle progression seen in tumor cells. Thus the upregulation of the p21 gene by p53 acts in two different ways causing a cascade of events. p53 is linked directly to homologous recombination processes via its interaction with the RAD51/RecA protein (St端rzbecher et al, 1996).

I. p53 gene bombs that explode in cancer cells Exogenous genes encoding "weapons" (suicide genes) and "triggers" have been devised whose delivery to somatic cells will affect only cancer cells. The production of mutated forms of p53 at high levels by cancer cells (normal cells do not have adequate amounts of wt p53 protein) is being exploited to pull a molecular trigger resulting in the transcriptional activation of a toxic gene and in the death of cancer cells (da Costa et al, 1996). This invention is based on the fact that ( i ) powerful chimeric transcription factors can be engineered consisting of a DNA-binding domain (DBD) and a transactivation domain (TAD) and ( i i ) prokaryotic or viral enzymes are able to convert nontoxic prodrugs into toxic derivatives (suicide genes, see HSV-tk, CD and PNP further below); the toxic derivative produced in tumor cells which are transfected can diffuse to surrounding cells causing their killing even in 55

Boulikas: An overview on gene therapy

F i g u r e 2 2 . Involvement of p53 and Pax5 in B cell apoptosis. Adapted from Stuart et al (1995).

normal cells do not express the p53 gene and those who do express the wt p53 are destined for programmed cell death; therefore, cancer cells containing elevated amounts of mutant forms of p53 were amenable to this strategy. The 55- kDa E1B protein of adenovirus binds to and inactivates the p53 gene; ONYX-015 is an E1B, 55-kDa gene-attenuated adenovirus unable to replicate and show cytopathogenicity in tumor cell lines which express a wt p53 such as in RKO and U20S carcinoma lines but can cause cytolysis in cell lines expressing a mutated form of p53; a wide range of human tumor cells, including numerous carcinoma lines with either mutant or normal p53 gene sequences (exons 5-9), were efficiently destroyed following intratumoral or intravenous administration of ONYX-015 to nude mouse-human tumor xenografts; furthermore, combination therapy with ONYX-015 plus chemotherapy (cisplatin, 5-fluorouracil) was significantly greater than with either agent alone. On the contrary, normal human cells were highly resistant to cytolysis by the adenovirus (Heise et al, 1997).

the absence of transfection of these cells, a phenomenon known as "bystander effect". Trigger genes in plasmids were made up of the DNAbinding domain of GAL4 (aa 1-147) fused in frame to a protein domain that could interact with p53; the p53binding domain was the 84-708 aa region of SV40 T antigen or of the 305-393 aa TAD domain of p53 (which acts as a tetramer) and which is similar between wt and mutant p53 (mutations on p53 are within the DBD). The constructs included the E. coli (DeoD) gene which encodes the purine nucleoside phosphorylase (PNP) under control of the GAL4 response element (known as upstream activating sequence or UAS); the PNP gene can convert the 6-methylpurine deoxyribose (MeP-dR) prodrug into the diffusible, toxic 6-methylpurine (see page 69) and can become a powerful suicide gene under these conditions (Sorscher et al, 1994). Transfection of cells in culture with these constructs followed by treatment of the cells with MeP-dR resulted in the death of the cells (da Costa et al, 1996). The mechanism was based on the fact that most 56

Gene Therapy and Molecular Biology Vol 1, page 57 animals subjected to asanguineous portal perfusion was examined by Drazan et al (1994). The gene transfer rate in whole liver and after hepatectomy ranged from 20% to 40%; liver regeneration and hepatocyte function were unaffected by overexpression of p53. Delivery of the p53 gene to malignant human breast cancer cells in nude mice using DOTMA:DOPE 1:1 cationic liposomes (400 nmoles liposomes/35 Âľg DNA) resulted in regression (60% reduction in tumor cell volume) in 8 out of 15 animals treated; animals were receiving one injection every 10 days (Lesoon-Wood et al, 1995). It was thought that wild-type p53 expression (tumor cells were expressing mutant forms of p53) upregulated p21 gene to inhibit cell growth by inhibition in cyclin-dependent kinases but also via induction of apoptosis preferentially in cancer cells. When a recombinant adenovirus encoding wild-type p53 under the control of the human CMV promoter was introduced into SK-OV-3 human ovarian carcinoma cells it increased by more than 50% the life span of nude mice injected with these cells; control animals in this highly aggressive ovarian xenograft model died between 25-45 days from injection time (Mujoo et al, 1996). Adenoviral transfer of a functional p53 gene into a radiation-resistant SCCHN cell line that harbors mutant p53 restored the G1 block and apoptosis in these cells in vitro and sensitized SCCHN-induced mouse xenografts to radiotherapy in vivo (Chang et al, 1997). The efficacy of a replication-deficient p53 adenovirus construct was tested against three human breast cancer cell lines expressing mutant p53, MDA-MB-231, -468, and 435 and was found to be highly effective against 231 and 468 cells as well as their tumor xenografts in nude mice but not against 435 cells probably due to their low adenovirus transduction. 37% of growth inhibition of 231 cells was due to p53, while 49% was adenovirus-specific (Nielsen et al, 1997). Cytotoxic T lymphocytes (CTLs) recognizing a murine wild-type p53 were able to discriminate between p53-overexpressing tumor cells and normal tissue and caused complete and permanent tumor eradication without damage to normal tissue after adoptive transfer into tumorbearing p53+/+ nude mice. CTLs, presented by the MHC class I molecule H-2Kb, were generated by immunizing p53 gene deficient (p53-/- ) C57BL/6 mice with syngeneic p53-overexpressing tumor cells (Vierboom et al, 1997).

J. Transfer of the p53 gene in cell culture Preclinical studies have shown that both viral and plasmid vectors able to mediate high efficiency delivery and expression of wild-type tumor suppressor p53 gene can cause regression in established human tumors, prevent the growth of human cancer cells in culture, or render malignant cells from human biopsies non-tumorigenic in nude mice. Inhibition in cell proliferation was observed in cell culture and in tumors after induction of p53 expression with adenovirus vectors (Bacchetti and Graham, 1993; Wills et al, 1994; Zhang et al, 1994). Transfer of the wild-type p53 gene using a defective HSV vector into a human medulloblastoma cell line containing a mutant copy of p53 resulted in p53 expression, increased the levels of mdm2 proteins and induced cell cycle arrest of the majority of transduced cells (Rosenfeld et al, 1995). Apoptosis can be induced in cultured NCI-H 596 human non-small cell lung cancer cells, which have a wild-type p53 gene, by EGF signaling in a p53-dependent manner; whereas treatment of these cells with EGF plus p53 sense oligonucleotides induced EGF-dependent and p53-dependent apoptosis within 8 hours, antisense p53 gene therapy suppressed the induction of apoptosis. A new nucleic acid drug was developed based on a mutated p53 antisense with a mutation at three bases immediately 5' and 3' from the CG dinucleotides which potentiated the induction of apoptosis and failed to suppress the induction of EGF-dependent apoptosis (Murayama and Horiuchi, 1997). Infection of the androgen-independent human prostate Tsu-pr1 cell line lacking functional p53 alleles with recombinant adenovirus vectors (replication-deficient) carrying the p53 gene under control of the CMV promoter resulted in expression of p53 and induced striking morphological changes: the cells were detached from the substratum, condensed, and exhibited breakdown of the nuclear DNA into nucleosome-size fragments characteristic of apoptosis; whereas control cells were able to elicit tumors in nude mice, the AdCMV/p53-infected cells failed to form tumors (Yang et al, 1995).

K. Animal studies using p53 gene transfer Intratracheal injection of a recombinant retrovirus containing the wt p53 gene was shown to inhibit the growth of lung tumors in mice nu/nu models inoculated intratracheally with human lung cancer H226Br cells whose p53 gene has a homozygous mutation at codon 254 (Fujiwara et al, 1994). A number of other studies have shown suppression in tumor cell growth and metastasis after delivery and expression of the wtp53 gene (Diller et al, 1990; Chen et al, 1991; Isaacs et al, 1991; Wang et al, 1993). The safety of the adenovirus-mediated p53 transduction of the liver in normal rats and in 50%-hepatectomized

L. Transfer of the p53 tumor suppressor gene to prostate cancer cells Although primary prostate tumors have few mutations in the p53 gene (Voeller et al, 1994; Isaacs et al, 1994), specimens from advanced stages of the disease and metastases as well as their cell lines frequently display mutations or deletions at both alleles of the p53 gene (Chi et al, 1994; Dinjens et al, 1994). Three of five prostate cancer cell lines examined (TSUPr-1, PC3, DU145) and one out of two primary prostate cancer specimens were 57

Boulikas: An overview on gene therapy found to harbor mutations altering the amino acid sequence of the conserved exons 5-8 of the p53 gene; transduction of the p53-defective cell lines with the wt p53 gene using lipofectin showed reduction in tumorigenicity assayed from reduced colony formation and the cells became growth arrested (Isaacs et al, 1991). Endocrine therapy is ineffective once the prostate cancer becomes androgen-independent; these cancers remain unresponsive to conventional chemotherapy. Androgenindependent and metastatic prostate cancers were established in athymic male mice by co-inoculation with the LNCaP human prostate cancer cell line and the MS human bone stromal cell line; these tumors became necrotic and were successfully eradicated by intratumoral injection of a recombinant p53/adenovirus; the p53 gene was driven by the CMV promoter and the SV40 poly(A) signal placed in the E1 region of Ad5 (Ko et al, 1996). It was suggested that in addition to the tumor suppressor, apoptotic, and antiangiogenesis function of p53, tumor necrosis was induced by a bystander effect or a general immune response which attracted immune cells to cause tumor cell killing (Ko et al, 1996).

patients with advanced and metastatic bladder cancer), and #156 (adeno p53 for non-small cell lung cancer).

XVIII. p21 and p16 in cancer gene therapy A. Molecular action of p21 p53 upregulates the p21/CIP1/WAF1 gene (simply called p21) (ElDeiry et al, 1993). Induction of the p21/Waf-1/Cip-1 gene causes growth arrest via inhibition of cyclin-dependent kinases (CDKs). CDKs are upregulated by cyclins which act as positive regulators of cell cycle cdk2 progression. Cdk2, also called p33 , is the master regulator of the cell cycle at the G1/S transition point. Whereas cdk2 is expressed at constant levels throughout the cell cycle, its activation by phosphorylation is first detected a few hours before the onset of DNA synthesis; furthermore, antibodies directed against Cdk2 blocked mammalian cells from entering S phase. D1 cyclin associates with Cdk2, Cdk4, and Cdk5 to control the G1)S transition point; the genes of cyclins D1 and E are overexpressed or rearranged in malignancies and conditional overexpression of human cyclins D1 and E in Rat-1 fibroblasts causes a decrease in the length of G1 and an acceleration of the G1/S phase transition. D1 appears to be specialized in the emergence of cells from quiescence (Go)G1 transition) whereas cyclin E is more oriented toward control of the G1/S transition. Cdc2, a close relative of Cdk2 and whose pattern of phosphorylation is cell cycle-regulated, becomes associated with cyclin B to regulate the G2)M transition (see Boulikas, 1995a). CDK activity is essential for the phosphorylation of RB at the G1/S checkpoint of the cell cycle resulting in the release of E2F transcription factor from RB-E2F complexes and in the up-regulation of genes required for DNA synthesis by the released E2F. p21 levels are reduced considerably in tumor cells that have lost the p53 protein or contain a nonfunctional mutated form of p53 (El-Deiry et al, 1993). Induction of the p21/Waf-1/Cip-1 gene also causes growth arrest via inactivation of PCNA; indeed, the p21 inhibitor of cyclin-dependent kinases associates with PCNA, the accessory of DNA polymerases " and ( , thus blocking its ability to activate these DNA polymerases; this could give rise to the abnormal control in DNA replication or to the loss of coordination between DNA replication and cell cycle progression seen in tumor cells (Li et al, 1994).

M. Clinical trials using p53 gene transfer A human clinical trial at M.D. Anderson Cancer Center uses transfer of the wild-type p53 gene, in patients suffering with non-small cell lung cancer and shown to have p53 mutations in their tumors, using local injection of an Ad5/CMV/p53 recombinant adenovirus at the site of tumor in combination with cisplatin (Roth, 1996; Roth et al, 1996; protocols #29 and 124, Appendix 1). A retroviral vector containing the wild-type p53 gene under control of a #-actin promoter was used for multiple percutaneous injections or direct thoracoscopic injections at the site of the tumor into nine patients, all in advanced stages, with non-small cell lung cancers. Patients whose conventional treatments failed were selected for a p53 mutation in the lung tumor. Reduction in tumor volume was achieved via apoptosis (assayed in posttreatment biopsies) in three patients, and arrest in tumor growth in three other patients (Roth et al, 1996). RAC-approved clinical trials (Appendix 1) using p53 cDNA transfer are #29 (treatment of non-small cell lung cancer with p53 and antisense K-ras), #124 (intratumoral delivery of adenoviral p53 cDNA plus cisplatin), #130 (intratumoral injection of adenoviral p53 to treat head and neck squamous cell carcinoma), #131 (primary and metastatic malignant tumors of the liver), #147 (percutaneous injections of adenovirus p53 for hepatocellular carcinoma), #148 (advanced or recurrent adenocarcinoma of the prostate), #152 (intra-tumoral injections of ad5cmv-p53 to patients with recurrent squamous cell carcinoma of the head and neck), #153 (intralesional delivery of adenovirus p53 in combination with chemotherapy in breast cancer), #154 (intratumoral injection of adeno p53 to patients with advanced prostate cancer), #155 (intratumoral injection of adeno p53 to

B. p21 and p16 gene transfer Introduction of the wt p53 or of the p21 downstream mediator of p53-induced growth suppression into a mouse prostate cancer cell line, deficient in p53, led to an association of p21 with Cdk2; this interaction was sufficient to downregulate Cdk2 by 65% (Eastham et al, 1995). The p21 gene, driven by CMV promoter into an 58

Gene Therapy and Molecular Biology Vol 1, page 59 Adenovirus 5 vector, was more effective than the AD5CMV-p53 vector, (harboring the p53 gene under control of the same elements as p21), in reducing tumor volume in syngeneic male mice with established s.c. prostate tumors; tumors were induced by injection of 2 million cells in each animal. These studies suggested that p21 expression might have more potent growth suppressive effect than p53 in this tumor model and that p21 may be seriously be included in the constellation of anticancer arsenals. Transfer of p21 is an effective tool to lead carcinoma cells with inactivated p53 into less malignant phenotypes. p53 is frequently inactivated by papilloma viruses in carcinomas of the uterine cervix. Transfer of the p21 gene to HeLa cells, a widely used uterine cervix cell line, resulted in a significant growth retardation by blockage of G1 to S transition, reduced anchorage-independent growth and attenuated telomerase activity (Yokoyama et al, 1997). Introduction of p21 with adenoviral vectors into malignant cells completely suppressed their growth in vivo and also reduced the growth of established pre-existing tumours (Yang et al, 1997). Transfer of p21 was used to suppress neointimal formation in the balloon-injured porcine or rat carotid arteries in vivo (Yang et al, 1996; Ueno H et al, 1997a). A combination therapy in mice with simultaneous transfer of the p21 gene and of the murine MHC class I H-2Kb gene, which induces an immune response that stimulates tumor regression, was more effective than treatment with either gene alone (Ohno et al, 1997). Malignant gliomas extensively infiltrate the surrounding normal brain and their diffuse invasion is one of the most important barriers to successful therapy; one of the most frequent abnormalities in the progression of gliomas is the inactivation of the tumor-suppressor gene p16, suggesting that loss of p16 is associated with acquisition of malignant characteristics. Restoring wildtype p16 activity into p16-null malignant glioma cells modified their phenotype. Adenoviral transfer of the p16/CDKN2 cDNA in p16-null SNB19 glioma cells significantly reduced invasion into fetal rat-brain aggregates and reduced expression of matrix metalloproteinase-2 (MMP-2), an enzyme involved in tumor-cell invasion (Chintala et al, 1997).

upregulation of a number of genes required for DNA replication; ( i i ) from the direct association of RB protein with a number of viral oncoproteins or key regulatory proteins including E1A of adenovirus (Whyte et al., 1988), SV40 large T (Ludlow et al., 1990) and the human papilloma virus E7 protein (Dyson et al., 1989). Normal cellular targets of RB, such as the transcription factor E2F (Bagchi et al., 1991; Chellapan et al., 1991) become dissociated from the RB protein in the presence of these viral proteins in the cell (E1A, T antigen, E7), leading to cell cycle progression. This constitutes a mechanism (also the interaction of viral proteins with p53, see above) viruses use to render infected cells continuously cycling. ( i i i ) RB is able to repress directly c-fos gene expression (Robbins et al., 1990) and has been proposed to have a similar effect on c-myc expression (Pietenpol et al., 1990). ( i v ) RB also suppresses cell growth by directly repressing transcription of the rRNA and tRNA genes by blocking the activity of RNA polymerase I transcription factor UBF (Cavanaugh et al, 1995; reviewed by White, 1998). Hypophosphorylated RB, but not mutant RB, was associated with the nuclear matrix, particularly concentrated at the nuclear periphery and in nucleolar remnants, only during early G1; the peripheral matrix proteins lamin A and C bound RB in vitro. This association was thought to be important for the ability of RB to regulate cell cycle progression (Mancini et al, 1994). It is interesting that mutated p53 but not wtp53 interacts with specific types of MARs (Will et al, 1998); nuclear matrix is an essential structure for replication transcription recombination and repair processes intimately connected to mechanisms of carcinogenesis. The tumor suppressor function of RB is believed to occur by complex formation between E2F and RB or the RB-related proteins p107 and p130, a complex that downregulates the DNA-binding activities of E2F; the transcription activating capacity of E2F on the genes it regulates can be repressed by interaction with RB (Nevins, 1992). Cyclin A, believed to facilitate DNA replication, also associates with E2F; both types of complexes, E2FRB and E2F-cyclin A, can be dissociated by the adenovirus E1A protein. The release of E2F by E1A results in cell cycling and this constitutes an additional mechanism of interference of adenoviruses with the proliferation of the infected cells; release of E2F from RB induced by E1A is critical for transformation of cells by E1A (for references see Hiebert et al, 1995). The p107 protein with similarities in structure and DNA-binding properties to RB also binds cyclin A; whereas RB is complexed to E2F during G1 the p107cyclin A complex interacted with E2F as cells entered S phase (Shirodkar et al., 1992). E2F is a transcription factor that activates the adenovirus E2 gene and a number of cellular genes that respond to proliferation signals and that control the passage of the cell cycle through S phase such as myc and DHFR genes and contributes to the uncontrolled

XIX. Gene therapies based on transfer of the retinoblastoma (RB) gene A. RB and E2F proteins in the control of the cell cycle and apoptosis Retinoblastoma protein is a transcription factor (Lee et al, 1987) involved in the regulation of cell cycle progression genes (reviewed by White, 1998, this volume). The role of RB on cell proliferation and tumor suppression arises ( i ) from its association with E2F, an association disrupted by RB phosphorylation at the G1/S checkpoint resulting in release of E2F and in the 59

Boulikas: An overview on gene therapy proliferation of adenovirus-transformed cells (Mudryj et al., 1991; see White, 1998, this volume). It has been speculated that the physiological function of RB (and also of its similar protein p107) in negatively-regulating cell growth and in acting as a tumor suppressor protein are exerted via its ability to down-regulate the activity of E2F (Shirodkar et al., 1992); this has been subsequently confirmed by numerous studies. RNA ligands that bind to E2F1 were selected from RNA libraries and were used to inhibit the induction of S phase in cultured cells (Ishizaki et al, 1996). Such molecules might find applications in cancer therapy because of the important role of E2F proteins in the regulation of cell cycling. Retinoblastoma protein has a functional domain (the pocket) for binding to transcription factor E2F implicated in cell growth control. The same domain is responsible for the association of RB with the adenovirus E1A, the SV40 large T, and the human papilloma virus E7 proteins (Kaelin et al., 1992). Using an approach for screening 'gt11 expression libraries, clones encoding for RB-binding proteins were identified; among those are RBAP-1 and 2, or retinoblastoma-associated proteins 1 and 2 (Kaelin et al., 1992) and RBP3 (Helin et al., 1992). RBAP-1 binds to the RB pocket, copurifies with E2F, contains a functional transactivation domain, and binds to E2F cognate sequences (Kaelin et al., 1992). E2F contains a RB-binding domain in its C-terminus (Helin et al., 1992; Shan et al., 1992). RB binds directly to the activation domain of E2F1 and silences it, thereby preventing cells from entering S phase. To induce complete G1 arrest, RB requires the presence of the hbrm/BRG-1 proteins, which are components of the coactivator SWI/SNF complex. This cooperation was mediated through a physical interaction between RB and hbrm/BRG-1. RB can contact both E2F1 and hbrm at the same time, thereby targeting hbrm to E2F1 (Trouche et al, 1997). E2F cooperates with p53 to induce apoptosis (Wu and Levine, 1994) and high levels of wild-type p53 potentiate E2F-induced apoptosis in fibroblasts (Qin et al, 1994). The physiological relevance of E2F in the apoptotic mechanism was thought to arise from the ability of E2F to act as a functional link between p53 and RB; p53 levels increase in response to high levels of E2F (DP is required for the association of E2F with RB); overexpression of both E2F-1 and DP-1 led to a rapid death of (IL-3)dependent 32D.3 myeloid cells even in the presence of survival factors (Hiebert et al, 1995). Overexpression of exogenous E2F-1 using a tetracycline-controlled expression system in Rat-2 fibroblasts promoted S-phase entry and subsequently led to apoptosis (Shan and Lee, 1994).

Work from several groups has shown that RB is un- or under-phosphorylated in G0/G1 and becomes phosphorylated in its N-terminal domain during S and G2/M (Buchkovich et al., 1989; Chen et al., 1989; DeCaprio et al., 1989; Mihara et al., 1989). Only under-phosphorylated RB interacts with E2F (Chellappan et al., 1991). Treatment with TGF-#1 maintained RB protein in its active dephosphorylated form, thus providing a link between RB growth suppression and growth inhibition by TGF-#1. Interleukin-6 (IL-6), known to mediate autocrine and paracrine growth of multiple myeloma (MM) cells and to inhibit tumor cell apoptosis was determined to exert this function via phosphorylation of RB protein; this finding could explain the abnormalities of RB protein and mutations of RB gene associated with up to 70% of MM patients and 80% of MM-derived cell lines. Culture of MM cells with RB antisense, but not RB sense, oligonucleotide triggered IL-6 secretion and proliferation in MM cells; phosphorylated pRB was constitutively expressed in MM cells and IL-6 shifted pRB from its dephosphorylated to its phosphorylated form (Urashima et al, 1996). Interleukin-1 (IL-1) causes G0/G1 phase growth arrest in human melanoma cells, A375-C6 via hypophosphorylation of RB protein. Exposure to IL-1 caused a timedependent increase in hypo-phosphorylated RB that correlated with an accumulation of cells arrested in the G0/G1 phase; this was abrogated by the SV40 large T antigen which binds preferentially to hypo-phosphorylated RB, but not by the K1 mutant of the T antigen, which is defective in binding to RB (Muthukkumar et al, 1996).

C. Genes regulated by RB protein RB represses a number of genes by sequestering or inactivating the positive transcription factor E2F and seems to activate some other genes by interacting with factors like Sp1 or ATF-2 (Rohde et al, 1996). RB protein is a master regulator of a complex network of gene activities defining the difference between dividing and resting or differentiated cells. Using the method of differential display Rohde et al (1996) detected a number of genes which were upregulated by ectopic expression of the RB gene in RB-deficient mammary carcinoma cells including the endothelial growth regulator endothelin-1 and the proteoglycans versican and PG40. Introduction of the wild-type RB gene via retrovirusmediated gene transfer has provided several RBreconstituted retinoblastoma cell lines (Huang et al., 1988; + Chen et al., 1992). These RB cell lines showed little difference in their growth rates in culture when compared + to the parental or revertant RB cells; however, RB cells invariably lost their tumorigenicity in nude mice assays (Chen et al., 1992). RB protein down-regulates its own gene and this negative autoregulation is mediated by the transcription factor E2F; this was shown by inserting the promoter of the RB gene 5' of the bacterial CAT reporter

B. Phosphorylation of RB: the TGF- 1, IL-1, and IL-6 connection 60

Gene Therapy and Molecular Biology Vol 1, page 61 + gene followed by its transfection into RB and RBretinoblastoma cells: RB promoter activity was signifi+ cantly decreased in RB cells (Shan et al., 1994).

through the ATF binding site in a variety of different cell types (Park et al, 1994). The candidate oncoprotein Bcl-3, previously characterized as a member of the I& B family, activated transcription of the RB gene, whose promoter has no typical NF-&B sites, via binding to a DNA element identical to E4TF1/GABP site; Bcl-3 promoted tetramerization of E4TF1. Expression of the antisense bcl3 RNA in myoblasts suppressed induction of RB and myogenic differentiation whereas transient expression of bcl-3 in myoblasts was shown to induce expression of the endogenous RB (Shiio et al, 1996). Two oncogenic point mutations at the Sp1 and ATF sites of the RB gene promoter were identified in two separate hereditary RB families. The Sp1 consensus site mutation was blocking the action of RBF-1, recently identified as the human GABP/E4TF1, a transactivator from the adenovirus early-region 4 promoter. The human GABP/E4TF1 protein enhanced the core RB promoter activity, whereas it did not stimulate a mutant RBF-1 site and was proposed to be the most essential transcription factor for human RB gene activation (Clark et al, 1997). Whereas binding of the Sp1 transcription factor is not significantly affected by methylation of the CpG dinucleotide within its binding site, 5'-GGGCGG (lower strand, 5'-CCGCCC) methylation of the outer C is inhibitory (mammalian cells also have the capacity to methylate cytosines at CpNpG sites) and in particular methylation of both cytosines m Cp m CpG inhibited binding by 95%; endogenous m Cp m CpG methylation of an Sp1 site in the CpG island promoter of the RB gene was identified by genomic sequencing in a proportion of retinoblastoma tumors which were extensively CpG methylated in the RB promoter (Clark et al, 1997).

D. Transcription factors (TFs) that regulate the RB gene Several mutations have been found in the promoter region of the RB gene, suggesting that inappropriate transcriptional regulation of this gene contributes to tumorigenesis. The presence of E2F recognition sites in promoters of a number of growth-related genes suggested that expression of these genes might be affected by RB. Understanding the nature and availability of TFs which regulate the RB gene in particular cell types is instructive for a successful gene therapy application involving transfer of RB. An E2F recognition site lies within a region critical for RB gene transcription; binding of E2F-1 at this site transactivates the RB promoter; striking back, the resulting overexpression of RB suppresses E2F-1-mediated stimulation of RB promoter activity and, thus, the expression of RB is negatively autoregulated through E2F1 (Shan et al, 1994). Up-regulation of the RB gene by E2F was shown by co-transfection of RB- osteosarcoma Saos2 cells in culture with a plasmid expressing E2F-1 under the control of the CMV immediate-early gene promoter-CAT construct: expression of E2F-1 stimulated RB promoter activity 10-fold under conditions where E2F1 had little effect on c-jun, c-myc, and EGR-1 gene expression (Shan et al., 1994). The autoregulation of RB gene by RB may be accomplished via a direct protein-DNA complex formation, via protein-protein interaction regulating the activity of other transcription factors on the promoter of the RB gene, or both. Two distinct DNA-binding factors, RBF-1 and ATF, play an important part in the transcription of the human RB gene. The promoter of the human RB gene and of the mouse RB1 gene (Zacksenhaus et al., 1993) contain binding sites for ATF, and a Sp1-like transcription factor (Mitchell and Tijan, 1989) where the RBF-1 (retinoblastoma binding factor 1) may bind (Sakai et al., 1991). Human RB gene is also regulated by AP-1 (Linardopoulos et al, 1993), as well by the early response transcription factor, nerve growth factor inducible A gene (NGFI-A) which is expressed in prostate cells and binds to the site GCGGGGGAG at -152 to -144 within the RB gene promoter (Day et al, 1993). The ATF site of the RB promoter is a responsive element during myogenic differentiation; RB promoter activity increased about 4-fold during differentiation and was reduced when a point mutation was designed in the ATF site (Okuyama et al, 1996). pRB activates expression of the human transforming growth factor-#2 gene through ATF-2; the human RB gene promoter is autoregulated by RB protein via an ATF-2-like binding site at the carboxyl-terminal domain of pRB; overexpression of RB stimulates RB promoter activity

E. RB gene transfer Functional loss of the RB gene has been implicated in the initiation or progression of several human tumor types including cancer of the eye, bone, bladder, and prostate. The cancer suppressor activity of RB was directly demonstrated by the introduction of a normal RB gene into retinoblastoma cells that have lost the RB function (inability to be phosphorylated because of mutations at the appropriate sites) by mutation at both alleles; this led to the suppression of the neoplastic phenotype and loss of the tumorigenicity of RB cells in nude mice (Huang et al, 1988). Expression of the normal RB gene into the human prostate carcinoma cell line DU145, mediated by recombinant retrovirus integration, also resulted in loss of its tumorigenic ability in nude mice (Bookstein et al, 1990). Studies with tumor cells reconstituted with RB ex vivo and implanted into immunodeficient mice, as well as with germline transmission of a human RB transgene into tumor-prone Rb+/- mice have demonstrated cancer suppression (see Riley et al, 1996). DU145 cells express a shorter protein lacking 35 amino acids from exon 21 due to a 105 nucleotide in-frame 61

Boulikas: An overview on gene therapy deletion (Bookstein et al, 1990). The human bladder carcinoma cell line J82 contains a mutated RB protein with exactly these features (Horowitz et al, 1989); this 35 amino acid stretch is required for complexation with T antigen and E1A. However, the two cell lines have lost exon 21 of RB because of a different type of mutation: J82 cells have a point AG to GG mutation in the intron 20splice acceptor site but the type of mutation in DU145 leading to exon 21 loss is different (Bookstein et al, 1990). Intratumoral infection of spontaneous pituitary melanotroph tumors arising in immunocompetent Rb+/mice with a recombinant adenovirus carrying the RB cDNA inhibited the growth of tumors, re-established innervation by growth-regulatory dopaminergic neurons, and prolonged the life spans of treated animals (Riley et al, 1996). Retrovirus-mediated gene transfer of RB to the breast carcinoma cell lines MDA-MB468 and BT549, both of which harbor partial RB gene deletions as well as point mutations of their p53 genes, restored its expression in cells, reduced their ability to grow in soft agar, and their tumorigenicity in nude mice, although it did not significantly altered growth rate in culture (Wang et al, 1993). Future therapeutic approaches using the RB gene are directed toward inhibition in cell proliferation (such as to inhibit neointima formation and smooth muscle cell proliferation in arterial diseases, see Arterial injury below and Chang et al, 1995) rather that aggressive suppression and apoptosis of solid tumors; p53 is a better gene than RB for tumor eradication.

formed; tadpoles delete their tails by apoptosis (reviewed by Duke et al, 1996). Virus-transformed as well as severely X-ray-damaged or UV-damaged cells are similarly eliminated from the tissue via apoptosis; if they are left they can form malignant cells. Initiated cancer cells may lead to tumor development only when a dysfunction in their apoptotic pathway takes place. Although the biochemical aspects of cell death are fraught with the problem of cause versus effect, the role of apoptosis in neoplasia and its regulation by a number of oncogenes and p53 has emerged. Apoptosis is essential for normal development and homeostasis; deregulation in the positive control of apoptosis is associated with cancer and autoimmune disease whereas deregulation in the negative control of apoptosis is associated with degenerative diseases (reviewed by White, 1993; Duke et al, 1996).

B. Molecular mechanisms for apoptosis: p53, Bax, Bcl-2, c-Myc and other proteins Apoptosis is of special interest in gene therapy not only of cancer but of other diseases such as arterial disease. Apoptosis is a complex process involving a significant number of apoptotic and antiapoptotic mechanisms. The cytotoxic (killer) T lymphocytes of the immune system of the infected organism bind to virus-infected cells inflicting their eradication with two different type of proteins: Perforin is a transmembrane molecule transferred from the killer T cell to the membrane of the infected cells forming holes on the membrane of the target cell allowing uptake of proteases called granzymes that activate ICE-like proteases to induce apoptosis. A number of antiviral drug development strategies are based on blockage of the activity of antiapoptotic viral proteins. Expression of a number of genes induce apoptosis; their protein products include adenovirus E1A (Debbas and White, 1993; Lowe and Rudley, 1993) and c-Myc (Hermeking and Eick, 1994; Wagner et al, 1994). A number of proteins when expressed at sufficient amounts block apoptosis; these include Bcl-2 and E1B 19 kDa protein of adenovirus (Debbas and White, 1993; Chiou et al, 1994). Exposure of cells to a variety of growth factors including IL-3, IL-6, and erythropoietin, acting as survival factors, inhibit induction of apoptosis (Johnson et al, 1993; Yonish-Rouach et al, 1993; Canman et al, 1995). The role of p53 in these molecular processes has been discussed in previous pages in this review. The involvement of p53 in apoptosis is thought to occur via upregulation of bax and downregulation of bcl-2 genes by wt p53 but not by mutated p53 proteins; Bax protein induces apoptosis and its upregulation triggers the apoptotic mechanism in cells which display elevated levels of p53 as a result, for example, of DNA damage. Downregulation of Bcl-2 has a similar effect on the induction of apoptosis. p53 may induce apoptosis independently of transcription, although the G1 arrest by p53 requires transcription of p53 targets (reviewed by Ko and Prives, 1996). Induction of the apoptotic pathway by p53 was

XX. Induction of apoptosis for cancer gene therapy A. Apoptosis as an essential process Apoptosis has become a basic tool in developing cancer research in establishing new anticancer strategies. The health of a multicellular organism depends both on the ability of the body to produce new cells but also on the ability of certain type of cells to perish, self-destruct, when they become superfluous or severely damaged. Apoptosis, or programmed cell death, is a biological process associated with pronounced morphological changes, chromatin condensation, drop in pH, and intranucleosomal DNA degradation by which a cell actively commits suicide. Virtually all tissues have apoptotic cells; salient examples in the adult are: the eye lenses which consist of apoptotic cells that replaced their cytoplasm with crystallin; intestinal wall cells which migrate to the tip of the finger-like projections over several days where they die; ineffectual T cells which mature in thymus and which would attack the bodyâ&#x20AC;&#x2122;s own tissues are eliminated by apoptosis before entering the bloodstream; skin cells migrate from the deepest layers to the surface where they commit suicide forming the outer layer of the skin. Apoptosis is an essential process during embryogenesis: mammals eliminate neuron cells as the nervous system is 62

Gene Therapy and Molecular Biology Vol 1, page 63 proposed to involve: (i ) transcriptional induction of redoxrelated genes; (i i ) formation of reactive oxygen species; and (i i i ) the oxidative degradation of mitochondrial components (Polyak et al, 1997). The potential of p53 in cancer gene therapy is discussed above. While p53 and E1A activate apoptosis, Bcl-2 and E1B 19k proteins inhibit apoptosis. All four protein molecules act upstream of Bax which is a potent inducer of apoptosis: both the cellular Bcl-2 and the 19 kDa protein E1B of adenovirus are able to interact with Bax inhibiting its involvement in induction of apoptosis (Han et al, 1996; Figure 1 on page 9). E1A acts upstream of p53 by increasing the half-life of p53 resulting in an accumulation of p53 molecules in the nucleus (Lowe and Ruley, 1993); increased levels of p53 are then believed to upregulate the bax gene (Figure 1). The survival factors IL-3 and IL-6 appear to prevent p53-dependent apoptosis (see White, 1993). p53 induces apoptosis after exposure to UV irradiation (Ziegler et al, 1994) and hypoxia (Graeber et al, 1996); this acts as a protective mechanism for the removal of severely damaged cells from the body which could become initiated cancer cells and progress to tumors. Spontaneous or radiation-induced apoptosis mediated by p53 has been shown to act for the removal of cells from the gastrointestinal tract in mice (Merritt et al, 1994) and the skin after sunburn (Ziegler et al, 1994). Epidermal growth factor (EGF) has induced apoptosis in various cancer cell lines via a novel signal transduction pathway of EGF mediated through p53 (Murayama and Horiuchi, 1997). c-myc expression, normally induced in proliferating hematopoietic cells by mitogens, drops dramatically by mitogen withdrawal leading to cell arrest in G1. During deregulated c-myc expression, c-myc levels were not downregulated upon mitogen withdrawal; instead, DNA synthesis continued resulting in apoptosis but not in growth arrest. The transforming segment of c-Myc was responsible for induction of apoptosis (see White, 1993). Pax5 is a repressor of expression of the p53 gene interacting directly with a regulatory region within exon 1 of the p53 gene. At early stages during pre-B cell development the levels of Pax5 are high and p53 is downregulated; however, later in development Pax5 levels drop and the p53 gene is activated; this process was proposed to lead to the decision of B cells to enter apoptosis or differentiate into plasma cells (Stuart et al, 1995). 2+

death; this could be effected by overexpression of thebax gene, by suppression of the endogenous bcl-2 gene (see below), or by transfer of the wt p53 gene.

C. Role of tumor necrosis factor (TNF) The tumor necrosis factor-" (TNF-") is a cytokine produced by macrophages, monocytes, lymphoid cells, fibroblasts and other cell types in response to inflammation and infection. TNF-" is produced by lipopolysaccharide (LPS)-stimulated macrophages; the molecular pathways leading to TNF-" production in these specialized cells involves activation by LPS of several kinases including the extracellular-signal-regulated kinases 1 and 2 (ERK1 and ERK2), p38, Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK), as well as activation of the immediate upstream MAPK activators MAPK/ERK kinases 1 and 4 (MEK1 and MEK4) and of MEK2, MEK3, and MEK6 (Swantek et al, 1997). TNF-" binds to two type of specific receptors, TNFR1 and TNFR2, causing their trimerization and leading to activation of a number of kinases (ceramide-activated kinase, I&B kinase, Raf-1, Jun N-terminal kinases or JNKs, p38/Mpk2). Activation of Raf-1, JNK, and p38/Mpk2 contribute to the induction of AP-1 whereas activation of I&B kinase is leading to the activation of the transcription factors NF-&B. This activation leads further to upregulation of genes and induction of other cytokines, metalloproteinases, and immunoregulatory proteins (see Liu et al, 1996 and the references cited therein). TNF can induce apoptotic death or necrosis in some tumor cells; this effect of TNF could be mediated by activation of sphingomyelinases and phospholipases, synthesis of metabolites of arachidonic acid, generation of free radicals, changes in intracellular calcium, generation of DNA strand breaks and activation of poly(ADPribosyl)ation, or activation of ICE-like proteases. TNF-", IL-1#, IFN-! , and vitamin D3 after binding to their transmembrane receptors stimulate the production of the second messager ceramide from sphingomyelin in the plasma membrane by activating sphingomyelinase; this results in a cascade of signal transduction events that result in down regulation of c-myc and induction of apoptosis, to terminal differentiation, or to RB-mediated cell cycle arrest (Figure 23). IL-1 signaling leads to NF-&B activation and to protection against TNF-induced apoptosis. The IL-1Rassociated kinase (IRAK) is homologous to Pelle of Drosophila. Two additional proximal mediators, both associating with the IL-1R signaling complex, were required for IL-1R-induced NF-& B activation: IRAK-2, a Pelle family member, and MyD88, an adaptor molecule containing a death domain (Muzio et al, 1997). Treatment of different cell types with TNF-" results in the activation of the MEKK1 pathway of protein kinases ultimately resulting in AP-1 transcription factor activation and in the upregulation of several cytokine genes. TNF-"-

Down regulation of the Cu /Zn superoxide dismutase (SOD1) induced oxidative stress and apoptosis (Troy et al, 1996). A great deal of oxidative damage during the procedures for ex vivo-modification of cells induces their 2+ 2+ apoptosis; transfer of the Cu /Zn superoxide dismutase to ex vivo modified cells increased their survival after implantation (see Nakao et al, 1995). This demonstrates the importance of blocking apoptotic pathways during cell manipulation for successful ex vivo gene therapy. Gene therapy for cancer could involve restoration of the apoptotic pathway in cancer cells leading to their suicidal 63

Boulikas: An overview on gene therapy stimulation also results in the activation of NF-&B and inhibition of apoptosis (Figure 24). A TNF-responsive

serine/threonine protein kinase termed GCK-related (GCKR) most likely signals via mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) kinase kinase 1 (MEKK1) to activate the SAPK pathway (Shi and Kehrl, 1997).

D. NF- B as anti-apoptotic molecule and TNF- signaling Activation of NF-&B is believed to lead to the activation of antiapoptotic genes that have not been fully identified. The antiapoptotic role of NF-&B at the molecular level and the TNF-" connection consists of the following events; signaling by TNF-" induces trimerization of its receptors, an event causing three different cascades: ( i ) Activation of I&B kinase and activation of NF-&B, a pathway which prevents cell death. A key step for NF-&B activation leading to the activation of the stress-activated protein kinase (SAPK, also called cJun N-terminal kinase or JNK) is the recruitment to the TNF receptor of TNF receptor-associated factor 2 (TRAF2). ( i i ) induction of apoptosis via a different pathway involving activation of sphingomyelinase in plasma membrane and generation of ceramide leading to EGFR activation and induction of apoptosis; ( i i i ) activation of MEKK1 and JNK protein kinases which is not linked to apoptotic death but to AP-1 activation (Figure 24). The antiapoptotic function of NF-&B may involve activation of the manganese superoxide dismutase and of the zinc finger protein A20; expression of these genes is induced by TNF and each of them provides protection against apoptosis (Liu et al, 1996). bcl-2 upregulation during progression of prostate cancer was implicated in the acquisition of the androgenindependent growth; a strong antioxidant that interferes with activation of NF-&B in prostate carcinoma cells, potentiated TNF-"-stimulated apoptosis signaling through a bcl-2-regulated mechanism; based on these studies, modulation of the NF-&B survival signaling was proposed to be used to clinical advantage in the treatment of prostate cancer patients (Herrmann et al, 1997). Transgenic mice lacking the p65 (RelA) subunit of NF-&B displayed increased apoptosis and degeneration in the liver providing further support to an apoptotic function of NF-&B (Beg et al, 1995). The TNF-induced death of mouse primary fibroblasts expressing deregulated c-Myc was inhibited by transient overexpression of the p65 subunit of NF-&B, which increased NF-&B activity in the cells (Klefstrom et al, 1997). Rel (a protooncogene, member of the NF-&B family) is implicated in both positive and negative regulation of GM-CSF expression in a variety of cell types (Gerontakis et al, 1996). The elucidation of IL-1, TNF, IFN and other signaling pathways would lead to the discovery of new drugs causing specific inhibition; for example, members of the IL-1 signaling cascade may provide therapeutic targets for inhibiting IL-1-induced inflammation (Muzio et al, 1997).

F i g u r e 2 3 . A pathway leading to the induction of growth arrest and apoptosis by the cytokines TNF-", IL-1#, and IFN!. The pathway is conserved between mammalian cells and yeast. Adapted from Nickels and Broach (1996). From Boulikas T (1 9 9 7 ) Gene therapy of prostate cancer: p53, suicidal genes, and other targets. Anticancer Res 17, 1471-1506. Reproduced with the kind permission from Anticancer Research.

Gene Therapy and Molecular Biology Vol 1, page 65 At least 10 ICE-like proteases have been identified which mediate apoptotic death after their induction by a number of stimuli (see Martin and Green, 1995); these are divided into three families: ( i ) the ICE/CED3 family, including ICE itself; ( i i ) the CPP32/Yama family; and ( i i i ) the Ich-1/Nedd2 family; they all contain the conserved QACRG pentapeptide in which the central cysteine participates in proteolytic catalysis (see J채nicke et al, 1996). Activation of these proteases by induction of apoptosis results in the cleavage of a large number of key regulatory proteins including among others poly(ADPribose) polymerase or PARP (Lazebnik et al, 1994), RB (J채nicke et al, 1996), PKCd (Emoto et al, 1995), Gas2 affecting microfilament reorganization (Brancolini et al, 1995), the DNA-dependent protein kinase (Casciola-Rosen et al, 1995), and the sterol regulatory element binding proteins (SREBPs) catalyzed by CPP32 ICE-like protease (PARP et al, 1996). Since cleavage of a single protein has not been shown to cause cell death it is not clear how many substrate protein molecules need to be cleaved. In addition different apoptotic pathways may exist and may operate in different cell types. Expression of the murine ICE cDNA in Rat-1 cells induced programmed cell death and this phenomenon could be reversed by overexpression of thebcl-2 oncogene (Miura et al, 1993). Expression of members of the family of cysteine proteases related to ICE have been shown to be necessary for programmed cell death in a number of organisms (Yuan et al, 1993). Overexpression of murine ICE or of the ICE-like proteases NEDD-2/ICH-1 and Yama/apopain induced apoptosis (Miura et al, 1993). Mice lacking ICE were resistant to apoptosis induced by Fas antibody (Kuida et al, 1995).

F i g u r e 2 4 . TNF-" signaling via trimerization of its receptors (TNF-"R1), is causing: ( i ) activation of I&B kinase and activation of NF-&B, a pathway which prevents cell death via activation of the manganese superoxide dismutase and of the zinc finger protein A20. ( i i ) induction of apoptosis via second message ceramide (see F i g u r e 2 3 ) and ( i i i ) activation of JNK leading to AP-1 activation and upregulation of cytokine genes.

F. Role of poly(ADP-ribose) polymerase (PARP) PARP is a central mediator of genome integrity and transmits signals from DNA damage to recruit locally DNA repair activities (Zardo et al, 1998; Quesada, 1998, this volume). An additional role of PARP is its involvement in apoptosis causing suppression of an apoptotic endonuclease; PARP is cleaved by an ICE-like protease during TNF-induced apoptosis (Lazebnik et al, 1994). Cleavage of PARP would abort these pathways resulting in loss of recruitment of DNA repair enzymes at damaged sites but also in loss in the inhibitory function of poly(ADP-ribose) groups on key regulatory enzymes (DNA ligase, topoisomerase). It is unlikely that PARP proteolysis by an ICE-like protease is a primary event since PARP-deficient mice show normal resistance to DNA damaging agents (Wang et al, 1995).

E. Interleukin-1# converting enzyme (ICE) and apoptosis The human interleukin-1# converting enzyme (ICE) is a cysteine-rich protease that can cleave the inactive 31 kDa precursor of IL-1# to generate the active cytokine; it has similarities to the C. elegans CED-3 protein. This protease plays a central role in apoptosis; the exact role and the involvement of IL-1# have not been elucidated; it is believed that signals from IL-1#, TNF-", vitamin D3, and interferon-! , which induce an antiproliferative response, converge on sphingomyelin of plasma membrane activating a sphingomyelinase which generates a ceramide second messenger (F igure 23); in S. cerevisiae, this leads to signal transduction via activation both of a cytoplasmic protein phosphatase 2A and a protein kinase leading to down-regulation in c-myc expression and induction of apoptosis as well as RBmediated cell cycle arrest via EGFR activation (Nickels and Broach, 1996).

G. Apoptosis in autoimmune disease and ischemic heart disease T cells are produced by bone marrow and then migrate to the thymus gland where they mature. The cytotoxic or killer T cells directed against foreign bodies are released in 65

Boulikas: An overview on gene therapy the bloodstream; a specific apoptotic mechanism eliminates T cells directed against specific antigens on healthy cells. However, the body allows some mildly selfreactive lymphocytes to circulate; although harmless, exposure to a microbe or food antigen can stimulate them causing an expansion in their proliferation and resulting in a mild autoimmune disease. Such mild autoimmune reactions usually disappear when the stimulating antigen is cleared away; in more severe autoimmune disease, however, these lymphocytes survive longer inducing apoptosis and self-destruction in healthy cells in various tissues. A number of classical diseases may originate by autoimmune mechanisms including initiation of atherosclerosis by apoptotic death of the epithelial cells in the arterial wall, diabetes by destruction of the pancreatic cells, lupus erythematosus, rheumatoid arthritis, and others. The mechanism via which T lymphocytes directed against self antigens defy apoptosis is not known; the mechanism might involve overexpression of the Bcl-2 gene in these lymphocytes or down-regulation of a gene encoding for the Fas ligand that sends a death message to the lymphocyte (Weih et al, 1996; reviewed by Duke et al, 1996). Excessive necrotic death in cells of the coronary artery wall results by oxygen and glucose deprivation after blockage of a blood vessel feeding a segment of the heart (also the brain in stroke). Destructive free radicals are then produced during inflammation of the area which can cause apoptotic or necrotic death in cells in the surroundings. Since both brain and heart cells in the adult are not regenerated, Biotech Companies (for example Genentech) are focusing in developing drugs that block free radical formation, inhibit ICE-like proteases, or inhibit apoptosis via other mechanisms. The progressive loss of neuron cells in senile or other brain diseases such as Alzheimerâ&#x20AC;&#x2122;s disease, Parkinsonâ&#x20AC;&#x2122;s disease, Huntingtonâ&#x20AC;&#x2122;s disease, and amyotrophical lateral sclerosis may ensue by apoptosis. Etiologic factors may include excessive levels of neurotransmitters, low levels of NGF, free radical-mediated damage, and deregulation in the expression of genes encoding apoptotic regulators during aging. Deregulation in apoptosis may also have a share in the induction of osteoporosis.

apoptotic pathways is desirable in the gene therapy of heart disease and degenerative brain disease (see below).

A. Gene therapy that targets bcl-2 Bcl-2 protein is overexpressed in a variety of human leukemias because of translocation of its gene to the immunoglobulin locus; Bcl-2 is associated with the outer surface of the mitochondrion and appears to be involved in scavenging oxygen radicals. Overexpression of thebcl-2 gene in tumors is thought to be responsible for the poor response of the tumors to antineoplastic drugs and radiation therapy blocking apoptosis of the tumor cells. Bcl-2 can interact with members of the Bcl-2 family including Bax, Bcl-X-S, Bcl-X-L, and Mcl-1 but also with heterologous protein molecules including BAG-1, Raf-1, and R-Ras. Introduction of the bcl-2 gene into human diploid breast epithelial MCF10A cells (containing the wild-type p53 gene) resulted in suppression in p21 gene expression although the level of expression of p53 was not affected; these studies suggested that Bcl-2 may inhibit the functional activity of p53 protein and might regulate the commitment of cells to commit suicide or proliferate (Upadhyay et al, 1995). Overexpression of the bcl-2 gene in tumors is thought to be responsible for the poor response of the tumors to antineoplastic drugs and radiation therapy blocking apoptosis of the tumor cells; therefore, down-regulation of the bcl-2 gene specifically in tumor cells could induce apoptosis. Primary untreated human prostate cancers were found to express significant levels of this apoptosissuppressing oncoprotein; this is a striking difference with normal prostate secretory epithelial tissue not expressing Bcl-2 (Raffo et al, 1995). Transfection of LNCaP human prostate cancer cells with a plasmid expressing bcl-2 rendered these cells highly resistant to a variety of apoptotic stimuli (serum starvation or treatment with phorbol ester) and induced earlier and larger tumors in nude mice. The ability of Bcl-2 to protect prostate cancer cells from apoptotic stimuli correlated with the ability of the cells to form hormone-refractory prostate tumors in nude mice (Raffo et al, 1995). The Bcl-2 oncoprotein suppresses apoptosis and, when overexpressed in prostate cancer cells, makes these cells resistant to a variety of therapeutic agents, including hormonal ablation. Overexpression of BCL-2 is common in non-Hodgkin lymphoma leading to resistance to apoptosis and promoting tumorigenesis. Therefore, bcl-2 provides a strategic target for the development of gene knockout therapies to treat human prostate cancers (Dorai et al, 1997) and non-Hodgkin lymphomas (Webb et al, 1997). Down-regulation of Bcl-2 can be accomplished with antisense. In patients with relapsing non-Hodgkin lymphoma, BCL-2 antisense therapy led to an improvement in symptoms; antisense oligonucleotides targeted at the open reading frame of the BCL-2 mRNA

XXI. Genes involved in the regulation of apoptosis as targets for gene therapy Many of the molecular controllers of apoptosis including cytokine signaling pathways (TNF-", IL-1#), tumor suppressor proteins (p53), viral proteins (E1A of adenovirus), cellular oncoproteins (Myc), proteins that control the cell cycle (E2F), apoptosis inducers (Bax) and antiapoptotic molecules (Bcl-2, NF-&B) could constitute potential targets for pharmacological intervention for the treatment not only of cancer but of other human disease. Although for cancer treatment it is desirable to induce apoptosis, the opposite effect, that is inhibition of 66

Gene Therapy and Molecular Biology Vol 1, page 67 showed effectiveness against lymphoma grown in laboratory animals and has entered human clinical trials. The first study was conducted on nine patients with BCL2-positive relapsed non-Hodgkin lymphoma using a daily subcutaneous infusion of 18-base, fully phosporothioated antisense oligonucleotide administered for 2 weeks (Webb et al, 1997). A local inflammation at the infusion site was noted. A reduction in tumour size was observed in two patients (one minor, one complete response) using computed tomography scans; in two other patients, the number of circulating lymphoma cells decreased during treatment. In four patients, serum concentrations of lactate dehydrogenase fell, and in two of these patients symptoms improved (Webb et al, 1997). A divalent hammerhead ribozyme, constructed by recombining two catalytic RNA domains into an antisense segment of the coding region for human bcl-2 mRNA was able to rapidly degrade bcl-2 mRNA in vitro; it was then tested for its ability to eliminate bcl-2 expression from hormone-refractory prostate cancer cells. When this hammerhead ribozyme was directly transfected into cultured prostate cancer cells (LNCaP derivatives), it significantly reduced bcl-2 mRNA and protein levels within 18 hr of treatment and induced apoptosis in a low-bcl-2-expressing variant of LNCaP, but not in a high-bcl-2-expressing LNCaP line (Dorai et al, 1997).

in fibroblasts. The physiological relevance of E2F in the apoptotic mechanism is thought to arise from the ability of E2F to act as a functional link between p53 and RB; p53 levels increased in response to high levels of E2F. Targeted disruption of the E2F-1 gene yields transgenic animals with an excess of mature T cells due to a defect in lymphocyte apoptosis (Field et al, 1996). Overexpression of the transcription factor E2F-1 could induce apoptosis in quiescent rat embryo fibroblasts in a p53-dependent manner; however, Hunt et al (1997) have shown that overexpression of the E2F-1 gene after adenoviral transfer can mediate apoptosis in the absence of wild-type p53: adenovirus-mediated transfer of the E2F-1 gene under control of the CMV promoter to human breast and ovarian carcinoma cell lines resulted in the induction of significant morphological changes in four of the five cell lines that had mutations in the p53 gene within 48 h of transduction characteristic of apoptosis. Retroviral vector-mediated transfer of the TNF-" gene into the DNA of human tumor cells induced apoptosis in high- TNF-"-producing clones generated from a human lymphoma T-cell line (ST4); the apoptotic death of the cells was associated with a downregulation of the apoptosis-preventing gene, bcl-2, while the expression of bax and p53 genes persisted (Gillio et al, 1996).

D. E6, E7 of human papillomavirus (HPV)

B. Bcl-xs Many cancers overexpress a member of the Bcl-2 family of inhibitors of apoptosis, such as Bcl-2 and BclxL. Members of the Bcl-2 family were found to be essential for survival of cancer cells derived from solid tissues including breast, colon, stomach, and neuroblasts (Clarke et al, 1995). On the contrary, Bcl-xs is a dominant negative repressor of Bcl-2 and Bcl-xL; thus, Bcl-xs induces apoptosis. Transient overexpression of Bcl-xs in MCF-7 human breast cancer cells, which overexpress BclxL, with a replication-deficient adenoviral vector induced apoptosis in vitro; intratumoral injection of the bcl-xs adenovirus on solid MCF-7 tumors in nude mice showed a 50% reduction in size with evident apoptotic cells at sites of injection (Ealovega et al, 1996). An adenovirus vector expressing bcl-xs specifically and efficiently killed carcinoma cells arising from multiple organs including breast, colon, stomach, and neuroblasts even in the absence of an exogenous apoptotic signal such as x-irradiation. In contrast, normal hematopoietic progenitor cells and primitive cells capable of repopulating SCID mice were not killed by the bcl-xs adenovirus. Thus, transfer of the bcl-xs gene could be used in killing cancer cells contaminating the bone marrow of patients undergoing autologous bone marrow transplantation (Clarke et al, 1995).

E6 and E7 of HPV possess transforming ability, have been shown to interact with the cellular tumor suppressors p53 and RB (Werness et al, 1990; Dyson et al., 1989) and are believed to play a central role in HPV-induction of cervical carcinogenesis as well as in the maintenance of the malignant phenotype. Viruses have developed strategies to shut down protein synthesis in the host and subdue its protein synthesizing machinery to produce progeny virus when infecting cells. Because virus-infected cells commit suicide to protect the organism from further infection viruses have evolved mechanisms to prevent apoptosis of the host cell ensuring their propagation; E6 protein interacts with p53 to exclude p53 molecules from their apoptotic functions and to inhibit apoptosis in HPVinfected cells thus giving to HPV a proliferation advantage. 27-mer phosphorothioate oligodeoxynucleotides (oligos) targeting the ATG translational start region of HPV-16 E6 and E7 sequences showed antiproliferative effects in all HPV-16-positive cell lines tested and in primary cervical tumor explants while the endometrial and two ovarian primary tumors as well as the HPV-negative C33-A cell line and HPV-18-positive cell line HeLa were relatively insensitive to the HPV-16 oligos (Madrigal et al, 1997).

C. E2F-1 and TNF- gene transfer E2F cooperates with p53 to induce apoptosis; high levels of wild-type p53 potentiate E2F-induced apoptosis 67

Boulikas: An overview on gene therapy HER-2/neu transcription and functions as a tumor suppressor gene in HER-2/neu-overexpressing cancer cells. Breast cancer cells that overexpress HER-2/neu are more resistant to chemotherapeutic agents such as paclitaxel (Taxol) and docetaxel (Taxotere) than those that do not overexpress HER-2/neu; paclitaxel sensitivity correlated with HER-2/neu expression level in a panel of mouse fibroblasts expressing different levels of HER-2/neu; downregulation of HER-2/neu expression by E1A sensitized the cells to paclitaxel. Transfer the E1A gene into two human breast cancer cell lines that overexpress HER-2/neu and E1A gene transfer sensitized these cells to the drug by repressing HER-2/neu expression (Ueno NT et al, 1997). Increased HER-2/neu expression led to more severe ovarian malignancy and increased metastatic potential in animal models; the adenovirus 5 E1A gene repressed HER2/neu gene expression and suppressed growth of human ovarian cancer SKOV-3 cells, which overexpress HER2/neu, in cell culture (Yu et al, 1995). Intraperitoneal injection of SKOV-3 cells into female nu/nu mice elicited tumors and the animals died within 160 days of severe tumor symptoms; cationic liposome-mediated delivery of the E1A gene into adenocarcinomas that developed in the peritoneal cavity and on the mesentery of the mice significantly inhibited growth and dissemination of ovarian cancer cells; about 70% of the treated mice survived at least for 365 days (Yu et al, 1995). Regulatory regions derived from the 5' flank of the human prostate-specific antigen (PSA) gene were inserted into adenovirus type 5 DNA to drive the expression of the E1A gene; infection of cells in culture with this recombinant adenovirus was able to drive the expression of the E1A gene only in cell lines which expressed PSA such as the human LNCaP cells but not in human DU145 cells which do not express PSA; the recombinant adenovirus 9 destroyed large LNCaP tumors (1x10 cells) and abolished PSA production in nu/nu mouse xenograft models after a single intratumoral injection (Rodriguez et al, 1997). A replication-deficient adenovirus containing the E1A + gene, Ad.E1A , was used to transduce E1A into HER2/neu-overexpressing and low expressing human lung cancer cell lines and shown a better therapeutic efficacy in HER-2/neu-overexpressing cells. The cell culture studies were then extended to animal studies: tumor-bearing mice established by intratracheal injection of lung cancer cells overexpressing HER-2/neu and treated by i.v. tail + injections of Ad.E1A showed suppression of the intratracheal lung cancer growth. However, no significant tumor suppression effect was observed in mice bearing a low HER-2/neu$expressing cell line with the same regimen (Chang et al, 1996).

E. Prevention of apoptosis for gene therapy of heart disease and for ex vivo manipulations of therapeutic cells As induction of apoptosis is the desired effect for the gene therapy of cancer, prevention of apoptosis by gene therapy can fight heart disease. Cardiomyocyte death results from heart ischemia proceeding via necrosis and from reperfusion which induces additional cardiomyocyte death by apoptosis; prevention of apoptosis would constitute an important target for fighting heart disease. Prevention of apoptosis should also solve a major problem in cell culture cells which are subject to oxidation damage during their manipulation for ex vivo gene transfer and most important during the step of reimplantation, encapsulation in biopolymer membranes for surgical implantation, and similar processes. Prevention of apoptosis could be effected by transfer and overexpression of the bcl-2 gene. Also prevention of oxidative damage during reimplantation of ex vivo-modified cells could be reduced by transfer and overexpression of the Cu/Zn superoxide dismutase gene (Nakao et al, 1995). Overexpression of bcl-2 delayed onset of motor neuron disease and prolonged survival in a transgenic mouse model of familial amyotrophic lateral sclerosis (Kostic et al, 1997).

XXII. E1A and HER-2/neu (c-erbB-2) in cancer gene therapy A. HER-2/neu The human epidermal growth factor receptor-2 (HER2), a membrane tyrosine kinase highly expressed in many epithelial tumors, could be a target for cancer gene therapy. The HER-2/neu (also called c-erbB-2) proto-oncogene is overexpressed in many human cancer cells, including those of breast cancer and ovarian cancer correlating with lower survival rate in ovarian cancer patients; amplification or overexpression of HER-2/neu has also been observed in human lung cancer and has been correlated with poor prognosis and chemoresistance. A reversible transformation of NIH3T3 fibroblasts by overexpression of the HER2/c-erbB2 receptor tyrosine kinase under control of a tetracycline-responsive promoter has been demonstrated in tissue culture; induction of HER2 expression resulted in cellular transformation in vitro and treatment of transformed cells with the effector anhydrotetracyline switched-off HER2 expression and induced morphological and functional changes characteristic for non-transformed cells (Baasner et al, 1996).

C. Clinical trials with E1A and c-Erb-B2

B. E1A-based gene therapy

Liposome-mediated E1A gene transfer suppressed tumor development and prolonged survival of mice bearing human breast cancer cells overexpressing HER-2/neu.

E1A-based gene therapy approaches are now in clinical trials (see below); the molecular mechanism behind this approach is that the E1A protein of Adenovirus 5 represses 68

Gene Therapy and Molecular Biology Vol 1, page 69 These studies resulted in the initiation of a phase I clinical trial using an E1A-liposome complex administered to patients with HER-2/neu-overexpressing breast or ovarian cancer (Protocol 205 in Table 4 of following article, pages 203-206). The principal investigators are Drs. Hortobagyi, Lopez-Berstein, and Hung at MD Anderson Cancer Center, Houston, Texas). The safety of this regimen was shown by intraperitoneal injection of E1A/liposomes in normal mice and at cumulative doses 5 to 40 times the DNA-lipid starting dose proposed for the phase I clinical trial (Xing et al, 1997). A Phase I multicenter study of intratumoral E1A gene therapy using cationic liposome gene transfer is also in course for patients with unresectable or metastatic solid tumors that overexpress HER-2 /neu (protocol 209, see page 205). Delivery of an anti-erbB-2 single chain (sFv) antibody gene for previously treated ovarian and extraovarian cancer patients is in clinical trials using adenoviral gene delivery (protocol #133). A clinical trial for tumor vaccination with HER-2 /Neu using a B7 expressing tumor cell line prior to treatment with HSV-tk gene-modified cells is in phase I for ovarian cancer (protocol #96, page 165).

cell proliferation is detrimental and has also been used to restrict intimal hyperplasia of the arterial wall and smooth muscle cell growth to limit restenosis after artery angioplasty (see below). Cancer cells can be induced to be conditionally sensitive to the antiviral drug ganciclovir after their transduction with the thymidine kinase (tk) gene from the herpes simplex virus (HSV); ganciclovir (GCV) is the 9{[2-hydroxy-1-(hydroxymethyl)-ethoxy]methyl}guanine (Field et al, 1983); it is converted by HSV-tk into its monophosphate form which is then converted into its triphosphate form by cellular enzymes and is then incorporated into the DNA of replicating mammalian cells leading to inhibition in DNA replication and cell death (Moolten, 1986; Borrelli et al, 1988; Moolten and Wells, 1990). It is only viral TK, not the mammalian enzyme, that can use efficiently ganciclovir as a substrate and this drug has been synthesized to selectively inhibit herpes virus replication (Field et al, 1983); indeed, the mammalian TK has a very low affinity for this guanosine analog. The toxicity of ganciclovir is manifested only when cells undergo DNA replication and it is not harmful to normal nondividing cells. This treatment strategy has been used for hepatocellular carcinoma (Huber et al, 1991; Su et al, 1996), fibrosarcoma, glioma (Culver et al, 1992, see below), adenocarcinoma (Osaki et al, 1994), prostate cancer (Eastham et al, 1996) and many other cancers.

XXIII. Suicidal genes for cancer therapy (prodrug gene therapy) A. Molecular mechanism of cell killing with HSV-tk gene and ganciclovir (GCV)

B. Treatment gliomas in rats with HSV-tk plus ganciclovir

Expression of genes encoding prodrug-activating enzymes can increase the susceptibility of tumor cells to prodrugs, and may ultimately achieve a better therapeutic index than conventional chemotherapy (Table 3). Direct suppression of tumor growth by cytotoxic gene therapy is a successful gene transfer approach. This approach has promise for a variety of other applications where excess

Brain tumors have the privilege of escaping immunologic rejection; therefore brain tumors are inaccessible to cancer immunotherapy. Culver and cowor-

Table 3. Prodrugs and enzymes used for their activation Prodrug-activating enzyme

Prodrug

Toxic substance it is converted to

Thymidine kinase from HSV

9-{[2-hydroxy-1-(hydroxymethyl)ethoxy]methyl}guanine or ganciclovir (GCV)

GCV monophosphate

Cytosine deaminase (CD) from E. coli

5-fluorocytosine (5FC)

5-fluorouracil (5FU)

Purine nucleoside phosphorylase (PNP) from E. coli

6-methylpurine-2â&#x20AC;&#x2122;-deoxyriboside (MePdR)

6-methylpurine (a very toxic adenine analog)

Purine nucleoside phosphorylase (PNP) from E. coli

Arabinofuranosyl-2-fluoroadenine monophosphate (F-araAMP) commercially known as fludarabine

A very toxic adenine analog

Human deoxycytidine kinase (dCK)

Cytosine arabinoside (ara-C)

A toxic drug inducing lethal strand breaks in DNA

Nitroreductase from E. coli

5-(aziridin-1-yl)-2,4-dinitrobenzamide (CB1954)

A potent dysfunctional alkylating agent which crosslinks DNA

fibroblasts were transduced with a retroviral vector expressing the HSV-tk gene (see above); the tumor cell mass was then infiltrated by intratumoral injection of the

kers (1992) took advantage of the fact that retroviral vectors require DNA synthesis for stable integration into the host genome to target gliomas in rats. Murine 69

Boulikas: An overview on gene therapy HSV-tk producing fibroblasts. This treatment gave a continuous local infusion of retroviral vector from the injected fibroblasts, integrating into the dividing cells of the growing brain tumor but not into the nondividing normal cells in the surroundings. Treatment of rats at day 5 after transplantation with ganciclovir (GCV) resulted in the complete regression of the tumor cell mass; this was thought to be induced by killing of cells that respond to signals promoting angiogenesis in the immediate vicinity of the tumor; vascular endothelial cells in the normal brain tissue, exhibiting cycling at a low rate, apparently were not affected. Other proliferating tissues, such as intestinal epithelium, thymus, and bone marrow, which might also uptake the retroviral HSV-tk vector and then be destroyed during GCV treatment were not affected by this approach over a 30 day period of treatment with GCV (Culver et al, 1992). A replication-defective, highly purified retroviral vector at titers of 10 8 colony forming units/mL was used to treat 9L gliosarcoma cells in rat brain. Animals with established 9L tumors treated with intralesional injection of the HSV-tk retrovirus followed by GCV treatment showed at day 26 that 29% (4/14) had no tumor and 50% (7/14) of the animals had < 1% tumor volume; substantial + + numbers of CD4 and CD8 lymphocytes infiltrated the tumors of animals treated with HSV-tk and GCV; the former tumor bed in cured animals contained cell debris, immune cells, and fibroblasts without signs of damage to the adjacent brain tissue (Kruse et al, 1997).

In a similar experiment, set to assess the “bystander effect” in vivo, mixtures of HSV-tk transduced and nontransduced oral squamous carcinoma cells were implanted subcutaneously in the left flank of nude mice, and naive HSV tk$ cells were implanted subcutaneously in the right flank. Treatment with GCV eradicated the tumors in the left flank consistent with a predicted bystander effect but also resolved or arrested the growth of the naive tumors in the right flank. The histology of regressing tumors from the right flank showed an infiltration of lymphoid cells suggesting that an immune-related antitumor response accounted for the distant bystander effect (Bi et al, 1997; see also Ramesh et al, 1998 this volume). The induction of higher levels of HSV-tk expression does not augment the sensitivity to GCV: adenoviral vectors that expressed HSV-tk at different efficiencies from CMV versus RSV promoters did not display a significant difference in antitumor effects; thus, increasing the HSVTK enzyme levels per cell above a minimal threshold level will not be effective in cell killing with GCV. To enhance the therapeutic responses of the HSV-tk/GCV system one needs to improve other parameters such as to use higher doses of GCV, to enhance the "bystander effect," to engineer mutant HSV-tk genes with higher substrate affinities, or to discover vectors with increased transduction efficiencies (Elshami et al, 1997). Suicide gene therapy may be useful not only for shortterm tumor regression mediated by direct cell killing and bystander effect, but may also exert a therapeutic vaccination effect resulting in long-term tumor regression and prevention of recurrence (Yamamoto et al, 1997).

C. The bystander effect of HSV-tk/GCV During HSV-tk/GCV treatment of brain tumors products from the dying cells in the brain tumor killed nearby non-HSVtk-transduced cancer cells without affecting normal cells, an effect described as "bystander" antitumor effect (Culver et al, 1992). The bystander effect of the HSV-tk plus GCV system appears to be powerful and significant, circumventing the low efficiency of transduction in vivo with recombinant retroviruses. Because of this effect, the low-level percentage of cells that can be transduced with a retrovirus can cause the elimination of a much larger percentage of proliferating cells in their surroundings (Kimura et al, 1996). In vitro, the “bystander” effect works by transfer of cytotoxic small molecules between cells via gap junctions. In order to understand the “bystander effect” mechanism during which adjacent nontransduced tumor cells are killed, Yamamoto et al (1997) used Renca cells from a renal carcinoma cell line transduced with a retroviral vector bearing the HSV-tk gene to inoculate BALB/c mice. After complete regression of inoculated tumors with GCV treatment, the animals were challenged with nontransduced tumor cells. In these animals, tumor-specific cytotoxic + CD8 T cells were efficiently induced which promoted the rejection or significant growth inhibition of challenged tumor cells.

D. Additional examples of tumor eradication with HSV-tk/GCV Chen et al (1996) used a recombinant adenoviral vector containing the HSV-tk gene for the treatment of metastatic colon carcinoma in the mouse liver; the HSV-tk alone exhibited substantial regression, although all treated animals suffered from subsequent relapses. Delivery of the HSV-tk + mouse IL-2 genes in adenoviral vectors to the hepatic tumors induced an effective antitumor immune response which nevertheless waned with time, and the treated animals eventually succumbed to hepatic tumor relapse; however, after combination treatment with HSVtk, mouse IL-2, and mouse GM-CSF a fraction of the animals developed long-term antitumor immunity and survived for more than 4 months without tumor recurrence (Chen et al, 1996). Microinjection of the HSV-tk gene, under control of " fetoprotein enhancer and albumin promoter, in a linear form flanked by the adeno-associated virus ITRs into pronuclei of mouse embryos led to transgenic animals expressing preferentially HSV-tk into adult liver cells; this led to an approach for the treatment of hepatocellular carcinomas (Su et al, 1996). Subcutaneous tumors induced 70

Gene Therapy and Molecular Biology Vol 1, page 71 by injection of RM-1 (mouse prostate cancer) cells in mice followed by injection of HSV tk in an adenovirus vector and treatment with ganciclovir for 6 days showed reduction in tumor volume (16% of control) and higher apoptotic index in tumor cells (Eastham et al, 1996). Recombinant adenoviruses carrying the HSV-tk gene under control of the CMV promoter displayed a significant cell killing efficiency for the eradication of brain tumors and leptomeningeal metastases in rats (Vincent et al, 1997). Pancreatic cancer is the fifth leading cause of cancer death in the United States. In order to treat peritoneal dissemination, one of the most common complications of the malignancies of the digestive system such as gastric or pancreatic cancers, mice were intraperitoneally (i.p.) inoculated with the human pancreatic cancer cell line PSN1; i.p. transfer of the HSV-tk suicidal gene under control of the potent hybrid CAG promoter was achieved with a DNA-lipopolyamine complex given eight days from the injection of cancer cells; animals were treated with GCV for 8 days; 8 out of 14 mice treated with HSV-tk and GCV were free of tumors on day 24. The gene transfer method resulted in the transduction of tumor nodule cells and not in normal organs as shown by reverse transcription polymerase chain reaction (RT-PCR) analysis as well as by transfer of the lacZ gene under similar conditions and localization of the blue staining; HSV-tk was expressed in about 10% of tumor cells but not in the normal pancreas or in the small intestine (Aoki et al, 1997). A murine pancreatic ductal adenocarcinoma cell line was used to induce intrahepatic solid tumors into the left lateral liver lobe; intratumoral injection of an adenovirus vector carrying the HSV-tk gene under control of the RSV promoter in combination with intraperitoneal administration of ganciclovir caused a significant reduction in tumor volume and necrosis; because pancreatic cancer patients have an overall low survival since metastases have already taken place at the time of diagnosis and because surgical resection of pancreatic cancers does not significantly change the clinical outcome even in combination with chemotherapy, gene therapy might offer an effective approach in the near future (Block et al, 1997). HSV-tk gene transfer was successfully used to eradicate adenocarcinoma-derived peritoneal carcinomatosis, a common clinical situation which, in most cases cannot be controlled by surgery or chemotherapy. DHD/K12 colon carcinoma cells stably expressing the HSV-tk gene were injected intraperitoneally to rats leading to the development of peritoneal carcinomatosis within 2-3 weeks from injection (Figure 25A). Treatment of these animals with GCV (Figure 25C) resulted in the eradication of the peritoneal tumor nodes. It ought to be emphasized, however, that the same spectacular results are not expected when treating tumors in patients; tumor cells in patients need first to be transduced with the HSV-tk gene whereas

the cells used to elicit these tumors in animals were already transduced with the HSV-tk gene in cell culture and most or all cells were expressing the viral thymidine kinase. Retrovirus-mediated transfer of HSV-tk was used to kill proliferating cells in rabbit models of proliferative vitreoretinopathy (PVR); traction retinal detachment results from proliferation of retinal pigment epithelial, glial, macrophages, and fibroblast cells in the vitreous cavity of the eye forming contractile membranes on both surfaces of the retina; PVR may ensue after retinal surgery or trauma and can be induced in rabbit models by surgical vitrectomy to facilitate cell attachment to the retina. Injection, into the vitreous cavity, of rabbit dermal fibroblasts transduced in vitro with retroviral vectors carrying the HSV-tk gene was used to preferentially kill proliferating cells for PVR in rabbit models; all eyes received 0.2 mg GCV on the following day and on day 4; significant inhibition of PVR was observed thus providing a novel therapeutic strategy for this disease (Kimura et al, 1996).

E. Expression of cytosine deaminase (CD) gene from E. coli and treatment with 5fluorocytosine Another suicide gene approach has been the expression of the cytosine deaminase (CD) from E. coli; mammalian cells, unlike certain bacteria and fungi, do not posses this enzyme. The CD protein normally catalyzes the conversion of cytosine to uracil but has been exploited for the conversion of the prodrug 5-fluorocytosine (5FC) into the toxic 5-fluorouracil (5FU); treatment of cells, transfected with this construct, with 5FC resulted in the conversion of the 5FC into the antitumor drug 5FU into CD-expressing tumor cells (Mullen et al, 1992; Austin and Huber, 1993; Huber et al, 1993; 1994; Richards et al, 1995). This approach has been used for the treatment of primary and metastatic hepatic tumors based on the overexpression of the suicidal CD gene under control of the regulatory regions of the tumor marker gene carcinoembryonic antigen (Richards et al, 1995, see below). Szary et al (1997) have developed a model for tumor radiosensitization using the CD gene/5FC system; when melanoma cells were transfected with the CD gene, subsequent treatment with 5FC sensitized the cells to radiation damage; 5FC did not change the radiosensitivity of parental, nontransfected cells; increased toxicity to radiation damage was thought to arise from 5-fluorouracil generated by CD.

Boulikas: An overview on gene therapy

F i g u r e 2 5 . Eradication of peritoneal carcinomatosis with HSV-tk plus GCV. Intraperitoneal injection to rats of DHD/K12 colon carcinoma cells stably expressing the HSV-tk gene caused peritoneal carcinomatosis at day 21 (A). The animal whose intraperitoneal cavity is shown in (B ) was treated with HBSS buffer alone and the animal shown in (C) was treated with GCV for 5 days at 150mg/Kg. The letter “T” indicates the peritoneal tumor nodes. From Lechanteur C, Princen F, Bue SL, Detroz B, Fillet G, Gielen J, Bours V, and Merville M-P (1 9 9 7 ) HSV-1 thymidine kinase gene therapy for colorectal adenocarcinoma-derived peritoneal carcinomatosis. Gene Ther 4, 1189-1194. Reproduced with the kind permission of the authors (Vincent Bours, University of Liège, Belgium) and of Stockton Press.

and can become a powerful suicide gene under these conditions (Sorscher et al, 1994). The significant advantages in eradicating experimentally-induced human tumors in nude mice with this system were: (i ) the bystander effect was 2-3 orders of magnitude higher than with HSV-tk/GCV and tumor eradication could be seen only after 3 doses of PNP/MePdR treatment, (i i ) the MeP-dR and F-araAMP crossed readily the cell membrane unlike GCV, and (i i i ) PNP/MeP-dR could kill both proliferating and nonproliferating tumor cells as has been demonstrated by eradication of the slowly-growing D54MG glioma tumors expressing the bacterial PNP gene in nude mice after treatment with MeP-dR (Figure 26; Parker et al, 1997).

Infection of the human breast cancer cell line, MDAMB-231, with a recombinant adenovirus expressing the Escherichia coli CD resulted in high levels of cytosine deaminase enzyme activity and infected cells became 1000fold more sensitive to 5-FC than cells infected with a control adenovirus; only 10% of infected cells in a population were needed to induce complete cytotoxicity of noninfectious cells exposed to 5-FC via bystander effects. Direct injection of the CD-adenovirus into human breast tumor xenografts in nude mice, followed by daily intraperitoneal injection of 5-FC was sufficient to inhibit tumor growth (Li et al, 1997).

F. Bacterial purine nucleoside phosphorylase (PNP) gene

G. Deoxycytidine kinase/ara-C and nitroreductase/5-(aziridin-1-yl)-2,4dinitrobenzamide

Another suicide gene/prodrug couple is the E. coli DeoD gene which encodes the purine nucleoside phosphorylase (PNP). The E. coli PNP, unlike the mammalian endogenous PNP, can utilize certain adenosine analogs as substrates including nontoxic purine nucleosides converting them to very toxic adenine analogs; these substrates include 6-methylpurine-2’-deoxyriboside (MeP-dR) and arabinofuranosyl-2-fluoroadenine monophosphate (F-araAMP) commercially known as fludarabine. This enzyme converts the 6-methylpurine deoxyribose (MeP-dR) prodrug into the diffusible, toxic 6-methylpurine

The human deoxycytidine kinase (dCK) can phosphorylate the prodrug cytosine arabinoside (ara-C), a cytidine analog, and catalyze its conversion into a toxic drug inducing lethal strand breaks in DNA. Although ara-C is a potent antitumor agent for hematologic malignancies it is ineffective against solid tumors; transduction of the dCK cDNA with adenovirus and retrovirus into the 9L gliosarcoma cell line followed by establishing intradermal and intracerebral gliomas in syngeneic rats demonstrated 72

Gene Therapy and Molecular Biology Vol 1, page 73 the efficacy of systemic ara-C treatment of the animals in eradicating these tumors (Manome et al, 1996). The prodrug 5-(aziridin-1-yl)-2,4-dinitrobenzamide (CB1954) is a weak, monofunctional alkylating agent which can be activated by Escherichia coli nitroreductase to a potent dysfunctional alkylating agent which crosslinks DNA. Transduction of colorectal and pancreatic cancer cell lines with the nitroreductase gene using a retroviral vector rendered them 50 to 500-fold more sensitive than parental cells to CB1954; concentrations of CB1954 which were minimally toxic to nontransduced cells achieved 100% cell death in a 50:50 mix of parental cells with transduced cells expressing nitroreductase due to "bystander" cell killing (Green et al, 1997).

H. Preferential expression of suicidal genes in cancer cells using promoters/ enhancers from tumor-specific genes The principle of VDEPT (virus-directed enzyme /prodrug therapy) was used to target hepatocellular carcinoma using the regulatory region from the tumorspecific "-fetoprotein gene to drive the Varicella zoster thymidine kinases gene (Huber et al, 1991). A similar gene therapy approach has been developed for the treatment of primary and metastatic hepatic tumors based on the overexpression of the suicidal gene cytosine deaminase (CD) from E. coli under control of the regulatory regions of the tumor marker gene carcinoembryonic antigen (CEA) (Richards et al, 1995); this created a chimeric gene that was specifically expressed in neoplastic cells. Development of this strategy has necessitated the identification of the regulatory regions of

F i g u r e 2 6 . A nude mouse xenograft model was developed bearing malignant gliomas by s.c. injection of D54MG human cells or D54MG human cells transduced and expressing E. coli PNP which are called D54-PNP cells; tumors were successfully eradicated with MeP-dR treatment. Representative animals from each of 4 groups at completion of the study (62 days) are shown: Group 1: nude mice were injected with D54MG cells, vehicle treated. Group 2: nude mice were injected with D54MG cells, MePdR treated. Group 3: nude mice were injected with D54-PNP cells, vehicle treated. Group 4: nude mice were injected with D54PNP cells, MeP-dR treated. From Parker WB, King SA, Allan PW, Bennett LLJr, Secrist JAIII, Montgomery JA, Gilbert KS, Waud WR, Wells AH, Gillespie GY, and Sorscher EJ (1 9 9 7 ) In vivo gene therapy of cancer with E. coli purine nucleoside phosphorylase. Hum Gene Ther 8, 1637-1644. With the kind permission from the corresponding author (Eric Sorscher, University of Alabama at Birmingham) and Mary Ann Liebert, Inc.

Gene Therapy and Molecular Biology Vol 1, page 74 cytidine deaminase catalytic subunit that creates a new termination codon and produces a truncated version of apoB (apo-B48); the cytidine deaminase catalytic subunit (apoB mRNA-editing enzyme catalytic polypeptide 1) of the multiprotein editing complex has been identified (Yamanaka et al, 1995).

the CEA gene; isolation of 14.5 kb of 5' flanking sequences for this gene followed by subcloning into luciferase pGL2 basic vectors and testing for luciferase activity in transfected LoVo, SW1463, Hep3B, and HuH7 cell lines (the first two express CEA whereas the other two do not) has identified the CEA promoter between bases -90 and +69, and two enhancers one at -13.6 to -10.7 and the other at -6.1 to -4.0 kb (Richards et al, 1995); these sequences were able to sustain high levels of expression of the CD gene into CEA-expressing cell lines. Regulatory sequences from the CEA gene (-322 to +111 bp) were also used to express the HSV thymidine kinase gene in pancreatic and lung neoplasms (Dimaio et al, 1994; Osaki et al, 1994).

B. Mechanism of MDR1 resistance A great deal of our knowledge of basic insights on drug uptake and molecular mechanisms of drug action were elucidated from the study of resistance of tumor cells to chemotherapeutic agents. The P-glycoprotein or p170 encoded by the multidrug resistanceMDR1 gene uses the energy of ATP to extrude a variety of drugs apparently unrelated; the only chemical similarity is that they contain condensed aromatic rings and have a positive charge at neutral pH; these drugs, most of which are effective against a variety of human tumors, include molecules found in nature such as colchicine, doxorubicin (also called adriamycin, member of the anthracycline family), actinomycin D, vinblastine, etoposide, taxol, vinca alcaloids, and epipodophyllotoxins collectively called MDR-type of drugs (reviewed by Gottesman and Pastan, 1988; see Lee et al, 1998 this volume). Cell lines resistant to drugs accumulate far less amounts of drug compared with parental cells because of overexpression of the MDR1 gene; development of multidrug resistance by tumor cells poses a major impediment to successful cancer chemotherapy. A number of cell lines with multidrug resistance have been derived like KB and K562 cells (Marie et al, 1991; Fardel et al, 1995). The P-glycoprotein is a 1280 amino acid molecule in human cells (Chen et al, 1986) or 1276 amino acid molecule in mouse cells with 80% sequence similarity to the human protein (Gros et al, 1986). P-glycoprotein has 12 hydrophobic domains grouped into pairs representing transmembrane domains. The molecule has a 500 amino acid duplication; each duplicated segment possesses an ATP-binding site on the cytoplasmic side; it also has several site of glycosylation near the N-terminus to the exterior side. Its gene is amplified in multidrug resistant

XXIV. Transfer of drug resistance genes A. Principles and genes used An attractive approach to circumvent chemotherapyinduced myelosuppression is the use of gene-transfer technology to introduce new genetic material into hematopoietic cells. Protection of bone marrow progenitor cells by introduction of a drug resistance gene allows larger and curative doses of chemotherapy to be administered to the patient as was shown in several pre-clinical studies. Drug resistance genes under experimental consideration are shown on Table 4. Clinical trials are now under way to evaluate the potential use of two gene sequences: MDR1 (protocols #43, 44, 59, 89, and 100) and O6methylguanine DNA methyltransferase (#101 see Appendix 1) (see also Lee et al, 1998, this volume). Dose-limiting hematopoietic toxicity produced by the cytosine nucleoside analogue cytosine arabinoside (Ara-C) is one of the major factors that limit its use in the treatment of neoplastic diseases. Deamination of Ara-C by cytidine deaminase results in a loss of its antineoplastic activity. Transfer of human cytidine deaminase into murine fibroblast and hematopoietic cells conferred drug resistance to Ara-C protecting them from drug toxicity (Momparler et al, 1996). It is worth mentioning that apolipoprotein B mRNA editing involves the deamination of cytidine by the Table 4. Drug resistance gene designs Dr ug r e si s t an c e g e n e

Confers resistance to

MDR1 (multidrug resistance)

Daunomycin, doxorubicin, taxol

Galski et al, 1989; Podda et al, 1992; Sorrentino et al, 1992 (see below)

Mutant dihydrofolate reductase

Methotrexate (MTX)

Williams et al, 1987; Corey et al, 1990; Li et al, 1994; Zhao et al, 1997

Glutathione transferase

DNA alkylating agents

reviewed by Maze et al, 1997

O6 -methyl guanine transferase

Nitrosoureas

Allay et al, 1995

Cytidine deaminase

Cytosine arabinoside (Ara-C)

Momparler et al, 1996

Aldehyde dehydrogenase

Cyclophosphamide

reviewed by Koc et al, 1996

Reference

Gene Therapy and Molecular Biology Vol 1, page 75 marrow stem cells during treatment of cancer patients with antineoplastic drugs for killing tumor cells. Transfer of the MDR1 cDNA into primary human hematopoietic progenitor cells of cancer patients undergoing high-dose chemotherapy will protect the bone marrow from the doselimiting cytotoxicity of cytostatic agents. Transgenic mice expressing the human MDR cDNA in their bone marrow cells were resistant to doxorubicin (Galski et al, 1989; Mickisch et al, 1991). Retroviral transfer of MDR1 resulted in high level expression of both RNA and P-glycoprotein; taxol-treatment of mouse bone marrow cells killed those that had not been transfected and resulted in an enrichment of the cells containing the human gene (Sorrentino et al, 1992; Podda et al, 1992). Transfer of the MDR1 gene via a retrovirus into human CD34+ cells, isolated from bone marrow and stimulated with IL-3, IL-6, and stem cell factor, showed that 20-70% of the CFU-GM or BFU-E cells contained the transferred MDR1 gene by PCR analysis (Ward et al, 1994). AAV and cationic liposomes have been used for the transfer of the human MDR1 cDNA to NIH-3T3 cells followed by selection of successfully transfected cells based on the drug-resistant phenotype conferred by the Pglycoprotein efflux pump; a single intravenous injection of the bicistronic vector complexed to cationic liposomes into recipient mice, achieved delivery of MDR1 and human glucocerebrosidase cDNAs in all the organs tested (Baudard et al, 1996). Eckert et al (1996) have designed novel retroviral vectors termed SF-MDR and MP-MDR which significantly elevated survival of transduced primary human hematopoietic progenitor cells under moderate doses of colchicine and paclitaxel in vitro when compared with a conventional MoMuLV-based vector; the novel vectors were based on the spleen focus-forming virus or the myeloproliferative sarcoma virus for the enhancer DNA sequence and the murine embryonic stem cell virus for the leader. A bicistronic retroviral vector (HaMID) containing a modified human MDR-1 cDNA and a mutant human dihydrofolate reductase cDNA bearing a leucine to tyrosine substitution at codon 22 was constructed and used to transduce the human CEM T lymphoblastic cell line as well as primary murine myeloid progenitors; HaMIDtransduced cells were highly resistant in the presence of 25 nM taxol and 100 nM trimetrexate simultaneously while control cells were entirely growth inhibited (Figures 27, 28; Galipeau et al, 1997). Several human clinical trials, approved by RAC and FDA, are under way with the long-term goal of transferring the MDR1 gene into bone marrow cells of advanced cancer patients using retroviral infection. A human gene therapy protocol (#100) for chemoprotection of patients treated for testicular cancer with high doses of carboplatin and etoposide proposes to use transplantation of these patients with autologous peripheral blood stem cells (drawn, purified and cryopreserved prior to chemotherapy treatment) and transduced with the MDR1

cell lines accompanied by an increased expression of the 4,500 to 5,000-nt in size mRNA for P-glycoprotein (Chen et al, 1986). Rates of drug influx for lipid-soluble drugs are proportional to drug concentrations in the medium; Pglycoprotein alone or in conjunction with other cellular components seems to transport drugs to the exterior of the cell, a mechanism pronounced in drug-resistant cell lines. Consistent with the presence of a membrane-bound, exchangeable pool of drug and a cytoplasmic, non exchangeable pool, P-glycoprotein was proposed to directly interact via its hydrophobic transmembrane domains with the membrane-associated drug molecules (anthracyclins, vinca alcaloids) to mediate their efflux to the extracellular milieu (Gros et al, 1986). Doxorubicin, an inhibitor of topoisomerase II which is a major nuclear matrix component, has been shown to interact with hydrophobic regions in calmodulin; calmodulin is also a nuclear matrix protein. Photoaffinity-labeled analogs of vinblastine showed direct binding of this drug to Pglycoprotein (Safa et al, 1986). Expression of P-glycoprotein is consistently low in bone marrow cells rendering them particularly sensitive to certain MDR-type of anticancer drugs; chemotherapy with these drugs largely depletes or wipes off bone marrow pluripotent stem cells from patients (myelosuppression). One approach to this problem has been removal and deepfreezing of bone marrow samples from cancer patients prior to chemotherapy; in a second phase CD34+ cells are isolated from the frozen bone marrow specimen using negative selection on soybean agglutinin plates followed by a positive selection on plates coated with anti-CD34 + antibody (Ward et al, 1994) which are then reimplanted to the patient or are simply injected intravenously and find their way to the bone marrow where they implant; this is a costly undertaking. Gene therapy approaches are being aimed at transferring the MDR1 gene under the control of a strong promoter/enhancer into bone marrow stem cells; transfected stem cells, from which all B and T cells are derived, would be rendered resistant to chemotherapeutic drugs used to treat cancer patients and allow administration of higher doses of these drugs. Furthermore, even if a small percentage of cells are successfully transfected, these cells could be expanded by selection with MDR-drug. The same approach could be used to express a nonselectable gene such as the #-globin gene to treat sickle cell anemias and thalassemias inserted in the same construct with the MDR1 gene as has been suggested by Ward and coworkers (1994).

C. Transfer of the MDR1 gene into bone marrow cells The purpose of this approach is to overexpress the MDR1 gene in bone marrow cells in ex vivo or in vivo protocols in order to render stem cells resistant to cancer chemotherapy; this will prevent destruction of the bone 75

Gene Therapy and Molecular Biology Vol 1, page 76

F i g u r e 2 7 . Structure of the retroviral vectors used to deliver the MDR1 and DHFR genes. The vectors are based on the Harvey murine sarcoma virus. A single transcript (arrow) is initiated in the retroviral 5â&#x20AC;&#x2122; LTR promoter. HaMDR1sc (top) contains the MDR1sc cDNA and HaDHFR(L22Y) (middle) contains a mutant DHFR cDNA. The bicistronic (two-gene) vector HaMID (bottom) contains both MDR1 and DHFR genes. From Galipeau J, Benaim E, Spencer HT, Blakley RL, Sorrentino BP (1 9 9 7 ) A bicistronic retroviral vector for protecting hematopoietic cells against antifolates and P-glycoprotein effluxed drugs. Hum Gene Ther 8, 1773-1783. Reproduced with kind permission from the authors and Mary Ann Liebert, Inc. Figure 2 8 . Growth inhibition assays Inc. comparing the effect of 25 nM taxol, 100 nM trimetrexate (TMTX) alone and in combination on CEM cells transduced with HaDHFR(L22Y), HaMDR1sc, or HaMID. Drug-selected CEM cells were washed and seeded at 1x105 cells/ml in 2 ml of media containing the indicated concentrations of drugs. After 72 hr, the percentage of growth was calculated by dividing the number of cells at each drug concentration by the number of cells present in control medium (100% growth). Quadruplicate experiments are shown. aCells preselected in 100 nM trimetrexate. b Cells preselected in 25 nM taxol. From Galipeau J, Benaim E, Spencer HT, Blakley RL, Sorrentino BP (1 9 9 7 ) A bicistronic retroviral vector for protecting hematopoietic cells against antifolates and P-glycoprotein effluxed drugs. Hum Gene Ther 8, 1773-1783. Reproduced with kind permission from the authors and Mary Ann Liebert,

Gene Therapy and Molecular Biology Vol 1, page 77

metastasis. An antisense IGF-IR construct, under control of the ZnSO 4-inducible metallothionein-1 promoter, was engineered by reverse transcription-PCR on total RNA with primers specific for the 0.7 kb cDNA of IGF-IR and subcloned into episomal vectors in the antisense orientation. Transfection of the construct into prostate cancer PA-III cells in culture was able to reduce dramatically the expression of IGF-IR after induction of the cells with ZnSO4 (Burfeind et al, 1996). This inhibition resulted in reduction in expression of both uPA and tPA; whereas PA-III cells were able to induce large tumors in nude mice, PA-III cells transfected with the antisense vector either developed tumors 90% smaller or remained tumor -free for long times postinjection (Burfeind et al, 1996). Lafarge-Frayssinet et al (1997) have developed a strategy for inducing a protective immunity by tumor cells transfected by the IGF-I antisense vector: the hepatocarcinoma cell line LFCI2-A, expressing both IGF I and II, produces voluminous tumors when injected subcutaneously into syngeneic rats; when LFCI2-A cells were transfected with an episomal vector expressing IGF-I antisense RNA, the cells became poorly tumorigenic exhibited a 4-fold increase of the MHC class I antigen, and, when injected subcutaneously, inhibited the growth of the parental tumoral cells or induced regression of established tumors; this loss of tumorigenicity and protective immunity was not observed after transfection with the IGF-II antisense vector (Lafarge-Frayssinet et al, 1997). Cationic lipid-mediated transfer of antisense cDNA for IGF I is in clinical trial for glioblastomas (protocol #189 in Table 4 in Martin and Boulikas, 1998, this volume, page 203).

cDNA. Similar protocols (#43, 44, 59, 89) use CD34+ autologous bone marrow cells retrovirally-transduced with MDR1 cDNA for hemoprotection of patients treated for ovarian, brain, or breast cancers (Appendix 1).

XXV. Antisense gene therapy of cancer. Among a variety of approaches to gene therapy of cancer, antisense oncogene gene therapy is a strategy aiming at correcting genetic disorders of cancer through correction of the abnormal expression of oncogenes implicated in signal transduction and control of proliferation. A number of protocols have been approved using antisense gene or oligonucleotide delivery. Protocol 29 uses a combination of p53 cDNA and K-ras antisense for non-small cell lung cancer. Protocol 41 uses antisense Rev for AIDS, protocol 91 antisense RRE decoy gene and protocol 168 uses antisense TAR and transdominant Rev protein genes for HIV infections. Protocol 64 uses antisense c-fos or antisense c-myc for breast cancer. Protocol 82 uses intraprostate injection of antisense c-myc for advanced prostate cancer. Protocol 162 uses TGF-Ă&#x;2 antisense genemodified autologous tumor cells for malignant glioma. And, protocol 189 uses antisense Insulin-like Growth Factor I for glioblastoma (see below).

A. Antisense c-fos and c-myc Because c-fos proto-oncogene has been implicated as a regulator of estrogen-mediated cell proliferation, antisense c-fos has been used to cause an inhibition of s.c. tumor growth and invasiveness of cells the growth of which depends on estrogen. Ex vivo transduction of MCF-7 human breast cancer cells with antisense c-fos, regulated by mouse mammary tumor virus control elements and delivered by a retroviral vector, produced expression of anti-fos RNA, decreased expression of the c-fos target mRNA, induced differentiation, and inhibited s.c. tumor growth and invasiveness in breast cancer xenografts in nude mice; a single injection of anti-fos inhibited i.p. MCF-7 tumor growth in athymic mice with a corresponding inhibition of c-fos and TGF-#1 (Arteaga and Holt, 1996). A phase I clinical study for the treatment of metastatic breast cancer uses in vivo infection with breasttargeted retroviral vectors expressing antisense c-fos or antisense c-myc RNA (Holt et al, 1996; protocol #64, Appendix 1, page 163).

C. Antisense ras gene transfer for pancreatic tumors K-ras point mutations occurs at a characteristically high incidence in human pancreatic cancers. Stable expression of a plasmid expressing antisense K-ras RNA into pancreatic cancer cells with K-ras point mutations (AsPC-1 and MIAPaCa-2) resulted in a significant suppression of cell growth; the effect of antisense treatment was not found in cells with a wild-type K-ras gene (BxPC-3). When the AsPC-1 cells with the K-ras point mutation were inoculated into the intraperitoneal cavity of nude mice, followed 3 days later by i.p. treatment with the antisense K-ras in a liposome complex, only 2 of 12 mice showed any evidence of tumors on day 28 compared with 9 out of 10 control mice that developed peritoneal dissemination and/or solid tumors on the pancreas (Aoki et al, 1995).

B. Antisense insulin-like growth factors I and II and their receptors Insulin-like growth factors I and II (IGF-I and -II) are expressed preferentially in bone tissue and contribute to bone metastases of cancer cells expressing IGF receptors. Prostate cancer cells express IGF-I receptor; this favors metastasis to bone, the most frequent tissue for prostate

D. Antisense oligonucleotides to metallothionein 77

Boulikas: An overview on gene therapy plasmid encompassing the human MT-IIA cDNA, constitutively driven by #-actin promoter and this was associated with a 2-fold increase in cell multiplication (Abdel-Mageed and Agrawal, 1997)

Abdel-Mageed and Agrawal (1997) have inhibited the expression of metallothionein (MT) gene using an 18-mer MT antisense phosphorothioate oligomer (complementary to a region 7 bases downstream from the AUG translational start site of the human MT-IIA gene) to elicit antiproliferative effects in breast carcinoma MCF7 cells; indeed, there is an increased MT gene expression in breast cancer which is associated with metastasis and poor prognosis of the disease; overexpression of MT potentiated the growth of MCF7 cells, whereas downregulation of MT elicited antiproliferative effects. Transfection of MCF7 cells with the antisense oligomer inhibited cell growth by 50-60% and induced morphological changes suggestive of apoptotic cell death at 72 hours posttransfection compared to cells transfected with a random 18-mer; the antisense oligomer induced chromatin cleavage into oligonucleosomal fragments, a 2-fold increase in the levels of cfos and p53 transcripts, a 2.5-fold decrease in c-myc transcripts, and a decrease in Bcl-2 protein levels compared to random oligomer-transfected cells. On the contrary, the expression of MT was 2.5-fold elevated after transfection of the cells with an expression

E. Other antisense approaches Transfer of an antisense cyclin G1 construct was used to inhibit osteosarcoma tumor growth in nude mice. Overexpression of the cyclin G1 gene is frequently observed in human osteosarcoma cells, and its continued expression is essential for their survival. This modality resulted in a decrease in the number of cells in S and G2/M phases of the cell cycle concomitant with an accumulation of cells in the G1 phase (Chen et al, 1997). Figure 29 shows that nude mice treated with the antisense cyclin G vector (panel A) have smaller tumors that animal treated with a control vector (panel B). The results of the measurements of the size of the tumor in treated and control animals are shown in C.

Fi g u r e 2 9 . Photographs of nude mice treated with antisense cyclin G vector (panel A) have smaller tumors that animal treated with a control vector (p a n e l B ). Panel C: the relative tumor size (% of day 0 tumor size divided by 100) is plotted, on the vertical axis, as a function of time (days), plotted on the horizontal axis. From Chen DS, Zhu NL, Hung G, Skotzko MJ, Hinton DR, Tolo V, Hall FL, Anderson WF, Gordon EM (1 9 9 7 ) Retroviral vector-mediated transfer of an antisense cyclin G1 construct inhibits osteosarcoma tumor growth in nude mice. Hum Gene Ther 8, 1667-1674. Reproduced with kind permission from the authors and Mary Ann Liebert, Inc.

Gene Therapy and Molecular Biology Vol 1, page 79 Formation of triple helical DNA was found to take place on AT- and GC-rich stretches. A pyrimidine third strand oligonucleotide, studied by NMR and other approaches, interacts with purine residues in the major groove of the target duplex in a parallel orientation (Moser and Dervan, 1987; Rajagapol and Feigon, 1989; de los Santos et al, 1989) whereas a purine oligonucleotide binds in an antiparallel orientation relative to the purine strand in the duplex (Cooney et al, 1988; Kohwi and KowhiShigematsu, 1988; Beal and Dervan, 1991). In this case G can recognize GC pairs and A or T can recognize AT pairs. + Specificity is provided from T. AT and C GC base triplets where the bases of the third polypyrimidine strand establish Hoogsteen base pairing with the purine strand of the duplex (Hoogsteen, 1959; Rajagopal and Feigor, 1989). The H form is the structural basis for S1-nuclease hypersensitivity (Mirkin et al, 1987). A restriction fragment from a human U1 gene containing the sequence d(C-T)18.d(A-G)18 under supercoiling and pH less than or equal to 6.0 showed S1 hyperreactivity in the center and at one end of the (C-T)n tract, and continuously from the center to the same end of the (A-G)n tract providing strong support for a triple-helical model (Johnston, 1988). Homopyrimidine oligodeoxyribonucleotides with EDTA-Fe attached at a single position bound the corresponding homopyrimidine-homopurine tracts within large double-stranded DNA by triple helix formation and cleaved at that site (Moser and Dervan, 1987). Studies from the group of Claude HĂŠlĂ¨ne have similarly focused on the development of artificial scissor oligonucleotides based on triplex technology (Praseuth et al, 1988; Perrouault et al, 1990). However, the feasibility of employing this exciting in vitro technology to animal studies has not yet been demonstrated. Intramolecular or intermolecular triple helices could be recognized by specific proteins that stabilize triplex structures and might play a role in gene regulation; a protein from HeLa cell nuclear extracts was identified that binds to a 55 nucleotide-long DNA oligomer that could fold on itself to form an intramolecular triple helix of the Py Pu x Py motif (Guieysse et al, 1997). Triplex-forming oligophosphoramidates containing thymines and cytosines or 5-methyl cytosines (5' T4CT4C 6T 3') bind strongly to a 16 base pair oligopurine.oligopyrimidine sequence of HIV proviral DNA even at neutral pH and are remarkably stable compared to oligonucleotides with natural phosphodiester linkages. The phosphoramidate oligomers induced an efficient arrest of both bacteriophage and eukaryotic transcriptional machineries (SP6, T7 or Pol II) under conditions where the phosphodiester oligos had no inhibitory effect and blocked the RNA polymerases at the triplex site (Giovannangeli et al, 1996). Oligonucleotide-directed triplex formation has been shown to inhibit binding of transcription factors to their cognate DNA sequences. A 21 bp homopurine element

The replication and expression of hepatitis B virus (HBV) could be inhibited through antisense gene transfer and this could become a new method for clinical gene therapy against HBV; infection of the human hepatoblastoma cell line 2.2.15, which expresses HBV surface antigen and releases HBV particles, with retroviral vectors carrying an antisense preS/S or preC/C genes of HBV inhibited expression of the surface antigen (Ji and St 1997). Phosphorothioate antisense oligos directed against cmyc and p53 in different cell lines (CAOV-3, SKOV-3, and BG-1) were shown to have both antiproliferative and stimulatory activity, as single agents and in combination; it was concluded that further in vitro studies are needed before considering clinical trials with these agents in gynecologic cancers (Janicek et al, 1995). Transfection of antisense cDNA constructs encompassing different regions of the c-erbB-2 gene in the lung carcinoma cell line Calu3, which overexpresses the c-erbB2 oncogene, reduced significantly anchorage-independent growth and tumor size in nude mice (Casalini et al, 1997). Antisense oligonucleotides against PCNA and cdc2 kinase transferred into injured arterial walls by proteinliposomes greatly reduced mRNA levels for those genes and inhibited neointima formation of the injured artery for 8 weeks; double-stranded oligonucleotides containing the consensus sequence for E2F binding sites also inhibited the growth of smooth muscle cells and prevented neointima formation (Kaneda et al, 1997). Antisense oligonucleotides to angiotensinogen1-receptor mRNA and to angiotensinogen mRNA reduced blood pressure (Tomita et al, 1995; Phillips, 1997; Phillips et al, 1997).

XXVI. Triplex gene therapy A. Molecular mechanisms for triplex formation Natural purine.pyrimidine sequences in regulatory regions of genes in eukaryotic cells with a mirror symmetry can form triple-helical structures; in addition, purine-rich segments in DNA unable to form triple helices on their own can be targeted by DNA or RNA oligonucleotides able to form triplex structures with their target DNA and these unusual structures can inhibit transcription factor binding, transcription initiation, and nuclear enzymatic activities. Understanding the advantages, limitations and pitfalls for using oligonucleotides as gene bullets, development of strategies for boosting their therapeutic efficiency, their covalent linkage to DNA damaging molecules to hit a specific genomic DNA sequence, and improvements to the methods for their delivery to cells could make reality their use as tools of micro-targeting specific genomic sites and as pharmacogenomic drugs.

Boulikas: An overview on gene therapy insert flanking a single Sp1 site in the adenovirus E4 promoter was used to study the effect of oligo targeting on transcriptional efficiency in vitro; assembly of the triple helical complex repressed basal transcription by rendering the triplex target inflexible and by blocking assembly of the promoter into initiation complexes; Sp1 was unable to cause derepression (Maher et al, 1992). Thus DNA triplexes can inhibit transcription initiation not only when directed to a TF binding site occluding its binding but also to a flanking region by other possible repression mechanisms including stiffening of the double helix (Maher et al, 1992).

door to the clinical application of bone marrow gene therapy to humans (Van Beusechem and Valerio, 1996). Retroviral vectors pseudotyped with vesicular stomatitis G glycoprotein (VSV-G) and expressing a murine cell surface protein, B7-1, were used to infect the human T-cell line Jurkat and human peripheral blood lymphocytes (PBLs); the transduction efficiency of PBLs with the pseudotyped vector reached a maximum of 1632% at an moi of 40 (Sharma et al, 1996). Introduction of a mutant H-ras gene (along with a neomycin resistance gene) into normal human bone marrow progenitor cells with a retrovirus followed by selection in cell culture with G418 suggested that expression of mutant H12-ras resulted in enhanced proliferation of early myeloid cells at the expense of differentiation (Maher et al, 1994). Dendritic cells (DCs) which are the most potent antigen-presenting cells (APCs) for the initiation of antigen-specific T-cell activation can be highly enriched from peripheral blood-adherent leukocytes by short-term culture in the presence of IL-4 and GM-CSF; adenoviral vectors expressing luciferase, #-galactosidase, IL-2, and IL7 readily transduced human DCs compared to other methods (Arthur et al, 1997). Transduction of hematopoietic stem cells with human IL-1Ra cDNA was used to alleviate symptoms of RA; the HSCs were subsequently injected into lethally irradiated mice; all of the mice survived and over 98% of the white blood cells in these mice produced biologically active human IL-1Ra type from 2-13 months after transplantation; the animals had the human IL-1Ra protein in their sera for at least 15 months (Boggs et al, 1995).

B. Triplex targeting of IGF-I Oligonucleotide-directed triple helix formation targeted toward IGF-I to inhibit its expression was studied following stable transfection of C6 rat glioblastoma cells with a plasmid from which an RNA was transcribed that coded for the third strand of a potential triple helix. A plasmid encoding the oligopurine variant of the triple helix but not the oligopyrimidine or a control sequence caused a dramatic reduction of IGF-I RNA and protein levels in cultured cells, morphological changes, and increased expression of protease nexin I and MHC class I molecules; the transfected cells displayed a reduced capacity for tumor growth when injected in nude mice (Shevelev et al, 1997).

XXVII. Gene transfer to some characteristic tissues or cell types A. Transduction of hematopoietic stem cells (HSCs)

B. Gene transfer to the brain

Hematopoietic stem cells (HSCs), which can be isolated with high speed flow-cytometric cell sorting from fetal or adult bone marrow and cytokine-mobilized peripheral blood, have extensive self renewal and multilineage repopulating potential; HSCs are being used as an hematopoietic graft to treat cancer patients undergoing high dose chemotherapy which eradicates HSCs; GM-CSF treatment of the patient can enhance mobilization of true HSCs; furthermore, HSCs can be stably transduced at high efficiency (32-75%) by co-culture with a cell line producing recombinant retroviruses containing the neomycin-resistant gene and are targets for hematopoietic cell-based gene therapy especially for the treatment of patients with multiple myeloma (Chen et al, 1995). The efficiency of gene transfer into monkey pluripotent hematopoietic stem cells (PHSCs) is at least one order of magnitude lower than what has been achieved in mice because primate PHSCs seem to require quite different culture conditions for their maintenance and transduction than mouse PHSCs. Successful retroviral vector-mediated gene transfer into monkey PHSCs supported maintenance of the long-term repopulating ability of autologous monkey grafts and has closed the gap between gene transfer experiments in mouse models and primates opening the

Several molecular approaches, including gene transfer with retroviral, adenoviral and herpes simplex virus vectors, as well as antisense vectors, and antisense oligonucleotides have been shown to have in vitro and in vivo activities against brain tumor cells. These approaches are especially important for the treatment of glioblastomas which remain incurable despite an aggressive combination regimens using surgery, radiation, and chemotherapy (reviewed by Yung, 1994). Intrathecal transplantation of polymer-encapsulated cell lines genetically engineered to release the human ciliary neurotrophic factor (CNTF) provided a means to deliver CNTF continuously behind the blood-brain barrier and bypass the immunologic rejection of allogeneic cells; for example, transduction of mouse C2C12 myoblasts with human CNTF followed by membrane encapsulation and intrathecal implantation in adult rats could partially rescue motor neurons from axotomy-induced cell death (Deglon et al, 1997). Since adult brain cells are nonproliferative, they are refractory to retroviral infection that could deliver the tyrosine hydroxylase gene to the brain to alleviate degeneration at the nigrostriatal pathway in Parkinson disease (PD). Implantation of immortalized fibroblasts, 80

Gene Therapy and Molecular Biology Vol 1, page 81 primary fibroblasts, or myoblasts, stably transfected in culture with the TH gene (Jiao et al, 1993) or direct injection of lipofectin-plasmid DNA complexes containing the TH gene under the influence of the SV40 promoter/enhancer (Cao et al, 1995) reduced behavioral abnormalities in PD animal models. A 7 kb region encompassing the TH promoter was able to confer expression of #-galactosidase in catecholaminergic cell types in the substantia nigra pars compacta compared to other regions of the brain after HSV-1-mediated transfer to adult rat brains (Song et al, 1997).

correct genetic defects; several studies have shown that adenoviral or retroviral vector-mediated gene transfer during the ebryonic or neonatal period results in prolonged gene expression. Gene transfer (or gene disruption) has been extensively studied in preimplantation embryos giving rise to transgenic animals difficient in a specific protein (e.g. Smith et al, 1995; Fong et al, 1995; Shalaby et al, 1995). Gene transfer to the embryo has shown the importance of the promoter, large genomic regulatory regions, cell-cell interactions and gene switch taking place during embryogenesis in maintaining transgene expression in different tissues; results obtained in embryos reflect the in vivo patterns of tissue-specific expression which could be useful to direct efforts in promoter choices for somatic gene transfer to the adult (as is the case for most gene therapy applications). Furthermore these studies provide the foundation of a new era where genetic manipulation of the embryo could permanently correct monogenic genetic disorders such as hemophilias, thalassemias and others. The promoter of the tie gene, which encodes a receptor tyrosine kinase that is expressed in the endothelium of blood vessels, was used to drive the expression of a luciferase reporter gene construct; in cultured cells the luciferase activity was not restricted to endothelial cells. In contrast, in transgenic mice expression of the reporter #galactosidase was restricted to endothelial cells undergoing vasculogenesis and angiogenesis; in adult transgenic mice, tie promoter activity in lung and many vessels of the kidney was as high as in the vessels of the corresponding embryonic tissues, whereas in the heart, brain and liver, tie promoter activity was downregulated and restricted to coronaries, cusps, capillaries, and arteries (Korhonen et al, 1995). A retroviral VEGF expression vector was used to infect quail ebryo and to increase the level of VEGF during critical periods of avian limb bud growth and morphogenesis. Overexpression of VEGF in the limb bud exclusively resulted in hypervascularization as reflected by an increase in vascular density from an augmentation of the VEGF signaling mechanism in a permissive environment; vascular permeability was also dramatically increased leading to local edema (Flamme et al, 1995). An avian leukosis virus (ALV)-based retroviral vector system was used for the efficient delivery of genes into preimplantation mouse embryos; a subset of the integrated proviruses expressed the delivered chloramphenicol acetyltransferase (CAT) reporter gene either from the constitutive viral promoter contained in the long terminal repeat or from the internal nonviral tissue-specific promoter in different sets of experiments. Thus, many of the sites that are accessible to viral DNA insertion in preimplantation embryos were thought to be incompatible with expression in older animals (Federspiel et al, 1996). Baldwin and coworkers (1997) have found that the expression of lacZ gene under control of CMV or RSV promoter transferred to early, postgastrulation mouse embryos gave tissue-specific patterns of expression which depended on the type of promoter used. Embryos were

C. Gene transfer to hepatocytes Hepatocytes are responsible for the production of many therapeutically important proteins such as LDL-R which clears LDL from the serum and the blood clotting Factors VIII and IX which are defective in hemophiliacs. Portal vein, rather than systemic intravenous injection, has been used to transduce preferentially hepatocytes (or liver macrophages, known as Kuppfer cells). For example, the Factor IX gene was delivered to a portal vein cannulated into a splenic vein in animals previously subject to twothird hepatectomy and resulted in the expression of low levels of factor IX for up to about 5 months; 0.3-1% of hepatocytes were found to be transduced (Kay et al, 1993). An adenovirus LDL-R cDNA, infused into the portal vein of rabbits deficient in LDL receptor, resulted in the expression of human LDL-R protein in the majority of hepatocytes that exceeded the levels found in human liver by at least 10-fold (Kozarsky et al, 1994). According to an ex vivo protocol, cultured hepatocytes from a FH patient were transduced ex vivo with the LDL receptor gene and transplanted by infusion into the portal vein of the patient (Wilson et al, 1992; Grossman et al, 1994). Delivery of a 5.6 kb genomic clone or of a 834-bp cDNA clone encoding the kallikrein gene into the portal vein or tail vein of spontaneously hypertensive rats resulted in significant reduction of their blood pressure for about 5-6 weeks (Chao et al, 1996). Portal vein injection of the human kallistatin cDNA in an adenoviral vector into spontaneously hypertensive rats resulted in a significant reduction of blood pressure for 4 weeks; this method resulted in the transduction not only of liver but also of spleen, kidney, aorta, and lung (Chen et al, 1997). Hepatocyte Growth Factor (HGF) is the most potent mitogen of mature hepatocytes; transfer of the human HGF gene into a recombinant retroviral cell line produced HGF in the supernatant which was correctly processed and biologically active; primary mouse and human hepatocytes could be transduced with the supernatant of transfected cells and, thus, this cell line should be useful for in vivo liver gene therapy (Pages et al, 1996a).

D. Gene transfer to the embryo Introduction of normal genes in utero or in the early postnatal period could become a successful approach to 81

Boulikas: An overview on gene therapy injected into the mesoderm of the neural fold (A in Figure 30) and #-galactosidase activity was detected in

the head

F i g u r e 3 0 .(A) E7.25 (1-2 somite) mouse embryos were injected via a micropipette inserted directly into the mesoderm of the neural fold (n f ). Thus, all head fold mesoderm and neural epithelium were directly exposed to the recombinant adenovirus carrying the LacZ reporter gene (a i p , anterior intestinal portal; y s , yolk sac; e p c , ectoplacental cone). (B ) Following 36 h in culture, #-galactosidase activity was detected in the head process and pharyngeal arches of the embryo; a smaller amount of #-galactosidase activity was detected within the outflow tract (c t ) of the developing heart (1 and 2 , first and second pharyngeal arches, v , ventricle). (C) Low and (D) high magnification dual immunofluorescent photomicrographs of a sagittal section through the head and heart of an embryo stained with a polyclonal antibody to #-galactosidase (red) detected by a rhodamine-conjugated second antibody as well as a monoclonal antibody to PECAM-1 (CD-31) (gr e e n ) which is specific for endothelial cells and is detected by a fluorescein-conjugated secondary antibody. Despite exposure of all cell types within the head fold of the embryo #-galactosidase activity is restricted to a subpopulation of endothelial cells within the aortic sac (a s ) and first pharyngeal arch artery (pha); (m, myocardium; e , endocardium). From Baldwin HS, Mickanin C, and Buck C (1 9 9 7 ) Adenovirus-mediated gene transfer during initial organogenesis in the mammalian embryo is promoter-dependent and tissue-specific. Gene Ther 4, 1142-1149. Reproduced with the kind permission of the authors (H Scott Baldwin, Childrenâ&#x20AC;&#x2122;s Hospital of Philadelphia) and Stockton Press.

Gene Therapy and Molecular Biology Vol 1, page 83 alleviated the symptoms of arthritis (Bandara et al, 1993). Similarly, ex vivo retroviral transfer of the secreted human IL-1Ra cDNA to primary synoviocytes followed by engraftment in ankle joints of rats with induced arthritis significantly suppressed the severity of the disease (Makarov et al, 1996; see also Ghivizzani et al, 1997). To demonstrate feasibility of the ex vivo FH therapy, three baboons underwent a partial hepatectomy, their hepatocytes were isolated, cultured, transduced with a retrovirus containing the human LDL-R gene, and infused via a catheter (Grossman et al, 1992). An important number of studies on cancer immunotherapy have been performed on animal models (For example, see Vieweg et al, 1994; Wiltrout et al, 1995; Caruso et al, 1996; Bramson et al, 1996; Tahara et al, 1996; Zhang et al, 1996; Rakhmilevich et al, 1997; Aruga et al, 1997; Clary et al, 1997; Ju et al, 1997) Studies with tumor cells reconstituted with RB ex vivo and implanted into immunodeficient mice have demonstrated cancer suppression (see Riley et al, 1996). 2+ 2+ Transfer of the Cu /Zn superoxide dismutase into ex vivo modified cells protected the cells from oxidative damage during manipulation and increased their survival after implantation (Nakao et al, 1995). Ex vivo transfer of the MDR1 gene in bone marrow cells has been used to render stem cells resistant to cancer chemotherapy (Lee et al, 1998, this volume). Ex vivo transduction of MCF-7 human breast cancer cells with antisense c-fos produced expression of antifos RNA, and inhibited s.c. tumor growth and invasiveness in breast cancer xenografts in nude mice (Arteaga and Holt, 1996). Direct in vivo injection of a gene (intratumoral, intravenous, etc) must be distinguished from ex vivo gene therapy methods. Some representative direct in vivo studies to animals using genes are summarized on Table 6.

process (B in Figure 30); sagittal sections through the head and heart of the embryos (c , d) were stained for #galactosidase (red) and for endothelial cells (green); the micrographs show that while not all endothelial cells demonstrated #-galactosidase activity (green only), #galactosidase was restricted to endothelial cell populations (yellow).

DIVISION THREE: GENE THERAPY OF HUMAN DISEASE OTHER THAN CANCER XXVIII. Ex vivo gene therapy A. Ex vivo (and in vivo) gene therapy on animal models A number of experimental approaches for the gene therapy of human disease are first being tested on animal models (preclinical trials) before receiving approval for phase I clinical trials on humans. A number of animal models have been developed such as hemophiliac dogs, rats with high blood pressure, rabbits with coronary heart disease, nude or SCID mice bearing a variety of human cancers, mice with symptoms resembling those of Parkinsonâ&#x20AC;&#x2122;s disease patients etc. Then, gene transfer has been used to treat these animals and alleviate the symptoms. The success of these studies is a prelude for their approval as a gene therapy technology on human patients. Ex vivo techniques although cumbersome are safer, because all genetic manipulations occur outside the body and cells may be extensively screened prior to implantation. According to ex vivo protocols cells from the mammalian body are removed, cultured, transduced with therapeutically important genes, and reimplanted into the body of the same individual. A representative number of such studies on animal models are summarized on Table 5. Some examples will be mentioned here. More information can be found in the specialized sections of this review. Ex vivo gene therapy for PD was performed on animal models with TH deficiency using implantation of immortalized rat fibroblasts releasing L-dopa (Wolff et al, 1989), or using primary fibroblasts (Fisher et al, 1991) and myoblasts (Jiao et al, 1993) stably transfected in culture with the TH gene. Retroviral vectors have successfully treated mucopolysaccharidosis VII by implantation of ex vivo modified mouse skin fibroblasts to mice (Moullier et al, 1993). Surgical implantation of factor VIII gene-transduced primary mouse fibroblasts into the peritoneal cavity in SCID mice resulted in correction of hemophilia A (Dwarki et al, 1995). Ex vivo transduction of primary myoblasts in mice with the factor IX gene followed by transplantation of the transduced cells led to partial correction of hemophilia B (Dai et al, 1992; Yao et al, 1994). Intraarticular injection of syngeneic synovial cells transduced with the IL-1R antagonist protein gene

B. Ex vivo gene therapy on humans The first person to be treated ex vivo was a 4-year-old suffering with ADA deficiency in 1990 (see ADA deficiency below). The US Patent Office has issued in 1995 a patent covering all ex vivo gene therapy to French Anderson, Steven Rosenberg, and Michael Blaese; the technique was developed at NIH in the 1980s and an exclusive license to this work has been awarded to Gene Therapy Inc, (Rockville, Maryland). Of the 220 protocols for Clinical Trials approved by NIH's Recombinant DNA Advisory Committee (RAC), a significant number (over 100) use ex vivo gene therapy applications (see Gavaghan, 1995). Ex vivo protocols are marked In Vitro in Appendicx 1 and Table 4 in following article (pages 203-206). Also protocols proposing immunotherapy use ex vivo transduction of cells from cancer patients with cytokine genes and immunization of the patient with the transduced cells (Appendix 1). Transduction of cells in vitro with adenoviruses makes the patients own cells antigenic leading to their destruction

Boulikas: An overview on gene therapy by T lymphocytes thus eliminating the therapeutic effect after reimplantation (e.g. Yang et al, 1994). It was thought that this antigenicity arises from the adenoviral proteins expressed in transduced cells; however, recent data have demonstrated that antigenicity could also arise from the expression of the therapeutic recombinant protein (see above). Ex vivo approaches have concentrated on correction of mutated genes involved in purine metabolism including adenosine deaminase (ADA) deficiency in severe combined immunodeficiency (SCID) patients, PNP (purine nucleoside phosphorylase) deficiency, and the therapy of Lesh-Nyhan syndrome caused by a deficiency in hypoxanthine-guanine phosphoribosyltransferase (HGPRT). The first human trial to be approved for ex vivo gene therapy was for the treatment of ADA deficiency which began in 1990 (Karlsson, 1991; Ferrari et al, 1991). Ex vivo studies include transfer of factor IX gene in skin fibroblasts from hemophilia B patients in China followed by subcutaneous injection of the cells to the patient (Wilson et al, 1992; reviewed by Anderson, 1992). From 1990-1992, a clinical trial was initiated using retrovirus mediated transfer of the 1.5 kb ADA gene cDNA to T cells from two children with severe combined immunodeficiency following multiple transplantations of ex vivo modified

blood cells; the vector was integrated and the ADA gene was expressed for long periods (Blaese et al, 1995; Bordignon et al, 1995). A clinical protocol for the therapy of amyotrophic lateral sclerosis uses a semipermeable membrane to enclose the ex vivo modified xenogenic BKH cells; the membrane is implanted intrathecally to provide human ciliary neurotrophic factor (Deglon et al, 1996; Pochon et al, 1996). An ex vivo clinical trial on humans, homozygous for mutations in the LDL receptor gene, is performed using cultured hepatocytes from the patient which are transduced ex vivo with the LDL receptor gene and transplanted by infusion into the portal vein of the patient (Wilson et al, 1992; Grossman et al, 1994). Cancer immunotherapy uses transfer of cytokine genes (IL-2, IL-7, IFN-! , GM-CSF) to autologous (cancer patientâ&#x20AC;&#x2122;s) cells followed by immunization of the patient to elicit activation of tumour-specific T lymphocytes capable of rejecting tumour cells from the patient, especially on immunoresponsive malignancies such as melanomas, colorectal carcinomas, and renal cell carcinomas (Uchiyama et al, 1993; Chang et al, 1996; Finke et al, 1997; Das Gupta et al, 1997; Mahvi et al, 1997).

Table 5. Ex vivo studies on animal models Gene target ADA

Human disease SCID (severe combined immunodeficie ncy)

Method

Animal model, objective, and method

Results

Reference

Retrovir us

Immunodeficiient mice were injected with peripheral blood lymphocytes from ADA- patients transduced with a retroviral vector for human ADA bcl-2 expressing LNCaP human prostate cancer cells are rendered highly resistant to apoptotic stimuli Injection of transduced primary myoblasts into the muscle

Restoration of immune functions (presence of human immunoglobulin and antigen-specific T cells)

Ferrari et al, 1991

bcl-2

prostate cancer

LNCaP-bcl-2 cells induced earlier, larger, and hormone-refractory prostate tumors in nude mice Factor IX was being synthesized and delivered to the circulation for over 6 months Human factor IX was detected in the bloodstream of nude mice in small quantities for one week Sustained expression of factor IX for over six months without any apparent adverse effects on the recipient mice; however, the levels of the factor IX protein secreted into the plasma (10 ng/ml for 10 7 injected cells) were not sufficient to be of therapeutic value; 100 times higher amounts of factor IX were needed Therapeutic levels of factor VIII in the blood of the animals for 24 hours

Rafo et al, 1995

Factor IX

hemophilia B

Factor IX

hemophilia B

retr

Transplantation of retrovirustransduced keratinocytes

Factor IX

hemophilia B

retrovir us

Mouse primary myoblasts were infected with retrovirus expressing the canine factor IX under control of mouse muscle creatine kinase and human CMV promoter; myoblasts were injected into the hindlegs of recipient mice; secreted canine factor IX was monitored in the plasma

Factor VIII

Hemophilia A

transfer rin

Factor VIII

Hemophilia A

Retrovir us

Transfection of fibroblasts and myoblasts with B-domain-deleted factor VIII gene followed by implantation into mice Mouse primary fibroblasts infected with a recombinant retrovirus containing factor VIII gene deleted at the B domain

Therapeutic levels of factor VIII in blood of animals for 1 week after surgical implantation into the peritoneal cavity in SCID mice of 15 million cells in the form of neoorgans

Dwarki et al, 1995

Dai et al, 1992 Gerrard et al, 1993, Dai et al, 1992; Yao et al, 1994

Zatloukal et al, 1994

Gene Therapy and Molecular Biology Vol 1, page 85 Growth hormone (human)

none

mice electrop oration

Growth hormone (human, hGH)

general

retr

HSV-tk

glioma

Retr

To directly transfer HSV TK gene and kill transduced proliferating brain tumor cells with ganciclovir without affecting nondividing normal cells

HSV-tk

pancreatic cancer

retrovir us

HSV-tk

proliferative vitreoretinopat hy (PVR)

IL-1 receptor antagonist protein gene

Rheumatoid arthritis (RA)

retrovir ustransduc ed rabbit dermal fibrobla sts Retr

BXPC3 primary human pancreatic adenocarcinoma cells were transduced with retroviral vector carrying the HSV-tk gene driven by the CEA promoter; engrafted subcutaneously into nude mice eliciting pancreatic tumors Traction retinal detachment results from proliferation of retinal pigment cells in the vitreous cavity of the eye; PVR may ensue after retinal surgery or trauma and can be induced in rabbit models by surgical vitrectomy.

IL-1 receptor antagonist (IL-1Ra)

Rheumatoid arthritis (RA)

IL-2; GMCSF

prostate cancer

LDL receptor

Familial hypercholester olemia (FH)

Retr

Nerve Growth Factor (NGF) XPD (ERCC2)

Alzheimer's disease

rat and primate

xeroderma pigmentosum (XP)

Retr

Ex vivo modified C2C12 cells with the hGH gene under control of the inducible UAS promoter and a synthetic hybrid steroid receptor (TAXI), activating transcription from the inducible promoter after treatment with the synthetic nontoxic drug inducer RU486; transplanted in mouse muscle Injection of genetically engineered myoblasts into mouse muscle

Synovial cells were surgically removed from joints of animals with experimental arthritis, cultured, transduced with the IL-1 -receptor antagonist protein gene and reimplanted into the respective donors by intraarticular injection Degradation of cartilage in RA is stimulated by IL-1; to inhibit IL-1; RA synovial fibroblasts transfected with the IL-1Ra gene were coimplanted with normal human cartilage in SCID mice Dunning rat R3327-MatLyLu prostate tumor model (an anplastic androgendependent, nonimmunogenic tumor that metastasizes to the lymph nodes and the lung); cytokine (IL-2)secreting human tumor cell preparations (tumor vaccines) were used for the treatment of advanced human prostate cancer in rats Watanabe heritable hyperlipidemic rabbit (deficient in both alleles of LDL receptor gene); establish hepatocyte culture from animal liver; transduce with LDL receptor gene responsible for LDL internalization into hepatocytes to reduce blood serum cholesterol; transplant hepatocytes into the animal Delivery of NGF by ex vivo-modified allogeneic cells surrounded by a semipermeable membrane and implanted intrathecally To transduce ex vivo human keratinocytes and produce skin grafts on immunodeficient mice; use it on XP patients as reconstructive surgery to alleviate cancers in UV-exposed areas

This model allows up to 100-fold induction of the hGH gene and can be finely tuned to lower levels of induction

Delort and Capecchi, 1996

hGH could be detected in serum for 3 months; myoblasts were fused into preexisting multinucleated myofibers that were vascularized and innervated Murine fibroblasts transduced ex vivo with HSV TK retroviral vectors caused complete regression of gliomas in rat brain after intratumor injection Animals treated with 0.1 mg/Kg ganciclovir exhibited a significant reduction in tumor growth

Dhawan et al, 1991

Significant inhibition of PVR (killing of proliferating cells in the retina) was observed in rabbit PVR models after injection into the vitreous cavity of rabbit dermal fibroblasts transduced in vitro; all eyes received 0.2 mg GCV on the following day and on day 4; Improvement in RA symptoms

Kimura et al, 1996

IL-1Ra expression protected the cartilage from chondrocytemediated degradation.

Otani et al, 1996; Makarov et al, 1996; Muller-Ladner et al, 1997b

All animals with subcutaneously established tumors were cured; the cancer vaccine induced immunological memory that protected the animals from subsequent tumor challenge; GMCSF was less effective than IL-2.

Vieweg et al, 1994

30-40% decrease in serum cholesterol that persisted for 4 months

Chowdhury et al, 1991

Release of NGF by the implant which is not subject to immunologic rejection due to the membrane

Kordower et al, 1994

The retroviral vector carrying the XPD gene and neoR under control of SV40 enhancer fully complemented the DNA repair deficiency of primary skin fibroblasts

GĂśzĂźkara et al, 1994; Carreau et al, 1995

Culver et al, 1992

DiMaio et al, 1994

Bandara et al, 1993

Boulikas: An overview on gene therapy TH ( Tyrosine

Parkinson's disease (PD)

Parkinson disease (PD)

Parkinson's disease (PD)

HSV

Parkinson's disease (PD)

Lipofect in

hydroxylase)

Retrovir us

Rat fibroblasts transduced with tyrosine hydroxylase (TH) produced and released L-dopa to the culture medium;

Overexpress TH that converts tyrosine to L-DOPA to alleviate degeneration of dopaminergic nigrostriatal neurons (DNN) in PD rat models; unilateral destruction of DNN in animals with 6hydroxydopamine and administration of apomorphine caused PD rats to turn contralaterally (7 or more rotations/min). Infection of 6-hydroxydopaminelesioned rats, used as a model of PD, with a defective herpes simplex virus type 1 vector expressing TH To alleviate the symptoms of PD in TH-deficient rats (PD animal models; perform colateral rotations at 15 rounds/min upon administration of apomorphine)

When grafted to the striatum of Fischer rats with a prior 6hydroxydopamine lesion, primary fibroblasts containing the TH transgene survived for 10 weeks, continued to express the transgene, and reduced rotational asymmetry. Implantation of transgenic immortalized fibroblasts and myoblasts intracerebrally improved rotational behavior

Wolff et al, 1989

Conversion of endogenous striatal cells into L-dopa-producing cells

During et al, 1994

Primary muscle cells were transduced with TH cDNA under control of CMV promoter; 10 million cells were injected into brains of TH-deficient rats; this resulted in 75% decrease in the number of rotations/min for more than 6 months

Jiao et al, 1993

Fischer et al, 1991

Table 6. In vivo somatic gene transfer strategies to animal models Gene target or delivered HSV TK and ganciclovir

Human disease hepatocellular carcinoma

Method

Animal model, objective, and method

Results

Reference

AAV mice

To preferentially kill hepatocellular carcinoma cells by the suicadal gene HSV TK (driven by the "-fetal protein (AFP) enhancer and albumin promoter) with ganciclovir

Su et al, 1996

Prostaglandi n G/H synthase

acute lung injury

Cat lipid rabbit

Rabbits intravenously transfected with the PGH synthase gene

Ornithine transcarbam ylase (OTC) "1antitrypsin

OTCdeficiency

Adenovi rus

iv injection of recombinant adenovirus to spf-ash mice (OTC-deficient)

Selective killing of AFP-positive cells in culture; transgenic mice were established by injection of AAV ITRs, neoR, and HSV TK genes as a linear DNA fragment; HSV TK was expressed predominantly in adult liver. Increased plasma levels of prostacyclin and PGE2; protection of lungs in rabbits against endotoxininduced inflammation, pulmonary edema, release of thromboxane B2, and pulmonary hypertension Correction of enzyme deficiency in OTC-deficient mice for over 1 year.

"1-antitrypsindeficiency in lung

Adenovi rus

"1-antitrypsindeficiency in liver

Cat lipid mice

Both in vitro and in vivo infections have shown production and secretion of "1-antitrypsin by the lung cells for over 1 week Small liposomes were much more effective in delivering the "1antitrypsin gene to mouse hepatocytes in vivo.

Rosenfeld et al, 1991

"1antitrypsin

"1antitrypsin (AT, human)

acute and chronic lung diseases.

Cat lipid

The adenovirus major late promoter was linked to a human "1-antitrypsin gene for its transfer to lung epithelia of cotton rat respiratory pathway Protect connective tissue from the lytic action of the leukocyte neutrophil elastase; plasmid was encapsulated into negatively-charged liposomes containing phoshpatidylcholine Aerosol and intravenous transfection to lungs of rabbits

Canonico et al, 1994

CFTR

Cystic fibrosis (CF)

Adenovi rus

Human "1AT mRNA and protein were detected for at least 7 days; immunohistochemical staining showed "1AT protein in the pulmonary endothelium following intravenous administration, in alveolar epithelial cells following aerosol administration, and in the airway epithelium by either route Expression of CFTR after intratracheal instillation into lungs of cotton rats; expression between days 2-10

To alleviate the symptoms of CF

Conary et al 1994

Stratford-Perricaudet et al, 1990

Ali単o et al, 1996

Rosenfeld et al, 1992

Gene Therapy and Molecular Biology Vol 1, page 87 CFTR (cystic fibrosis transmembr ane conductance regulator) CFTR

Cystic fibrosis (CF)

Cat lipid

To express the normal CFTR gene in lungs of Edinburgh insertional mutant mouse (cf/cf) after delivering CFTR cDNA-liposome complexes into the airways by nebulization.

Full restoration of cAMP related chloride responses in some animals; human CFTR cDNA expression in the same tissues

Alton et al, 1993

Cystic fibrosis (CF)

Lipofect in

To express the human CFTR gene in lungs in CFTR-deficient transgenic mice by tracheal instillation of lipofectin-plasmid

Hyde et al, 1993.

CFTR

Lipofect in

CFTR cDNA (human)

Cystic fibrosis (CF)

AAV

Transduction of airway epithelial cells in normal mice by intratracheal instillation of a plasmid carrying the CFTR gene under control of the Rous sarcoma virus promoter Intratracheal instillation into neonatal New Zealand white rabbits

Successful transfer of the CFTR gene to epithelia and to alveoli deep in the lung leading to correction of the ion conductance defects found in the trachea of transgenic mice Airway epithelial cells were the major target and site of expression of CFTR

Rubenstein et al, 1997

p53

lung cancer

retrovir us

Lung tumors were elicited in nu/nu mice after intratracheal inoculation with human lung cancer H226Br cells whose p53 gene has a homozygous mutation at codon 254

Epithelial expression of the human CFTR fusion protein was detected using antisera to both the human CFTR R domain and the aminoterminal epitope at up to 6 weeks after vector inoculation, a time coinciding with the completion of the alveolar phase of lagomorph lung development Intratracheal injection of a recombinant retrovirus containing the wt p53 gene was shown to inhibit the growth of the tumor

Cat lipid:DN A 1:5 mice

To give a complete Class I molecule with heavy and light chains; MHC class I HLA-B7 heavy chain gene has an internal ribosome entry site; the #2microglobulin was driven by RSV promoter

Experiments in mice showed rapid (1 day) destruction of the plasmid in tissues; femtogram amounts only in muscle at 6 months postinjection

Lew et al, 1995

To suppress audiogenic epileptic seizures by cholecystokinin octapeptide (CCK-8) injected intracerebroventricularly (i.c.v.)

AS in rats was markedly reduced between day 3 and 4.

Zhang et al, 1992

Virus-injured rat carotid arteries; hirudin (from medicinal leech) is a potent protein inhibitor of thrombin; thrombin converts fibrinogen to fibrin and also stimulates smooth muscle cell proliferation during neointima formation in the arterial walls To kill preferentially the smooth muscle cells and reduce neointima formation in the arterial wall To inhibit vascular smooth muscle cell (VSMC) proliferation after arterial injury To inhibit vascular smooth muscle cell (VSMC) proliferation after arterial injury; p21 protein functions both by inhibiting cyclin dependent kinases (CDKs) required for the initiation of S phase and by binding to and inhibiting PCNA Transfer of ras transdominant negative mutants to rats in which the common carotid artery was subjected to balloon injury TGF-# accelerates wound healing and inhibits epithelial and smooth cell proliferation; loss of functional TGF-# receptors in cancer cells Mice with induced arthritis

35% reduction in neointima formation in hirudin cDNAtransduced arterial wall cells in vivo

Rade et al, 1996

47% reduction in I/M area ratio following local delivery of HSV-tk and systemic ganciclovir therapy 42% reduction in I/M area ratio

Ohno et al, 1994; Guzman et al, 1994

Over-expression of human p21 inhibited growth factor-stimulated VSMC proliferation and neointima formation in the rat carotid artery model of balloon angioplasty in vitro by arresting VSMCs in the G1 phase of the cell cycle Reduced neointimal formation

Chang et al, 1995b

Inhibition in epithelial and smooth cell proliferation

Grainger et al, 1995

Effective in lowering inflammation of joints with already established arthritis and inhibiting the spreading of the disease to other joints in mice

Chernajovsky et al, 1997

MHC class I HLA-B7 heavy chain gene plus #2microglobuli n CCK (cholecystok inin) hirudin

restenosis; arterial injury

Lipofect in (DOTM A:DOP E) adenovi rus

HSV-tk

arterial injury; restenosis

Adenovi rus

atherosclerosis , restenosis, arterial injury atherosclerosis , restenosis, arterial injury

Adenovi rus

p21

congenital audiogenic epileptic seizures (AS)

ras

arterial injury

TGF-#

arterial injury

TGF-#1

Rheumatoid arthritis (RA)

Adenovi rus

Retr

Yoshimura et al, 1992

Fujiwara et al, 1994

Chang et al, 1995a

Indolfi et al, 1995

Boulikas: An overview on gene therapy Gene target or delivered

Human disease

Method

Goal/rationale

Results

Reference

LDL receptor

Familial hypercholester olemia (FH)

Adeno

Infusion of adenovirus human LDL-R cDNA into the portal vein of rabbits deficient in LDL receptor

Kozarsky et al, 1994

Very low density lipoprotein receptor (VLDL-R)

Familial hypercholester olemia (FH)

Adeno

LDL-R knockout mice

HSV TK+gancicl ovir (GCV)

Gliomas

Retr

HSV TK plus ganciclovir

prostate cancer

Adenovi rus mouse

Subcutaneous tumors induced by injection of RM-1 (mouse prostate cancer) cells followed by injection of HSV TK and treatment with ganciclovir for 6 days showed reduction in tumor volume (16% of control) and higher apoptotic index in tumor cells

Eastham et al, 1996

HSV TK plus ganciclovir TH

adenocarcino ma

Treatment of rats with cerebral glioma; intratumoral stereotaxic injection of murine fibroblasts (G418selected) producing a retroviral vector with the HSV TK gene Ganciclovir is converted by HSV TK into its triphosphate form which is then incorporated into the DNA of replicating mammalian cells leading to inhibition in DNA replication and cell death; it is only viral TK, not the mammalian enzyme, that can use efficiently phosphorylated ganciclovir as a substrate Carcinoembryonic antigen-producing human lung cancer cells

Human LDL receptor protein was produced in the majority of hepatocytes that exceeded the levels found in human liver by at least 10fold A single intravenous injection resulted in reduction in total cholesterol by approximately 50% at days 4 and 9; marked reduction in the intermediate density lipoprotein/low density lipoprotein (IDL/LDL) Tretament with GCV 5 days postinjection resulted in complete regression of gliomas

rat glial cell line-derived neurotrophic factor (rGDNF),

Cat lipos Overexpress TH to alleviate rat degeneration of dopaminergic nigrostriatal neurons (DNN) in PD rat models AAV To protect nigral dopaminergic neurons in the progressive Sauer and Oertel 6-hydroxydopamine (6-OHDA) lesion model of Parkinson's disease (back-labeled fluorogold-positive neurons in the substantia nigra)

Factor IX

Hemophilia B

Adenovi rus mice

Correction of FIX defect by injection of recombinant adenovirus hosting the canine FIX gene into hind leg muscle of mice

Factor IX

Hemophilia B

Correction of the factor IX gene

Factor IX

Hemophilia B

Adenovi rus mice in vivo Retrovir us dogs in vivo

Factor IX

Hemophilia B

Adenovi rus dogs in vivo

Establishment of the blood serum factor IX levels in hemophilic B dogs by infection with recombinant adenovirus vectors delivering the Factor IX gene and treatment with cyclosporin A

Factor IX

Hemophilia B

AAV

Expression of factor IX gene in liver in hemophilia B dogs

Kobayashi et al, 1996; Kozarsky et al, 1996

Culver et al, 1992

Cell type-specific expression of Osaki et al, 1994 herpes simplex virus thymidine kinase gene Direct injection of lipofectin-TH Cao et al, 1995 expression plasmid on nigra-lesioned side generated L-DOPA locally and decreased contralateral rotations. 94% cell protection; 85% of tyrosine Mandel et al, 1997 hydroxylase-positive cells

Establishment of factor IX levels in the blood in nude mice for 300 days; only for 10 days in normal mice; transduced cells were removed by cell-mediated as well as humoral immune responses; cyclophosphamide or cyclosporin A immunosuppression maintained FIX protein for 5 months Therapeutic plasma levels of factor IX in mice

Dai et al, 1995

1% of hepatocytes were transduced with a retroviral vector carrying the canine factor IX gene injected into the portal vein (2/3 hepatectomy was required); low therapeutic levels of factor IX Treatment with cyclosporin A suppressed T cell activation; T cells attack the adenovirus-transduced cells eliminating them from the body; Cycl A tretment led to prolonged Factor IX levels in the blood of hemophilic dogs Successful transduction of the mouse liver in vivo after a single administration; persistent, curative concentrations of functional human factor IX can be achieved

Kay et al, 1993

Smith et al, 1993

Kay et al, 1994; Fang et al, 1995

Snyder et al, 1997

Gene Therapy and Molecular Biology Vol 1, page 89 Gene target or delivered

Human disease

Method

Goal/rationale

Results

Reference

Factor IX

Hemophilia B

AAV

Intramuscular injection into hindlimb muscles of C57BL/6 mice and Rag 1 mice

Herzog et al, 1997

Factor X

Hemophilia B

Retr

Delivery to rat hepatocytes in vivo during liver regeneration; under control of "1-antitrypsin promoter

p53

breast carcinoma MDA-MB-435 cells

DOTM A:DOP E

Nude mice inoculated with breast carcinoma cells (have mutated p53)

Cdc2 kinase and PCNA

Restenosis

liposom eSendai virus

Delivery to rat carotids after balloon injury; inhibition of Cdc2 kinase and PCNA with antisense oligonucleotides using PS:PC:Chol liposomes

vascular endothelial growth factor (VEGF)

restenosis

naked plasmid

VEGF promotes endothelial cell proliferation to accelerate reendothelialization of the artery reducing intimal thickening

Kallikrein

hypertension

naked plasmid

Tissue kallikrein is a serine proteinase cleaving the kininogen to produce the vasoactive kinin peptide; kinin causes smooth muscle contraction and relaxation, increase in vascular permeability, and vasodilatation

Presence of hF.IX protein by immunofluorescence staining of muscles harvested 3 months after injection in both strains of mice; no plasma FIX in immunocompetent mice; Rag 1 mice which lack functional B and T cells, displayed therapeutic levels (200-350 ng/ml) of F. IX in the plasma in addition to F.IX in muscle cells 10% to 43% of normal human factor X levels in 4 rats; expression remained stable for more than 10 months in two rats Iv injection of p53 gene under control of #-actin promoter and intron every 10-12 days resulted in more than 60% reduction in tumor volume Whereas antisense cdc2 kinase or PCNA alone failed to have an effect, combination of the two antisense oligos significantly reduced neointima formation and smooth muscle cell proliferation after balloon injury VEGF gene expression using ELISA or RT-PCR was detected for 3-14 days after a single transfer using a hydrogel/polymer-coated ballon angioplasty catheter to induce simultaneous injury and delivery of plasmid to the femoral artery in rabbits. Significant reduction in blood pressure in spontaneously hypersensitive rats after a single delivery of naked DNA to portal vein which lasted for 5-6 weeks.

Kallikrein

hypertension

Adeno

Tissue kallikreinbinding protein (HKBP) or kallistatin Human endothelial NO synthase (eNOS) gene Antisense oligonucleoti des to AT1receptor mRNA and to angiotensino gen mRNA Endothelial basic FGF (bFGF) Atrial natriuretic peptide (ANP) gene

hypertension

Adeno

Kallistatin, a serine proteinase inhibitor, may function as a vasodilator in vivo

hypertension

Blood pressure is controled by the endothelium-derived nitric oxide (NO) in peripheral vessels

hypertension

Liposom Angiotensinogen, produces angiotensin es I in the liver (component of the reninangiotensin system); mutations in the angiotensinogen (AT) gene are associated with hypertension

hypertension hypertension

Subphysiological amounts in blood vessels of spontaneously hypertensive rats Chronic infusion of ANP causes natriuresis, diuresis, and hypotension

Le et al, 1997

Lesoon-Wood et al, 1995

Morishita et al, 1993

Isner et al, 1996

Chao et al, 1996

Sustained delay in the increase in Jin et al, 1997 blood pressure from day 2 to day 41 post injection (iv) into spontaneously hypertensive rats; human tissue kallikrein mRNA was detected in the liver, kidney, spleen, adrenal gland, and aorta. Delivery of the human kallistatin Chen et al, 1997 cDNA/CMV by portal vein injection resulted in a significant reduction of blood pressure of hypertensive rats for 4 weeks. Significant reduction of systemic blood pressure for 5 to 6 weeks.

Lin et al, 1997

Antisense oligonucleotides delivered to rat liver via the portal vein diminished the expression of hepatic angiotensinogen mRNA and reduced blood pressure.

Tomita et al, 1995; Phillips, 1997; Phillips et al, 1997

Restored the physiological levels levels of bFGF in the vascular wall and corrected hypertension Significant reduction of systemic blood pressure in young hypertensive rats (4 weeks old); the effect continued for 7 weeks.

Cuevas et al, 1996 Lin et al, 1995

Boulikas: An overview on gene therapy IL-2

prostate cancer

liposom es

Direct transfer of the IL-2 gene under control of the CMV promoter with or without the AAV inverted terminal repeats

mouse leptin cDNA

obesity

Adeno

rat leptin cDNA/CMV

obesity

Adeno

ob/ob mouse (which is genetically deficient in leptin and exhibits both an obese and a mild non-insulindependent diabetic phenotype) Wistar rats infused with 8 ng/ml adeno/leptin cDNA for 28 days

VEGF165

cancer (vascularizatio n)

calcium phospha te

Expression of VEGF 165 in rat C6 glioma cells and subcutaneous injection of the transduced cells in athymic mice.

AAV

To treat retinal degeneration due to recessive mutation in the endogenous gene

cGMP retinal phosphodiest degeneration erase-# (PDE-#) gene

Plasmid DNA containing the AAV inverted terminal repeats showed 310 fold higher levels of gene transfer and IL-2 expression compared with constructs lacking the AAV sequences. Dramatic reductions in both food intake and body weight, as well as in normalization of serum insulin levels and glucose tolerance Animals became hyperleptinemic; 30-50% reduction in food intake; gained only 22 g over the experimental period versus 115-132 gained by control animals Tumors from cells expressing VEGF grew slower than tumors developed from nontransduced C6 cells, were highly vascularized, and contained varying degrees of necrosis and eosinophilic infiltrate Intraocular injection of AAV-PDE-# cDNA increased retinal expression of immunoreactive PDE protein; treated eyes showed increased numbers of photoreceptors and a two-fold increase in sensitivity to light

Vieweg et al, 1995

Muzzin et al, 1996

Chen et al, 1996

Saleh, 1996

Jomary et al, 1997

exposures; such individuals have a homozygous defect in the CCR-5 receptor gene which consists of a 32-bp deletion in the region encoding the second extracellular loop of the receptor; the defective protein is not detected at the cell surface; this defect prevents the proper interaction and entry of HIV-1 into their T cells and macrophages (Liu et al, 1996). This defect precludes infection from HIV-1 from all routes. The disease progresses much slower in heterozygotes for the 32-bp deletion (18% in populations of European descent) who are not protected from HIV-1 infections. This finding offers the hope of reconstituting the immune system of HIV-infected individuals with CD34+ stem cells from fetal cord blood or stem cells and lymphoid tissue from individuals who carry the homozygous deletion in the CCR-5 gene, an approach to deal with the immune rejection problems in patients with heart and kidney transplants. The identification of chemokine receptors and their role in HIV-1 infections has closed a major gap in AIDS research; transgenic animals can now be produced expressing both human CD4 and chemokine receptors to evaluate the efficacy of AIDS therapeutics and testing vaccines; new prophylactic or therapeutic vaccines can be designed by immunization with portions or the entirety of CCR-5 and/or gp120 to generate appropriate antibodies (D’Souza and Harden, 1996). Inactivation of the CCR-5 co-receptor to mimic the natural resistance of the CCR-5defective individuals, in cultured lymphocytes, rendered them viable and resistant to macrophage-tropic HIV-1 infection (Yang et at, 1997).

XXIX. Gene therapy of HIV A. Mechanism of HIV-1 entry into T cells and macrophages Targets of human immunodeficiency virus (HIV) are helper T cells and macrophages; macrophage-tropic HIV-1 isolates represent the most prevalent phenotype isolated from individuals shortly after seroconversion during the asymptomatic period of the disease; tropism is determined by specific sequences in the third variable loop (V3 domain) of gp120 coat protein of HIV-1. The CD4 receptor on both macrophages and T cells is the primary receptor mediating HIV-1 entry into the cell; however, HIV-1 was unable to infect CD4+ T cells of mice engineered to express human CD4 (reviewed by D’Souza and Harden, 1996). Thus, a second chemokine receptor was thought to be necessary for HIV entry into immortalized T cell lines. The second receptor for entry of HIV-1 into T cells and macrophages is CCR-5, a #-chemokine receptor; CCR-5 is a seven transmembrane-domain glycoprotein of the chemokine superfamily of receptors related to the receptor of IL-8 (G protein-coupled proteins that transduce signals from the cell surface to the interior of cells). The #chemokines RANTES, MIP-1", and MIP-1# inhibited replication of M-tropic isolates of HIV-1 and were found to be major HIV-suppressive factors produced by CD8+ T cells. The V3 loop of gp120 determines interaction with the chemokine receptor. CD4, in addition to providing a docking surface for the gp120 glycoprotein of HIV-1 promotes exposure of the V3 domain on gp120 that can interact with the chemokine receptor CCR-5 (Scarlatti et al, 1997; reviewed by D’Souza and Harden, 1996). A small number of individuals (1% in populations of European descent but much lower in non-Caucasian populations) remain uninfected despite multiple high risk

B. Therapeutic strategies against HIV A number of therapy strategies emerged soon after identification of HIV as the etiologic agent of AIDS. Virtually every stage in the viral life cycle and every viral gene product is a potential target. Albeit major efforts for 90

Gene Therapy and Molecular Biology Vol 1, page 91 the combat of HIV have focused on the development of antiviral drugs and preventive vaccines, a number of studies have been aimed at eliminating HIV with gene therapy. The intracellular immunization approach (Baltimore, 1988) has prompted the advent of molecular tactics for inhibiting replication and infection of HIV (Trono et al, 1989; Malim et al, 1989). Four main targets have been defined in HIV therapeutics:( i ) viral RNAs using ribozymes and antisense RNAs; ( i i ) viral proteins using RNA decoys, trans-dominant viral proteins, intracellular single-chain antibodies, and soluble CD4; ( i i i ) infected cells aimed at eliminating those with transfer and expression of suicide genes; and( i v ) the immune system by in vivo immunization (see Corbeau et al, 1996). Such gene therapeutic approaches can be combined with potent antiretroviral drugs especially the potent reverse transcriptase and protease inhibitors (Junker et al, 1997). The elucidation of the chemokine receptor mechanisms for entry of HIV-1 into T cells and macrophages provides new tactics for intervention at the level of interaction of gp120 with CCR-5. Inactivation of the CCR-5 gene might be achieved ( i ) with triplex oligonucleotide technologies once critical transcription factor binding sites in the regulatory region of the gene have been determined; ( i i ) with saturation of the blood of infected individuals with ligands, selected from peptide libraries, that block the extracellular domains of the CCR-5 receptor and preclude interaction with gp120; ( i i i ) with antagonists of RANTES (see above) that lack chemotactic activity but can block HIV infections (Arenzana-Seisdedos et al, 1996). Specificity for the ablation of HIV Tat-expressing cells has been achieved through the use of the promoter element from the long terminal repeat (LTR) of HIV (Venkatesh et al, 1990; Caruso et al, 1992).

since the HIV-2 promoter can sustain a considerable level of basal expression in the absence of its activator, Tat, a number of modifications were made to the HIV-2 promoter in order to minimize toxicity to non-infected cells. Retroviruses export unspliced, intron-containing RNA to the cytoplasm of infected cells despite the fact that intron-containing cellular RNAs cannot be exported; this export pathway is a critical step in the HIV-1 life-cycle. In HIV-1 this is accomplished through an interaction between the viral regulatory Rev protein and the Rev response element (RRE) RNA. In the absence of Rev, these introncontaining HIV-1 RNAs are retained in the nucleus. The nuclear export sequence (NES) LQLPPLERLTL has been identified on Rev that is responsible for its export to the cytoplasm (see Boulikas, 1998, this volume for more details). Targeting of Rev has provided a framework for novel interventions to reduce virus production in the infected host. Because disruption of either Rev or the RRE will completely inhibit HIV-1 replication, an anti-HIV-1 intracellular immunization strategy was developed based on RRE region-specific hammerhead ribozymes and on the intracellular expression of an anti-HIV-1 Rev single chain variable fragment (Sfv), which specifically targets the Rev activation domain. This combination resulted in a potent inhibition of HIV-1 replication in cell culture that holds promise as a future therapeutic regimen (Duan et al, 1997). To disable Rev function, primary T cells or macrophages were transduced with a recombinant AAV carrying an antiRev single-chain immunoglobulin (SFv) gene or an RRE decoy gene or with combinations of the two genes to disrupt the interaction between Rev and the RRE; when the transfected cells were then challenged by either clinical or laboratory HIV-1 isolates, this genetic antiviral strategy effectively inhibited infection (Inouye et al, 1997). A synergic effect of anti-Tat and anti-Rev molecules was found when the RRE sequence was cloned 3' to a tat transdominant negative mutant (tat22/37) gene; for this strategy Jurkat cells were transduced with the recombinant retroviruses containing the tat22/37 gene and an RRE decoy in different positions or the tat22/37 and the RevM10 transdominant negative mutant genes to produce monoclonal and polyclonal cultures expressing the integrated genes; none of these recombinant constructs inhibited virus replication at a high multiplicity of infection (MOI) and combination of tat and rev mutants was ineffective in inhibiting HIV-1 replication at both low and high MOIs; however, at a low MOI, two cell clones containing tat22/37 and the RRE decoy in 3' position showed a long lasting protection against virus replication and in two cell clones, expressing the RevM10 mutant alone, the HIV-1 replication was efficiently blocked (Caputo et al, 1997). IL-16 is secreted by activated CD8+ T lymphocytes and acts on CD4 + T lymphocytes, monocytes and eosinophils. Recently, the C-terminal 130-amino acid portion of IL-16 was shown to suppress HIV-1 replication in vitro. HIV replication was inhibited by as much as 99% in HIV-1susceptible CD4+ Jurkat cells following transfection and expression of the C-terminal 130-amino acid portion of IL-

C. Gene therapy against HIV in cell culture Strategies for HIV gene therapy include the inactivation of the CCR-5 coreceptor which is accomplished by targeting a modified CC-chemokine to the endoplasmic reticulum to block the surface expression of newly synthesized CCR-5 (Yang et al, 1997). A different gene therapy strategy proposed is targeting Tat, an early regulatory protein that is critical for viral gene expression and replication and which transactivates the LTR of HIV-1 via its binding to the transactivation response element (TAR); Tat also superactivates the HIV-1 promoter via activation of NF-&B in a pathway involving protein kinase C and TNF-"; combinations of the NF-&B inhibitors, pentoxifylline and Go-6976, with a stably expressed anti-Tat single-chain intracellular antibody suppressed HIV-1 replication and LTR-driven gene expression (Mhashilkar et al, 1997). Production of recombinant retrovirus containing the HSV-tk gene coupled to the HIV-2 promoter and Tat responsive region (TAR) has been used on human and mouse cells in culture for the specific elimination of HIV Tat-expressing cells; 91

Boulikas: An overview on gene therapy 16; the mechanism of HIV-1 inhibition by IL-16 was not at the level of viral entry or reverse transcription, but at the expression of mRNA (Zhou et al, 1997). The in vitro antiviral efficacy of two gene therapy strategies (trans-dominant RevM10 and Gag antisense RNA) were tested in combination with the clinically relevant reverse transcriptase inhibitors AZT and ddC or the protease inhibitor indinavir by Junker et al (1997). The combination of RevM10 or Gag antisense RNA with antiviral drugs inhibited HIV-1 replication 10-fold more effectively than the single antiviral drug regimen alone in retrovirally transduced human T cell lines after inoculation with high doses of HIV-1HXB3 in the presence or absence of inhibitors. The level of anti-HIV-1 activity of the psigag antisense sequence correlated with the length of the antisense transcript and maximal anti-HIV efficacy was observed with complementary sequence more than 1,000 nucleotides long, whereas transcripts less than 400 nucleotides long failed to inhibit HIV-1 replication in a Tcell line and in primary peripheral blood lymphocytes (Veres et al, 1996). The HSV-1 and HSV-2 virion host shutoff gene (vhs), each of which encodes a protein that accelerates the degradation of mRNA molecules leading to inhibition of protein synthesis, was used as a suicide gene for HIV gene therapy to inhibit replication of HIV; an infectious HIV proviral clone was cotransfected into HeLa cells together with the vhs gene under control of the CMV IE promoter; HSV-1 vhs gene driven by the HIV LTR inhibited HIV replication more than 44,000-fold in comparison to a mutant vhs gene (Hamouda et al, 1997). The specificity of the Vpr protein for the HIV-1 virus particle was exploited to develop an anti-HIV strategy targeting the events associated with virus maturation; nine cleavage sites of the Gag and Gag-Pol precursors were added to the C terminus of Vpr and the chimeric Vpr genes were introduced into HIV-1 proviral DNA to assess their effect on virus infectivity; the chimeric Vpr containing the cleavage sequences from the junction of p24 and p2 completely abolished virus infectivity (Serio et al, 1997).

A safe strategy to gene therapy of AIDS aimed at reducing the virus load in HIV-1-infected individuals was developed by Nakaya et al (1997). The Rev protein shifts RNA synthesis to viral transcripts by binding to the RRE within the env gene. Anti-Rev chimeric RNA-DNA oligonucleotides, consisting of 29 or 31 nucleotides, were designed to inhibit the Rev regulatory function and as decoys on HIV-1 replication; anti-Rev oligonucleotides containing an RNA "bubble" structure of 13 oligonucleotides (that bound to Rev with high affinity) were found to reduce more than 90% of the HIV-1 production from infected human T-cell lines and from healthy donor-derived peripheral blood mononuclear cells; control oligonucleotides without the bubble structure, that bound to Rev with considerably less affinity, did not reduce HIV-1 production (Nakaya et al, 1997).

E. Gene therapy of HIV with ribozymes Antisense, ribozyme, or RNA aptamers, must be efficiently transcribed, stabilized against rapid degradation, folded correctly, and directed to the part of the cell where they can be most effective. Among (i) antisense RNA, (ii) hairpin and hammerhead ribozymes, and (iii) RNA ligands (aptamers) for Tat and Rev RNA binding proteins, Revbinding RNAs but not the others, efficiently blocked HIV1 gene expression when tested in expression cassettes based on the human tRNA(met) and U6 snRNA promoters. In situ localization of both tRNA and U6 promoter transcripts revealed primarily punctate nuclear patterns (Good et al, 1997). Isolation of an RNA aptamer that can bind with high affinity to Tat protein, including two TAR-like RNA motifs for higher-affinity binding to Tat peptides provided a novel therapeutic strategy against HIV (Yamamoto et al, 1998, this volume). A hairpin ribozyme specific for simian immunodeficiency virus (SIV) and HIV-2 was used to inhibit viral replication in T lymphocytes derived from transduced CD34+ progenitor cells. Retroviral transduction of rhesus macaque CD34+ progenitor cells with the SIV-specific ribozyme â&#x20AC;&#x153;geneâ&#x20AC;? and the selectable marker neomycin phosphotransferase (NeoR) gene, followed by expansion and selection with the neomycin analog G418, rendered CD4+ T cells (derived from the transduced CD34+ hematopoietic cells) highly resistant to challenge with SIV; CD4 + T cells exhibited up to a 500-fold decrease in SIV replication, even after high multiplicities of infection (Rosenzweig et al, 1997). Monomeric (targeting one site) and multimeric (targeting nine highly conserved sites) hammerhead ribozyme genes both directed against the HIV-1 envelope (Env) mRNA were stably expressed in a human CD4+ T lymphocyte cell line; whereas the monomeric ribozyme caused a delay in HIV-1 replication, the multimeric ribozyme caused complete inhibition in HIV-1 replication for up to 60 days after infection (Ramezani et al, 1997).

D. Gene therapeutic strategies for AIDS A gene therapy strategy to combat acquired immunodeficiency syndrome (AIDS) in individuals already infected with HIV-1 has been directed toward GM-CSF mobilized peripheral blood CD34+ cells isolated from HIV1-infected individuals and transduced with retroviral vectors containing three different anti-HIV-1-genes:(i ) the Rev binding domain of the RRE (RRE decoy) carrying also the NeoR gene, (i i ) a double hammerhead ribozyme vector targeted to cleave the tat and rev transcripts (L-TR/TATneo), and (i i i ) the RevM10 transdominant negative mutant gene. After selection with G418, transduced cultures displayed up to 1,000-fold inhibition of HIV-1 replication following challenge with HIV-1 (Bauer et al, 1997).

Gene Therapy and Molecular Biology Vol 1, page 93 patients with one abnormal LDL receptor allele suffer premature coronary disease and myocardial infarction whereas patients with two abnormal alleles have extraordinarily high levels of cholesterol and accelerated atherosclerosis developing life-threatening coronary artery disease in early childhood. One type of mutation in the LDL receptor gene has incurred by Alu-Alu recombination deleting several exons and thus producing a truncated receptor molecule with loss of function (Lehrman et al, 1987). Treatment of patients with FH is accomplished through the administration of drugs that stimulate the expression of LDL receptor from the normal allele in order to lower the plasma level of LDL; however this regimen is not effective for the treatment of homozygous deficient patients, especially those that retain less than 2% of residual LDL-R activity. A more direct approach has been to correct the deficiency of hepatic LDL receptor by transplanting a liver that expresses normal levels of LDL receptor; three patients that survived this procedure normalized their serum LDL-cholesterol (see Wilson et al, 1992 for references).

A multitarget ribozyme of an unusually large size (3.7 kb) had a notable antiviral potential which may lead to a gene therapy approach; this ribozyme, directed against multiple sites within the gp120 coding region of HIV-1 RNA, co-localized with unspliced HIV-1 pre-mRNA and/or genomic HIV-1 RNA in the nucleus catalyzing the reduction of all spliced and unspliced HIV-1 RNAs; the same ribozyme functioned as a mRNA for a chimeric CD4/Env protein in the cytoplasm (Paik et al, 1997). Antisense oligonucleotides for HIV (anti-TAT) coupled with the influenza HA-2 protein-derived N-terminal fusogenic peptide have improved 5- to 10-fold their antiviral potency (Bongartz et al, 1994).

F. Novel therapies based on HIV vectors A major drawback in the gene therapy of HIV has been the poor efficiency of gene transfer in vivo; especially important for HIV, most recombinant retroviruses transduce poorly cells harboring HIV, such as monocytes and macrophages, which are nondividing. Transfection of a fibroblast cell line with a HIV vector, bearing a deletion of the major packaging sequence has provided an HIV-1 packaging cell line which produced a large amount of HIV1 structural proteins and non-infectious mature particles with normal reverse transcriptase activity but lacked RNA. When this cell line was stably transfected with an HIV-1based retroviral vector virions were produced capable of 5 transducing CD4-positive cells with efficiencies up to 10 cells per ml (Corbeau et al, 1996). For more details see VI. HIV vectors for gene transfer.

B. Gene therapy of FH: experiments in cell culture A transmissible retroviral vector containing a fulllength human cDNA for the LDL-R was used to infect fibroblasts from the Watanabe heritable hyperlipidemic (WHHL) rabbit which expressed the human receptor efficiently, as indicated by RNA and ligand blotting studies (Miyanohara et al, 1988). The number of hepatocytes that could be transduced by retroviruses bearing the therapeutic gene was one of the limiting steps that could impair the success of this strategy; addition of human hepatocyte growth factor (HGF) to hepatocytes allowed marked increase in the transduction efficiency in mouse (up to 80%) and human (40%) hepatocytes (Pages et al, 1995). Transduction of the human LDL-R cDNA under the transcriptional control of the liver-type pyruvate kinase promoter allowed high and tissue specific expression of the gene in primary hepatocytes; a second vector with a housekeeping promoter corrected the LDL-R deficiency in fibroblasts from a FH patient (Pages et al, 1996b).

E. Clinical trials involving HIV Gene therapy approaches directed against viral targets have been successful at inhibiting HIV-1 replication in cultured human cells; however, clinical trials involving gene therapy directed at HIV-1 are still in their infancy (reviewed by Gottfredsson and Bohjanen, 1997). Treatment of HIV-1 infections using gene therapy for intracellular immunization strategies is currently being tested in clinical trials. The first RAC-approved phase I study on HIV was to evaluate the safety of cellular adoptive immunotherapy using genetically modified CD8+ HIVspecific T cells in HIV seropositive individuals (protocol #15, Appendix 1). The significant number of ongoing clinical trials directed against HIV can be found on protocols 24, 40, 47, 52, 55, 73, 78, 79, 81, 85, 86, 88, 91, 94, 105, 108, 116, 117, and 168 in Appendix 1.

C. Gene therapy of FH: experiments on animals Liver is the preferred target organ for gene transfermediated treatment of FH. The presence of unique receptors at the cellular membrane of hepatocytes forms the basis for transfer strategies based on receptor targeting (reviewed by Sandig and Strauss, 1996). An authentic animal model used in FH gene therapy is the Watanabe heritable hyperlipidemic rabbit which is homozygous for FH and has a deletion in a cysteine-rich region of the LDL receptor gene; this renders the receptor completely dysfunctional (Yamamoto et al, 1986); these animals display high levels of serum cholesterol, diffuse atherosclerosis, and die

XXX. Familial hypercholesterolemia (FH) A. Molecular mechanisms for FH FH is an autosomal dominant disorder caused by a defect in the low density lipoprotein (LDL) receptor gene. LDL receptor on hepatocytes clears LDL from the serum; 93

Boulikas: An overview on gene therapy prematurely. Liver tissue was removed from such animals and the cultured hepatocytes were transduced with retroviruses carrying the rabbit LDL receptor gene; the genetically corrected cells were transplanted into the animal from which they were derived. This treatment resulted in a 30-40% reduction in serum cholesterol that lasted for at least 4 months (Chowdhury et al, 1991). The portal vein has been used for liver targeting in New Zealand White (NZW) rabbits. Expression of lacZ was obtained in virtually all hepatocytes within 3 days (but was undetectable by 3 weeks) after transfer of the lacZ reporter gene under the control of different promoters using recombinant, replication-defective adenoviruses which were infused into the portal circulation. An adenovirus human LDL-R cDNA was then infused into the portal vein of rabbits deficient in LDL receptor and demonstrated human LDL receptor protein in the majority of hepatocytes that exceeded the levels found in human liver by at least 10fold. Transgene expression diminished to undetectable levels within 3 weeks (Kozarsky et al, 1994). To demonstrate feasibility of the ex vivo FH therapy, three baboons underwent a partial hepatectomy, their hepatocytes were isolated, cultured, transduced with a retrovirus containing the human LDL-R gene, and infused via a catheter that had been placed into the inferior mesenteric vein at the time of liver resection (Grossman et al, 1992). Gene replacement therapy of human LDL receptor gene into the murine model of FH transiently corrected the dyslipidaemia; long-term expression of the therapeutic gene was extinguished by humoral and cellular immune responses to LDL receptor which developed and possibly contributed to the associated hepatitis (Kozarsky et al, 1996). As an alternative strategy, expression in the liver of the very low density lipoprotein (VLDL) receptor, which is homologous to the LDL receptor but has a different pattern of expression, using recombinant adenoviruses corrected the dyslipidaemia in the FH mouse; transfer of the VLDL receptor gene circumvented immune responses to the transgene leading to a higher duration of metabolic correction (Kozarsky et al, 1996). Replication-defective adenovirus-mediated gene transfer of the very low density lipoprotein receptor (VLDL-R) driven by a cytomegalovirus promoter in LDL-R knockout mice by a single intravenous injection resulted in reduction in total cholesterol by approximately 50% at days 4 and 9 and returned toward control values on day 21. Lowering in total cholesterol was mediated by a marked reduction in the intermediate density lipoprotein/low density lipoprotein (IDL/LDL) fraction. In treated animals, there was also an approximately 30% reduction in plasma apolipoprotein (apo) E accompanied by a 90% fall in apoB-100 on day 4 of treatment. Thus, adenovirus-mediated transfer of the VLDLR gene induced high-level hepatic expression of the VLDL-R, resulted in a reversal of the hypercholesterolemia, and enhanced the ability of these animals to clear IDL (Kobayashi et al, 1996).

The human apolipoprotein E (apoE) gene driven by the cytochrome P450 1A1 promoter produced transgenic mice where robust expression of apoE depended upon injection of the inducer #-naphthoflavone; a transgenic line exhibiting basal expression of apoE in the absence of the inducer upon breeding with hypercholesterolemic apoEdeficient mice produced animals which were as hypercholesterolemic as their nontransgenic apoE-deficient littermates in the basal state. When injected with the inducer, plasma cholesterol levels of the transgenic mice decreased dramatically. The inducer could pass transplacentally and via breast milk from an injected mother to her suckling neonatal pups, giving rise to the induction of human apoE in neonate plasma (Smith et al, 1995). Other potential approaches for the treatment of FH include transfer of the apolipoprotein (apo) B mRNA editing protein which is an essential catalytic component of the apoB mRNA editing enzyme complex. This enzyme deaminates a cytidine residue at nucleotide position 6666 in apoB mRNA, converting it to uridine leading to the production of apoB-48 in place of apoB-100. The editing protein exists as a homodimer and can be used as a therapeutic agent to reduce apoB-100; somatic gene transfer of the editing protein cDNA was highly effective in lowering plasma low density lipoproteins (Chan et al, 1996).

D. Clinical trials on FH The first clinical trial for gene therapy in the liver, based on ex vivo gene delivery, has shown both the feasibility and the limits of the current technology. According to this protocol, cultured hepatocytes from patients homozygous for mutations in the LDL receptor gene were proposed to be transduced ex vivo with the LDL receptor gene and transplanted by infusion into the portal vein of the patient (Wilson et al, 1992). A 29 year old woman, with homozygous FH, was transplanted with autologous hepatocytes that were genetically corrected with recombinant retroviruses carrying the LDL receptor gene. She tolerated the procedures well and in situ hybridization of liver tissue four months after therapy revealed evidence for engraftment of transgene-expressing cells. The patient's LDL/HDL ratio declined from 10-13 before gene therapy to 5-8 following gene therapy, an improvement which remained stable for the 18-month duration of the treatment (Grossman et al, 1994). Five patients, ranging in age from 7 to 41 years, with homozygous FH, underwent hepatic resection and placement of a portal venous catheter; primary hepatocyte cultures from the resected liver were transduced with the human LDL-R gene with a retrovirus and the cells were then transplanted into the liver through the portal venous catheter. The liver-directed ex vivo gene therapy was accomplished safely and, in a child patient, normalization of cholesterol levels took place; LDL-R expression was detected in a limited number of hepatocytes of liver tissue 94

Gene Therapy and Molecular Biology Vol 1, page 95 four months after hepatocyte implantation from all five patients whereas significant and prolonged reductions in LDL cholesterol were demonstrated in three of five patients. The major obstacle in this first trial was the level and duration of LDL-R expression which â&#x20AC;&#x153;precluded a broader application of liver-directed gene therapy without modifications supporting substantially greater gene transferâ&#x20AC;? (Grossman et al, 1995; Raper et al, 1996).

Protein Acidic and Rich in Cysteine) (reviewed by Jendraschak and Sage, 1996). Additional angiogenic factors include bFGF, angiopoietin-1, and thymidine phosphorylase whereas naturally occurring inhibitors of angiogenesis, other than angiostatin and vasculostatin include thrombospondin (see below, reviewed by Bicknell and Harris, 1996). Tissue factor (TF), which is the principal cellular initiator of coagulation and its deregulated expression has been implicated in cancer and inflammation has a documented role in tumor-associated angiogenesis (reviewed by Carmeliet et al, 1997). In the adult, angiogenesis accompanies the pathological processes of neovascularization during tumor growth, wound healing, and diabetic retinopathy and the normal processes of ovulation and placental development. There is a difference between vasculogenesis and angiogenesis: vasculogenesis occurs only during early embryogenesis resulting in the formation of the primordia of the heart and large vessels; on the other hand, angiogenesis is required for both the embryonic and postnatal tissues (for references see Davis et al, 1996). These processes involve two families of receptor tyrosine kinases, the Flt-1, Flk-1 receptors which interact with VEGF and the TIE (or Tek) receptor tyrosine kinases which are stimulated by angiopoietin-1 (see below).

XXXI. Angiogenesis and human disease A. Formation of new blood vessels (angiogenesis) Blood vessels, named in anatomy on the basis of their luminar diameter, branching, position and organ supplied, are formed with their proper diameter in the embryo before the heart starts beating. During a complex developmental program leading to formation of the cardiovascular system angioblasts are derived from mesoderm; this process requires the action of basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF). Angioblasts then differentiate into endothelial cells which undergo proliferation, migration, and morphologic organization in the context of their surrounding tissues to form the blood vessels (reviewed by Folkman and Dâ&#x20AC;&#x2122;Amore, 1996). Angiogenesis virtually never occurs physiologically in adult tissues except in the ovary, the endometrium and the placenta. During this process, which is also essential in pathological situations such as wound healing and inflammation, formation of new microvessels from parent microvessels takes place. The process involves remodeling of the basement membrane and interstitial extracellular matrix (ECM) using degrading proteases produced by the endothelial cells and other adjacent cells, and the synthesis of ECM. The endothelial cells are able to synthesize and secrete cytokines. The angiogenesis is strictly controlled by a redundancy of pro- and anti-angiogenic paracrine peptide molecules. The tumor suppressor p53 protein has been shown to control the expression and synthesis of two anti-angiogenic factors (reviewed by Norrby, 1997). Blood vessels in embryogenesis are formed in two stages: ( i ) during vasculogenesis newly differentiated endothelial cells coassemble into tubules that further fuse forming the primary vasculature of the embryo; ( i i ) angiogenesis, involves sprouting of new capillary vessels from preexisitng vasculature, also occurring into initially avascular organs such as the brain and kidney, and remodeling of the primary vascular network into large and small vessels (Davis et al, 1996). During tumor angiogenesis endothelium gives rise to new vessels which requires (i) dissolution of the basement membrane, (ii) migration and (i ii) proliferation of endothelial cells, (iv) formation of the vascular loop, and (v) formation of a new basement membrane; VEGF and PDGF act directly on endothelial cells and may also activate inflammatory cells to synthesize angiogenic factors such as SPARC (Secreted

B. Vascular endothelial growth factor (VEGF) VEGF (also known as vascular permeability factor, VPF) is a secreted protein related to PDGF which has been cloned (Keck et al, 1989; Leung et al, 1989; Abraham et al, 1991). VEGF is a mitogen for endothelium required for the differentiation of mesoderm-derived angioblasts into endothelial cells which form de novo vessels during embryogenesis, and in pathological states such as cancer and wound healing. VEGF is required for both vasculogenesis and angiogenesis. Targeted disruption of the VEGF gene in mice resulted in delayed differentiation of endothelial cells, impairment of vasculogenesis and angiogenesis and death at 8.5 to 9 days of gestation (Carmeliet et al, 1996; Ferrara et al, 1996). VEGF is a tumor secreted protein but also is synthesized in specialized endothelial and epithelial cells (primarily in alveolar walls of the lung, kidney glomeruli, heart, and adrenal gland, and secondarily in liver, spleen, and in autonomic nerves supplying the smooth muscle layers of the gastrointestinal tract) and in embryonic tissues; placenta expresses a related protein, placenta growth factor (PGF). The expression of VEGF in corpus luteum in primate ovaries is under hormonal control (reviewed by Senger et al, 1993). The positive staining for VEGF in normal corpus luteum is coincidental with the angiogenesis incurring concurrently with development and differentiation in this tissue; on the other hand, the expression of VEGF in lung, kidney, heart, GI tract, and 95

Boulikas: An overview on gene therapy adrenals, where angiogenesis is not normally occurring, might serve to maintain a certain density of endothelial cells in these tissues as well as for inducing and maintaining the base-line vascular permeability for plasma proteins, including antibodies and lipoproteins (reviewed by Senger et al, 1993). VEGF is produced in four isoforms by alternative splicing giving polypeptides of 210, 189, 165, and 121 amino acid residues (Leung et al, 1989; Keck et al, 1989; Abraham et al, 1991); VEGF is overexpressed in about 70% of hepatocellular carcinomas and other tumors (Suzuki et al, 1996). At least three pathophysiological roles for VEGF have been unraveled: ( i ) Primarily, VEGF induces angiogenesis (Plate et al, 1992; Shweiki et al, 1992), i.e. sprouting of capillaries from preexisting blood vessels, a process occurring during embryonic development but also under pathological conditions such as wound healing, eye disease, and tumor growth; VEGF expression is an early event during carcinogenesis and is also involved in metastasis (Liotta et al, 1991). ( i i ) VEGF is a mitogenic factor primarily for vascular endothelial cells stimulating phospholipase C after high affinity binding to the receptor. ( i i i ) VEGF also stimulates migration of monocytes across the endothelial cell monolayer. Other endothelial growth factors with angiogenic activity are the plateletderived growth factor (PDGF) and fibroblast growth factor (FGF) (reviewed by Folkman and Klagsbrun, 1987).

levels of hybridization in the endothelial lining of the atrium in heart, endothelial cells in the peribronchial capillaries (but not bronchial epithelium) in the lung, the menings, and at the inner surface of the atrium and the aorta; this expression seemed to be restricted to endothelial cells in blood vessels and capillaries. Flk-1 was also expressed in capillaries in brain at postnatal day 4 (Millauer et al, 1993). (i i i ) The Flt-4 (or VEGF-R3) (for references see Suri et al, 1996). Transgenic mouse embryos with targeted disruption of the VEGF-R1 and R2 genes have unraveled the different roles these receptors undertake during development: Flt-1 regulates normal endothelial cell-cell or cell-matrix interactions during vascular development (Fong et al, 1995) and Flk-1 is required for the formation of blood islands and vascularization of the embryo; disruption of the flk-1 gene interfered with differentiation of endothelial cells leading to death of embryos at day 8.5 to 9.5 (Shalaby et al, 1995 ). The temporal and spatial expression of VEGF exactly correlated with the expression pattern of Flk-1 in all tissues and developmental stages in mice examined suggesting a pivotal role of the growth factor/receptor duet in development and differentiation of the vascular system; their expression was high during development and declined in adulthood (Millauer et al, 1993). Although VEGF expression showed a moderate increase during tumor development in pancreatic islets of Langerhans, the expression of the flt-1 and flk-1 receptor genes remained unchanged during pancreatic carcinogenesis in an animal model (transgenic mice having a targeting expression of the SV40 T antigen gene under control of the insulin gene regulatory regions in #-cells of the pancreatic islets) (Christofori et al, 1995).

C. VEGF receptors The potential targets of secreted VEGF are proximal cells expressing VEGF receptors. Three VEGF receptors have been identified: (i ) A fms-like tyrosine kinase, the Flt-1 protein, (also called VEGF-R1), which is a transmembrane receptor with seven immunoglobulin-like domains in the extracellular region, a single transmembrane-spanning region, and a tyrosine kinase sequence (de Vries et al, 1992); Flt-1 is expressed in endothelial cells but is not found in nonendothelial cells (Shweiki et al, 1992). Disruption of the flt-1 gene permits differentiation of endothelial cells but interferes with a later stage of vasculogenesis causing thinwalled blood vessels with larger diameter and the death of mouse embryos at day 9 (Fong et al, 1995). (i i ) The Flk-1, (also called VEGF-R2), also a tyrosine kinase receptor. Flk-1 is endothelial cell-specific, already expressed in the angioblasts of the blood islands in early mouse embryos, progenitors of vascular endothelial cells. The Flk-1, (fetal liver kinase-1) was cloned independently and had been suggested to be involved in hematopoietic stem cell renewal. Flk-1 exhibits a high affinity for VEGF -10 (Kd=10 M) and is a major regulator of angiogenesis and vasculogenesis (Millauer et al, 1993). Hybridization of a parasagittal section of mouse embryos, at 14.5 days of development, with a single-stranded antisense DNA probe comprising the Flk-1 extracellular domain showed high

D. Angiopoietin and its receptors Although the TIE1 and TIE2 receptor tyrosine kinases were found to be involved in vasculogenesis already in 1992, angiopoietin-1, the natural ligand of TIE2 was only recognized in 1996 by secretion-trap expression cloning; according to this method entire cDNA libraries were transfected into large numbers of cells and the cell that contained the desired cDNA was uniquely marked on its surface by expression of the desired ligand; this rare cell was isolated within a background of millions of cells and was expanded leading to the isolation of the ligandencoding cDNA (Davis et al, 1996). This finding represents a significant milestone in the effort to understand the molecular mechanisms that govern formation of blood vessels with important implications for cancer targeting by inhibition of angiogenesis. Targeted disruption of the angiopoietin-1 gene in mice (Suri et al, 1996) gave defects in the blood vessels reminiscent of those caused by disruption in its receptor TIE2 (Dumont et al, 1994); these defects were mainly in 96

Gene Therapy and Molecular Biology Vol 1, page 97 the endocardium and myocardium but also generalized defects in vascular complexity leading to the death of mice by day 12.5 of gestation. These studies have established the important role of angiopoietin in mediating reciprocal interactions between the endothelium and surrounding matrix and mesenchyme (Suri et al, 1996). However, angiopoietin-1 does not display the mitogenic activities of VEGF on endothelial cells. The efficacy of disrupting the binding of angiopoietin to its ligand in tumor cell growth remains to be established. A missense mutation in the receptor tyrosine kinase TIE2, resulting in a Arg to Trp substitution at position 849 of the kinase domain, was found in patients from two unrelated families suffering with venous malformations; this disorder is characterized by the presence of veins with large lumens lined by a monolayer of endothelial cells but with thin walls because of a reduction in smooth muscle layers. Expression of the wild-type and mutant TIE2 in insect cells has shown that the mutation caused a 6 to 10fold increase in autophosphorylation activity of TIE2; thus, this mutation causes a defect in vascular remodeling and the overproliferation of endothelial cells without a complementary increase in smooth muscle layers (Vikkula et al, 1996). This finding demonstrates the significance of the TIE2 signaling pathway for endothelial cell-smooth muscle cell communication in venous morphogenesis.

determined by fibrinogen concentration and Factor XIII crosslinking of " and ! chains. Tumor cells as well as inflammatory cells in healing wounds produce a higher concentration of degradative proteases for extracellular matrix but also protease inhibitors which explains the resistance toward degradation of the fibrin gel in solid tumors (reviewed by Senger et al, 1993). The mechanism of overexpression of VEGF in solid tumors might involve its induction by hypoxia (Shweiki et al, 1992); the rapid proliferation of the cells in the center of the tumor induces an increase in the interstitial pressure and may lead to closure of capillaries by compression; inefficient vascular supply, including the compensatory development of collateral blood vessels in ischaemic tissues, leads to neovascularization via production of VEGF. A clustering of capillaries alongside VEGF-producing cells in a subset of glioblastoma cells immediately proximal to necrotic foci have been observed in intracranial brain neoplasms obtained from surgical specimens; this was thought to be the result of a local angiogenic response elicited by VEGF (Shweiki et al, 1992). VEGF expression by hypoxia was also induced in skeletal muscle myoblasts, in the fibroblast mouse L cell line, and in cells from rat heart muscle (Shweiki et al, 1992). Targeting of VEGF gene leading to its transcriptional inactivation (e.g. via triplex-forming oligonucleotides or antisense vectors) is expected to limit growth in solid tumors via inhibition in neo-vascularization; the prolonged sustenance of hypoxia in the center of the tumor is also expected to induce p53. An important concept to understand is that tumor angiogenesis results from a balance between angiogenic and anti-angiogenic factors. Expression of VEGF165 in rat C6 glioma cells and subcutaneous injection of the transduced cells in athymic mice has shown that tumors from cells expressing VEGF grew slower than tumors developed from nontransduced C6 cells, were highly vascularized, and contained varying degrees of necrosis and eosinophilic infiltrate (Saleh, 1996). VEGF plays an important role not only in carcinogenesis but also restenosis (see below). VEGF promotes endothelial cell proliferation to accelerate reendothelialization of the artery reducing intimal thickening; up-regulation of VEGF is the desired effect for treatment of restenosis (Asahara et al, 1996; Isner et al, 1996a). The transfer of the VEGF gene demonstrates a special mission of gene therapy: how to treat one human disease by upregulating the expression of a specific gene while treating a different disease by downregulating the expression of the same gene. Targeting is important. Also, exploring the molecular mechanisms affected by the transfer and overexpression of the cDNA of a gene, in all aspects and at their entire spectrum, is essential for a successful gene therapy application.

E. Involvement of VEGF in tumor angiogenesis Important for tumor growth are alterations in extracellular matrix. Solid tumors are composed of the malignant cells and the supportive vascular and connective tissue stroma whose synthesis is induced by the malignant cells. Fibrin is an essential component of the connective tissue stroma upon which tumor cells depend for their oxygen/nutrient supply and waste disposal. Fibrin gel provides solid tumors with a matrix which favors the ingrowth of macrophages, fibroblasts, and endothelial cells; these compose, along with neoangiogenesis vessels and elements found in normal connective tissue, the mature tumor stroma. Fibrinogen and other plasma proteins are found in increased amounts in tumor stroma and in healing wounds compared with normal stroma of most tissues; deposition of fibrin at the extravascular site requires a previous increase of permeability of the microvasculature thought to be mediated by VEGF (Senger et al, 1993). VEGF is the factor largely responsible for leakiness and hyperpermeability of tumor blood vessels; leakage of plasminogen (which is converted to plasmin) and of fibrinogen and clotting factors II, V, VII, X, and XIII (responsible for the formation of the extravascular fibrin gel) through postcapillary venules in tumor cells is responsible for generating the supporting matrix for fibroblast migration, angiogenesis, and fibroplasia (reviewed by Senger et al, 1993). Migration of macrophages and fibroblasts in the fibrin gel in tumors is

F. Transfer of the VEGF gene in ischemia 97

Boulikas: An overview on gene therapy VEGF gene transfer can improve blood supply to the ischaemic limb and is a promising approach for the treatment of acute limb ischemia. In a gene therapy approach for tissue ischemia, the VEGF165 cDNA under the transcriptional control of the HSV immediate-early 4/5 promoter was used to transduce BLK-CL4 fibroblasts resulting in the secretion of high levels of biologically active VEGF; when the transduced cells were resuspended in basement membrane extract (matrigel) and were injected subcutaneously into syngeneic C57BL/6 mice they showed a strong angiogenic response (Mesri et al, 1995). Regional angiogenesis was induced in nonischemic retroperitoneal adipose tissue by adenoviral VEGF gene transfer supporting a 123% increase in vessel number compared to control (Magovern et al, 1997). Treatment of a 71 year-old patient with an ischaemic leg with 2 mg phVEGF165 plasmid applied to the hydrogel polymer coating of an angioplasty balloon and reaching the distal popliteal artery resulted in an increase in collateral vessels at the knee, mid-tibial, and ankle levels, which persisted for 12-weeks (Isner et al, 1996a). Ischemia, induced in the hindlimb of rats by excision of the femoral artery, was experimentally treated by transfer of the VEGF gene; therapeutic angiogenesis produced morphologically similar, but significantly more extensive, networks of collateral microvessels (Takeshita et al, 1997). Direct i.m. injection of naked VEGF165 plasmid DNA into the ischemic thigh muscles in rabbits resulted in more angiographically recognizable collateral vessels at 30 days posttransfection (Tsurumi et al, 1996, 1997).

Angiostatin is a potent naturally occurring inhibitor of angiogenesis and growth of tumor metastases, which is generated by cancer-mediated proteolysis ofplasminogen to a 38 kDa plasminogen fragment; angiostatin selectively instructs endothelium to become refractory to angiogenic stimuli (O'Reilly et al, 1994, 1996). A number of enzymes including metalloelastase, pancreas elastase, plasmin reductase, and plasmin collaborate in the conversion of plasminogen to angiostatin. Systemic administration of angiostatin, but not intact plasminogen, inhibited neovascularization in vitro and in vivo and suppressed the growth of Lewis lung carcinoma metastases (O'Reilly et al, 1994; Gately et al, 1997); human angiostatin inhibited almost completely the growth of three human and three murine primary carcinomas in mice without detectable toxicity or resistance (O'Reilly et al, 1996); these studies have developed the â&#x20AC;&#x153;dormancy therapyâ&#x20AC;? for cancer based on that malignant tumors are regressed by prolonged blockade of angiogenesis to microscopic dormant foci in which tumor cell proliferation is balanced by apoptosis (O'Reilly et al, 1996). Human angiostatin, administered to mice with s.c. hemangioendothelioma and associated disseminated intravascular coagulopathy (Kasabach-Merritt syndrome), significantly reduced tumor volume and increased survival (Lannutti et al, 1997). PC-3 human prostate carcinoma cells release uPA and free sulfhydryl donors that converted plasminogen to angiostatin; these two components were sufficient for angiostatin generation (Gately et al, 1997); reduction of one or more disulfide bonds in the serine proteinase, plasmin, by a reductase secreted by Chinese hamster ovary cells triggered proteolysis of plasmin, generating fragments with the domain structure of angiostatin; two reductases (protein disulfide isomerase and thioredoxin) although able to produce biologically active angiostatin from plasmin and to inhibit proliferation of human dermal microvascular endothelial cells, were not the reductases secreted by cultured cells; instead the plasmin reductase factor secreted was a different one requiring reduced glutathione for activity (Stathakis et al, 1997). Two members of the human matrix metalloproteinase (MMP) family, matrilysin (MMP-7) and gelatinase B/type IV collagenase (MMP-9), hydrolyzed human plasminogen to generate angiostatin fragments; 58-, 42- and 38-kDa angiostatin fragments were generated; these studies implicated MMP-7 and MMP-9 in regulation of new blood vessel formation by cleaving plasminogen and generating angiostatin molecules (Patterson and Sang, 1997). Recent studies exploring the mechanism responsible for the in vivo production of angiostatin that inhibits growth and metastasis in Lewis lung carcinoma have shown that angiostatin is produced by tumor-infiltrating macrophages whose metalloelastase expression is stimulated by tumor cell-derived GM-CSF (Dong et al, 1997). Endostatin is a 20 kDa C-terminal fragment of collagen XVIII produced by hemangioendotheliomas which

G. Cancer treatment with angiogenesis inhibitors On November 20, 1997 the first exciting data on clinical trials using the TNP-470, a drug extracted from fungi which inhibits angiogenesis, were reported in a speech before the National Institutes of Health by Judah Folkman (Harvard University). A woman in Texas with cervical cancer and metastasis to lungs had been tumor-free for months after treated with TNP-470; and a young girl with a slow-growing bone tumor in her jaw was cancerfree after treatment with IFN-". Both TNP-470 and IFN-" are relatively weak inhibitors of blood vessel formation compared with angiostatin (O'Reilly et al, 1994, 1996), endostatin (O'Reilly et al, 1997), and vasculostatin which eliminated tumors in small animals. A combination therapy with angiostatin and endostatin was even more effective in tumor eradication; furthermore, these drugs have no apparent side effects and there is virtually no resistance of tumors to these drugs (see below). However, the number of human patients treated with angiogenesis inhibitors is too small and a larger number of cases need to be examined.

H. Angiostatin and endostatin

Gene Therapy and Molecular Biology Vol 1, page 99 specifically inhibits endothelial cell proliferation, angiogenesis and tumor growth (O'Reilly et al, 1997). Reports on the transfer of the angiostatin cDNA for the treatment of malignancies are about to appear (Toshihide Tanaka, personal communication).

concentrations of HA in vivo resulted in significant inhibition of neointimal formation (Savani and Turley, 1995). Intervening with the HA and receptor genes could provide potential molecular targets for restenosis. A number of drugs have been developed to inhibit neointima formation such as the drug CVT-313, identified from a purine analog library; CVT-313 is a specific and potent inhibitor of CDK2 reducing hyperphosphorylation of RB (Brooks et al, 1997). The angiotensin-converting enzyme inhibitor, cilazapril, also prevented myointimal proliferation after vascular injury (Powell et al, 1989). However, most of these drugs are toxic and need continuous administration to the artery. Gene therapy could result in stable transfection of the arterial wall cells with the gene of a therapeutic protein circumventing these problems. Significant progress has been made in the area of coronary restenosis, particularly in identifying target genes to reduce neointima formation in vein grafts used in coronary bypass surgery. Targets of gene therapy include the prevention of postangioplasty restenosis, postbypass atherosclerosis, peripheral atherosclerotic vascular disease and thrombus formation (reviewed by Malosky and Kolansky, 1996; Yl채-Herttuala, 1996).

XXXII. Gene therapy of restenosis A. Pathophysiology of restenosis The pathological situation, described as recurrent narrowing of a blood vessel after a successful revascularization procedure, has been termed restenosis (from the Hellenic stenos=narrow); the most frequent revascularization procedure has been the percutaneous transluminal angioplasty (PTA) used to treat atherosclerotic obstructions in the coronary and peripheral vascular circulations; PTA is achieved using a tiny balloon mounted on a catheter which is advanced under x-ray guidance to the site of a blocked artery. The most frequent artery suffering restenosis is the superficial femoral artery (SFA)/popliteal artery of the leg and the iliac arteries. One of the factors contributing to restenosis is the intimal hyperplasia of the arterial wall; among others, the mechanism for intimal hyperplasia involves increasing the tissue levels of TGF-# following injury; injection of antibodies directed against TGF-# has blocked restenosis in a rat model (reviewed by Border and Noble, 1995). Transfer of the TGF-# gene into porcine arteries caused restenosis (Nabel et al, 1993a,b). Arterial injury has pleiotropic effects at the molecular level; for example, injury of rat arteries led to an increase in FGF receptors in vascular smooth muscle cells. Atherosclerosis and restenosis following balloon angioplasty are characterized by two steps: during the thrombotic phase in the arterial wall following injury fibrin networks are synthesized with platelet depositions; this phase is followed by smooth muscle cell proliferation. Both the synthesis of fibrin from fibrinogen, as well as the proliferation of platelet and smooth muscle cells are upregulated by the protease thrombin. Inhibition of the action of thrombin in the arterial wall is a potential target against arterial disease. The most potent and specific inhibitor of thrombin known today is the polypeptide hirudin, an anticoagulant derived from the medicinal leech, Hirudo medicinalis; especially important is a local delivery of hirudin to prevent thrombosis circumventing the systemic coagulopathy associated with systemic administration of the purified protein (for references see Rade et al, 1996). The pathogenesis of both atherosclerosis and restenosis involves the migration of medial smooth-muscle cells across the internal elastic lamina to form a neointima; inflammatory reactions involving T cells and other leukocytes maintain smooth-muscle cell migration, proliferation and matrix deposition. The stenotic response involves the expression of HA (hyaluronan) receptors on both the infiltrating white cells and on smooth-muscle cell populations; exposure of injured arteries to high

B. VEGF gene transfer for restenosis Thickening of the arterial intima, composed of smooth muscle cells, is an important area for intervention by gene transfer to alleviate the syptoms of restenosis following balloon treatment. Gene therapy for restenosis may be achieved following transfer of the gene encoding VEGF; this therapy has been applied to animal models and has now entered clinical trials (Isner et al, 1996a). Previous studies using administration of recombinant, 165 amino acid, VEGF protein to rabbits in vivo has shown a significant augmentation in collateral vessel development by angiography after excision of the ipsilateral femoral artery in the animal to induce severe hind limb ischemia (Takeshita et al, 1994a). The rationale behind this approach is that VEGF promotes endothelial cell proliferation (Leung et al, 1989) to accelerate reendothelialization of the artery reducing intimal thickening and thrombogenicity (Asahara et al, 1996); the inner lining of the blood vessels is made up of endothelial cells which are important in preventing the formation of blood clots or atherosclerotic plaques. Animal studies have shown that endothelial cells require a relatively long time for growth after injury. "Naked" plasmid encoding the 165 amino acid VEGF isoform is being delivered using a hydrogel /polymer-coated balloon angioplasty catheter without use of liposomes or recombinant viruses to humans (Isner et al, 1996a). Yl채-Herttuala and coworkers (Laitinen et al, 1997a) have examined and compared the efficiency of lacZ delivery to the rabbit carotid artery (using a collar method) with (i ) plasmid/liposome complexes (Lipofectin), (i i ) replicationdeficient Moloney murine leukemia virus (MMLV)-derived 99

Boulikas: An overview on gene therapy retroviruses, (i i i ) pseudotyped vesicular stomatitis virus protein G (VSV-G)-containing retroviruses and (i v ) adenoviruses. Transfer of the gene to the adventitia took place with all gene transfer systems tested except MMLV; the adenovirus gave the highest gene transfer efficiency and up to 10% of the cells displayed #-galactosidase activity compared with 0.05% of cells using VSV-G retrovirus, 0.05% with Lipofectin, and less than 0.01% with MMLV retrovirus (Figure 31). During the collar application procedure extravascular gene transfer took place without intravascular manipulation; the model is suited for gene transfer studies involving difussible or secreted gene products (such as VEGF, see Figure 32) that act primarily on the endothelium; effects on medial smooth muscle cell (SMC) proliferation and even endothelium can be achieved from the adventitial side of the artery (Laitinen et al, 1997a). Transfer of the VEGF gene was performed on rabbits using a silicone collar inserted around the carotid arteries. The collar acted as an agent that caused intimal SMC growth and as a reservoir for the VEGF gene; the model preserved the integrity of endothelial cells and permitted extravascular gene transfer without intravascular manipulation. A plasmid carrying the mouse VEGF164 gene under control of the CMV promoter in a mixture with Lipofectin was injected under anesthesia by gently opening the collar (Laitinen et al, 1997b). As a control, arteries were injected with the lacZ cDNA; animals after 3, 7, or 14 days from the operation were sacrificed and the arteries were examined by electron microscopy using SMC-specific immunostaining (Figure 32A,B). One week after VEGF transfer with cationic liposomes there was a significant reduction in intimal thickening (Figure 32B) compared to control arteries (A). When the nitric oxide synthase inhibitor L-NAME was administered to the animals it abolished the therapeutic efficacy of VEGF and there was no VEGF-induced reduction in intimal thickening of the arteries (Laitinen et al, 1997b).

C. Transfer of other genes against restenosis and arterial injury A number of vascular disorders can be treated by arterial gene transfer (Nabel et al, 1990, 1993a-c; Ohno et al, 1994; Takeshita et al, 1994b; Chang et al, 1995a,b). One of the drawbacks has been the low percentage of the cells transfected which were in the order of 1% to less than 0.1% using naked plasmid delivery with a balloon catheter (e.g. Isner et al, 1996a), cationic liposomes (Nabel et al, 1990, 1993a-c), or retroviruses (Flugelman et al, 1992). A variety of genes have been transferred into arterial wall cells. Transfer of the human growth hormone gene, a secreted protein, into rabbit arterial organ culture using lipofectin permitted determination of hGH levels in the

100

culture medium using a sensitive immunoassay method (Takeshita et al, 1994b). The ADA gene has been transferred efficiently to vascular smooth muscle cells in rats (Lynch et al, 1992). Transfer of the fibroblast growth factor-1 gene (Nabel et al, 1993b), of the platelet-derived growth factor B gene (Nabel et al, 1993c), or of the transforming growth factor-#1 gene (Nabel et al, 1993a,b) into animal arteries promoted intimal hyperplasia and angiogenesis. The therapeutic induction of angiogenesis in ischemic tissues using recombinant cytokines is also promising for clinical application (Norrby, 1997). In vivo suppression of injury-induced vascular smooth muscle cell (VSMC) accumulation is a widely used approach (Ohno et al, 1994). Other than VEGF gene transfer (Isner et al, 1996a, see above), additional approaches for the treatment of restenosis after injuryinduced VSMC accumulation is via delivery of the HSV-tk gene followed by ganciclovir treatment in order to kill preferentially the smooth muscle cells (Guzman et al, 1994; Ohno et al, 1994); by transfer of the cytosine deaminase (CD) gene the product of which is capable of metabolizing 5-fluorocytosine (5-FC) to 5-fluorouracil in a rabbit femoral artery model of balloon-induced injury (Harrell et al, 1997); by transfer of the RB (Chang et al, 1995a; Smith et al, 1997), or p21 genes (Chang et al, 1995b); by transfer of ras (Indolfi et al, 1995), TGF gene (Grainger et al, 1995); and, by transfer of the nitric oxide synthase gene (von der Leyen et al, 1995). At the molecular level, arterial injury results in exposure of vascular smooth muscle cells (VSMC) and fibroblasts to multiple growth factors that activate second messengers and induce expression of immediate-early genes within minutes to hours after stimulation resulting in the exit of VSMC from the quiescent G0 state. A series of CDKs are activated and tumor suppressor genes need to be down-regulated for VSMC proliferation including p53, p21, p16, and RB (reviewed by Muller, 1997). Therapeutic strategies to restrict neointima formation in the injured artery include (i) inhibition in the expression of protooncogenes (c-myc); (ii) transfer of suicide genes (HSV-tk, CD); (iii) use of molecular decoys or drugs to block specific steps required for cell cycle progression,(iv) transfer of tumor suppressor genes (p21, RB); (v) treatment with antisense oligonucleotides to down regulate genes required for cell proliferation or DNA synthesis (antisense cyclin G1, PCNA); (vi) transfer of a number of unrelated genes such as of gax, TGF-#, hirudin, PKC(, #interferon (Table 7). It is worth considering that delivery of recombinant adenoviruses themselves causes (i) a pronounced infiltration of T cells throughout the artery wall; (ii) upregulation of intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 in arterial smooth muscle cells; (iii) neointimal hyperplasia (Newman et al, 1995).

Gene Therapy and Molecular Biology Vol 1, page 101

F i g u r e 3 1 . #-galactosidase gene transfer into the collared rabbit carotid arteries using (i) plasmid/liposome complexes, (ii) replication-deficient Moloney murine leukemia virus (MMLV)-derived retroviruses, (iii) pseudotyped vesicular stomatitis virus protein G (VSV-G)-containing retroviruses and (iv) adenoviruses. Gene transfer was done on day 5 after the collar operation. Arteries were analyzed 5 days after the gene transfer for general histology, cell types and #-galactosidase activity using immunocytochemistry and X-gal staining. (A , B ) Immunostainings of serial sections of an artery transfected with plasmid/liposome complexes. A: Endothelium remained anatomically intact during manipulations as shown with endothelialspecific staining using the monoclonal antibody CD31. B : The majority of cells in intima and media were smooth muscle cells (SMC) as shown with the SMC-specific antibody HHF-35. C: Absence of staining with X-gal in an untransfected control artery. (D-L): Arteries transfected with various gene transfer constructs. D: Plasmid/liposome complexes (25 µg lacZ plasmid, 25 µg Lipofectin reagent in 600 µl Ringer solution. E: MMLV retroviruses (600 µl pLZRNL retrovirus, titer 5x105 cfu/ml). F: VSV-G pseudotyped retroviruses (600 µl pLZRNL+G retrovirus, titer 1x107 cfu/ml). G: E1$E3-deleted adenoviruses (600 µl nuclear targeted pCMVBA-LACZ Adenovirus 5, titer 1x109 pfu/ml). H: Higher magnification of G. I: Higher magnification of G showing intense staining of the nucleae (arrow) in the adventitia with the nuclear targeted lacZ construct and X-gal staining of some endothelial cells (arrow-head). (J,K): Inflammatory cells were seen in adventitia after the gene transfer with VSV-G retroviruses and adenoviruses: J: VSV-G pseudotyped retrovirus-transfected artery (macrophage-specific staining with the monoclonal antibody RAM-11). K: Adenovirus-transfected artery (macrophage-specific staining with the monoclonal antibody RAM-11). L: Nonimmune control (first antibody omitted). Original magnification X10 (G); X25 (A-C,F,H); X50 (D,J,L); X100 (E,I). From Laitinen M, Pakkanen T, Donetti E, Baetta R, Luoma J, Lehtolainen P, Viita H, Agrawal R, Miyanohara A, Friedmann T, Risau W, Martin JF, Soma M, Ylä-Herttuala S (1 9 9 7 a ) Gene transfer into the carotid artery using an adventitial collar: comparison of the effectiveness of the plasmid-liposome complexes, retroviruses, pseudotyped retroviruses, and adenoviruses. Hum Gene Ther 8, 1645-1650. Reproduced with the kind permission of the authors and Mary Ann Liebert, Inc.

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Boulikas: An overview on gene therapy

F i g u r e 3 2 . Representative micrographs showing the characteristics of rabbit carotid arteries 7 days after VEGF or lacZ gene transfer. A. Control artery transfected with lacZ-plasmid/liposomes using smooth muscle cell (SMC)-specific immunostaining (HHF-35) showing a typical intimal thickening. B . Artery transfected with VEGF plasmid/liposomes showing a limited intimal thickening after SMC-specific immunostaining (HHF-35). C. Serial section to A, but stained for endothelium with CD31 monoclonal antibodies; it shows the presence of endothelium in all vascular segments examined. D. Serial section to B, but stained for endothelium with CD31 monoclonal antibodies, showing an intact endothelium. E. In situ hybridization with a VEGF antisense riboprobe labeled with [35 S]UTP in VEGF-transfected artery; bright spots (arrows) indicate the expression of VEGF mRNA (dark-field image). Control hybridizations with sense riboprobes were negative (not shown). F. The absence of inflammation was shown in VEGF-transfected arteries with macrophage-specific immunostaining (RAM-11). G. Neovascularization (arrow) in the adventitia of VEGF-transfected artery 14 days after gene transfer using endothelium-specific immunostaining (CD31). No neovascularization was detectable in lacZ-transfected arteries (not shown). H. Nonimmune control for the immunostainings (first antibody omitted); sections were counterstained with hematoxylin. Magnification, 25X in A-E, G and 50X in F,H. From Laitinen M, Zachary I, Breier G, Pakkanen T, Hakkinen T, Luoma J, Abedi H, Risau W, Soma M, Laakso M, Martin JF, Yl채-Herttuala S (1 9 9 7 b ) VEGF gene transfer reduces intimal thickening via increased production of nitric oxide in carotid arteries. Hum Gene Ther 8, 1737-1744. Reproduced with the kind permission of the authors and Mary Ann Liebert, Inc.

was engineered upstream of the hirudin cDNA in order to achieve secretion of the protein in effectively transduced cells. The therapeutically important levels of hirudin which were secreted in vivo resulted in 35% reduction in neointimal hyperplasia as shown on histologic sections of the carotid arteries (Rade et al, 1996). Significant issues on toxicity and immunogenicity of the vector remain to be resolved for application of the method to humans (Rade et al, 1996).

Transfer of the bone morphogenetic protein-2 (BMP-2) gene inhibited serum-stimulated increase in DNA synthesis and cell number of cultured rat arterial SMCs as well as injury-induced intimal hyperplasia; the mode of BMP-2 action differed from that mediated by TGF-#; BMP-2 had the ability to inhibit SMC proliferation without stimulating extracellular matrix synthesis (Nakaoka et al, 1997). cDNA for hirudin has been delivered to smooth muscle cells of injured rat carotid arteries using an adenoviral vector; the coding region for the human growth hormone signal peptide (MATGSRTSLLLAFGLLCLPWLQEGSA) 102

Gene Therapy and Molecular Biology Vol 1, page 103 Table 7. Genes or antisense used to inhibit smooth muscle cell proliferation and neointima formation for the treatment of arterial injury and restenosis Gene or antisense

Reference

VEGF gene transfer

Isner et al, 1996a; Van Belle et al, 1997; Laitinen et al, 1997a,b Guzman et al, 1994; Ohno et al, 1994 Harrell et al, 1997 Chang et al, 1995a; Smith et al, 1997 Chang et al, 1995b Rade et al, 1996 Indolfi et al, 1995; Ueno H et al, 1997b Grainger et al, 1995

HSV-tk gene /GCV Cytosine deaminase (CD) gene /5-fluorocytosine RB gene p21 gene Hirudin gene Dominant-negative mutated form of c-H-ras gene TGF gene Nitric oxide synthase gene gax gene Protein kinase C( gene (by suppressing G1 cyclin expression) Bone morphogenetic protein-2 (BMP-2) gene

von der Leyen et al, 1995 Weir et al, 1995; Maillard and Walsh, 1996; Smith et al, 1997 Fukumoto et al, 1997

#-interferon gene

Nakaoka et al, 1997 Stephan et al, 1997

Antisense cdk2 oligonucleotides Antisense oligodeoxynucleotides to c-myc Antisense oligonucleotides to PCNA Antisense cyclin G1

Morishita et al, 1994 Bennettet al, 1994 Simons et al, 1994 Zhu et al, 1997

Inhibition in proliferation of the smooth muscle cells has also been achieved by an antibody against basic fibroblast growth factor (Lindner and Reidy, 1991), by antisense oligodeoxynucleotides to c-myc applied in a pluronic gel to the arterial adventitia (Bennettet al, 1994), and by antisense phosphorothioate oligonucleotides to PCNA in a rat carotid artery injury model (Simons et al, 1994). Transfer of the protein kinase C( gene to a rat clonal VSMC inhibited the proliferation of vascular smooth muscle cells by suppressing G1 cyclin expression and arrested the cells in the G0/G1 phase of the cell cycle; overexpression of PKC( caused reduction in the expression of cyclins D1 and E and RB phosphorylation, and increased the protein levels of p27 (Fukumoto et al, 1997). RB is implicated in the control of the cell cycle via its interaction with E2F (see above); a phosphorylation competent, amino-terminal-truncated RB protein (Rb56) was a more potent inhibitor of E2F-mediated transcription relative to the full-length Rb construct (Rb110); adenoviral transfer of either Rb56 or Rb110 inhibited neointima formation after balloon injury in the rat carotid artery (Smith et al, 1997). On the other hand, overexpression of the fibroblast growth factor-1 gene (Nabel et al, 1993b), platelet-derived growth factor B gene (Nabel et al, 1993c), and transforming growth factor #1 gene (Nabel et al, 1993a,b) into the arteries induced intimal hyperplasia in vivo.

p21 protein is a negative regulator of mammalian cell cycle progression that functions both by inhibiting cyclin dependent kinases (CDKs) required for the initiation of S phase and by binding to and inhibiting PCNA. p21 gene transfer has been used to inhibit vascular smooth muscle cell (VSMC) proliferation after arterial injury for the treatment of atherosclerosis and restenosis. Overexpression of human p21 inhibited growth factorstimulated VSMC proliferation and neointima formation in the rat carotid artery (a model of balloon angioplasty) by arresting VSMCs in the G1 phase of the cell cycle. p21associated cell cycle arrest was associated both with a significant inhibition of the phosphorylation of RB (see above) and with the formation of complexes between p21 and PCNA in VSMCs (Chang et al, 1995b). Transfer of p21 was also used by others to suppress neointimal formation in the balloon-injured porcine or rat carotid arteries in vivo; vascular endothelial and smooth muscle cell growth was arrested through the ability of p21 to inhibit progression through the G1 phase of the cell cycle (Yang et al, 1996; Ueno et al, 1997a). Gax is a growth arrest gene which regulates proliferation of VSMC; gax is an homeobox gene whose expression in the adult is largely confined to cardiovascular tissues. In contrast to a number of genes which are upregulated following balloon injury and/or angioplasty, such as the early response genes c-myc and c-fos, gax is down-regulated within hours of balloon injury; gax may be required to maintain the gene expression of proteins in VSMC that are associated with the nonproliferative or 103

Boulikas: An overview on gene therapy contractile phenotype. Gax is also rapidly down-regulated in cultured VSMC upon stimulation by serum or plateletderived growth factor (PDGF); like the genes in the gas and gadd families, gax is expressed at its highest levels in quiescent cells and is down-regulated following mitogen activation (Weir et al, 1995). Percutaneous gax adenovirusmediated gene transfer into normal and atherosclerotic rabbit iliac arteries suggested prevention of neointimal formation (Maillard and Walsh, 1996). The gax-induced growth inhibition correlated with a p53-independent upregulation of the cyclin-dependent kinase inhibitor p21; gax overexpression also led to an association of p21 with cdk2 complexes and a decrease in cdk2 activity and, thus, gax overexpression inhibited cell proliferation in a p21dependent manner. Ras proteins are key transducers of mitogenic signals from the membrane to the nucleus. DNA vectors expressing ras transdominant negative mutants, which interfere with ras function, reduced neointimal formation after injury in rats in which the common carotid artery was subjected to balloon injury (Indolfi et al, 1995). An adenoviral vector, expressing a potent dominant-negative mutated form of c-H-ras, in which tyrosine replaced aspartic acid at residue 57, completely inhibited serumstimulated activation of mitogen-activated protein kinase, and abolished the DNA synthesis in response to serum mitogens in infected smooth muscle cells in culture. Application of the adenoviral vector into balloon-injured rat carotid arteries from inside the lumen resulted in a significant reduction in neointima formation (Ueno H et al, 1997b). Nitric oxide-generating vasodilators inhibit vascular smooth muscle cell proliferation. S-nitroso-Nacetylpenicillamine (SNAP), a nitric oxide-releasing agent, inhibited the activity of cyclin-dependent kinase 2 (Cdk2) and the phosphorylation of RB but it did not inhibit the activities of cyclin D-associated kinases Cdk4 and Cdk6 (Ishida et al, 1997). Suppression of injury-induced vascular smooth muscle cell accumulation was also achieved by transfer of the endothelial cell nitric oxide synthase gene (von der Leyden et al, 1995). Based on the fact that administration of the nitric oxide (NO) synthase inhibitor L-NAME to rabbit carotids eliminated the difference in intimal thickening between VEGF and mock-transfected (lacZ) arteries it was proposed that VEGF may reduce smooth muscle cell proliferation via VEGF-induced NO production from the endothelium (Laitinen et al, 1997).

furthermore, transgenic mice overexpressing lipoprotein (a) exhibit decreased levels of TGF-# at sites of lipoprotein (a) accumulation such as in the aortic wall (Grainger et al, 1995). As prolonged overproduction of TGF-# may lead to tissue fibrosis by overproduction of extracellular cell matrix (Border and Noble, 1995) a balanced overexpression of TGF- might be of great therapeutic value for heart disease. The plasminogen system, via its triggers, t-PA and uPA and its inhibitor, plasminogen activator inhibitor-1 (PAI-1), has been implicated in thrombosis, arterial neointima formation, and atherosclerosis (reviewed by Carmeliet et al, 1997). A number of animal models have been used to induce atherosclerotic lesions such as the iliac arteries of New Zealand white rabbits fed with cholesterol. Substantial progress in vector development and the demonstration of efficacy in relevant animal models will be required before gene therapy for atherosclerosis becomes a clinical reality (Rader, 1997).

E. Acidic and basic fibroblast growth factors (aFGF, bFGF) Acidic and basic fibroblast growth factors and their receptors are involved in many fundamental biological processes but also in pathological processes (Webster and Donoghue, 1998, this volume). Whereas VEGF is a regulator of vascular permeability and an endothelial cell growth factor the acidic and basic fibroblast growth factor (aFGF, bFGF) and placenta growth factor (PGF) polypeptides are endowed with endothelial cell growth-promoting activity; however, FGFs have not been reported to be expressed in blood vessels in vivo. aFGF and bFGF seem to act as mitogens for a large number of different cell types; in situ hybridization analysis has shown that there is no expression of FGF receptors 1 and 2 (Flg and Bek) in capillary endothelial cells during embryonic development; it is only VEGF and PGF which are known to be specific for endothelial cells (for references see Millauer et al, 1993). Many of the studies that have demonstrated therapeutic efficacy using fibroblast (aFGF, bFGF, FGF-5), endothelial (VEGF) and other types of factors using the purified peptides can potentially be transferred to the gene level. This will not require repeated administration of the drug, especially whenever the somatic cell targets are transfected efficiently and the expression of the transgene lasts. Liposome-mediated gene transfer of antisense-oriented bFGF or fibroblast growth factor receptor-1 (FGFR-1) cDNAs in episomal vectors into human melanomas, grown as subcutaneous tumors in nude mice caused complete arrest or regression of the tumors as a result of blocked intratumoral angiogenesis and subsequent necrosis (Wang and Becker, 1997). Inhibition of bFGF synthesis in vivo using an antisense RNA strategy significantly

D. Atherosclerosis The atherotic plaque is formed by a complex mechanism initiated by the accumulation of lipid, macrophages and T cells at artery lesions leading to smooth muscle cell proliferation (Ross, 1993). TGF-# is involved in atherosclerosis via its activation by plasmin and via its inhibition by atherogenic lipoprotein deposited on the arterial wall. Inhibition of TGF-# would lead to smooth muscle cell proliferation; patients with advanced coronary disease have decreased serum TGF-# levels; 104

Gene Therapy and Molecular Biology Vol 1, page 105 inhibited intimal thickening after arterial balloon injury (Hanna et al, 1997). Infection of human umbilical vein endothelial cell cultures with a bFGF-expressing recombinant adenovirus enhanced the proliferation rate and tubular formation of these cells on reconstituted basement membrane (Takahashi et al, 1997). Low level expression of bFGF upregulated Bcl-2 and delayed apoptosis in NIH3T3 cells; on the other hand cells expressing from 8-15 times background levels of bFGF became phenotypically transformed (gave dense foci at confluence, had decreased adherence to tissue culture plates and grew colonies in soft agar) (Wieder et al, 1997). Blood vessels of spontaneously hypertensive rats were shown to be associated with subphysiological amounts of bFGF; transfer of the bFGF gene corrected hypertension, restored the physiological levels of bFGF in the vascular wall, and ameliorated endothelial-dependent responses to vasoconstrictors (Cuevas et al, 1996).

adulthood but were predisposed to spontaneous thrombotic lesions in many tissues and displayed fibrin deposition in the liver (Bugge et al, 1995). These animals showed severe impairment in the healing of skin wounds; thus, plasminogen plays a central role in extracellular matrix degradation during wound healing in rodents in vivo and most likely also in humans (Rømer et al, 1996). Fibrin dissolution allows keratinocyte migration from incisional wound edges; detriment in fibrin degradation slows down wound repair by the limited ability of epidermis cells to dissect their way through the extracellular matrix beneath the wound crust (Rømer et al, 1996). Fibrin is a major component of the extracellular matrix in wounds and solid tumors but not in normal embryonic or adult tissue.

G. TGF- in injury and wound healing Transforming growth factor-# (TGF-#) is a cytokine implicated in the pathogenesis of impaired wound healing but also in autoimmune disease, malignancy, and atherosclerosis (Grainger et al, 1995). TGF-# has gained interest for the treatment of autoimmune disease, multiple sclerosis (autoimmune encephalomyelitis), and arthritis; however, prolonged overproduction of TGF-# may lead to tissue fibrosis by overproduction of extracellular cell matrix affecting kidney, liver, lung and other organs. TGF#1 is involved in the pathogenesis of fibrosis by its matrix-inducing effects on stromal cells such as in activation of the pulmonary fibrotic process (Sime et al, 1997). Overexpression of TGF-#1 in the heart is thought to contribute to the development of cardiac hypertrophy and fibrosis (Villarreal et al, 1996). TGF-# can activate and then suppress T cells, macrophages, and leukocytes; transgenic mice with targeted disruption of the TGF- gene die with autoimmune symptoms (reviewed by Border and Noble, 1995). The preservation and architectural design of the extracellular matrix depends on the action of cytokine polypeptides. TGF-# controls the mitogenic action of platelet-derived growth factor (PDGF); in response to injury or disease, the production of TGF-# and PDGF are increased stimulating extracellular matrix production; this is accomplished by inhibition of proteases and stimulation of synthesis of extracellular matrix proteins by TGF-#. Failure of cells to produce enough TGF-# has been proposed to result in impaired wound healing in the elderly, glucocorticoid-treated individuals, and in diabetics; a single injection of TGF-# has been shown to accelerate wound healing (reviewed by Border and Noble, 1995). TGF-# inhibits epithelial and smooth cell proliferation; it is believed that one of the factors contributing to the unrestricted growth of cancer cells is their nonresponsiveness to TGF-# because of loss of functional TGF-# receptors; microsatellite instability in colon cancer cells inactivates the type II TGF-# receptors (Markowitz et al, 1995). Tamoxifen, an anticancer drug, stimulates TGF-# thus inhibiting cancer cell proliferation. Restoration of the TGF-# receptor genes might constitute

F. Wound healing and plasminogen A number of diseases including cancer, cancer metastasis, atherosclerosis, arthritis, hepatitis, dermatitis, inflammatory bowel disease, sickle cell anemia, and autoimmune disease result in severe tissue damage. Plasminogen has a profound importance in wound healing and might play a central role in many of these diseases (Rømer et al, 1996). Plasminogen is an inactive precursorprotease synthesized and secreted by the liver and converted into plasmin (trypsin-like serine protease) by the action of two different proteases: ( i ) tissue-type plasminogen activator (tPA) and ( i i ) urokinase-type plasminogen activator (uPA); in addition to uPA- and tPA-regulation, the activity of plasminogen is also controlled by plasminogen-specific cell surface receptors, by inhibitors of plasminogen activation (PAI-1 and PAI-2), and by the receptor of uPA (uPAR). Angiostatin is a 38 kDa plasminogen fragment generated by cancer-mediated proteolysis of plasminogen (O'Reilly et al, 1994, 1996, see angiostatin). Endostatin is a 20 kDa C-terminal fragment of collagen XVIII (O'Reilly et al, 1997); both angiostatin and endostatin inhibit angiogenesis and tumor growth. Plasmin passes to extravascular fluids and stimulates proteolytic activity in the extracellular matrix (such as degradation of fibrin) but also contributes to the activation of growth factors and other proteases (see Rømer et al, 1996). Fibrin is an important component of the wound healing and reepithelization process and is formed from fibrinogen by the action of the protease thrombin (see Rade et al, 1996). These processes take place during wound healing but also during the process of atherosclerosis, restenosis, response to vascular injury, and in tumorigenesis during formation of the tumor stroma. Plasminogen-deficient mice (transgenic animals produced by targeted disruption in the plasminogen gene) completed embryonic development and survived to 105

Boulikas: An overview on gene therapy a strategy for treating human cancers (Border and Noble, 1995). Transfer of the cDNA of porcine TGF-#1 to rat lung induced prolonged and severe interstitial and pleural fibrosis characterized by extensive deposition of the extracellular matrix (ECM) proteins collagen, fibronectin, and elastin (Sime et al, 1997). Particle-mediated delivery of mutant porcine constitutively active TGF-#1 cDNA to rat skin at the site of skin incisions increased tensile strength up to 80% for 14-21 days (Benn et al, 1996). Neurodegeneration associated with Alzheimer's disease is believed to involve toxicity to #-amyloid and related peptides; this neurotoxicity was significantly attenuated by single treatments with TGF-#1 and was prevented by repetitive treatments, a process associated with a preservation of mitochondrial potential and function (Prehn et al, 1996); this implies a potential avenue of TGF-#1 gene transfer for Alzheimer's disease. Transfer of TGF-#1 cDNA in vivo suppresses local T cell immunity and prolonged cardiac allograft survival in mice; TGF-#1 gene transfer may become a new type of immunosuppressant avoiding the systemic toxicity of conventional immunosuppression (Qin et al, 1996). GM-CSF overexpression induced TGF-#1 gene expression and secretion from macrophages purified from bronchoalveolar lavage fluid 7 days after GM-CSF gene transfer; these findings implicate GM-CSF in pulmonary fibrogenesis (Xing et al, 1997).

bacteria bound to ciliated CF epithelial cells (Davies et al, 1997). The architecture of the lung and the terminal differentiation of the defective cells imposes a serious hurdle for ex vivo gene therapy for CF: the epithelial cells on the airway surface cover the successively branching structures of the lung making impossible their removal and reimplantation (Yoshimura et al, 1992). Successful introduction of the entire 250 kb human CFTR gene locus and adjacent sequences into Chinese hamster ovary-K1 (CHO) cells which lack endogenous CFTR was achieved using yeast artificial chromosomes (YACs); integration of the human CFTR-containing YACs into the CHO genome took place on the order of one copy per genome; functional human CFTR was expressed from subclones and human CFTR expression in CHO cells was unexpectedly high (Mogayzel et al, 1997). This type of studies are very useful as a number of DNA control elements for CFTR may be â&#x20AC;&#x153;hiddenâ&#x20AC;? throughout the gene locus, including enhancers, ORIs, silencers, MARs, that participate in the tissue- and developmental stage-specific CFTR gene expression. The airway epithelium is in the process of injury and regeneration in CF; regenerating poorly differentiated cells of human airway epithelium in culture were efficiently transfected with CFTR cDNA using adenoviral vectors; CFTR expression and cAMP-regulated stimulation of the cell membrane chloride ion secretion took place on these cells as determined by light fluorescence microscopy and scanning laser confocal microscopy (Dupuit et al, 1997).

XXXIII. Cystic fibrosis (CF) A. Molecular mechanism of CF pathogenesis

B. CFTR gene transfer in animal models Direct transfer of the human CFTR gene was achieved using a replication-defective adenovirus vector by intratracheal instillation into cotton rat lungs; the presence of human CFTR mRNA transcripts was detected by in situ hybridization with a cRNA (antisense) probe as well as by immunohistochemical evaluation using antibodies directed against the CFTR protein (Rosenfeld et al, 1992). First generation adenovirus-mediated gene transfer of CFTR to the mouse lung resulted in the expression of viral proteins leading to the elimination of the therapeutic cells expressing CFTR by cellular immune responses; second generation E1-deleted viruses displayed substantially longer recombinant gene expression and induced a lower inflammatory response (Yang et al, 1994). Adenoviral vector constructs with an E1E3+E4ORF6+ backbone encoding CFTR (or #galactosidase) produced declining levels of expression while a similar vector with an E1-E3+E4+ backbone gave rise to sustained, long-term reporter gene expression in the lung in nude mice; CTLs directed against either adenoviral proteins or #-galactosidase reduced expression in nude mice stably expressing #-galactosidase from the E4+ vector (Kaplan et al, 1997).

CF is a lethal recessive hereditary disorder characterized by abnormalities of the airway epithelium; it affects 1 in 2,000 Caucasians. Inflicted individuals show secretion of thick mucus and chronic colonization of the lung epithelium with pathogens such as Pseudomonas aeruginosa. The defect arises from mutations in the 250-kb gene encoding a 12-transmembrane domain glycoprotein (1480 amino acids), called cystic fibrosis transmembrane conductance regulator (CFTR), that modulates the permeability of Cl - in response to elevation of intracellular cAMP. The defect is caused by deletion of three base pairs eliminating a single phenylalanine residue at the center of the first nucleotide-binding domain of the CFTR protein (Riordan et al, 1989). Much of the mortality seen in CF is related to chronic infection of the respiratory tract with Pseudomonas aeruginosa; Pseudomonas colonization has been attributed to increased numbers of specific cell-surface receptors and to the presence of mucus. Adherence of P. aeruginosa directly to the cell surface of CF airway epithelium (from noncultured nasal epithelial cells isolated from CF patients) was significantly increased over that in cells from healthy donors. Liposome-mediated CFTR gene transfer resulted in a significant reduction in the numbers of 106

Gene Therapy and Molecular Biology Vol 1, page 107 protocols approved use adenoviral delivery of CFTR (#118-123, 125, 128, 129) or cationic lipids (#193, 203, 212, and 214). The only two protocols using AAV in clinical trials are for CF (#165, 166) whereas no retroviral protocol has been approved for CF as of December 1997. Results of clinical trials have been reported and more will become available in the near future. A single dose of 400 Âľg pCMV-CFTR in complex with 2.4 mg DOTAP, administered to the nasal epithelium of eight CF patients resulted in partial, sustained correction of CFTR-related functional changes toward normal values in two treated patients; transgene DNA was detected in seven of eight treated patients for up to 28 days after treatment; vector derived CFTR mRNA was detected in two of the seven patients at 3 and 7 days from treatment using PCR (Porteous et al, 1997). Complexes of plasmids with DOTAP liposomes rendered their DNA resistant to DNaseI something relevant to clinical trials for gene therapy of CF, in which patients are normally removed from treatment with DNase before receiving administration of DNA (Crook et al, 1997). A formulation of plasmid encoding CFTR (pCF1CFTR) was at least as effective as complexes of DNA with lipid in partially correcting the Cl- transport defect in CF patients by administering complexes of DNA-lipid to one nostril and DNA alone to the other nostril in a randomized, double-blind study (Zabner et al, 1997). The safety of the nebulised cationic lipid formulation (GL-67/DOPE/DMPE-PEG5000) to be used for the transfer of CFTR to CF patients was first tested on 15 healthy volunteers in the absence of plasmid DNA; no adverse clinical events were seen (Chadwick et al, 1997).

Aerosol delivery of an adenoviral vector encoding CFTR to non-human primates showed human CFTR mRNA in lung tissue from all treated animals on days 3, 7, and 21 post-exposure; other than some rather mild complications on individual animals ranging from an increase in lavage lymphocyte numbers to bronchointerstitial pneumonia, the treatment was rather safe (McDonald et al, 1997). Adenoviruses elicit am immune response. Effective gene therapy for CF would ideally be accomplished with a vector capable of long-term expression of the CFTR in the absence of a host inflammatory response; in this respect AAV might be better suited. Administration of single doses of AAV-CFTR vector to 10 rhesus macaques by fiberoptic bronchoscopy to the right lower lobe of lungs showed that transfer of the CFTR gene occurred in bronchial epithelial cells of each animal by in situ DNA PCR; vector mRNA was detectable for 180 days after administration as detected by RT-PCR and there was no evidence of inflammation (Conrad et al, 1996). Transfer of CFTR gene was also achieved using retroviral vectors; sodium butyrate treatment of murine retrovirus packaging cells producing the vector increased the production of the retrovirus vector between 40- and 1,000-fold (Olsen and Sechelski, 1995). Yoshimura and coworkers (1992) have transduced airway epithelial cells in mice by intratracheal instillation of a plasmid carrying the CFTR gene under control of the Rous sarcoma virus promoter with cationic liposomes. Use of liposomes have successfully transferred theCFTR gene to epithelia and to alveoli deep in the lung leading to correction of the ion conductance defects found in the trachea of transgenic mice (Hyde et al, 1993). Complexes of cationic polymers and cationic lipids with adenovirus enhanced gene transfer to the nasal epithelium of cystic fibrosis mice in vivo (Fasbender et al, 1997). The novel cationic lipid EDMPC (1,2dimyristoylsn-glycero-3-ethylphosphocholine, chloride salt) mediated efficient intralobar DNA delivery of CFTR to rodents; there was no correlation between DNAEDMPC formulations that delivered DNA most efficiently in vitro and those that worked best in vivo (Gorman et al, 1997). The structures of several novel cationic lipids that were effective for CFTR gene delivery to the lungs of mice were investigated; an amphiphile (lipid #67) consisting of a cholesterol anchor linked to a spermine head-group in a "Tshape" configuration was shown to sustain a 1,000-fold increase in expression above that obtained in animals instilled with naked pDNA alone and was greater than 100fold more active than other cationic lipids and comparable to that of adenoviral vectors (Lee et al, 1996).

XXXIV. Rheumatoid arthritis (RA) A. Molecular mechanisms for development of RA RA is a systemic autoimmune disease caused by genetic and environmental factors; chronic inflammation and hyperactivation of synovial cells in the joints is the salient feature of RA resulting in the thickening (hyperplasia) of the synovial membrane lining the interior surface of the joint capsule. The mechanism involves synovial cell proliferation, infiltration by leukocytes, and excessive extracellular cell matrix deposition. The activated synovial cells in the hypertrophied synovium produce inflammatory cytokines and degradative enzymes that invade and erode the articular cartilage leading to partial or complete destruction of cartilage and bones. The inflamed synovium in RA is infiltrated by lymphocytes and monocytes, a process mediated by the enhanced binding of the very late antigen-4 (VLA-4) to vascular cell adhesion molecule-1 (VCAM-1) (Chen et al, 1995). Several well-characterized murine models of arthritis closely resemble RA immunologically, genetically, and histopathologically and have been developed to study RA. Collagen-induced arthritis in DBA/1 mice is one model of

C. Clinical trials on cystic fibrosis patients A significant number of clinical trials on CF have received RAC approval (Appendix 1 and Table 4 in Martin and Boulikas, following article). The clinical 107

Boulikas: An overview on gene therapy RA; the animals exhibit marked synovitis and erosions. The disease can be adoptively transferred to SCID mice using arthritogenic splenocytes from DBA/1 mice injected with bovine collagen type II (Chernajovsky et al, 1997). Bacterial cell wall-induced arthritis in rats is another model (Makarov et al, 1996).

C. Ex vivo gene therapy of RA using IL1Ra-transduced cells

B. Approaches to gene therapy of RA The current emphasis for RA gene therapy is on transferring genes encoding secreted proteins which possess antiarthritic properties. Genes may be delivered locally to individual diseased joints or systemically to extra-articular sites where the secreted gene products may enter the circulation. Gene transfer to the synovium would ensure local production of anti-inflammatory gene products directly in the articular space where they could exert a down-regulatory effect on the autoimmune process. Although adenoviral delivery appeared best suited for gene delivery to synovium, induction of an inflammatory response resulting in loss of gene expression may take place (Evans and Robbins, 1996). High efficiency lacZ gene transfer and expression was achieved in both type A and type B synoviocytes throughout the articular and periarticular synovium of the rabbit knee by Roessler et al (1993). Intra-articular administration of an E1a-E3-deleted adenoviral (Ad5) vector expressing the lacZ transgene into mouse joints showed lacZ expression in the articular synovium for at least 14 days. However, a gradual loss of transgene expression was caused by a predominantly neutrophilic, inflammatory response. Pretreatment with the anti-T cell receptor monoclonal antibody (mAb) H57 resulted in a significant reduction in lymphocytic infiltration and in persistence of transgene expression. Thus, anti-T cell mAbs may be useful in inhibiting adenovirus-induced immune responses that lead to the loss of therapeutically transduced cells (Sawchuk et al, 1996). Many new therapeutic approaches are currently being developed, including the use of soluble receptors to IL-1 or TNF, monoclonal antibodies to TNF-", and a specific IL1 receptor antagonist. A number of studies have assessed the impact of gene transfer on inflammatory and chondrodestructive effects during the acute phase of antigen-induced arthritis in RA joints. A promising therapy for RA involves delivery of the TNF-" and IL-1 proteins to the joints to inhibit the activity of proinflammatory cytokines (Bandara et al, 1993; Arend and Dayer, 1995). Angiogenesis is not only essential for the growth and metastatic spread of solid tumors but in diseases such as rheumatoid arthritis, psoriasis, liver cirrhosis and diabetic retinopathy (Norrby, 1997). Future approaches for the gene therapy of RA may thus include anti-angiogenesis approaches to the inflamed joints.

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Degradation of cartilage in RA in vitro is stimulated by IL-1, a proinflammatory cytokine, which is released from RA synovial fibroblasts (RA-SF). Synovial cells were surgically removed from joints of animals with experimental arthritis, cultured and transduced with the naturally occurring inhibitor of IL-1, IL-1-receptor antagonist (IL-1Ra) protein gene and reimplanted into the respective donors by intra-articular injection (Bandara et al, 1993). Retroviral transfer of the IL-1Ra gene to RA-SF which were then coimplanted with normal human cartilage in SCID mice protected the cartilage from chondrocytemediated degradation; the IL-1Ra-transduced RA-SF continued to secrete IL-1Ra over a 60-day period (MullerLadner et al, 1997a,b). Transfer the human IL-1Ra gene to rabbits' knees produced a marked chondroprotective effect although the anti-inflammatory effect was milder (Otani et al, 1996). Ex vivo retroviral delivery of the secreted human IL1Ra cDNA to primary synoviocytes followed by engraftment in ankle joints of rats with recurrent bacterial cell wall-induced arthritis significantly suppressed the severity of recurrence of arthritis as assessed by measuring joint swelling and by the gross-observation score; this ex vivo approach attenuated but did not abolish erosion of cartilage and bone; the level of locally expressed IL-1Ra was about four orders of magnitude higher than that attained from systemically administered recombinant IL1Ra protein (Makarov et al, 1996). These findings provide experimental evidence for the feasibility of antiinflammatory gene therapy for arthritis. Retroviral transduction of hematopoietic stem cells with human IL-1Ra cDNA was also used for the treatment of RA; HSCs were subsequently injected into lethally irradiated mice; all of the mice survived and over 98% of the white blood cells in these mice were arising from the transduced HSCs (donor type) from 2-13 months after transplantation; the animals had the human IL-1Ra protein in their sera for at least 15 months. These results demonstrated that systemic production of biologically active human IL-1Ra can be obtained by retrovirusmediated gene transfer to hematopoietic stem cells which could be useful in the treatment of chronic diseases such as rheumatoid arthritis as well as bone degeneration caused by aging (Boggs et al, 1995). Characterization of the interleukin-1/interleukin-1 receptor antagonist pathways in RA resulted in the first gene therapy trial in animals and humans for RA (Evans et al, 1996; reviewed by Evans and Robbins, 1996; MullerLadner et al, 1997a). Protocol #56 (page 162) involves removal of autologous synovial cells from the patient, their retroviral transduction with the IL-1Ra cDNA followed by injection of the transduced cells into the metacarpal phalangeal joints of RA patients.

Gene Therapy and Molecular Biology Vol 1, page 109 Infection of arthritogenic splenocytes from DBA/1 mice transferred to SCID mice with a recombinant retrovirus carrying the TGF- 1 gene was effective in lowering inflammation of joints with already established arthritis and inhibiting the spreading of the disease to other joints in mice (Chernajovsky et al, 1997).

D. Direct gene delivery for RA A retroviral vector based on a murine leukemia virus was used to deliver the human growth hormone and lacZ genes to the synovium of the rabbit knee to test the efficacy of gene transfer and to develop an approach for the gene therapy of RA (Ghivizzani et al, 1997). An effective treatment of arthritis is via eliminating most or all of the activated synovial cells. The death factor Fas/Apo-1 and its ligand (FasL) play pivotal roles in maintaining self-tolerance and immune privilege; Fas is expressed constitutively in most tissues and is dramatically upregulated at the site of inflammation. Unlike Fas, however, the levels of FasL expressed in the arthritic joints are extremely low, and most activated synovial cells survive despite high levels of Fas expression. Delivery of the FasL gene via a replication-defective adenovirus by injection into inflamed joints conferred high levels of FasL expression, induced apoptosis of synovial cells, and ameliorated collagen-induced arthritis in DBA/1 mice (Zhang et al, 1997). A strategy was developed for inhibiting T lymphocyte retention and activation within the rheumatoid synovium (Chen et al, 1995). The inflamed synovium in RA is infiltrated by lymphocytes and monocytes, a process mediated by the enhanced binding of the very late antigen-4 (VLA-4) to vascular cell adhesion molecule-1 (V CAM-1) expressed on microvascular endothelial cells; VLA-4 binding appears to play a role in T cell retention and activation within the inflamed synovial membrane. Therefore, blocking of VLA-4 binding by utilizing a soluble congener of the VCAM-1 molecule might be of therapeutic efficiency for RA. Adenoviral infection of human synoviocytes carrying the cDNA for a secreted form of VCAM-1 (sVCAM-1) showed secretion of transgenic sVCAM-1 by ELISA of tissue culture supernatants. In vivo, transgenic sVCAM-1 expression was determined by immunohistochemical analysis and in situ hybridization of synovial tissue, and secretion of transgenic sVCAM-1 was demonstrated by ELISA of tidal knee lavage fluid. The results showed that recombinant adenovirus can mediate the expression of a biologically active sVCAM-1 by synoviocytes in vivo and suggested that this strategy may be useful for inhibiting T lymphocyte retention and activation within rheumatoid synovium. Ex vivo studies using this strategy have not been reported.

XXXV. Adenosine deaminase (ADA) deficiency and severe combined immunodeficiency (SCID) 109

Severe combined immunodeficiency is secondary to the deficiency in adenosine deaminase; the enzyme is involved in purine catabolism. The syndrome is characterized by defective B and T cell function caused by the large amounts of deoxyadenosine which is preferentially converted into the toxic compound deoxyadenosine triphosphate in T cells disabling the immune system (see Blaese et al, 1995 and the references cited therein). Affected individuals experience recurrent infections and the disease is usually fatal unless affected children are kept in isolation. One therapeutic approach has been to partially reconstitute the immune system by bone marrow transplantation from a human leukocyte antigen (HLA)-identical sibling donor (Hirschorn et al, 1981). An enzyme replacement therapy consisted of introducing bovine ADA enzyme conjugated with polyethylene glycol (PEG-ADA) in order to increase the circulation time of the ADA enzyme in the blood and other extracellular fluids (Hershfield et al, 1987). The first person to be treated ex vivo was a 4-year-old suffering with ADA deficiency in 1990. Protocol #2 treating ADA deficiency with autologous lymphocytes transduced ex vivo with the ADA gene was the second RAC-approved protocol. Because of this innovative work, the US Patent Office has issued in 1995 a patent covering all ex vivo gene therapy to French Anderson, Steven Rosenberg, and Michael Blaese. From 1990-1992, a clinical trial was initiated using retrovirus mediated transfer of the 1.5 kb ADA gene cDNA to T cells from two children with severe combined immunodeficiency following multiple transplantations of ex vivo modified blood cells; the integrated ADA gene was expressed for long periods (Blaese et al, 1995; Bordignon et al, 1995). The success of this ex vivo approach probably arose from that the ADA gene-corrected T cells acquired a survival advantage compared with uncorrected cells when transplanted into immunodeficient but ADA normal BNX mice (Ferrari et al, 1991) and humans (Kohn et al, 1995). Three neonates with ADA deficiency have been successfully treated later with hematopoietic stem CD34+ cells, isolated from their umbilical cord blood, transduced with the ADA gene under control of the LTR of the MoMuLV using retroviral vectors, followed by autologous transplantation (Kohn et al, 1995). Ex vivo studies have shown correction of the severe combined immunodeficiency in ADA-deficient mice by transfer of human peripheral blood lymphocytes transduced with a retroviral vector in cell culture carrying the ADA gene; the injected human cells survived for long times in mice and restored the immune functions (presence of human immunoglobulin and antigen-specific T cells) (Ferrari et al, 1991). Ex vivo correction of the defect in T cells from ADA-deficient patients with retroviral vectors gave to these cells an advantage for cell division against a background of slowly dividing uncorrected T cells after their transplantation (Karlsson, 1991).

Boulikas: An overview on gene therapy enzyme activity mediating phenotypic correction to neighboring GAA-deficient cells (Zaretsky et al, 1997). Aspartylglucosaminuria (AGU, appearance of aspartylglucosamine in the urine) is the only known human disease caused by an amidase deficiency, in this case by deficiency of the enzyme aspartylglucosaminidase (AGA) in virtually all cell types of patients; AGA deficiency fails to perform the final breakdown of asparagine-linked glycoproteins leading to the intralysosomal accumulation of uncleaved glycoasparagines and to their abnormal urinary excretion. AGU patients display progressive psychomotor retardation starting in early childhood. The most common mutation in the Finnish population responsible for AGA deficiency is a point mutation resulting in the amino acid substitution Cys-163 to Ser in the AGA gene (Ikonen et al, 1991). Retrovirus-mediated gene transfer was successfully used to correct the AGA gene in cultured human primary fibroblasts and lymphoblasts from AGU patients as a prelude to ex vivo human gene therapy; enzyme correction

XXXVI. Gaucher disease, lysosomal storage disease, and mucopolysaccharidosis VII Many mutations affecting the glucocerebrosidase gene have been defined as causes of the glycolipid storage disorder, Gaucher disease; disease symptoms are a result of macrophage engorgement secondary to this enzyme deficiency. The recombinant from of glucocerebrosidase imiglucerase protein is effective in treating the disease as a replacement therapy (reviewed by Beutler, 1997). An amphotropic producer cell line that synthesized viral particles carrying a fusion of the selectable MDR1 cDNA encoding P-glycoprotein (P-gp) and the human glucocerebrosidase gene was constructed; complete restoration of glucocerebrosidase deficiency in Gaucher fibroblasts was achieved using this retrovirus; selection of the transduced Gaucher fibroblasts in colchicine (MDR1 function) raised their glucocerebrosidase activity from nearly undetectable to normal levels; combination of much lower concentrations of colchicine and inhibitors of the Pgp pump (verapamil) allowed to select for high-level expression of MDR1 and glucocerebrosidase; this regimen, in clinical use for the treatment of multidrug-resistant malignancies, may find application for high level selection of a nonselectable gene such as glucocerebrosidase (Aran et al, 1996; see also Migita et al, 1995). Allogenic bone marrow transplantation (Parkman, 1986) or intravenous infusion of glucocerebrosidase (enzyme replacement therapy) in a patient with Gaucher's disease (Barton et al, 1990), although has partially corrected deficiencies in lysosomal enzymes, was not amenable to brain cells because of the brain barrier and cannot alleviate symptoms in the central nervous system. Protocols #38, 39, and 51 use glucocerebrosidase cDNA to transduce CD34+ autologous peripheral blood cells followed by intravenous injection of the transduced cells into patients with Gaucher's disease. CD34+ cells obtained from G-CSF mobilized peripheral blood stem cells or from bone marrow (#51) are being transduced ex vivo and reinfused into the patient at the National Institutes of Health and Childrenâ&#x20AC;&#x2122;s Hospital of Los Angeles (Dunbar and Kohn, 1996). Type II Glycogen Storage Disease, a deficiency of acid "-glucosidase (GAA), results in the abnormal accumulation of glycogen in skeletal and cardiac muscle lysosomes; this can have devastating effects ultimately leading to death; the wild-type enzyme was produced in deficient myoblasts after gene transfer with a retroviral vector carrying the cDNA for GAA. The transduced cells secreted GAA that was endocytosed via the mannose-6phosphate receptor into lysosomes of deficient cells and digested glycogen; thus, the transduced cells provided phenotypic correction to distant cells in the culture by secretion. Figure 33 shows that the GAA-transduced myoblasts (red fluorescence) were able to fuse with deficient myoblasts (green fluorescence) and provide

F i g u r e 3 3 . Immunofluorescent detection of in vitro fusion of "-glucosidase (GAA)-deficient muscle cells with myoblasts transduced with the GAA gene using a retroviral vector. The GAA-transduced myoblasts (red fluorescence) were able to fuse (arrows) with deficient myoblasts (green fluorescence) and provide enzyme activity. From Zaretsky JZ, Candotti F, Boerkoel C, Adams EM, Yewdell JW, Blaese RM, Plotz PH (1 9 9 7 ) Retroviral transfer of acid "-glucosidase cDNA to enzyme-deficient myoblasts results in phenotypic spread of the genotypic correction by both secretion and fusion. Hum Gene Ther 8, 1555-1563. Reproduced with the kind permission of the authors and Mary Ann Liebert, Inc.

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Gene Therapy and Molecular Biology Vol 1, page 111 further took place by cell-to-cell interaction between transduced and nontransduced cells in culture suggesting that only partial cell transduction might be sufficient to correct AGA deficiency in vivo (Enomaa et al, 1995). Mucopolysaccharidosis type VII (Sly syndrome or MPS VII) is a result of an inherited deficiency of #glucuronidase in humans, mice, and dogs (see Wolfe et al, 1992 for references). The symptoms are progressive degeneration in several tissues resulting from storage in lysosomes of undegraded glycosaminoglycans affecting the spleen, liver, brain, cornea, kidney, and skeletal muscle. Affected individuals display a reduced life-span which is 5 months in mice. Retroviral vectors have successfully treated mucopolysaccharidosis VII by somatic cell gene transfer in mouse models (Wolfe et al, 1992) or by implantation of ex vivo modified mouse skin fibroblasts (Moullier et al, 1993). Retrovirus-mediated transfer of the iduronate-2-sulfatase cDNA into lymphocytes is being applied for the clinical treatment of mild Hunter syndrome (mucopolysaccharidosis type II, protocol #65 on page 163).

XXXVII. Hereditary 1-antitrypsin ( 1-AT) deficiency Destruction of components of the extracellular matrix of the lung by neutrophil elastase (NE) is believed to be a critical event in the development of obstructive lung disease. "1-antitrypsin (also known as "1-proteinase inhibitor) is an antiprotease that protects the lung from destruction by the powerful protease NE. The hereditary "1-antitrypsin deficiency is caused by mutations in the coding region of the 12.2 kb-gene resulting in decreased serum and lung levels of "1-antitrypsin; affected individuals develop emphysema at age 30-40 (Crystal, 1990). Lung-derived epithelial cells have the capacity to synthesize functional "1-antitrypsin but also to increase the rate of its production when stimulated by specific inflammatory mediators, including oncostatin M, IL-1, and dexamethasone (Cichy et al, 1997). The respiratory epithelium has been a potential site for somatic gene therapy because of the possibility of direct delivery of a functional gene by tracheal instillation. A drawback for retrovirus-mediated gene transfer arises from that the majority of alveolar and airway epithelial cells are terminally differentiated and only a small fraction of these cells are proliferating and amenable to recombinant retrovirus infection; however, lung epithelial cells are prone to adenovirus infections because host cell replication is not required for expression of adenoviral proteins (see Rosenfeld et al, 1991). The adenovirus major late promoter was linked to a human "1-antitrypsin gene for its transfer to lung epithelia of cotton rat respiratory pathway as a model for the treatment of "1-antitrypsin deficiency; the cotton rat is an animal commonly used to evaluate the pathogenesis of adenoviral respiratory tract infections. Both in vitro and in

vivo infections have shown production and secretion of "1-antitrypsin by the lung cells: cells, removed by brushing the epithelial surface of the tracheobronchial tree from the lungs of cotton rats demonstrated the possibility of their infection in culture and secretion of the therapeutic protein; following direct infection of the animals in vivo, demonstrated that the protein was synthesized and secreted in the epithelial lining fluid of the lung for over 1 week (Rosenfeld et al, 1991). i.v. administration of a full-length cDNA encoding human "1-antitrypsin (100 ng/mouse) encapsulated in small liposomes resulted in expression in liver parenchymal cells as shown on immunohistochemical liver sections; this effect remained for at least 2 weeks and some enzyme could be detected in plasma (AliĂąo et al, 1996). The human "1-antitrypsin gene was transferred to lungs of rabbits; immunohistochemical staining showed "1-AT protein in the pulmonary endothelium following intravenous administration, in alveolar epithelial cells following aerosol administration, and in the airway epithelium by either route (Canonico et al, 1994). A recombinant adenovirus vector was also used for "1-AT cDNA transfer (Gilardi et al, 1990). "1-AT cDNA in an adenoviral vector was administered by retrograde ductal instillation to the submandibular glands of male rats; transient expression took place in salivary glands (Kagami et al, 1996). Guo et al (1996) have evaluated the transcriptional activities of 5 viral and cellular enhancer/promoter elements, showing either high-level or hepatocyte-specific expression following transient transfection into hepatoma cells using recombinant adenoviruses expressing human "1-antitrypsin; the human elongation factor 1" gene promoter produced 2 Âľg/ml serum level of human "1antitrypsin, which is physiologic in humans and will be therapeutic for patients with "1-antitrypsin deficiency.

XXXVIII. Approaches to the gene therapy of Parkinsonâ&#x20AC;&#x2122;s disease (PD) A. Etiology and mechanisms of destruction of neurons in PD First described by James Parkinson in 1817 this neurodegenerative disorder is characterized by resting tremor, postural instability and bradykinesia (slow movement); surviving neurons display intracytoplasmic inclusions known as Lewy bodies. PD symptoms ensue when the pars compacta region of the substantia nigra (black substance) at the base of the brain loses neurons that normally issue motion-controlling signals (dopamine) to the striatum (divided into caudate nucleus and putamen). The death of neurons is believed to be caused by oxygen free radical damage; brain contains unusually low levels of antioxidants. This damage might be caused by a decline in the activity of the mitochondrial complex I. PD can be induced in experimental animals by selective destruction of the dopaminergic neurons of the substantia nigra by the neurotoxic drug 1-methyl-4-phenyl111

Boulikas: An overview on gene therapy 1,2,3,6-tetrahydropyridine (MPTP) through inhibition of complex I of the mitochondrial respiratory chain (see Polymeropoulos et al, 1996 and the references cited therein). MPTP was found as an impurity in heroin and explained some earlier observations of addicts who became almost completely immobile after making use of the drug, a symptom characteristic of severe PD. MPTP crosses the brain-blood barrier and is converted by mitochondrial monoamine oxidase B into a reactive molecule that inhibits the complex I enzyme resulting in energy deficit and increase in free radicals in the cell (reviewed by Youdim and Riederer, 1997). There is substantial evidence which implicates immune mechanisms in the destruction of neurons. The substantia nigra of Parkinsonâ&#x20AC;&#x2122;s patients contains active microglia which, after stimulation by cytokines, could produce the free radical nitric oxide which can penetrate the cell membrane of vicinal neurons, inhibit the complex I mitochondrial enzyme and activate signal transduction pathways. Furthermore, NO with superoxide, emitted by hyperactive microglia, can free iron ions from intracellular stores which can oxidize dopamine into neuromelanin, a molecule that acts as an oxidant when complexed with transition metals. These oxidative stress mechanisms could trigger apoptosis in neurons. Excessive release of the neurotransmitter glutamate (known to occur in stroke) into the striatum and substantia nigra could induce a similar cascade of NO and free radical damage. These mechanisms suggest that excessive stressful conditions in predisposed individuals might precipitate the onset of PD symptoms.

factors in the brain is under investigation on humans. Also in clinical trials are strategies of direct implantation of dopamine-producing cells into the brain of patients (Youdim and Riederer, 1997). Ex vivo and in vivo gene therapy strategies for PD have a promising future (see below).

C. Candidate genes for PD A susceptibility gene for PD has been mapped to chromosome 4q21-q23 by genotyping genomic DNA from a large family in Contursi in the Salemo province of Southern Italy where 60 individuals out of 592 members are affected by PD at an average age of 46. A total of 140 genetic markers were typed in the pedigree and only those associated with this chromosomal region were altered showing recombination events in PD patients in this family; this type of recombination does not involve expansions of the CAG trinucleotide repeat (Polymeropoulos et al, 1996). The neurologic abnormalities associated with PD were thought to result from a severe reduction in L-DOPA as a consequence of degeneration of dopaminergic neurons of the nigrostriatal pathway. L-DOPA is synthesized from tyrosine by the enzyme tyrosine hydroxylase (TH); LDOPA is then converted into the neurotransmitter dopamine by a decarboxylase. Besides TH, other genes whose malfunction has been linked to PD include glutathione peroxidase, a brain-derived neurotrophic factor, catalase, amyloid precursor protein, Cu/Zn superoxide dismutase, and debrisoquine 4-hydroxylase; however, none of these candidate genes are found in the 4q21-q23 region to be linked as etiologic agents of PD; instead, candidate genes in the 4q21-q23 region include alcohol dehydrogenase, formaldehyde dehydrogenase, synuclein, and UDP-N-acetylglycosamine phosphotransferase (Polymeropoulos et al, 1996). A mutation was identified in the "-synuclein gene, which codes for a presynaptic protein thought to be involved in neuronal plasticity, in the Italian kindred with autosomal dominant inheritance for the PD phenotype (Polymeropoulos et al, 1997). The missense mutation in the "-synuclein gene suggested that at least some fraction of familial PD with diffuse Lewy bodies is the result of an abnormal protein that interferes with normal protein degradation leading to the development of inclusions and ultimately neuronal cell death. Furthermore, a peptide fragment of "-synuclein is known to be a constituent of Alzheimer's disease plaques; there may be common pathogenetic mechanisms involved in "-synuclein mutations in PD and #-amyloid and presenilin gene mutations in Alzheimer's disease (Nussbaum and Polymeropoulos, 1997).

B. Drug treatment of PD The first medicament in the mid-1900s included extracts of the deadly nightshade plant which inhibited the activity of acetylcholine in the striatum; acetylcholine overexcites striatal neurons that projected to higher motor regions of the brain, an effect normally counteracted by dopamine. Later in 1960s L-DOPA, which is converted into dopamine, proved valuable for treatment of PD patients; dopamine itself cannot cross the blood-brain barrier (a network of specialized blood vessels that control which substances are allowed to pass from the blood into the central nervous system). Drugs that mimic the actions of dopamine (agonists) have also been used. Selegiline (also called deprenyl), an inhibitor of monoamine oxidase B, the enzyme that breaks down dopamine in the astrocytes and microglia, is of therapeutic potential (reviewed by Youdim and Riederer, 1997). Amantadine is used to block the effects of glutamate in substantia nigra. Antioxidants able to cross the brain-blood barrier could have a protective effect on the destruction of neurons. Unfortunately, the first indications show that vitamin E in the low doses tested, which can cross to some extent the brain-blood barrier, is ineffective; however, the effect of higher doses of vitamin E need to be investigated. The efficacy of glial-derived neurotrophic factor (GDNF) injected into the brain of PD patients is in trials. Rasagiline, which could activate neuronal growth

D. Gene and cell therapy for PD 112

Gene Therapy and Molecular Biology Vol 1, page 113 concluded that there is sufficient aromatic L-amino acid decarboxylase near striatal grafts producing L-DOPA and that the close proximity of L-amino acid decarboxylase to TH-producing cells is detrimental for optimal dopamine production (Wachtel et al, 1997).

1. Grafting of dopamine neurons Transplantation of human embryonic dopamine neurons have been performed on patients with Parkinson's disease but the amelioration of the symptoms is transient; death of therapeutic cells was thought to arise from hypoxia, oxidative stress, and trauma during preparation and grafting of the cells. Grafting of dopamine neurons into transgenic mice overexpressing the Cu/Zn superoxide dismutase increased 4-fold the survival of the transplanted cells providing a direct support to the free radical-mediated death of dopaminergic neurons in brain tissue grafts (Nakao et al, 1995). Cells transduced with tyrosine hydroxylase and GTP cyclohydrolase I were grafted alone or in combination with cells transduced with aromatic L-amino acid decarboxylase into the 6-hydroxydopamine-denervated rat striatum; it was

2. Tyrosine hydroxylase (TH) Since adult brain cells are nonproliferative, they are refractory to retroviral infection that could deliver theTH gene to the brain to alleviate degeneration at the nigrostriatal pathway. Gene therapy of PD has been approached ex vivo using PD animal models with TH deficiency. Unilateral destruction of dopaminergic nigrostriatal neurons in PD animal models with 6hydroxydopamine and administration of apomorphine causes PD rats to turn contralaterally (7-15 rotations/min).

F i g u r e 3 4 . Transfer of the tyrosine hydroxylase gene to primary muscle cells followed by transplantation of these cells to brains of TH-deficient rats has alleviated the number of collateral rotations of the animals which are models for Parkinsonâ&#x20AC;&#x2122;s disease. Adapted from Jiao et al, 1993. Reproduced from Boulikas T (1 9 9 6 b ) Gene therapy to human diseases: ex vivo and in vivo studies. Int J Oncol 9, 1239-1251. With the kind permission from the International Journal of Oncology.

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Gene Therapy and Molecular Biology Vol 1, page 114 peptide; the 256 kDa single-chain protein composed of the homologous domains A1-A2-B-A3-C1-C2 is processed by proteolysis to a heterodimer composed of a heavy chain (90-200 kDa) and a light chain (80 kDa) which circulates in plasma. Both the 186 kb gene encoding human FVIII and the 7.2 kb cDNA sequences are known; recombinant FVIII has been expressed from both intact cDNA and a cDNA lacking the B domain; however, the expression of the protein from cell culture was 100-1000 times lower than the expression of other recombinant proteins. This is due to the large size of the protein, its required proteolytic processing, instability of mRNA, abnormal secretion from inefficient transport from the endoplasmic reticulum to the Golgi, and the required N- and O-linked glycosylation for biological activity. Recombinant FVIII is being administered to approximately 50% of the patients. Biologically active FVIII has been produced recently in the milk of transgenic pigs by targeting expression of human FVIII cDNA to the mammary gland of the animals; the expression of the transgene was driven by regulatory sequences from the mouse whey acidic protein gene (Paleyanda et al, 1997). Infusion of purified factor VIII is the most widely used therapy; however, protein replacement suffers from transfusion-associated complications (AIDS and hepatitis B and C infections); over 50% of the patients treated from 1977 to 1985 were infected with HIV. Improved manufacturing procedures and production of recombinant factor VIII have reduced infectious complications. Hemophilia A is particularly amenable to gene therapy because even a slight increase in blood plasma of factor VIII can convert a severe form of the disease to a mild form. Application of retrovirus-mediated transfer of factor VIII gene has been hampered by a 100- to 1000-fold reduction in mRNA accumulation and protein production in cells, as well as in retrovirus titer, because of the presence of a secondary structure (arising from inverted repeats) within the DNA coding region of clotting factor VIII gene (Lynch et al, 1993; Chuah et al, 1995). Partial solution to this problem has been provided by Chuag and coworkers (1995) who determined that insertion of a 5' intron in the retrovirus vector increased 20-fold gene expression and 40-fold virus titer after transfection of the human T cell line SupT1, human Raji Burkitt B lymphoblastoma and other cell lines. An more exciting approach has been the use of a Bdomain-deleted form of factor VIII; at the protein level, this domain is not required for pro-coagulant activity and retains the thrombin-cleavage sites. Transferrin-mediated transfection of fibroblasts and myoblasts with B-domaindeleted factor VIII gene followed by implantation into mice gave therapeutic levels of factor VIII in the blood of the animals for 24 hours (Zatloukal et al, 1994). A similar ex vivo transfection procedure used retroviral vectors for the introduction of B-domain$deleted factor VIII gene into primary mouse fibroblasts in culture; use of the MFG vector, which utilizes authentic viral splicing

Implantation of immortalized rat fibroblasts releasing L-dopa into the cell culture medium (Wolff et al, 1989), of primary fibroblasts (Fisher et al, 1991) and myoblasts (Jiao et al, 1993), stably transfected in culture with the TH gene, reduced behavioral abnormalities in PD animal models and the number of contralateral rotation dropped to 4 rotations/min (Figure 34). Direct injection of lipofectin-plasmid DNA complexes containing the TH gene under the influence of the SV40 promoter/enhancer (pSVK3 plasmid of Pharmacia) has also shown expression of TH into striatal cells compensating for the loss of the intrinsic striatal dopaminergic input reducing quickly and significantly the rotational abnormalities in rat models (Cao et al, 1995). A different approach has been aimed at converting endogenous striatal cells into L-dopa-producing cells; this was obtained by infection of 6-hydroxydopamine-lesioned rats, used as a model of PD, with a defective herpes simplex virus type 1 vector expressing TH (During et al, 1994). Recombinant adenovirus are attractive delivery vehicles of genes to alleviate PD symptoms because they can transduce both quiescent and actively dividing cells, thereby allowing both direct in vivo gene transfer and ex vivo gene transfer to neural cells; because the brain is partially protected from the immune system, the expression of adenoviral vectors can persist for several months with little inflammation (reviewed by Horellou and Mallet, 1997). 3. Glial cell line-derived neurotrophic factor The rat glial cell line-derived neurotrophic factor (rGDNF), a putative central nervous system dopaminergic survival factor, was evaluated for its ability to protect nigral dopaminergic neurons in the progressive Sauer and Oertel 6-hydroxydopamine (6-OHDA) lesion model of Parkinson's disease. Perinigral injections to rats of rGDNF protected a significant number of cells when compared with cell counts of rats injected with a recombinant AAV carrying the lacZ gene (94% vs. 51%, respectively); this treatment gave 85% of tyrosine hydroxylase-positive cells (vs. only 49% in the lacZ group) (Mandel et al, 1997; see also Bohn and Choi-Lundberg, 1998, this volume).

XXXIX. Gene therapy of hemophilia A and B A. Gene therapy of hemophilia A Hemophilia A, characterized by hemorrhagic episodes of which the spontaneous intracranial bleeding could result in crippling or death, affects 1 in 10,000 males. It is caused by a deficiency in Factor VIII (FVIII), crucial in blood coagulation, responsible for accelerating activation of factor X by factor IXa in the presence of calcium and phospholipids. Human FVIII is synthesized as a 2351amino acid precursor protein with a 19- amino acid signal

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Gene Therapy and Molecular Biology Vol 1, page 115 signals and lacks a selectable marker gene was crucial in producing high titer viral stocks (Dwarki et al, 1995). This procedure was followed by surgical implantation into the peritoneal cavity in SCID mice of 15 million cells in the form of neo-organs formed on expanded poly(tetra fluoroethylene) (PTFE) fibers after they were coated with type I collagen from rat tail. Coated fibers were arranged to the bottom of tissue culture dishes and genetically modified cells were allowed to solidify on the PTFE fibers for 1-2 days; the levels of factor VIII obtained were 501000 ng/ml which are 10-fold higher than those required for correction of hemophilia A (Dwarki et al, 1995). Efficient delivery of factor VIII was also observed after direct i.v. or i.p. injection but not after i.m. or s.c. injection of genetically-modified cells; this difference might arise from protease levels in the extracellular space of muscle and skin (Dwarki et al, 1995). The subject is being reviewed in depth by Connelly and Kaleko (1998) and Hoeben (1998) in this volume.

factor IX gene in adenovirus vectors by injection into the splenic veins of 1.6 to 2.2 pfu/Kg adenovirus (Kay et al, 1994). The therapy, however, obtained was transient and although these animals displayed 300% factor IX levels in their blood at day 2 from treatment, the levels dropped to 1% by three weeks and to 0.1% by 2 months (Kay et al, 1994). Since T cell immunity was responsible for attacking and eliminating virally-transduced cells from the body, clearing the corrected cells from the liver of animals, daily administration of 19.5 mg/Kg cyclosporin A led to prolongation of the therapeutic effect and to the persistence of adenovirus-transduced cells (10% of factor IX levels by two months (Fang et al, 1995; Figure 35). Cyclosporin A (CsA) is a cyclic peptide, fungal metabolite, displaying low myelotoxicity but toxic to T cell lymphocytes; it is widely used for immunosuppression of individuals receiving renal and other organ transplants but also for the therapy of autoimmune diseases. CsA inhibits activation of IL-2 gene, an early event required for T cell activation and this mechanism is thought to govern its immunosuppressive effects. However, CsA also inhibits the activity of TBP required for transcription from the adenovirus major late promoter. Recombinant adenovirus-transduced cells were able to elute host immune surveillance in dogs by cyclosporin A treatment (Fang et al, 1995). Similar conclusions were reached by Dai and coworkers (1995) using adenoviral vectors for delivering the canine factor IX gene into the hind leg muscle of mice: whereas in nude mice a high level of expression of FIX protein was detectable for 300 days, expression of FIX protein lasted for 7-10 days in normal mice. CD8 + lymphocytes were localized in the site of injection; both cell-mediated and humoral immune responses were found to be responsible for eliminating the adenovirus-infected cells from the organism. Recently, successful transduction of the mouse liver in vivo after a single hepatic gene transfer of F.IX cDNA in an AAV vector was achieved; persistent and curative concentrations of functional human factor IX were detected in the blood of the animals (Snyder et al, 1997). Intramuscular injection of a recombinant AAV vector expressing human factor IX (hF.IX) into hindlimb muscles of C57BL/6 mice and Rag 1 mice demonstrated the presence of hF.IX protein by immunofluorescence staining of muscles harvested 3 months after injection; however, no hF.IX was detected in the plasma of immunocompetent C57BL/6 mice because these animals had developed circulating antibodies to hF.IX. Rag 1 mice on the other hand, which carry a mutation in the recombinase activating gene-1 and thus lack functional B and T cells, displayed therapeutic levels (200-350 ng/ml) of F.IX in the plasma in addition to muscle cells; F.IX levels gradually increased over a period of several weeks before reaching a plateau that was stable 6 months after injection. Furthermore, these studies have demonstrated colocalization of hF.IX and collagen IV in interstitial spaces between muscle fibers; this was explained following identification of collagen IV as a F.IX-binding protein (Herzog et al, 1997).

B. Gene therapy of hemophilia B Hemophilia B is caused by a defect in the blood clotting factor IX (FIX) affecting about 1 in 30,000 males. The therapy consists on administration of factor IX concentrates prepared from human plasma, a fact that led to the infection of hemophiliacs with HIV and hepatitis B virus in the 80s. Current research efforts are focused on the delivery of factor IX gene using ex vivo transduction of primary myoblasts in mice with factor IX gene followed by transplantation of the transduced cells (Dai et al, 1992; Yao et al, 1994). Mouse primary myoblasts were infected with retrovirus expressing the canine factor IX under control of mouse muscle creatine kinase and human CMV promoter; successfully infected myoblasts, selected in the presence of G418, were injected into the hindlegs of recipient mice; secreted canine factor IX was monitored in the plasma. Sustained expression of factor IX for over six months without any apparent adverse effects on the recipient mice was obtained; however, the levels of the factor IX protein secreted into the plasma (10 ng/ml for 7 10 injected cells) were not sufficient to be of therapeutic value but 100 times below the desired levels (Dai et al, 1992). Dogs, lacking a functional factor IX gene, have been used as animal models for hemophilia B. The liver of the animals is the organ responsible for the production of factor IX; direct infusion of recombinant retroviral vectors, carrying the canine factor IX gene, into the portal vein cannulated into a splenic vein in animals previously subject to two-thirds hepatectomy resulted in the expression of low levels of factor IX for up to about 5 months; about 0.3-1% of hepatocytes were found to be transduced and stained blue with X-Gal in liver sections when the #-galactosidase gene of E. coli was delivered with the same retroviral vector (Kay et al, 1993). A sustained partial correction of the defect was succeeded in hemophilia B dogs by directly delivering the 115

Gene Therapy and Molecular Biology Vol 1, page 116

F i g u r e 3 5 . Transfer of the Factor IX gene for the treatment of hemophilia B. The FIX gene under control of RSV LTR in a recombinant adenovirus was infused into the portal vein of hemophiliac dogs (see text for details). Reproduced from Boulikas T (1 9 9 6 b ) Gene therapy to human diseases: ex vivo and in vivo studies. Int J Oncol 9, 1239-1251. With the kind permission from the International Journal of Oncology.

circulation for at least up to 10 months (Wang JM et al, 1997).

A mouse model for hemophilia B was generated by homologous recombination-mediated disruption of the clotting factor IX gene; the factor IX coagulant activities for wild-type (+/+), heterozygous (+/-), and homozygous (/-) mice were 92%, 53%, and <5%, respectively. Plasma factor IX activity in the deficient mice (-/-) was restored by introducing wild-type murine FIX gene via adenoviral vectors (Wang L et al, 1997). Primary skeletal myoblast-mediated gene transfer was tested for achieving a long-term stable systemic production of human factor IX in SCID mice; a hFIX minigene under the control of a #-actin promoter with the muscle creatine kinase enhancers was used; myotubes derived from the myoblasts produced 1,750 ng hFIX/106 cells/24 hours in culture; intramuscular injection of 5-20x 106 myoblasts to SCID mice stably produced hFIX into the systemic

XL. Gene therapy of hypertension A. Molecular mechanisms of high blood pressure Blood pressure can be altered by mutations in at least 10 genes which alter blood pressure through a common pathway, affecting salt and water reabsorption in the kidney. Probably the most promising lead has involved the genes governing the structure of angiotensinogen, the substrate in the renin reaction. Disorders associated with hypertension are the glucocorticoid-remediable aldosteronism, the syndrome of apparent mineralocorticoid

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Gene Therapy and Molecular Biology Vol 1, page 117 excess, and Liddle's syndrome. Syndromes linked with hypotension are the pseudohypoaldosteronism type 1, and Bartter's, and Gitelman's syndromes (reviewed by Hollenberg, 1996; Karet and Lifton, 1997). Identification of mutations in the gene encoding a subunit of the renal sodium channel in the Liddle syndrome has unraveled the mechanisms involved in this form of hereditary hypertension. Salt retention and secondary high blood pressure are the result of constitutive activation of the renal sodium channel by mutations in its gene. The pathophysiological basis of Liddle's syndrome (pseudoaldosteronism), a rare autosomal dominant form of arterial hypertension, has been found to rest on missense mutations or truncations of the #- and ! -subunits of the epithelial sodium channel (ENaC) which controls sodium reabsorption in the distal nephron; these mutations result in constitutive activation of the amiloride-sensitive distal renal epithelial sodium channel (Shimkets et al, 1994); its activity is under the control of aldosterone. The genes encoding ENaC have been identified and revealed an heteromultimeric structure of the protein composed of three homologous " # ! subunits. Most of the mutations on ENaC reported are either nonsense mutations or frame shift mutations which would truncate the cytoplasmic carboxyl terminus of the # or ! subunits of the channel. The original report was that Liddle's syndrome is caused by a premature stop codon that truncates the cytoplasmic carboxyl terminus of the encoded # subunit (Shimkets et al, 1994). Sequencing of the ENaC in a family with Liddle syndrome has also revealed a missense mutation in the # subunit which predicted substitution of Tyr by His at codon 618 (Tamura et al, 1996; reviewed by Schild, 1996); molecular variants of these genes might also contribute to the common polygenic forms of hypertension. Liddle's syndrome can also result from a mutation truncating the carboxy terminus of the ! subunit of this channel; this truncated subunit also activates channel activity. These findings indicate independent roles of # and ! subunits in the negative regulation of channel activity (Hansson et al, 1995). Expression of the mineralocorticoid receptor (MR) is restricted to some sodium-transporting epithelia and a few nonepithelial target tissues. The genomic structure of the human MR (hMR) revealed two different untranslated exons (1" and 1#), which splice alternatively into the common exon 2, giving rise to two hMR mRNA isoforms (hMR-" and hMR-#). Expression of the human MR transcripts in renal, cardiac, skin, and colon tissue samples was examined by in situ hybridization. Functional hypermineralocorticism was associated with reduced expression of hMR # in sweat glands of two patients affected by Conn's and Liddle's syndrom (Zennaro et al, 1997). Blood vessels and other tissues of hypertensive patients may have abnormal levels of several factors such as ( i ) the peptide kinin (produced from kininogen) and its specific receptor, ( i i ) kallikrein which processes

kininogen into kinin, ( i i i ) tissue kallikrein-binding protein (kallistatin), ( i v ) endothelial basic FGF, ( v ) the endothelium-derived nitric oxide (NO) and endothelial NO synthase, and ( v i ) the renin-angiotensin system (angiotensinogen, AT) and its receptor (AT1 receptor).

B. Gene therapy for hypertension The peptide kinin, after binding to its specific receptor, triggers a broad spectrum of biological effects such as vasodilatation, increase in vascular permeability, smooth muscle contraction and relaxation, and electrolyte and glucose transport; it plays an important role in homeostasis of blood pressure, sodium excretion in the kidney, and inflammatory disorders. It is produced from a larger oligopeptide precursor, kininogen, after cleavage by a specific protease calledkallikrein. Low levels of this protease in urine have been associated with hypertension. Repeated oral administration of swine pancreatic kallikrein can lower the blood pressure of hypertensive patients albeit in a temporal manner. Delivery of a 5.6 kb genomic clone or of a 834-bp cDNA clone encoding the kallikrein gene under control of the albumin promoter, CMV, RSV, or metallothionein promoters into the portal vein or tail vein of spontaneously hypertensive rats resulted in significant reduction of their blood pressure for about 5-6 weeks (Chao et al, 1996). Intravenous injection of an adenoviral vector containing the human tissue kallikrein gene under the control of a CMV promoter, into spontaneously hypertensive rats caused a sustained delay in the increase in blood pressure from day 2 to day 41 post-injection. The therapeutic effect was a result of transfection of the human gene into several rat tissues and human tissue kallikrein mRNA was detected in the liver, kidney, spleen, adrenal gland, and aorta (Jin et al, 1997). Treatment of hypertension with gene therapy has also been attained by transfer of the human tissue kallikreinbinding protein (HKBP) or kallistatin, a serine proteinase inhibitor (serpin). Transgenic mice overexpressing rat kallikrein-binding protein are hypotensive; kallistatin may function as a vasodilator in vivo. Delivery of the human kallistatin cDNA under control of the RSV 3' LTR in an adenoviral vector into spontaneously hypertensive rats by portal vein injection resulted in a significant reduction of blood pressure for 4 weeks; human kallistatin mRNA was detected in liver, spleen, kidney, aorta, and lung (Chen et al, 1997). Blood pressure is also controlled by other factors such as by the endothelium-derived nitric oxide (NO) in peripheral vessels. Transfer of the human endothelial NO synthase (eNOS) gene to spontaneously hypertensive rats gave a continuous supply of eNOS which caused a significant reduction of systemic blood pressure for 5 to 6 weeks; the effect continued for up to 10 weeks after a second injection (Lin et al, 1997). Angiotensinogen, a substrate for angiotensin I generation, is mainly produced in the liver, and is a unique 117

Boulikas: An overview on gene therapy component of the renin-angiotensin system. Mutations in the angiotensinogen gene are associated with hypertension. It is unclear whether circulating angiotensinogen is a ratelimiting step in blood pressure regulation. Transfer of antisense oligonucleotides against rat angiotensinogen into the rat liver via the portal vein diminished the expression of hepatic angiotensinogen mRNA and resulted in a transient decrease in plasma angiotensinogen levels in spontaneously hypertensive rats from day 1 to day 7 after the injection. Liposomes were used for the transfer of oligonucleotides containing viral agglutinins to promote fusion with target cells. This treatment resulted in a decrease in plasma angiotensin II concentration; transfection of sense and scrambled oligonucleotides did not show any changes in plasma angiotensinogen level, blood pressure, or angiotensinogen mRNA level (Tomita et al, 1995). The renin-angiotensin system plays an important role in blood pressure regulation; Phillips and coworkers (1997) have targeted the renin-angiotensin system at the level of synthesis (angiotensinogen, AT) and the receptor (AT1 receptor). Antisense oligonucleotides to AT1receptor mRNA and to angiotensinogen mRNA reduced blood pressure. The cDNA for the AT1 receptor was inserted in the antisense direction under control of CMV promoter in AAV (which was the system of choice among adeno, retrovirus, naked DNA and liposomes tested) and injected either directly in the hypothalamus (1 ÂľL) or in the lateral ventricles (5 ÂľL). A prolonged decrease in blood pressure in spontaneously hypertensive rats was achieved via delivery of antisense DNA for AT1-R causing a significant reduction in AT1 receptors. After a single injection there was a significant decrease of blood pressure (approximately 23 +/- 2 mm Hg) for up to 9 weeks (Phillips, 1997; Phillips et al, 1997). Blood vessels of spontaneously hypertensive rats were shown to be associated with sub-physiological amounts of endothelial basic FGF (bFGF); this decrease correlated both with hypertension and with a decrease in the endothelial content of nitric oxide synthase. As a consequence, transfer of thebFGF gene corrected hypertension, restored the physiological levels of bFGF in the vascular wall, significantly enhanced the number of endothelial cells with positive immunostaining for nitric oxide synthase, and ameliorated endothelial-dependent responses to vasoconstrictors (Cuevas et al, 1996). Gene therapy of hypertension has been achieved via transfer of the atrial natriuretic peptide (ANP) gene to genetically hypertensive rats; chronic infusion of ANP has been shown to cause natriuresis, diuresis, and hypotension in rats and humans. Intravenous delivery of the human ANP gene fused to the RSV 3'-LTR (shown to be expressed in heart, lung, and kidney) caused a significant reduction of systemic blood pressure in young hypertensive rats (4 weeks old), and the effect continued for 7 weeks; a maximal blood pressure reduction of 21 mm Hg in young hypertensive rats was observed 5 weeks after injection along with significant increases in urinary volume and urinary potassium output (Lin et al, 1995). 118

XLI. Gene therapy for obesity A. Molecular mechanisms of obesity Obesity results from an imbalance in the mechanisms which control storage of energy as triglycerides in adipose cells versus energy expenditure. The identification of the ob gene, and its encoded protein leptin, as subfunctional in obesity (Zhang et al, 1994) has advanced our understanding on the mechanisms of receival and integration of a feedback signaling reflecting the amount of adipose energy stores (reviewed by Spiegelman and Flier, 1996). Further advancement was the identification of thedb gene (also known as OB-R gene) on mouse chromosome 4 encoding the receptor of leptin which is expressed primarily in the hypothalamus and choroid plexus; OB-R is a single membrane-spanning receptor most related to the gp130 signal-transducing component of the IL-6 receptor, the GCSF receptor, and the LIF receptor (Tartaglia et al, 1995). The leptin is a hormone which is secreted from the white adipose tissue as a plasma protein, that acts in the hypothalamus to regulate the size of the body fat depot; the leptin with its receptor constitute a hormone-receptor pair that signals the status and magnitude of energy (fat) stores to the brain serving as an adipostatic signal to reduce food intake and body weight. Leptin might have evolved to inform the brain that energy stores in adipose tissue are sufficient but also to trigger a neuroendocrine response to fasting and limitation of food intake (Ahima et al, 1996). Leptin also acts acutely to increase glucose metabolism after intravenous and intracerebroventricular administrations; both intravenous or intracerebroventricular infusion of leptin into wild-type mice increased glucose turnover and glucose uptake (the plasma levels of insulin and glucose did not change), but decreased hepatic glycogen content; thus, the effects of leptin on glucose metabolism are mediated by the central nervous system (Kamohara et al, 1997). Plasma leptin was found to be highly correlated with body mass index (BMI) in rodents and in 87 lean and obese humans. In humans, there was variability in plasma leptin at each BMI group suggesting that there are differences in its secretion rate from fat. Weight loss due to food restriction was associated with a decrease in plasma leptin in samples from mice and obese humans (Maffei et al, 1995).

B. Animal models for obesity A number of rodent models for obesity are being used in the laboratories including db/db, fa/fa, yellow (Ay/a) VMH-lesioned, and those induced by gold thioglucose, monosodium glutamate, and by transgenic ablation of brown adipose tissue. The ob/ob mouse is genetically deficient in leptin. The expression of leptin mRNA and the level of circulating leptin are increased in these animal models, suggesting resistance to one or more of the actions of leptin. High-fat diet was found to evoke a

Gene Therapy and Molecular Biology Vol 1, page 119 sustained increase in circulating leptin in normal FVB mice and FVB mice with transgene-induced ablation of brown adipose tissue; leptin levels were found to accurately reflect the amount of body lipid across a broad range of body fat. However, despite increased leptin levels, animals fed a high-fat diet became obese without decreasing their caloric intake, suggesting that a high content of dietary fat limits the action of leptin (Frederich et al, 1995). Peripheral and central administration of microgram doses of OB (leptin) protein reduced food intake and body weight of ob/ob and diet-induced obese mice but not in db/db obese mice (Campfield et al, 1995). Body weight and adiposity appear to play a critical role in the timing of puberty in humans and rodents. Leptin is the signal that informs the brain that energy stores are sufficient to support the high energy demands of reproduction, and may be a major determinant of the timing of puberty. Indeed, injections of recombinant leptin (once daily) in female mice showed an earlier onset of three classic pubertal parameters (i.e., vaginal opening, estrus, and cycling) compared with saline-injected controls. In addition to its effects on body weight, chronic leptin treatment restored puberty and fertility to ob/ob mice with total leptin deficiency, and acute treatment with leptin substantially corrected hypogonadism in mice starved for 2 days without affecting body weight (Ahima et al, 1997). In a different study leptin was found to play a significant role in sustaining the male mouse reproductive pathways: all leptin-treated ob/ob males fertilized normal females mice that carried out normal pregnancies and deliveries, demonstrating that the reproductive capacity of sterile ob/ob males was corrected only with leptin treatment (Mounzih et al, 1997).

C. Glucocorticoids and obesity The crucial role of glucocorticoids in obesity and insulin resistance and the actions of the OB protein leptin on the hypothalamic-pituitary-adrenal axis suggest that there is an important interaction of leptin with the glucocorticoid system. Leptin inhibits cortisol production in adrenocortical cells and therefore appears to be a metabolic signal that directly acts on the adrenal gland (Bornstein et al, 1997). Glucocorticoids play a key inhibitory role in the action of leptin: the permissive role of glucocorticoids in the establishment and maintenance of obesity syndromes in rodents arises from that glucocorticoids restrain the effect of leptin. Leptin injected intracerebroventricularly in normal rats induced modest reductions in body weight and food intake. In marked contrast, the same dose of leptin had very potent and longlasting effects in decreasing both body weight and food intake when administered to adrenalectomized rats (Zakrzewska et al, 1997).

result of leptin insensitivity. To test this hypothesis Halaas et al (1997) have used subcutaneous infusion of leptin to lean mice; this resulted in a dose-dependent loss of body weight at physiologic plasma levels. Chronic infusions of leptin intracerebroventricularly (i.c.v.) at doses of 3 ng/hr or greater resulted in complete depletion of visible adipose tissue, which was maintained throughout 30 days of continuous i.c.v. infusion. Direct measurement of energy balance indicated that leptin treatment prevented the energy decrease that follows reduced food intake but did not increase total energy expenditure (Halaas et al, 1997). In New Zealand Obese (NZO) mice, which were unresponsive to peripheral leptin but were responsive to i.c.v. leptin, obesity was the result of leptin resistance most likely arising from a decreased transport of leptin into the cerebrospinal fluid(Halaas et al, 1997). Leptin administration reduced obesity in leptindeficient ob/ob mice. Van Heek et al (1997) examined whether diet-induced obesity in mice produces resistance to peripheral and/or central leptin treatment. In a diet-induced obesity model, mice exhibited resistance to peripherally administered leptin, while retaining sensitivity to centrally administered leptin (by a single intracerebroventricular infusion). Whereas C57BL/6 mice initially responded to peripherally-administered leptin with a marked decrease in food intake, leptin resistance developed after 16 days on high fat diet; however, central administration of leptin to peripherally leptin-resistant mice resulted in a robust response to leptin. Thus, the effects of additional leptin administration in obese humans who have high circulating leptin levels, especially after intravenous injection (peripheral) versus intracerebroventricular infusion, remain to be determined. This study also implies the importance of the tissue target for the delivery of the leptin gene for treatment of obesity in humans.

E. The leptin and leptin receptor genes Cloning of the gene encoding leptin (ob gene) and its receptor (db gene) has provided spectacular insights in elucidating the mechanisms involved in the control of food intake and body weight maintenance in obese and lean individuals. Transgenic mice lacking both alleles of either ob or db genes showed early onset obesity from excessive food intake and decreased energy expenditure, and in addition showed severe insulin resistance, diabetes, and sterility; administration of recombinant leptin had weight reducing effects (Campfield et al, 1995; Halaas et al, 1995; Pellymounter et al, 1995). However, the vast majority of obese humans appear to have excessively high levels of leptin and absence of mutations in the OB gene (Considine et al, 1995; Maffei et al, 1996). The nonsense mutation in the ob mouse which results in the conversion of arginine 105 to a stop codon of leptin gene was not present in human obesity. The defect in humans is localized in the signaling pathway in the brain which might involve: ( i ) the leptin receptor; ( i i ) the Tub protein, expressed in the hypothalamus, that

D. Therapy of obesity with leptin infusion High leptin levels are observed in obese humans and rodents, suggesting that, in some cases, obesity is the 119

Boulikas: An overview on gene therapy mediates the signaling to the interior of the hypothalamus cell; ( i i i ) the Agouti protein which is expressed in all tissues in obese mice but in the skin in normal mice, supposed to antagonize melanocortin signal to the CNS; and ( i v ) carboxypeptidase E (product of thefat gene) expressed in endocrine and neuroendocrine tissues (reviewed by Spiegelman and Flier, 1996). Using a reverse transcription PCR product of the coding region of the Obese (ob) gene from five lean and five obese subjects it was determined that there was 72% more ob gene expression in eight obese subjects compared to eight lean controls; thus, ob gene expression is increased in human obesity (Considine et al, 1995). Limitation in food intake reduced the reproductive competence, reduced the levels of thyroid hormone, and activated the adrenal-pituitary stress axis; these starvation adaptations were reversed with administration of recombinant leptin (Ahima et al, 1996). Obesity can result from a promotion in adipocyte differentiation. A number of mitogens (PDGF, EGF, FGF, tumor promoters), cytokines (TNF-", IL-1, IL-6, TGF-#, IFN-! ) and oncogenes inhibit adipocyte differentiation and, therefore, inhibited adipogenesis and obesity. Insulin plays a positive role in the differentiation of adipocyte precursors and stimulates lipogenesis in adipose cells but exerts a negative lipogenic response in fibroblasts; preadipocytes which express small amounts of insulin receptors require insulin or insulin-like growth factor-1 for optimal differentiation. The mechanism of inhibition of adipogenesis by mitogens involves activation of the mitogen-induced MAP kinase which phosphorylates at serine-112 the adipogenic transcription factor PPAR! (peroxisome proliferatoractivated receptor ! ). PPAR! acts as a dimer with RXR" to regulate adipocyte differentiation and sensitivity of the adipose cells to insulin. PPAR! is the high affinity receptor for the thiazolidinedione class of insulinsensitizing drugs and the PPAR! -drug binding results in a powerful adipogenic response; thus, factors which stimulate the MAP kinase phosphorylation of PPAR! , could cause resistance to insulin (Hu et al, 1996).

F. Gene transfer of the leptin gene This field is in its infancy; however, animal studies showed that treatment of obese persons is one of the future prospects of gene therapy. Adenovirus-mediated transfer of the mouse leptin cDNA in the ob/ob mouse (which is genetically deficient in leptin and exhibits both an obese and a mild non-insulin-dependent diabetic phenotype) resulted in dramatic reductions in both food intake and body weight, as well as the normalization of serum insulin levels and glucose tolerance (Muzzin et al, 1996). Thus, transfer of the leptin gene can correct the obese and diabetic phenotypes in the adult ob/ob mice; these studies also

provided confirming evidence that the control in body weight may be critical in the long-term management of non-insulin-dependent diabetes mellitus in obese patients. Infusion of a recombinant adenovirus containing the rat leptin cDNA under control of the CMV promoter to Wistar rats (8 ng/ml for 28 days) made them hyperleptinemic; these animals exhibited a 30-50% reduction in food intake and gained only 22 g over the experimental period versus 115-132 gained by control animals. Body fat was absent and plasma triglycerides and insulin levels were significantly lower in hyperleptinemic compared to control rats (Chen et al, 1996). On the contrary, delivery of the rat neuropeptide Y (NPY) cDNA with AAV and Sendai virosomes into the rat hypothalamic para-ventricular nucleus increased body weight and food intake for 21 days (Wu et al, 1996).

XLII. Profile of Biotech companies: the big race Research and drug development in Biotech Industry have played and continue to play an important role in advancing molecular medicine. A number of Biotechnology Companies sponsor ongoing gene therapy Clinical Trials led by Genetic Therapy Incorporated (part of Novartis, Gaithersburg, MD) with the phase III trial in glioblastoma. Biotech Companies have the expertise and resources in large scale production, regulatory affairs, and clinical development to take off the basic science of gene therapy into the skies of commercialization and clinical trials. This is more evident in USA where strong bridges between the academia and industry have been established and numerous biotechnology companies dedicated to gene therapy have been founded. As of 1997 Europe lags some 3 to 5 years behind USA in translating basic science into commercial technology; however, European Pharmaceutical Companies have invested $1.4 billion in the American gene and cell therapy industry (compared with a modest $140 million investment from USA biotech firms to European partners). It is certain that both USA and Europe will ultimately benefit from the successful gene therapy development (Martin, 1997). Table 8 gives a glimpse of the role of Biotech Industry in drug discovery related to gene therapy and the clinical trials sponsored.

Table 8. Biotech companies and drug development related to genes or cancer targeting 120

Gene Therapy and Molecular Biology Vol 1, page 121 Company

Drug/Gene type

Calydon (San Francisco, CA) Canji/Schering-Plough (San Diego, CA) Canji/Schering-Plough (San Diego, CA)

Prostate cancer gene therapy p53 to restore p53-mediated tumor suppression or apoptosis RB to restore RB-mediated cell cycle arrest

Cell Genesys, Inc

CC49-Zeta T cell receptor

Cell Genesys, Inc Chiron Corporation (Emerville & San Diego CA) IDUN Pharmaceutical (San Diego, CA) Incyte Pharmaceuticals (Palo Alto, CA)

CD4-zeta Chimeric Receptor HIV-1IIIB envelope protein (#108)

Introgen Therapeutics (Austin and Houston, TX)

p53 gene therapies to restore p53-mediated tumor suppression or apoptosis "1-Antitrypsin cDNA/cationic lipids Human insulin-like growth factor-1(hIGF-1) HSV-tk

Gene Medicine, Inc. (The Woodlands, Texas) Gene Medicine, Inc. Genetic Therapy, Inc. /Novartis (Gaithersburg, MD)

Genetic Therapy, Inc. /Novartis Genetic Therapy, Inc. /Novartis Genset Genta (La Jolla, CA) Genzyme Corporation (Framingham, MA) GenVec, Inc. Glaxo Wellcome Inc. LXR (Richmond, CA) Mitotix (Cambridge, MA) Myriad Genetics (Salt Lake City, UT) Onyx (Richmond, CA) Ribozyme Pharmaceuticals, Inc (Boulder, CO) Ribozyme Pharmaceuticals, Inc (Boulder, CO) Rh么ne-Poulenc Rorer Gencell (Vitry-sur-Seine, France &

Goal/Disease

Colon with hepatic metastases(#131); prostate (#148) Bladder(#140), non-small cell lung cancer (#156), lung, ovarian, and liver cancers Colorectal carcinomas expressing the tumorassociated antigen, TAG72 (#110) HIV (#85, 116) HIV

Status of drug d e v e l o p m e n t (end of 1997) Preclinical trials Phase I Early clinical trials Phase I /II

Phase I-II Phase I

bcl-2 inhibition

Preclinical trials

Gene sequence databases

License access to database for drug discovery to different companies Phase I-II

Lung, head and neck cancers, prostate cancer (#154)

Phase I

CFTR/adenovirus vector

"1-antitrypsin deficiency (#194) Cubital Tunnel syndrome (#164) brain tumors (#17), recurrent pediatric brain tumors (#45), recurrent glioblastoma (#32, 97, 99), astrocytoma (#42, 68), leptomeningeal carcinomatosis (#49), multiple myeloma (#75) Cystic fibrosis (#121)

Glucocerebrosidase cDNA

Gaucher disease (#39)

Phase I

Isolation of regulatory regions cancer

Early clinical trials

bcl-2 inhibition with antisense CFTR/adenovirus vector/cationic lipids VEGF121 cDNA to the ischemic myocardium CFTR/Cationic Liposome Complex Antiapoptotics Cell cycle inhibitors Diagnostic genetic testing p53 gene therapies killing cells that lack p53 Tat and Rev Hammerhead Ribozyme Growth factor inhibition

Phase I Phase I

Phase I

Cystic fibrosis (#120, 128, 129, 203) Coronary artery disease (#157)

Phase I

Cystic fibrosis

Phase I

BRAC analysis kit for breast cancer

Phase I

Advanced clinical trials Preclinical trials In the market Early clinical trials

HIV replication inhibition (#117)

Phase II Preclinical trials

p53

Head and neck squamous cell carcinoma (#152)

121

Phase II

Boulikas: An overview on gene therapy Santa Clara, California) SEQUUS Pharmaceuticals (Menlo Park, CA) SUGEN (Redwood City, CA) Targeted Genetics Corporation Targeted Genetics Corporation

Doxorubicin encapsulated into “stealth” liposomes Tyrosine kinase inhibitor to inhibit PDGF receptor HSV-tk E1A/ DC-Chol-DOPE

Targeted Genetics Corporation Vical, Inc.

CFTR/AAV Tumor idiotype

Vical, Inc. (San Diego, California)

#-2 Microglobulin

Vical, Inc.

Interleukin-2 cDNA

Vical, Inc.

HLA B7 cDNA

cDNA/cationic lipids

Kaposis sarcoma Gliomas HIV (#81) Metastatic breast or ovarian cancer, metastatic solid tumors that overexpress HER-2 /neu Cystic fibrosis Non-Hodgkin’s B-cell lymphoma (#161) Immunotherapy of advanced colorectal carcinoma, renal cancer (#195), melanoma (#196), metastatic malignancies (#201) Lymphoma (#198), solid tumor immunotherapy (#211) Renal cancer immunotherapy

In the market (Doxil); clinical trials for other cancers Clinical trials Phase I/II Phase I

Phase I Phase I/II Phase I/II

Phase I Phase I

substitution at an important domain of the encoded protein, in amino acid deletions, or in protein truncation. For example, deletion of three nucleotides resulting in deletion of a single phenylalanine at the protein level of the CFTR molecule is responsible for cystic fibrosis (Riordan et al, 1989); an A to T transversion leading to a premature stop at amino acid 337 in one allele and a C to T transition triggering an erroneous splice event and to frameshift in the other allele are associated with mutations in the ERCC6 helicase in Cockayne's syndrome (Troelstra et al, 1992). Mutations could result in failure of the protein to interact with DNA (mutated p53), with other regulatory proteins, or in enzymatic dysfunction of the molecule. Defects at the nuclear localization signal of a nuclear protein resulting in its cytoplasmic retention have been identified in cancer cells (Chen et al, 1995). Mutations in regulatory regions (promoters, enhancers) of genes, poorly understood but expected to play an important role in human disease, could result in down-regulation of the gene they dictate; mutation in the DNA-binding or transactivation domains of transcription factors are expected to down-regulate the expression of their target genes. Most important, mutations in genes involved in DNA repair are expected to have a domino effect on the appearance of mutations in other regions of the genome, since it is these genes that are responsible for removal of premutagenic lesions incurring by a number of xenobiotics by patrolling the human genome (Boulikas, 1996c).

XLIII. Prospects A. Gene discovery: novel horizons in gene therapy By the year 2005 the human genome project will be completed and by the end of 1998 the entire cDNA repertoire of the 125,000 human genes will be sequenced completely (Incyte Pharmaceuticals, Palo Alto, CA). Every single open reading frame of the human genome will become known. Genes implicated in human disease are being identified by mapping the mutation to a chromosomal locus after examining the DNA from a number of patients; candidate genes residing in this locus are then examined in a large number of patients for inactivating mutations (e.g. Polymeropoulos et al, 1996, 1997). A number of other classical techniques are aimed at identifying novel tumor suppressor genes or genes involved in metastasis, adding new weapons to the fight against cancer. The elucidation of many of the pathways implicated in the regulation of the cell cycle, signaling pathways with cytokines, and activation and action of transcription factors on the regulatory regions of genes lead to the discovery of new drugs interfering with those pathways. The elucidation of a number of players in apoptosis provides also targets not only for cancer treatment but for a number of neurodegenerative diseases. All these studies will ultimately provide new targets and genes for gene therapy.

B. Mutations in DNA and human disease

C. Regulatory regions and the MAR project

A number of human disorders have been linked to mutations in specific genes that result in loss of function of a specific protein in all somatic cells of the body. In the majority of cases known today the mutation is at the coding region of the gene resulting in one amino acid

The identification of the regulatory regions from the human genome should also become a first priority. Regulatory regions will provide new DNA control 122

Gene Therapy and Molecular Biology Vol 1, page 123 elements for the tissue-specific expression, episomal replication, insulation, and silencing of genes in gene therapy protocols but also targets, using small oligonucleotides (e.g. triplex) to abort transcription of specific genes. Regulatory regions include enhancers (ENHs, at least two for each gene), promoters (about 125,000 total, a significant fraction of which might be known because of their proximity to the 5' end), origins of replication (ORIs, about 50,000 have been estimated), silencers, locus control regions (LCRs, perhaps several thousand), and matrix-attached regions (seem to coincide with enhancers and ORIs). 445,000 is a modest estimate of the total number of regulatory regions in the human genome. Their size ranges from 100-500 bp but much more for LCRs and some ORIs. Identification of strong regulatory regions from the human genome is expected to provide strong promoter and enhancer sequences for the universal and cell type-specific expression of transgenes in gene therapy but also strong human ORIs able to sustain extrachromosomal replication of plasmids loaded with therapeutic genes in human and animal model tissues. Transcription factor recognition sequence databases can be used in conjuction with other software methods (DNA curvature, inverted repeats, triplex DNA, Z-DNA, phased nucleosomes) to predict regulatory regions from the large DNA sequence information arising from the human genome project (Boulikas, 1995b; Bode et al, 1998, this volume). A technology developed in our laboratory based on isolation and cloning of matrix-attached regions, shown to harbor a large fraction of regulatory regions from the human and other genomes, is being applied for identifying human regulatory regions (MAR project). MAR libraries include tissue-specific and tumor-specific regulatory regions. One particular MAR clone that has been extensively characterized (Boulikas et al, in preparation) represents the ORI, enhancer, and MAR of the human choline acetyltransferase gene, of crucial importance in neurological disorders including Alzheimer's disease. When a subfragment of only 513 bp of this MAR/ORI/ENH was placed at the flanks of the luciferase gene it was able to sustain episomal replication in human culture cells (K562 erythroleukemia) for more than 4 months. The actual 3.6 kb ChAT ORI region comprises a 1.2 kb silencer whose presence inhibits the ORI function; thus, mammalian origins of replication are much more sophisticated than viral ORIs and contain a number of control elements, including silencers, for the cell type and developmental stage-specific regulation. Identification and elimination of silencers from human ORIs is of importance in the exploitation of ORI fragments in the episomal replication of therapeutic genes. MAR sequences sorted out into MAR/ORI, MAR/enhancer and MAR/insulators can be used to promote extrachromosomal replication, to enhance the transcription of genes or to insulate genes from position effects from chromatin surroundings after integration. A number of studies show that MARs act as insulators of genes shielding them from position effect variegation from 123

neighboring chromatin domains in transgenic studies; this shielding results in a 2 to 1000-fold increase in the expression level of transgenes when MARs are included on both sides of the foreign gene (see Boulikas 1995b). Identification of tumor-specific MARs, such as identification of the MARs of the carcinoembryonic antigen (CEA) gene, the breast cancer/ovarian cancer BRCA1 gene, and others can lead to the development of plasmid vectors able to drive the expression of therapeutic genes in specific tumor cell types. In the postgenomic era, identification of a reasonable fraction of regulatory regions will revolutionarize our approaches to human disease.

D. What is next on gene therapy? Theoretically, most human disorders could constitute targets for gene therapy, aimed at correcting the defect either by transferring the wild-type gene in all somatic cells of the body or to those specific cell types responsible mainly for the synthesis of the particular protein (e.g. factor IX gene in liver cells of hemophilia B patients). Nuclear localization signal (NLS) peptides hooked to triplex-oligonucleotides or to plasmids, or complexation of plasmids with nuclear proteins possessing multiple NLSs are expected to increase nuclear localization and enhanced expression of foreign genes. A significant number of discoveries in molecular biology of human diseases have opened doors to the development of strategies for gene therapy. New genes whose mutations are responsible for human disease, from mild to life threatening, are being discovered and the molecular mechanisms are being unraveled. Many pieces of the puzzle aimed at elucidating mechanisms leading to human disease and the genes implicated have been solved and lie as scattered pieces of knowledge in various publications, lab notebooks, or patent applications. Preexisting Biotech Companies redefine their missions and new Biotech Companies are being founded to explore new discoveries and develop new drugs; to win the race in the fight against human disease, especially cancer and AIDS, we need to gather the right components into a successful assemble. Retroviruses, adenoviruses, AAV, HSV, naked plasmid delivery, and liposomes all have a good share as delivery vehicles for genes and it seems that they will be developed independently, each with its own strengths and limitations for particular gene therapy protocols. For example, liposomes have a distinct advantage over other systems for the delivery of oligonucleotides, stealth liposomes could prove their strength in the systemic delivery of genes by intravenous injection, retroviruses and adenoviruses for their high transfection efficiency, AAV for not stimulating inflammation, HSV as a vehicle for gene therapy to the nervous system, HIV and HSV vectors for their high payload capacity. Furthermore, adenoviruses, AAV, HSV1, HIV-1 vectors can transduce nondividing cells (T able 1 on page 29).

Boulikas: An overview on gene therapy

References

A lot has been learned about the involvement of the tumor suppressor p53 protein in cancer etiology. The current view is that an initiated tumor cell in the body, having mutations in one or more oncogenes needs to acquire loss in function in both alleles of p53 or other tumor suppressor gene in order to expand into the tumor cell mass. Expression of the wild-type (non-mutated form) of p53 arrests the proliferation in tumor cells and induces apoptosis (suicidal programmed death) by boosting the expression of the genes of p21, bax, and Gadd45 and by repressing the bcl-2 gene. Transfer of the p53 gene with adenovirus or retrovirus after intratumoral injection has successfully led to eradication of tumors in animal models and in human patients at advanced stages of non small cell lung cancer. Intratumoral injection, however, is not expected to be applicable to metastases very frequently associated with advanced stages of cancer. Stealth liposomes might offer a solution to this problem. Anti-angiogenesis therapy, both drug-mediated and gene therapy, would bring important ammunition in the fight against cancer. Improvements in oligonucleotide delivery in vivo, a very promising field that is in its infancy at the delivery level, will advance the field of pharmacogenomics by providing triplex-forming oligonucleotide drugs to inhibit the transcription of specific genes or ribozyme drugs to lower the mRNA level of a specific target protein. We expect the final victory of the human race on cancer to be accomplished over the next 10 years. Gene therapy would, no doubt, have an important role to play. It is likely that a combination of gene therapy (p53, HSV-tk, angiostatin) along with the already existing antineoplastic drugs (doxorubicin, cisplatin) but at subtoxic doses and at much lower concentrations than those used today, as well as less severe doses of radiation, would become routine regimens in hospitals for the eradication of all cancer types. In this respect, the emerging priority in gene therapy is to improve the efficiency of tissue targeting and gene delivery.

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Appendix 1. RAC-approved human gene therapy protocols Using retrovirus, protocols: 1-117 Using adenovirus: 118-157 With naked plasmid: 158-164 Adeno-Associated virus: 165, 166 Immunotherapy or other: 167-186 Cationic lipids: 187-220 on page 203-206 of following article (Martin and Boulikas, 1998) (Last updated: 21 November 1997) RETROVIRAL GENE DELIVERY

Disease

Protocol title

Procedures

Principal investigator

Gene Marking /Cancer

The Treatment of Patients with Advanced Cancer Using Cyclophosphamide, Interleukin-2 and Tumor Infiltrating Lymphocytes.

In Vitro /Tumor Infiltrating Lymphocytes /Retrovirus /Neomycin Phosphotransferase cDNA /Intravenous

Rosenberg, Steven A

Gene Therapy /Phase I /Monogenic Disease /Severe Combined Immune Deficiency due to Adenosine Deaminase Deficiency

Treatment of Severe Combined Immune Deficiency (SCID) due to Adenosine Deaminase (ADA) Deficiency with Autologous Lymphocytes Transduced with the Human ADA Gene: An Experimental Study.

In Vitro /Autologous Peripheral Blood Cells /CD34+ Autologous Peripheral Blood Cells /Cord Blood /Placenta Cells /Retrovirus /Adenosine Deaminase cDNA /Neomycin Phosphotransferase cDNA /Intravenous

Blaese, R. Michael

Gene Therapy /Phase I /Cancer /Melanoma /Immunotherapy

Gene Therapy of Patients with Advanced Cancer Using Tumor Infiltrating Lymphocytes Transduced with the Gene Coding for Tumor Necrosis Factor.

In Vitro /Tumor Infiltrating Lymphocytes /Retrovirus /Cytokine /Tumor Necrosis Factor cDNA /Neomycin Phosphotransferase cDNA /Intravenous

Rosenberg, Steven A

Gene Marking /Cancer /Acute Myelogenous Leukemia

Autologous Bone Marrow Transplant for Children with Acute Myelogenous Leukemia in First Complete Remission: Use of Marker Genes to Investigate the Biology of Marrow Reconstitution and the Mechanism of Relapse.

In Vitro /Autologous Bone Marrow Cells /Retrovirus /Neomycin Phosphotransferase cDNA /Bone Marrow Transplant

Brenner, Malcolm K.

Gene Marking /Cancer /Neuroblastoma

A Phase I /II Trial of High Dose Carboplatin and Etoposide with Autologous Marrow Support for Treatment of Stage D Neuroblastoma in First Remission: Use of Marker Genes to Investigate the Biology of Marrow Reconstitution and the Mechanism of Relapse.

In Vitro /Autologous Bone Marrow Cells /Retrovirus /Neomycin Phosphotransferase cDNA /Bone Marrow Transplant

Brenner, Malcolm K.

Gene Marking /Cancer /Neuroblastoma

A Phase II Trial of High-Dose Carboplatin and Etoposide with Autologous Marrow Support for Treatment of Relapse /Refractory Neuroblastoma Without Apparent Bone Marrow Involvement.

In Vitro /Autologous Bone Marrow Cells /Retrovirus /Neomycin Phosphotransferase cDNA /Bone Marrow Transplant

Brenner, Malcolm K

Gene Marking /Cancer /Chronic Myelogenous Leukemia

Autologous Bone Marrow Transplantation for Chronic Myelogenous Leukemia in which Retroviral Markers are Used to Discriminate between Relapse which Arises from Systemic Disease Remaining after PreparativeTherapy Versus Relapse due to Residual Leukemic Cells in Autologous Marrow: A Pilot Trial.

In Vitro /Autologous Bone Marrow Cells /Retrovirus /Neomycin Phosphotransferase cDNA /Bone Marrow Transplant

Deisseroth, Albert B

Gene Marking /Acute Hepatic Failure

Hepatocellular Transplantation in Acute Hepatic Failure and Targeting Genetic Markers to Hepatic Cells.

In Vitro /Autologous Hepatocytes /Retrovirus /Neomycin Phosphotransferase cDNA /Intrahepatic

Ledley, Fred D.

Gene Marking /Cancer /Melanoma

The Administration of Interleukin-2 and Tumor Infiltrating Lymphocytes to Patients with Melanoma.

In Vitro /Tumor Infiltrating Lymphocytes /Retrovirus /Neomycin Phosphotransferase cDNA /Intravenous

Lotze, Michael T

10.

Gene Therapy /Phase I /Cancer /Melanoma /Renal Cell /Colon /Breast /Immunotherapy

Immunization of Cancer Patients Using Autologous Cancer Cells Modified by Insertion of the Gene for Tumor Necrosis Factor (TNF).

In Vitro /Autologous Tumor Cells /Lethally Irradiated /Retrovirus /Cytokine /Tumor Necrosis Factor cDNA /Neomycin Phosphotransferase cDNA /Subcutaneous Injection

Rosenberg, Steven A

11.

Gene Therapy /Phase I /Cancer /Melanoma /Renal Cell /Colon /Immunotherapy

Immunization of Cancer Patients Using Autologous Cancer Cells Modified by Insertion of the Gene for Interleukin-2 (IL-2).

In Vitro /Autologous Tumor Cells /Lethally Irradiated /Retrovirus /Cytokine /Interleukin-2 cDNA /Subcutaneous Injection

Rosenberg, Steven A

159

Boulikas: An overview on gene therapy 12.

Gene Marking /Cancer /Acute Myelogenous Leukemia /Acute Lymphocytic Leukemia

Retroviral-Mediated Gene Transfer of Bone Marrow Cells during Autologous Bone Marrow Transplantation for Acute Leukemia.

In Vitro /Autologous Bone Marrow Cells /Retrovirus /Neomycin Phosphotransferase cDNA /Bone Marrow Transplant

Cornetta, Kenneth

13.

Gene Marking /Cancer /Melanoma /Renal Cell

The Treatment of Patients with Metastatic Melanoma and Renal Cell Cancer Using In Vitro Expanded and Genetically-Engineered (Neomycin Phosphotransferase) Bulk, CD8+ and /or CD4 + Tumor Infiltrating Lymphocytes and Bulk, CD8+ and /or CD4 + Peripheral Blood Leukocytes in Combination with Recombinant Interleukin-2 Alone, or with Recombinant Interleukin-2 and Recombinant " Interferon.

In Vitro /CD4+ Autologous Peripheral Blood Lymphocytes /CD8+ Autologous Peripheral Blood Lymphocytes /CD4+ Autologous Tumor Infiltrating Lymphocytes /CD8 + Autologous Tumor Infiltrating Lymphocytes /Retrovirus /Neomycin Phosphotransferase cDNA /Intravenous

Economou, James S. and Belldegrun, Arie

14.

Gene Therapy /Phase I /Cancer /Ovarian /ProDrug

In Vitro /Allogeneic Tumor Cells /Lethally Irradiated /PA317 /Retrovirus /Herpes Simplex Virus Thymidine Kinase cDNA /Ganciclovir /Intraperitoneal Administration

Freeman, Scott M

15.

Gene Therapy /Infectious Disease /Human Immunodeficiency Virus

Phase I Study to Evaluate the Safety of Cellular Adoptive Immunotherapy Using Genetically Modified CD8+ HIV-Specific T Cells in HIV Seropositive Individuals.

In Vitro /CD8+ Allogeneic Cytotoxic T Lymphocytes /CD8+ Syngeneic Cytotoxic T Lymphocytes /Retrovirus /Hygromycin Phosphotransferase /Herpes Simplex Virus Thymidine Kinase cDNA /Intravenous

Greenberg, Philip D. and Riddell, Stanley

16.

Gene Therapy /Phase I /Cancer /RelapsedRefractory Neuroblastoma /Immunotherapy

Phase I Study of Cytokine-Gene Modified Autologous Neuroblastoma Cells for Treatment of Relapsed /Refractory Neuroblastoma.

In Vitro /Autologous Neuroblastoma Cells /Allogeneic Partially HLA-Matched /Retrovirus /Cytokine /Interleukin-2 cDNA /Subcutaneous Injection

Brenner, Malcolm K.

17.

Gene Therapy /Phase I /Cancer /Brain / ProDrug

Gene Therapy for the Treatment of Brain Tumors Using Intra-Tumoral Transduction with the Thymidine Kinase Gene and Intravenous Ganciclovir.

In Vivo /Autologous Tumor Cells /PA317 /Retrovirus /Herpes Simplex Virus Thymidine Kinase cDNA /Ganciclovir /Intratumoral /Stereotactic Injection

Oldfield, Edward

Gene Transfer for the Treatment of Cancer.

Sponsor: Genetic Therapy, Inc. /Novartis 18.

Gene Marking /Cancer /Chronic Myelogenous Leukemia

Use of Two Retroviral Markers to Test Relative Contribution of Marrow and Peripheral Blood Autologous Cells to Recovery After Preparative Therapy.

In Vitro /Autologous Bone Marrow Cells /Autologous Peripheral Blood Cells /Retrovirus /Neomycin Phosphotransferase cDNA /Bone Marrow Transplant

Deisseroth, Albert B

19.

Gene Therapy /Phase I /Cancer /Melanoma /Immunotherapy

Immunization with HLA-A2 matched Allogeneic Melanoma Cells that Secrete Interleukin-2 in Patients with Metastatic Melanoma.

In Vitro /Allogeneic Partially HLA-Matched /Retrovirus /Cytokine /Interleukin-2 cDNA /Subcutaneous Injection

Gansbacher, Bernd

20.

Gene Therapy /Phase I /Cancer /Renal Cell /Immunotherapy

Immunization with Interleukin-2 Secreting Allogeneic HLA-A2 Matched Renal Cell Carcinoma Cells in Patients with Advanced Renal Cell Carcinoma.

In Vitro /Allogeneic Partially HLA-Matched /Retrovirus /Cytokine /Interleukin-2 cDNA /Subcutaneous Injection

Gansbacher, Bernd

21.

Gene Marking /Cancer /Multiple Myeloma

Retroviral-Mediated Gene Transfer of Bone Marrow and Peripheral Blood Stem Cells During Autologous Bone Marrow Transplantation for Multiple Myeloma.

In Vitro /CD34+ Autologous Peripheral Blood Cells /Intravenous /Autologous Bone Marrow Cells /Retrovirus /Neomycin Phosphotransferase cDNA /Bone Marrow Transplant

Dunbar, Cynthia

22.

Gene Marking /Cancer /Breast

Retroviral-Mediated Gene Transfer of Bone Marrow and Peripheral Blood Stem Cells During Autologous Bone Marrow Transplantation for Metastatic Breast Cancer.

In Vitro /CD34+ Autologous Peripheral Blood Cells /Intravenous /Autologous Bone Marrow Cells /Retrovirus /Neomycin Phosphotransferase cDNA /Bone Marrow Transplant

Dunbar, Cynthia

23.

Gene Marking /Cancer /Chronic Myelogenous Leukemia

Retroviral-Mediated Gene Transfer of Bone Marrow and Peripheral Blood Stem Cells During Autologous Bone Marrow Transplantation for Chronic Myelogenous Leukemia.

In Vitro /CD34+ Autologous Peripheral Blood Cells /Intravenous /Autologous Bone Marrow Cells /Retrovirus /Neomycin Phosphotransferase cDNA /Bone Marrow Transplant

Dunbar, Cynthia

24.

Gene Marking /Infectious Disease /Human Immunodeficiency Virus

A Study of the Safety and Survival of the Adoptive Transfer of Genetically Marked Syngeneic Lymphocytes in HIV Infected Identical Twins.

In Vitro /Syngeneic Peripheral Blood Lymphocytes /Retrovirus /Neomycin Phosphotransferase cDNA /Intravenous

Walker, Robert E

25.

Gene Marking /Cancer

Study on Contribution of Genetically Marked Peripheral Blood Repopulating Cells to Hematopoietic Reconstitution after Transplantation.

In Vitro /G-CSF Mobilized CD34+ Autologous Peripheral Blood Cells /Retrovirus /Neomycin Phosphotransferase cDNA /Bone Marrow Transplant

Schuening, Friedrich G.

160

Gene Therapy and Molecular Biology Vol 1, page 161 26.

Gene Marking /Cancer /Lymphoid Malignancies

Evaluation of the Use of Recombinant Human GCSF Stimulated Peripheral Blood Progenitor Cell Supplementation in Autologous Bone Marrow Transplantation in Patients with Lymphoid Malignancies.

In Vitro /G-CSF Mobilized Autologous Peripheral Blood Cells /Retrovirus /Neomycin Phosphotransferase cDNA /Bone Marrow Transplant

Schuening, Friedrich G

27.

Gene Marking /Cancer

A Trial of G-CSF Stimulated Peripheral Blood Stem Cells for Engraftment in Identical Twins.

In Vitro /G-CSF Mobilized CD34+ Autologous Peripheral Blood Cells /Retrovirus /Neomycin Phosphotransferase cDNA /Bone Marrow Transplant

Schuening, Friedrich G

28.

Gene Marking /Cancer /Chronic Lymphocytic Leukemia /Follicular Non-hodgkins Lymphoma

Use of Retroviral Markers to Identify Efficacy of Purging and Origin of Relapse Following Autologous Bone Marrow and Peripheral Blood Cell Transplantation in Indolent B Cell Neoplasms (Follicular Non-Hodgkin's Lymphoma or Chronic Lymphocytic Leukemia) Patients.

In Vitro /Autologous Bone Marrow Cells /Autologous Peripheral Blood Cells /Retrovirus /Neomycin Phosphotransferase cDNA /Bone Marrow Transplant

Deisseroth, Albert B

29.

Gene Therapy /Phase I /Cancer /Non-small Cell Lung Cancer /Antisense /Tumor Suppressor Gene

Clinical Protocol for Modification of Oncogene and Tumor Suppressor Gene Expression in Non-Small Cell Lung Cancer (NSCLC).

In Vivo /Autologous Tumor Cells /Retrovirus /p53 cDNA /K-ras Antisense /Intratumoral /Bronchoscope

Roth, Jack A

30.

Gene Marking /Cancer /Neuroblastoma

A Phase II Trial of the Baxter Neuroblastoma Bone Marrow Purging System Using Gene Marking to Assess Efficacy.

In Vitro /Autologous Bone Marrow Cells /Retrovirus /Neomycin Phosphotransferase cDNA /Bone Marrow Transplant

Brenner, Malcolm K

31.

Gene Therapy /Phase I /Cancer /Renal Cell /Immunotherapy

A Pilot Study of IL-4 Gene Modified Antitumor Vaccines.

In Vitro /Autologous Fibroblasts /Lethally Irradiated /In Combination with Untransduced Autologous Tumor Cells /Retrovirus /Cytokine /Interleukin-4 cDNA /Subcutaneous Injection

Lotze, Michael T. and Rubin, Joshua T

32.

Gene Therapy /Phase I /Cancer /Glioblastoma /Pro-Drug

Gene Therapy for the Treatment of Recurrent Glioblastoma Multiforme with In Vivo Tumor Transduction with the Herpes Simplex Thymidine Kinase Gene /Ganciclovir System.

In Vivo /Autologous Tumor Cells /PA317 /Retrovirus /Herpes Simplex Virus Thymidine Kinase cDNA /Ganciclovir /Intratumoral /Direct Injection

Van Gilder, John C.et al

Sponsor: Genetic Therapy, Inc. /Novartis 33.

Gene Marking /Cancer /Leukemia /Nonmalignant Disorders

Administration of Neomycin Resistance Gene Marked EBV Specific Cytotoxic T Lymphocytes to Recipients of Mismatched-Related or Phenotypically Similar Unrelated Donor Marrow Grafts.

In Vitro /Epstein-Barr Virus Specific Allogeneic Cytotoxic T Lymphocytes /Retrovirus /Neomycin Phosphotransferase cDNA /Bone Marrow Transplant

Heslop, Helen E.

34.

Gene Marking /Cancer /Acute Myelogenous Leukemia

Assessment of the Efficacy of Purging by Using Gene-Marked Autologous Marrow Transplantation for Children with Acute Myelogenous Leukemia in First Complete Remission.

In Vitro /Autologous Bone Marrow Cells /Retrovirus /Neomycin Phosphotransferase cDNA /Bone Marrow Transplant

Brenner, Malcolm K

35.

Gene Therapy /Phase I /Cancer /Renal Cell /Immunotherapy

Phase I Study of Non-Replicating Autologous Tumor Cell Injections Using Cells Prepared With or Without Granulocyte-Macrophage Colony Stimulating Factor Gene Transduction in Patients with Metastatic Renal Cell Carcinoma.

In Vitro /Autologous Tumor Cells /Lethally Irradiated /Retrovirus /Cytokine /GranulocyteMacrophage Colony Stimulating Factor cDNA /Subcutaneous Injection

Simons, Jonathan

36.

Gene Therapy /Phase I /Cancer /Melanoma /Immunotherapy

A Phase I Trial of Human ! InterferonTransduced Autologous Tumor Cells in Patients With Disseminated Malignant Melanoma.

In Vitro /Autologous Tumor Cells /Lethally Irradiated /Retrovirus /! Interferon cDNA /Subcutaneous Injection

Seigler, Hilliard F and Merritt, James A

37.

Gene Therapy /Phase I /Cancer /Ovarian /Chemoprotection

Use of Safety-Modified Retroviruses to Introduce Chemotherapy Resistance Sequences into Normal Hematopoietic Cells for Chemoprotection During the Therapy of Ovarian Cancer: A Pilot Trial.

In Vitro /CD34+ Autologous Bone Marrow Cells /Retrovirus /Multi-Drug Resistance-1 cDNA /Bone Marrow Transplant

Deisseroth, Albert B.

38.

Gene Therapy /Phase I /Monogenic Disease /Gaucher Disease

Gene Therapy for Gaucher Disease: Ex Vivo Gene Transfer and Autologous Transplantation of CD34+ Cells.

In Vitro /CD34+ Autologous Peripheral Blood Cells /Retrovirus /Glucocerebrosidase cDNA /Bone Marrow Transplant

Barranger, John A

39.

Gene Therapy /Phase I /Monogenic Disease /Gaucher Disease

Retroviral Mediated Transfer of the cDNA for Human Glucocerebrosidase into Hematopoietic Stem Cells of Patients with Gaucher Disease.

In Vitro /CD34+ Autologous Peripheral Blood Cells /Retrovirus /Glucocerebrosidase cDNA /Bone Marrow Transplant

Karlsson, Stefan

In Vivo /Autologous Muscle Cells /Retrovirus /HIV-1IIIB Envelope Protein /Intramuscular Injection

Galpin, Jeffrey E

Sponsor: Genetic Therapy, Inc. /Novartis 40.

Gene Therapy /Phase I /Infectious Disease /Human Immunodeficiency Virus /Immunotherapy

A Preliminary Study to Evaluate the Safety and Biologic Effects of Murine Retroviral Vector Encoding HIV-1 Genes [HIV-IT(V)] in Asymptomatic Subjects Infected with HIV-1. Sponsor: Chiron Corporation

161

Boulikas: An overview on gene therapy 41.

Gene Therapy /Phase I /Infectious Disease /Human Immunodeficiency Virus /Replication Inhibition /Antisense

A Molecular Genetic Intervention for AIDS Effects of a Transdominant Negative Form of Rev.

In Vitro /CD4+ Autologous Peripheral Blood Cells /Retrovirus /Particle Mediated Gene Transfer (Accell速) /RSV-tar /Rev M10 /Intravenous

Nabel, Gary J

42.

Gene Therapy /Phase I /Cancer /Astrocytoma /Pro-Drug

Gene Therapy for the Treatment of Recurrent Pediatric Malignant Astrocytomas with In Vivo Tumor Transduction with the Herpes Simplex Thymidine Kinase Gene.

In Vivo /Autologous Tumor Cells /PA317 /Retrovirus /Herpes Simplex Virus Thymidine Kinase cDNA /Ganciclovir /Intratumoral /Ommaya Injection

Raffel, Corey

Sponsor: Genetic Therapy, Inc. /Novartis 43.

Gene Therapy /Phase I /Cancer /Ovarian /Brain /Chemoprotection

Human MDR Gene Transfer in Patients with Advanced Cancer.

In Vitro /CD34+ Autologous Bone Marrow Cells /Retrovirus /Multi-Drug Resistance-1 cDNA /Bone Marrow Transplant

Hesdorffer, Charles and Antman, Karen

44.

Gene Therapy /Phase I /Cancer /Breast /Chemoprotection

Retroviral Mediated Transfer of the Human MultiDrug Resistance Gene (MDR-1) into Hematopoietic Stem Cells During Autologous Transplantation after Intensive Chemotherapy for Breast Cancer.

In Vitro /CD34+ Autologous Peripheral Blood Cells /Retrovirus /Multi-Drug Resistance-1 cDNA /Intravenous

O'Shaughnessy, Joyce

45.

Gene Therapy /Phase I /Cancer /Brain Tumors /Pro-Drug

Gene Therapy for Recurrent Pediatric Brain Tumors.

In Vivo /Autologous Tumor Cells /PA317 /Retrovirus /Herpes Simplex Virus Thymidine Kinase cDNA /Ganciclovir /Intratumoral /Direct Injection

Kun, Larry E.

46.

Gene Therapy /Phase I /Cancer /Melanoma /Immunotherapy

Immunization of Malignant Melanoma Patients with Interleukin 2-Secreting Melanoma Cells Expressing Defined Allogeneic Histocompatibility Antigens.

In Vitro /Allogeneic Tumor Cells /Lethally Irradiated /Retrovirus /Interleukin-2 cDNA /Neomycin Phosphotransferase cDNA /Subcutaneous Injection

Das Gupta, Tapas K. and Cohen, Edward P

47.

Gene Therapy /Phase I /Infectious Disease /Human Immunodeficiency Virus-1 /Replication Inhibition /Hairpin Ribozyme

A Phase I Clinical Trial to Evaluate the Safety and Effects in HIV-1 Infected Humans of Autologous Lymphocytes Transduced with a Ribozyme that Cleaves HIV-1 RNA.

In Vitro /CD4+ Peripheral Blood Cells /Retrovirus /Hairpin Ribozyme /Intravenous

Wong-Staal, Flossie

48.

Gene Therapy /Phase I /Cancer /Melanoma /Immunotherapy

Genetically Engineered Autologous Tumor Vaccines Producing Interleukin-2 for the Treatment of Metastatic Melanoma.

In Vitro /Allogeneic Tumor Cells /Lethally Irradiated /In Combination with Untransduced Autologous Tumor Cells /Retrovirus /Interleukin2 cDNA /Subcutaneous Injection

Economou, James S. and Glasby, John A

49.

Gene Therapy /Phase I /Cancer /Leptomeningeal Carcinomatosis /ProDrug

Intrathecal Gene Therapy for the Treatment of Leptomeningeal Carcinomatosis.

In Vivo /Autologous Tumor Cells /PA317 /Retrovirus /Herpes Simplex Virus Thymidine Kinase cDNA /Ganciclovir /Intraventricular Injection /Subarachnoid Injection

Oldfield, Edward H. and Ram, Zvi

50.

Gene Therapy /Phase I /Cancer /Colon /Immunotherapy

Injection of Colon Carcinoma Patients with Autologous Irradiated Tumor Cells and Fibroblasts Genetically Modified to Secrete Interleukin-2.

In Vitro /Autologous Fibroblasts /Lethally Irradiated /In Combination with Untransduced Autologous Tumor Cells /Retrovirus /Interleukin2 cDNA /Subcutaneous Injection

Sobol, Robert E. and Royston, Ivor

51.

Gene Therapy /Phase I /Monogenic Disease /Gaucher Disease

Retrovirus-Mediated Transfer of the cDNA for Human Glucocerebrosidase into Peripheral Blood Repopulating Cells of Patients with Gaucher's Disease.

In Vitro /G-CSF Mobilized CD34+ Autologous Peripheral Blood Cells /Retrovirus /Glucocerebrosidase cDNA /Intravenous

Schuening, Friedrich

52.

Gene Therapy /Phase I /Infectious Disease /Human Immunodeficiency Virus /Immunotherapy

An Open Label, Phase I /II Clinical Trial to Evaluate the Safety and Biological Activity of HIVIT(V) (HIV-1 IIBenv /Retroviral Vector) in HIV-1 Infected Subjects.

In Vivo /Autologous Muscle Cells /Retrovirus /HIV-1IIIB Envelope Protein /Intramuscular Injection

Haubrich, Richard and Merritt, James A

53.

Gene Therapy /Phase I /Cancer /Melanoma /Immunotherapy

Adoptive Immunotherapy of Cancer with Activated Lymph Node Cells Primed In Vivo with Autologous Tumor Cells Transduced with the GM-CSF Gene.

In Vitro /Autologous Tumor Cells /Lethally Irradiated /Used in Combination with Anti-CD3 and Interleukin-2 Primed Autologous Lymph Node Cells to Prime Autologous Peripheral Blood Cells In Vitro /Retrovirus /GM-CSF cDNA /Intravenous

Chang, Alfred E

54.

Gene Therapy /Phase I /Cancer /Neuroblastoma /Immunotherapy

A Phase I Study of Immunization with ! Interferon Transduced Neuroblastoma Cells.

In Vitro /Autologous Tumor Cells /Allogeneic Tumor Cells /Lethally Irradiated /Retrovirus /! Interferon cDNA /Subcutaneous Injection

Rosenblatt, Joseph

Sponsor: Genetic Therapy, Inc. /Novartis

162

Gene Therapy and Molecular Biology Vol 1, page 163 In Vitro /CD8+ Syngeneic Peripheral Blood Cells /Retrovirus /CD4-zeta Chimeric Receptor /Intravenous /Concurrent Interleukin-2 Therapy

Walker, Robert

Clinical Trial to Assess the Safety, Feasibility, and Efficacy of Transferring a Potentially Anti-arthritic Cytokine Gene to Human Joints with Rheumatoid Arthritis.

In Vivo /Autologous Synovial Cells /Retrovirus /Interleukin-1 Receptor Antagonist Protein cDNA /Intrajoint /Metacarpal Phalangeal Joints

Evans, C. H. and Robbins, Paul

Gene Marking /Cancer /Ovarian

Use of a Retroviral Vector to Study the Trafficking Patterns of Purified Ovarian TIL Populations Used in Intraperitoneal Adoptive Immunotherapy of Ovarian Cancer Patients: A Pilot Study.

In Vitro /Autologous Peripheral Blood Cells /Autologous Tumor Infiltrating Lymphocytes /Retrovirus /Neomycin Phosphotransferase cDNA /Intraperitoneal

Freedman, Ralph

58.

Gene Marking /Cancer /Pediatric Malignancies

Use of Double Marking with Retroviral Vectors to Determine the Rate of Reconstitution of Untreated and Cytokine Expanded CD34+ Selected Marrow Cells in Patients Undergoing Autologous Bone Marrow Transplantation.

In Vitro /CD34+ Autologous Bone Marrow Cells /Retrovirus /Neomycin Phosphotransferase cDNA /Bone Marrow Transplant

Heslop, Helen

59.

Gene Therapy /Phase I /Cancer /Breast /Chemoprotection

Use of Safety-Modified Retroviruses to Introduce Chemotherapy Resistance Sequences into Normal Hematopoietic Cells for Chemoprotection During the Therapy of Breast Cancer: A Pilot Trial.

In Vitro /CD34+ Autologous Peripheral Blood Cells /Retrovirus /Multi-Drug Resistance-1 cDNA /Intravenous

Deisseroth, Albert

60.

Gene Therapy /Phase I /Monogenic Disease /Fanconi Anemia

Retroviral Mediated Gene Transfer of the Fanconi Anemia Complementation Group C Gene to Hematopoietic Progenitors of Group C Patients.

In Vitro /CD34+ Autologous Peripheral Blood Cells /Retrovirus /Fanconi Anemia Complementation Group C cDNA /Intravenous

Liu, Johnson, M. and Young, Neal S

61.

Gene Therapy /Phase I /Cancer /Glioblastoma /Immunotherapy

Injection of Glioblastoma Patients with Tumor Cells Genetically Modified to Secrete Interleukin-2 (IL-2): A Phase I Study.

In Vitro /Autologous Fibroblasts /Lethally Irradiated /In Combination with Untransduced Autologous Tumor Cells /Lethally Irradiated /Retrovirus /Cytokine /Interleukin-2 cDNA /Subcutaneous Injection

Sobol, Robert and Royston, Ivor

62.

Gene Therapy /Phase I /Cancer /Melanoma /Lymphoma /Breast /Head and Neck Cancer /Immunotherapy

IL-12 Gene Therapy Using Direct Injection of Tumor with Genetically Engineered Autologous Fibroblasts.

In Vitro /Autologous Fibroblasts /Lethally Irradiated /Retrovirus /Cytokine /Interleukin-12 cDNA /Neomycin Phosphotransferase cDNA /Intratumoral /Direct Injection

Lotze, Michael T

63.

Gene Therapy /Phase I /Cancer /Prostate /Immunotherapy

Phase I /II Study of Autologous Human GM-CSF Gene Transduced Prostate Cancer Vaccines in Patients with Metastatic Prostate Carcinoma.

In Vitro /Autologous Tumor Cells /Lethally Irradiated /Retrovirus /Cytokine /GranulocyteMacrophage Colony Stimulating Factor cDNA /Subcutaneous Injection

Simons, Jonathan

64.

Gene Therapy /Phase I /Cancer /Breast /Antisense

Gene Therapy for the Treatment of Metastatic Breast Cancer by In Vivo Infection with BreastTargeted Retroviral Vectors Expressing Antisense cfos or Antisense c-myc RNA.

In Vivo /Autologous Tumor Cells /Retrovirus /c-fos Antisense RNA /c-myc Antisense /Intrapleural /Intraperitoneal

Holt, Jeffrey, and Arteaga, Carlos B

65.

Gene Therapy /Phase I /Monogenic Disease /Hunter Syndrome

Retroviral-Mediated Transfer of the Iduronate-2Sulfatase Gene into Lymphocytes for Treatment of Mild Hunter Syndrome (Mucopolysaccharidosis Type II).

In Vitro /Autologous Peripheral Blood Cells /Retrovirus /Iduronate-2-Sulfatase cDNA /Intravenous

Whitley, Chester B

66.

Gene Marking /Cancer /Lymphoma /Breast

High Dose Chemotherapy and Autologous Bone Marrow plus Peripheral Blood Stem Cell Transplantation for Patients with Lymphoma or Metastatic Breast Cancer: Use of Marker Genes to Investigate the Biology of Hematopoietic Reconstitution in Adults.

In Vitro /CD34+ Autologous Bone Marrow Cells /CD34+ Autologous Peripheral Blood Cells /Retrovirus /Neomycin Phosphotransferase cDNA /Bone Marrow Transplant

Douer, Dan and Kenneth Norris

67.

Gene Therapy /Phase I /Cancer /Melanoma /Immunotherapy

A Phase I Study of Vaccination with Autologous, Irradiated Melanoma Cells Engineered to Secrete Human Granulocyte-Macrophage Colony Stimulating Factor.

In Vitro /Autologous Tumor Cells /Lethally Irradiated /Retrovirus /Cytokine /GranulocyteMacrophage Colony Stimulating FactorcDNA /Subcutaneous Injection

Dranoff, Glen

68.

Gene Therapy /Phase I /Cancer /Astrocytoma /Pro-Drug

Stereotaxic Injection of Herpes Simplex Thymidine Kinase Vector Producer Cells (PA317 /G1TkSvNa.7) and Intravenous Ganciclovir for the Treatment of Recurrent Malignant Glioma.

In Vivo /Autologous Tumor Cells /PA317 /Retrovirus /Herpes Simplex Virus Thymidine Kinase cDNA /Ganciclovir /Intratumoral /Stereotactic Injection

Fetell, Michael

In Vivo /Autologous Tumor Cells /PA317 /Retrovirus /Herpes Simplex Virus Thymidine Kinase cDNA /Ganciclovir /Intraperitoneal /Catheter

Link, Charles and Moorman, Donald

55.

Gene Therapy /Phase III /Infectious Disease /Human Immunodeficiency Virus /Immunotherapy

A Phase I /II Pilot Study of the Safety of the Adoptive Transfer of Syngeneic Gene-Modified Cytotoxic T-Lymphocytes in HIV-Infected Identical Twins.

56.

Gene Therapy /Phase I /Other /Rheumatoid Arthritis

57.

Sponsor: NIH /Cell Genesys, Inc.

Sponsor: Genetic Therapy, Inc. /Novartis 69.

Gene Therapy /Phase I /Cancer /Ovarian / ProDrug

A Phase I Trial of In Vivo Gene Therapy with Herpes Simplex Thymidine Kinase /Ganciclovir System for the Treatment of Refractory or Recurrent Ovarian Cancer.

163

Boulikas: An overview on gene therapy 70.

Gene Therapy /Phase I /Cancer /Melanoma /Immunotherapy

A Phase I Testing of Genetically Engineered Interleukin-7 Melanoma Vaccines.

In Vitro /Allogeneic Tumor Cells /Lethally Irradiated /Retrovirus /Cytokine /Interleukin-7 cDNA /Hygromycin Phosphotransferase /Herpes Simplex Virus Thymidine Kinase cDNA /Subcutaneous Injection

Economou, James

71.

Gene Therapy /Phase I /Cancer /Melanoma /Immunotherapy

Phase I /II Study of Immunization with MHC Class I Matched Allogeneic Human Prostatic Carcinoma Cells Engineered to Secrete Interleukin-2 and Interferon-!

In Vitro /HLA-Matched Allogeneic Tumor Cells /Lethally Irradiated /Retrovirus /Cytokine /Interleukin-2 cDNA /! Interferon cDNA /Subcutaneous Injection

Gansbacher, Bernd

72.

Gene Therapy /Phase I /Monogenic Disease /Chronic Granulomatous Disease

Gene Therapy Approach for Chronic Granulomatous Disease.

In Vitro /G-CSF Mobilized CD34+ Autologous Peripheral Blood Cells /Retrovirus /p47phox /Intravenous

Malech, Harry

73.

Gene Therapy /Phase II /Infectious Disease /Human Immunodeficiency Virus /Immunotherapy

A Repeat Dose Safety and Efficacy Study of HIVIT(V) in HIV-1 Infected Subjects with Greater Than or Equal to 100 CD4 + T Cells and No AIDS Defining Symptoms.

In Vivo /Autologous Muscle Cells /Retrovirus /HIV-1IIIB Envelope Protein /Intramuscular Injection

Parenti, David

74.

Gene Marking /Cancer /Chronic Myelogenous Leukemia

Autologous Marrow Transplantation for Chronic Myelogenous Leukemia Using Stem Cells Obtained After In Vivo Chemotherapy Cytokine Priming.

In Vitro /Autologous G-CSF and ATA-C Mobilized Bone Marrow Cells /Retrovirus /Neomycin Phosphotransferase cDNA /Bone Marrow Transplant

Verfaillie, Catherine

75.

Gene Therapy /Phase I /Cancer /Multiple Myeloma /Pro-Drug

Thymidine Kinase (TK) Transduced Donor Leukocyte Infusions as a Treatment for Patients with Relapsed or Persistent Multiple Myeloma after T-cell Depleted Allogeneic Bone Marrow Transplant.

In Vitro /Allogeneic T Lymphocytes /Retrovirus /Herpes Simplex Thymidine Kinase /Ganciclovir /Intravenous

Munshi, Nikhil C. and Barlogie, Bart

Sponsor: Genetic Therapy, Inc. /Novartis 76.

Gene Therapy /Phase I /Cancer /Ovarian /Immunotherapy

Treatment of Patients with Advanced Epithelial Ovarian Cancer using Anti-CD3 Stimulated Peripheral Blood Lymphocytes Transduced with a Gene Encoding a Chimeric T-cell Receptor Reactive with Folate Binding Protein.

In Vitro /Anti-CD3 Stimulated Autologous Peripheral Blood Lymphocytes /Retrovirus /Antibody /MOv-! (Reactive with Folate Binding Protein) /Intravenous /Intraperitoneal

Hwu, Patrick

77.

Gene Therapy /Phase I /Monogenic Disease /Purine Nucleoside Phosphorylase Deficiency

Gene Therapy for Purine Nucleoside Phosphorylase Deficiency.

In Vitro /Autologous Peripheral Blood Lymphocytes /Retrovirus /Purine Nucleoside Phosphorylase cDNA /Intravenous

McIvor, R. Scott

78.

Gene Therapy /Phase I /Infectious Disease /Human Immunodeficiency Virus / Replication Inhibition /Single Chain Antibody Gene

Intracellular Antibodies Against HIV-1 Envelope Protein for AIDS Gene Therapy.

In Vitro /CD4+ Autologous Peripheral Blood Lymphocytes /Retrovirus /sFv105 Anti-HIV-1 Envelope Protein(gp160)Gene /Intravenous

Marasco, Wayne A

79.

Human Immunodeficiency Virus-1 /Immunotherapy

A Randomized, Double Blinded, Phase I /II Dosing Study to Evaluate the Safety and Optimal CTL Inducing Dose of HIV-IT(V) in Pre-Selected HIV-1 Infected Subjects.

In Vivo /Autologous Muscle Cells /Retrovirus /HIV-1IIIB Envelope Protein /Intramuscular Injection

Conant, Marcus

80.

Gene Therapy /Phase I /Cancer /Glioma /Immunotherapy

Gene Therapy of Malignant Gliomas: A Phase I Study of IL-4 Gene -Modified Autologous Tumor to Elicit an Immune Response.

In Vitro /Autologous Tumor (Glioma) Cells /Non-Irradiated /Retrovirus /Cytokine /Interleukin-4 cDNA /Subcutaneous Injection

Bozik, Michael

81.

Gene Therapy /Phase I /Human Immunodeficiency Virus-1

Phase I Study to Evaluate the Safety of Cellular Adoptive Immunotherapy using Autologous Unmodified and Genetically Modified CD8+ HIVSpecific T Cells in HIV Seropositive Individuals.

In Vitro /CD8+ Allogeneic Cytotoxic T Lymphocytes /CD8+ Syngeneic Cytotoxic T Lymphocytes /Retrovirus /Neomycin Phosphotransferase /Herpes Simplex Virus Thymidine Kinase cDNA /Retrovirus /Intravenous

Riddell, Stanley R

Sponsor: Targeted Genetics Corporation 82.

Gene Therapy /Phase I /Cancer /Prostate /Antisense

Gene Therapy for the Treatment of Advanced Prostate Cancer by In Vivo Transduction with Prostate-Targeted Retroviral Vectors Expressing Antisense c-myc RNA.

In Vivo /Autologous Tumor Cells /Retrovirus /Antisense c-myc RNA /Intraprostate Injection

Steiner, Mitchell S

83.

Gene Marking /Cancer /EBV-Positive Hodgkin Disease

Administration of Neomycin Resistance Gene Marked EBV Specific Cytotoxic T Lymphocytes as Therapy for Patients Receiving a Bone Marrow Transplant for Relapsed EBV-Positive Hodgkin Disease.

In Vitro /EBV-Specific Cytotoxic T Lymphocytes /Retrovirus /Neomycin Phosphotransferase cDNA /Bone Marrow Transplant

Roskrow, Marie

84.

Gene Marking /Cancer

Administration of Neomycin Resistance Gene

164

In Vitro /EBV-Specific Cytotoxic T

Roskrow, Marie

Gene Therapy and Molecular Biology Vol 1, page 165 /EBV-Positive Hodgkin Disease

Marked EBV Specific Cytotoxic T Lymphocytes to Patients with Relapsed EBV-Positive Hodgkin Disease.

Lymphocytes /Retrovirus /Neomycin Phosphotranspherase cDNA /Intravenous Administration

Gene Therapy /Phase II /Infectious Disease /Human Immunodeficiency Virus

A Randomized, Controlled, Phase II Study of the Activity and Safety of Autologous CD4-Zeta GeneModified T Cells in HIV-Infected Patients.

In Vitro /Autologous CD8+ T Cells /Retrovirus /CD4-Zeta Chimeric Receptor /Intravenous

Connick, Elizabeth

86.

Gene Therapy /Phase I /Infectious Disease /Human Immunodeficiency Virus /Replication Inhibition

Phase I Study to Evaluate the Safety and In Vivo Persistence of Adoptively Transferred Autologous CD4+ T Cells Genetically Modified to Resist HIV Replication.

In Vitro /Autologous CD4+ T Cells /Retrovirus /Neomycin Phosphotransferase Gene /PolyTAR Decoy Gene /RRE-polyTAR Decoy Gene

Greenberg, Philip D

87.

Gene Therapy /Phase I /Cancer /Metastatic Melanoma /Immunotherapy

Phase I Study to Evaluate the Safety of Cellular Adoptive Immunotherapy Using Autologous Unmodified and Genetically Modified CD8+ Tyrosinase-Specific T Cells in Patients with Metastatic Melanoma.

In Vitro /Autologous CD8+ TyrosinaseSpecific TCells /Retrovirus /Hygromycin Phosphotranspherase /Intravenous Administration

Yee, Cassian and Greenberg, Philip D

88.

Gene Therapy /Phase I /Infectious Disease /Human Immunodeficiency Virus-1 /Replication Inhibition

Intracellular Immunization Against HIV-1 Infection Using an Anti-Rev Single Chain Variable Fragment (SFv).

In Vitro /Autologous CD4+ Peripheral Blood Lymphocytes /Retrovirus /Anti-Rev SFv /Intravenous

Pomerantz, Roger J

89.

Gene Therapy /Phase I /Cancer /Breast /Chemoprotection

Antimetabolite Induction, High-Dose Alkylating Agent Consolidation, and Retroviral Transduction of the MDR1 Gene Into Peripheral Blood Progenitor Cells Followed by Intensification Therapy with Sequential Paclitaxel and Doxorubicin for Stage 4 Breast Cancer.

In Vitro /Autologous CD34+ Peripheral Blood Lymphocytes / /Retrovirus /Multi-Drug Resistance-1 cDNA /Neomycin Phosphotransferase cDNA /Intravenous

Cowen, Kenneth H

90.

Gene Therapy /Phase I /Cancer /Hematologic Malignancies Following Allogeneic Bone Marrow Transplant /Pro-Drug /Elimination of Graft Versus Host Disease

Adoptive Immunotherapy for Leukemia: Donor Lymphocytes Transduced with the Herpes Simplex Thymidine Kinase Gene for Remission Induction.

In Vitro /Allogeneic Peripheral Blood Lymphocytes /Retrovirus /Herpes Simplex Virus Thymidine Kinase cDNA /Ganciclovir /Intravenous

Link, Charles J

91.

Gene Therapy /Phase I /Infectious Disease /Human Immunodeficiency Virus /Replication Inhibition /Antisense

Transduction of CD34+ Cells from the Bone Marrow of HIV-1 Infected Children: Comparative Marking by and RRE Decoy.

In Vitro /CD34+ Autologous Bone Marrow Cells /Retrovirus /RRE Decoy Gene, and Retrovirus /Neomycin Phosphotransferase Gene /Intravenous

Kohn, Donald B

92.

Gene Therapy /Phase I /Cancer /Ovarian /Tumor Suppressor Gene

Ovarian Cancer Gene Therapy with BRCA-1.

In Vivo /Autologous Tumor Cells /Retrovirus /BRCA-1 Gene /Intraperitoneal Administration (Ultrasound Guided)

Holt, Jeffrey T

93.

Gene Therapy /Phase I /Inherited Genetic Disorder /Monogenic Disease / X-Linked Severe Combined Immune Deficiency /Correction

Gene Therapy for X-linked Severe Combined Immune Deficiency using Retroviral Mediated Transduction of the c cDNA into CD34+ Cells.

In Vitro /CD34+ Autologous Umbilical Cord Blood or Bone Marrow /Retrovirus /cDNA for Common Chain of Multiple Cytokine Receptors /Intravenous

Weinberg, Kenneth I

94.

Gene Therapy /Phase I /Infectious Disease /Human Immunodeficiency Virus /Replication Inhibition /Hammerhead Ribozyme

Transduction of CD34+ Autologous Peripheral Blood Progenitor Cells from HIV-1 Infected Persons: a Phase I Study of Comparative Marking Using a Ribozyme Gene and a Neutral Gene.

In Vitro /CD34+ Autologous Peripheral Blood Cells /Retrovirus /Tat and Rev Hammerhead Ribozyme /Intravenous

Kohn, Donald B

95.

Gene Therapy /Phase I /Cancer /Brain Tumors /Pro-Drug

Phase I Study of Retroviral-Mediated Incorporation of the HSV Thymidine Kinase Gene and Ganciclovir in Malignant Gliomas.

In Vivo /Autologous Tumor Cells /psiCRIPMFG-S-TK1-67 Cells /Retrovirus /Herpes Simplex Thymidine Kinase cDNA /Ganciclovir /Intratumoral /Direct Injection

Harsh IV, Griffith R.

96.

Gene Therapy /Phase I /Cancer /Ovarian /Pro-

Tumor Vaccination With HER-2 /Neu Using a B7 Expressing Tumor Cell Line Prior To Treatment With

In Vitro /Allogeneic Tumor Cells /Cationic Liposome Complex /B7(CD80) cDNA

Freeman, Scott M., and

85.

Sponsor: Cell Genesys

165

Boulikas: An overview on gene therapy Drug /Immunotherapy

HSV-TK Gene-Modified Cells.

/Retrovirus /Herpes Simplex Thymidine Kinase /Ganciclovir /Intraperitoneal

Robinson III, William R

97.

Gene Therapy /Phase III of #9303-037 /Cancer /Glioblastoma /Pro-Drug

Prospective, Open-Label, Parallel-Group, Randomized Multicenter Trial Comparing the Efficacy of Surgery, Radiation, and Injection of Murine Cells Producing Herpes Simplex Thymidine Kinase Vector Followed by Intravenous Ganciclovir Against the Efficacy of Surgery and Radiation in the Treatment of Newly Diagnosed, Previoulsy Untreated Glioblastoma.

In Vivo /Autologous Tumor Cells /PA317 /Retrovirus /Herpes Simplex Virus Thymidine Kinase cDNA /Ganciclovir /Intratumoral /Direct Injection

Maria, Bernard

98.

Gene Marking /Cancer /Pediatric Malignancies

A Comparative Evaluation of the Utility of Hemopoietic Progenitor Cells Derived from Peripheral Blood vs Bone Marrow.

In Vitro /CD34+ Autologous Bone Marrow and Peripheral Blood /Retrovirus /Neomycin Phosphotransferase cDNA /Bone Marrow Transplant

Heslop, Helen E.

99.

Gene Therapy /Phase II /Cancer /Glioblastoma /Pro-Drug

Multicenter, Extension Trial for the Treatment of Recurrent Glioblastoma Multiforme with Surgery and Injection of Murine Cells Producing Herpes Simplex Thymidine Kinase Vector Followed by Intravenous Ganciclovir for Patients with Disease Progression Following Standard Treatment on Protocol GTI-0115.

In Vivo /Autologous Tumor Cells /PA317 /Retrovirus /Herpes Simplex Thymidine Kinase cDNA /Ganciclovir /Intratumoral /Direct Injection

Maria, Bernard, et.al.

Sponsor: Genetic Therapy, Inc. /Novartis

Sponsor: Genetic Therapy, Inc. /Novartis 100. Gene Therapy /Phase I /Cancer /Germ Cell Tumors (Testicular Cancer) /Chemoprotection

High Dose Carboplatin and Etoposide Followed by Transplantation with Peripheral Blood Stem Cells Transduced with the Multiple Drug Resistance Gene in the Treatment of Germ Cell Tumors - A Pilot Study.

In Vitro /G-CSF Mobilized Autologous CD34+ Peripheral Blood Cells /Retrovirus /Multi-Drug Resistance-1 cDNA /Bone Marrow Transplant

Cornetta, Kenneth

101. Gene Therapy /Phase I /Cancer /Brain Tumors /Chemoprotection

A Pilot Study of Dose Intensified Procarbazine, CCNU, Vincristine(PCV) for Poor Prognosis Pediatric and Adult Brain Tumors Utilizing FibronectinAssisted, Retroviral-Mediated Modification of CD34+ Peripheral Blood Cells with O6 -Methylguanine DNA Methyltransferase.

In Vitro /Peripheral Blood CD34+ Cells /Retrovirus /O6-Methylguanine DNA Methyltransferase cDNA /Intravenous Infusion

Williams, David A

102. Gene Therapy /Phase I /Cancer /Melanoma /Immunotherapy

A Pilot Study Using Interleukin-2 Transfected Irradiated Allogeneic Melanoma Cells Encapsulated in an Immunoisolation Device In Patients with Metastatic Malignant Melanoma.

In Vitro /Allogeneic Tumor Cells /Lethally Irradiated /Retrovirus /Interleukin-2 cDNA /Neomycin Phosphotransferase cDNA /Immunoisolation Device /Subcutaneous Implantation

Das Gupta, Tapas K

103. Gene Marking /Cancer /Chronic Myelogenous Leukemia

Autologous Marrow Transplantation for Chronic Myelogenous Leukemia Using Retrovirally Marked Peripheral Blood Progenitor Cells Obtained after In Vivo Cyclophosphamide /G-CSF Priming.

In Vitro /Autologous Peripheral Blood Cells Mobilized by Cyclophosphamide and G-CSF /Retrovirus /Neomycin Phosphotransferase cDNA /Autologous Bone Marrow Transplant

Verfaille, Catherine,

104. Gene Therapy /Phase I /Infectious Disease /Human Immunodeficiency Virus /Replication Inhibition

Phase I Clinical Trial of TREV Gene Therapy for Pediatric AIDS.

In Vitro /CD34+ Autologous Cord Blood Cells /Retrovirus /Transdominant Trev /Intravenous

Belmont, John W

105. Gene Therapy /Phase II /Infectious Disease /Human Immunodeficiency Virus

A Phase II Study of the Activity and Safety of Autologous CD4-Zeta Gene-Modified T Cells With or Without Exogenous Interleukin-2 in HIV Infected Patients.

In Vitro /Autologous CD8 + and CD4 + T Lymphocytes /Retrovirus /CD4-Zeta Chimeric Receptor /Intravenous /Concurrent Interleukin-2 Therapy

Connick, Elizabeth

106. Gene Marking /Cancer /EBV-Positive Hodgkin Disease

Administration of Neomycin Resistance Gene Marked EBV Specific Cytotoxic T-Lymphocytes To Patients With Relapsed EBV-Positive Hodgkin Disease.

In Vitro /EBV-Specific Hodgkin Disease /In Vitro /EBV-Specific Cytotoxic Lymphocytes /Retrovirus /Neomycin Phosphotransferase /Bone Marrow Transplant

Straus, Stephan E

107. Gene Therapy /Phase I /Cancer /Chronic Myelogenous Leukemia /Chemoprotection /Tyr22 Murine Dihydrofolate Reductase Gene /Antisense /Antib3a2BCR /ABL Gene

Autologous Transplantation for Chronic Myelogenous Leukemia with Stem Cells Transduced with a Methotrexate Resistant DHFR and Anti-BCR /ABL Containing Vector and Post Transplant Methotrexate Administration.

In Vitro /Autologous Peripheral Blood CD34+ Cells Mobilized by Cyclophosphamide and GCSF /Retrovirus /Autologous Bone Marrow Transplant

Verfaillie, Catherine

108. Gene Therapy /Phase II /Infectious Disease /Human

A Phase II, Randomized, Double Blind Placebo Controlled Study of Combination Drug AntiRetroviral Therapy to Include a Reverse

In Vivo /Autologous Muscle Cells /Retrovirus /HIV-1 IIIB Envelope Protein /Intramuscular Injection

Aboulafia, David

Sponsor: Cell Genesys, Inc.

166

Gene Therapy and Molecular Biology Vol 1, page 167 Immunodeficiency Virus /Immunotherapy

Transcriptase Inhibitor and a Protease Inhibitor Plus HIV-IT(V) or Placebo in HIV Patients with CD4+ Counts > 100, and HIV RNA > 1K, and < 10K Sponsor: Chiron Corporation

109. Gene Therapy /Phase I /Monogenic Disease /Chronic Granulomatous Disease

Fibronectin-Assisted, Retroviral-Mediated Transduction of CD34+ Peripheral Blood Cells with gp91 phox in Patients with X-Linked Chronic Granulomatous Disease: A Phase I Study

In Vitro /G-CSF Mobilized CD34+ Autologous Peripheral Blood Cells /Retrovirus /gp91phox /Intravenous Infusion

Smith, Franklin O., and Dinauer, Mary C

110. Gene Therapy /Phase I /II /Cancer /Colorectal Carcinoma Expressing TAG-72

A Phase I /II Study of Autologous CC49-Zeta Gene-Modified T Cells and a-Interferon in Patients with Advanced Colorectal Carcinomas Expressing the Tumor-Associated Antigen, TAG-72

In Vitro /Autologous CD8+ and CD4 + T Lymphocytes /Retrovirus /CC49-Zeta T Cell Receptor /Intravenous Infusion

Venook, Alan

Sponsor: Cell Genesys, Inc. 111. Gene Therapy /Phase I /Cancer /Melanoma /Breast /Head and Neck Cancer /Cutaneous TCell Lymphoma /Immumotherapy

IL-12 Gene Therapy Using Direct Injection of Tumors with Genetically Engineered Autologous Fibroblasts

In Vitro /Autologous Fibroblasts /Lethally Irradiated /Retrovirus /Cytokine /Interleukin-12 /Intratumoral Injection

Park, Chan H

112. Gene Therapy /Phase I /Monogenic Disease /Leukocyte Adherence Deficiency (LAD)

Retrovirus-Mediated Transfer of the cDNA for Human CD18 into Perpheral Blood Repopulating Cells of Patients with Leukocyte Adherence Deficiency

In Vitro /G-CSF Mobilized CD34+ Autologous Peripheral Blood Cells /Retrovirus /CD18 /Intravenous Infusion

Hickstein, Dennis

113. Gene Therapy /Phase I /II /Cancer /Prostate /Immunotherapy

Phase I /II Study of Allogeneic Human GM-CSF Gene Transduced Irradiated Prostate Cancer Cell Vaccines in Patients with Prostate Cancer

In Vitro /Allogeneic Tumor Cells /Lethally Irradiated /Retrovirus /Cytokine /GranulocyteMacrophage Colony Stimulating Factor /Subcutaneous Injection

Simons, Jonathan W

114. Gene Therapy /Phase I /II /Cancer /Chronic Myelogenous Leukemia /Adoptive Immunotherapy

Infusion of Polyclonal HyTK (hygromycin phosphotransferase and HSV thymidine kinase gene)-transduced Donor T Cells for Adoptive Immunotherapy in Patients with Relapsed CML after Allogeneic Stem Cell Transplant: Phase I-II Clinical Trial

In Vitro /Donor CD8+ and CD4 + Lymphocytes /Retrovirus /Hygromycin PhosphotransferaseHerpes Simplex Thymidine Kinase Fusion Gene /Intravenous Infusion

Flowers, Mary E. D. and Riddell, Stanley

115. Gene Therapy /Phase I /Cancer /Mesothelioma /Pro-Drug

The Treatment of Malignant Pleural Mesothelioma with aGene-Modified Cancer Vaccine: A Phase I Study

In Vivo /Allogeneic Tumor Cells /Lethally Irradiated /Retrovirus /Herpes Simplex Virus Thymidine Kinase /Ganciclovir /Intrapleural Administration

Schwarzenberg er, Paul

116. Gene Therapy /Phase II /Infectious Disease /Human Immunodeficiency Virus

A Phase II Study of Autologous CD4-Zeta GeneModified T Cells in HIV-Infected Patients with Undectable Plasma Viremia on Combination Antiretroviral Drug Therapy

In Vitro /Autologous CD8+ T Cells /Retrovirus /CD4-Zeta Chimeric Receptor /Intravenous

Deeks, Steven G

117. Gene Therapy /Phase II /Infectious Disease /Human Immunodeficiency Virus /Replication Inhibition /Hammerhead Ribozyme

High Dose Chemotherapy and Autologous Peripheral Stem Cell Transplantation for HIV Lymphomas: A Phase IIa Study of Comparative Marking Using a Ribozyme Gene and a Neutral Gene

In Vitro /CD34+ Autologous Peripheral Blood Cells /Retrovirus /Tat and Rev Hammerhead Ribozyme /Intravenous

Krishnan, Amrita and Zaia, John, A

118. ADENOVIRUS Gene Therapy /Phase I /Monogenic Disease /Cystic Fibrosis

A Phase I Study, in Cystic Fibrosis Patients, of the Safety, Toxicity, and Biological Efficacy of a Single Administration of a Replication Deficient, Recombinant Adenovirus Carrying the cDNA of the Normal Human Cystic Fibrosis Transmembrane Conductance Regulator Gene in the Lung.

In Vivo /Nasal Epithelial Cells /Respiratory Epithelial Cells /Adenovirus /Serotype 5 /Cystic Fibrosis Transmembrane Conductance Regulator cDNA /Intranasal /Respiratory Tract Administration (Bronchoscope)

Crystal, Ronald G

119. Gene Therapy /Phase I /Monogenic Disease /Cystic Fibrosis

Gene Therapy of Cystic Fibrosis Lung Diseases Using E1 Deleted Adenoviruses: A Phase I Trial.

In Vivo /Nasal Epithelial Cells /Respiratory Epithelial Cells /Adenovirus /Serotype 5 /E2a Temperature Sensitive Mutant /Cystic Fibrosis Transmembrane Conductance Regulator cDNA /Intranasal /Respiratory Tract Administration (Bronchoscope)

Wilson, James M

120. Gene Therapy /Phase I /Monogenic Disease /Cystic Fibrosis

Cystic Fibrosis Gene Therapy Using an Adenovirus Vector: In Vivo Safety and Efficacy in Nasal Epithelium.

In Vivo /Nasal Epithelial Cells /Adenovirus /Serotype 2 /Cystic Fibrosis Transmembrane Conductance Regulator cDNA /Intranasal

Welsh, Michael J and Smith, Alan E

Sponsor: Cell Genesys, Inc.

Sponsor: Ribozyme Pharmaceuticals, Inc.

167

Boulikas: An overview on gene therapy Sponsor: Genzyme Corporation 121. Gene Therapy /Phase I /Monogenic Disease /Cystic Fibrosis

A Phase I Study of Gene Therapy of Cystic Fibrosis Utilizing a Replication Deficient Recombinant Adenovirus Vector to Deliver the Human Cystic Fibrosis Transmembrane Conductance Regulator cDNA to the Airways.

Wilmott, Robert W

Sponsor: Genetic Therapy, Inc. /Novartis 122. Gene Therapy /Phase I /Monogenic Disease /Cystic Fibrosis

Gene Therapy for Cystic Fibrosis Using E1 Deleted Adenovirus: A Phase I Trial in the Nasal Cavity.

In Vivo /Nasal Epithelial Cells /Adenovirus /Serotype 5 /Cystic Fibrosis Transmembrane Conductance Regulator CDNA /Intranasal

Boucher, Richard C. and Knowles, Michael R

123. Gene Therapy /Phase I /Monogenic Disease /Cystic Fibrosis

Adenovirus-Mediated Gene Transfer of CFTR to the Nasal Epithelium and Maxillary Sinus of Patients with Cystic Fibrosis.

In Vivo /Nasal Epithelial Cells /Maxillary Sinus Epithelial Cells /Adenovirus /Serotype 2 /Cystic Fibrosis Transmembrane Conductance Regulator cDNA /Intranasal /Maxillary Sinus Administration

Welsh, Michael J

Sponsor: Genzyme Corporation 124. Gene Therapy /Phase I /Cancer /Non-small Cell Lung Cancer /Tumor Suppressor Gene

Clinical Protocol for Modification of Tumor Suppressor Gene Expression and Induction of Apoptosis in Non-Small Cell Lung Cancer (NSCLC) with an Adenovirus Vector Expressing Wildtype p53 and Cisplatin.

In Vivo /Autologous Tumor Cells /Adenovirus /Serotype 5 /p53 cDNA /Intratumoral /Bronchoscope

Roth, Jack A

125. Gene Therapy /Phase I /Monogenic Disease /Cystic Fibrosis

Evaluation of Repeat Administration of a Replication Deficient, Recombinant Adenovirus Containing the Normal Cystic Fibrosis Transmembrane Conductance Regulator cDNA to the Airways of Individuals with Cystic Fibrosis.

Crystal, Ronald G

126. Gene Therapy /Phase I /Cancer /Central Nervous System /ProDrug

Treatment of Advanced CNS Malignancy with the Recombinant Adenovirus H5.020RSVTK: A Phase I Trial.

In Vivo /Autologous Tumor Cells /Adenovirus /Serotype 5 /Herpes Simplex Virus Thymidine Kinase cDNA /Ganciclovir /Stereotactic Injection

Eck, Stephen L. and Alavi, Jane B

127. Gene Therapy /Phase I /Cancer /n /Pro-Drug

Treatment of Advanced Mesothelioma with the Recombinant Adenovirus H5.010RSVTK: A Phase I Trial.

In Vivo /Autologous Tumor Cells /Adenovirus /Serotype 5 /Herpes Simplex Virus Thymidine Kinase cDNA /Ganciclovir /Intrapleural

Albelda, Steven M

128. Gene Therapy /Phase I /Monogenic Disease /Cystic Fibrosis

Adenovirus Mediated Gene Transfer for Cystic Fibrosis: Safety of Single Administration in the Lung (lobar instillation).

In Vivo /Respiratory Epithelial Cells /Adenovirus /Serotype 2 /Cystic Fibrosis Transmembrane Conductance Regulator cDNA /Respiratory Epithelial Cells /Bronchoscope

Dorkin, Henry L and Lapey, Allen

In Vivo /Respiratory Epithelial Cells /Adenovirus /Serotype 2 /Cystic Fibrosis Transmembrane Conductance Regulator cDNA /Respiratory Epithelial Cells /Aerosol Administration

Dorkin, Henry L. and Lapey, Allen

Sponsor: Genzyme Corporation 129. Gene Therapy /Phase I /Monogenic Disease /Cystic Fibrosis

Adenovirus Mediated Gene Transfer for Cystic Fibrosis: Safety of a Single Administration in the Lung (aerosol administration). Sponsor: Genzyme Corporation

130. Gene Therapy /Phase I /Cancer /Head and Neck Squamous Cell /Tumor Suppressor Gene

Clinical Protocol for Modification of Tumor Suppressor Gene Expression in Head and Neck Squamous Cell Carcinoma (HNSCC) with an Adenovirus Vector Expressing Wild-type p53.

In Vivo /Autologous Tumor Cells /Adenovirus /Serotype 5 /p53 cDNA /Intratumoral /Bronchoscope

Clayman, Gary

131. Gene Therapy /Phase I /Cancer /Colon /Hepatic Metastases /Tumor Suppressor Gene

Gene Therapy of Primary and Metastatic Malignant Tumors of the Liver Using ACN53 Via Hepatic Artery Infusion: A Phase I Study.

In Vivo /Autologous Tumor Cells /Adenovirus /Serotype 5 /p53 cDNA /Intrahepatic /Hepatic Artery /Bolus Infusion

Venook, Alan and Warren, Robert

132. Gene Therapy /Phase I /Cancer /Central Nervous System Malignancies /ProDrug

Phase I Study of Adenoviral Vector Delivery of the HSV-TK Gene and the Intravenous Administration of Ganciclovir in Adults with Malignant Tumors of the Central Nervous System.

In Vivo /Autologous Tumor Cells /Adenovirus /Serotype 5 /Herpes Simplex Virus Thymidine Kinase cDNA /Ganciclovir /Intra- tumoral /Stereotactic Injection

Grossman, Robert and Woo, Savio

133. Gene Therapy /Phase I /Cancer /Ovarian and Extraovarian /AntierbB-2 Single Chain Antibody Gene

A Phase I Study of Recombinant Adenovirus Vector-Mediated Delivery of an Anti-erbB-2 Single Chain (sFv) Antibody Gene for Previously Treated Ovarian and Extraovarian Cancer Patients.

In Vivo /Autologous Tumor Cells /Adenovirus /Anti-erbB-2 (oncoprotein /extracellular domain) Single-chain Antibody Gene /Intraperitoneal Injection

Curiel, David T. and Alvarez, Ronald D

134. Gene Therapy /Phase I /Cancer /Colon Carcinoma (Hepatic

A Phase I Study of Direct Administration of a Replication-Deficient Adenovirus Vector Containing the E. coli Cytosine Deaminase Gene to Metastatic

In Vivo /Autologous Tumor Cells /Adenovirus /E. coli Cytosine Deaminase cDNA /Intratumoral (Hepatic) Injection /Combined with

Crystal, Ronald, G

Sponsor: Schering Plough Corporation (formerly Canji)

168

Gene Therapy and Molecular Biology Vol 1, page 169 Metastases) /Pro-Drug

Colon Carcinoma of the Liver in Association with the Oral Administration of the Pro-Drug 5Fluorocytosine.

Oral 5-Fluorocytosine

135. Gene Therapy /Phase I /Cancer /Neuroblastoma /Immunotherapy

Phase I Study of Cytokine Gene Modified Autologous Neuroblastoma Cells for Treatment of Relapsed /Refractory Neuroblastoma Using an Adenoviral Vector.

In Vitro /Autologous Tumor Cells (Nonirradiated) /Type 5 Adenovirus /Cytokine /Interleukin-2 cDNA /Subcutaneous Injection

Brenner, Malcolm K

136. Gene Therapy /Phase I /Cancer /Ovarian and Extraovarian Cancer /Single Chain Antibody

A Phase I Study of Recombinant Adenovirus Vector-Mediated Intraperitoneal Delivery of Herpes Simplex Virus Thymidine Kinase (HSV-TK) Gene and Intravenous Ganciclovir for Previously Treated Ovarian and Extraovarian Cancer Patients.

In Vivo /Autologous Tumor Cells /Adenovirus /Herpes Simplex Thymidine Kinase Gene /Intraperitoneal Injection /Combined with Intravenous Ganciclovir Administration

Alvarez, Ronald D. and Curiel, David T

137. Gene Therapy /Phase I /Monogenic Disease /Partial Ornithine Transcarbamylase (OTC) Deficiency

A Phase I Study of Adenoviral Vector Mediated Gene Transfer to Liver in Adults with Partial Ornithine Transcarbamylase Deficiency.

In Vivo /Autologous Peripheral Blood Cells /Adenovirus /Type 5 (E2a Temperature-Sensitive Mutant) /Ornithine Transcarbamylase cDNA /Intravenous

Batshaw, Mark

138. Gene Therapy /Phase I /Cancer /Melanoma /Immunotherapy

Phase I Trial in Patients with Metastatic Melanoma of Immunization with a Recombinant Adenovirus Encoding the MART-1 Melanoma Antigen.

In Vivo /Adenovirus /Type 2 /MART-1 Melanoma Antigen /Subcutaneous Injection /Immunization

Rosenberg, Steven A

139. Gene Therapy /Phase I /Cancer /Prostate / ProDrug

Phase I Study of Adenoviral Vector Delivery of the HSV-tk Gene and the Intravenous Administration of Ganciclovir in Men with Local Recurrence of Prostate Cancer after Radiation Therapy.

In Vivo /Autologous Tumor Cells /Adenovirus /Serotype 5 /Herpes Simplex Virus Thymidine Kinase cDNA /Ganciclovir /Intratumoral /Intraprostatic Tumor Injection

Scardino, Peter T

140. Gene Therapy /Phase I /Cancer /Bladder /Tumor Suppressor Gene

Gene Therapy of Bladder Cancer Using Recombinant Adenovirus Containing the Retinoblastoma Gene (ACNRB): A Phase IA Study.

In Vivo /Autologous Tumor Cells /Adenovirus /Serotype 5 /Retinoblastoma cDNA /Intravesical Catheter Administration

Small, Eric J. and Carroll, Peter R

141. Gene Therapy /Phase I /Cancer /Head and Neck Squamous Cell Carcinoma /Pro-Drug

Phase I Study of Adenoviral Vector Delivery of the HSV-tk Gene and the Intravenous Administration of Ganciclovir in Adults with Recurrent or Persistent Head and Neck Cancer.

In Vivo /Autologous Tumor Cells /Adenovirus /Serotype 5 /Herpes Simplex Virus Thymidine Kinase cDNA /Ganciclovir /Intratumoral Injection

O'Malley, Bert W

142. Gene Therapy /Phase I / Cancer /Melanoma /Immunotherapy

Phase I Trial in Patients with Metastatic Melanoma of Immunization with a Recombinant Adenovirus Encoding the GP100 Melanoma Antigen.

In Vivo /AutologousTumor Cells /Adenovirus /Serotype 2 /GP100 Melanoma Antigen /Subcutaneous or Intramuscular Injection /Concurrent Interleukin-2 Therapy

Rosenberg, Steven A

143. Gene Therapy /Phase I /Cancer /Liver(Hepatic)Metasta ses /Pro-Drug

Phase I Trial of Adenoviral Vector Delivery of the Herpes Simplex Thymidine Kinase Gene by Intratumoral Injection Followed by Intravenous Ganciclovir in Patients with Hepatic Metastases.

In Vivo /Autologous Tumor Cells /Adenovirus /Serotype 5 /Herpes Simplex Thymidine Kinase Gene /Ganciclovir /Intratumoral Injection

Sung, Max W., and Woo, Savio L.C

144. Non-Therapeutic

Immune Response to Intradermal Administration of an Adenovirus Type 5 Gene Transfer Vector (AdGVCD.10) in Normal Individuals.

In Vivo /Intradermal Cells /Adenovirus /Serotype 5 /E.coli Cytosine Deaminase /Intradermal Injection

Harvey, BenGary, and Crystal, Ronald G

145. Gene Therapy /Phase I /Cancer /Glioblastoma /Pro-Drug

Gene Therapy for Recurrent Glioblastoma Multiforme: Phase I Trial of Intraparenchymal Adenoviral Vector Delibvery of the HSV-TK Gene and Intravenous Administration of Ganciclovir.

In Vivo /Autologous Tumor Cells /Adenovirus /Serotype 5 /Herpes Simplex Thymidine Kinase cDNA /Ganciclovir /Intratumoral /Stereotactic Injection

Lieberman, Frank

146. Gene Therapy /Phase I /Cancer /Prostate / ProDrug

Phase I Trial of Adenoviral-Mediated Herpes Simplex Thymidine Kinase Gene Transduction in Conjuction with Ganciclovir Therapy as Neoadjuvant Treatment for Patients with Clinically Localized (Stage T1c and T2b&c)Prostate Cancer Prior to Radical Prostatectomy.

In Vivo /AutologousTumor Cells /Adenovirus /Serotype 5 /Herpes Simplex Thymidine Kinase Gene /Ganciclovir /Intratumoral Injection

Hall, Simon J. and Woo, Savio L.C

147. Gene Therapy /Phase I /Cancer /Hepatocellular Carcinoma /Tumor Supressor Gene

Phase I Study of Percutaneous Injections of Adenovirus p53 Construct (Adeno-p53) for Hepatocellular Carcinoma.

In Vivo /AutologousTumor Cells /Adenovirus /Serotype 5 /p53 cDNA /Intratumoral Injection

Belani, Chandra P

148. Gene Therapy /Phase I /Cancer /Prostate /Tumor suppressor Gene

A Phase I Study in Patients with Locally Advanced or Recurrent Adenocarcinoma of the Prostate Using SCH58500 (rAd /p53) Administered by Intratumoral Injection

In Vivo /Autologous Tumor Cells /Adenovirus /Serotype 5 /p53 cDNA /Intratumoral Injection

Belldegrun, Arie, and Figlin, Robert

In Vitro /Autologous Tumor Cells /Lethally Irradiated /Adenovirus /Serotype 5 /Cytokine /Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) /Subcutaneous Injection

Dranoff, Glenn and Soiffer, Robert

Sponsor: Schering Plough Corporation (formerly Canji)

Sponsor: Schering-Plough Corporation 149. Gene Therapy /Phase I /Immunotherapy /Cancer /Melanoma

A Phase I Study of Vaccination with Autologous, Lethally Irradiated Melanoma Cells Engineered by Adenoviral Mediated Gene Transfer to Secrete Human Granulocyte-Macrophage Colony Stimulating

169

Boulikas: An overview on gene therapy Factor 150. Gene Therapy /Phase I /Immunotherapy /Cancer /Non-Small Cell Lung Carcinoma (NSCLC)

A Phase I Study of Vaccination with Autologous, Lethally Irradiated Non-Small Cell Lung Carcinoma Cells Engineered by Adenoviral Mediated Gene Transfer to Secrete Human GranulocyteMacrophage Colony Stimulating Factor

In Vitro /Autologous Tumor Cells /Lethally Irradiated /Adenovirus /Serotype 5 /Cytokine /Granulocyte-Macrophage Colony Stimulating Factor ( GM-CSF) /Subcutaneous Injection

Dranoff, Glenn and Salgia, Ravi

151. Non-Therapeutic (under review)

Systemic and Respiratory Immune Response to Administration of an Adenovirus Type 5 Gene Transfer Vector (AdGVCD.10)

In Vivo /Bronchial Epithelial Cells /Adenovirus /Serotype 5 /E. coli Cytosine Deaminase /Intrabronchial Administration

Harvey, BenGary and Crystal, Ronald G

152. Gene Therapy /Phase II /Cancer /Head and Neck Squamous Cell Carcinoma /Tumor Suppressor Gene

A Phase II Multi-Center, Open Label, Randomized Study to Evaluate Effectiveness and Safety of Two Treatment Regimens of Ad5CMV-p53 Administered by Intra-Tumoral Injections in 78 Patients with Recurrent Squamous Cell Carcinoma of the Head and Neck (SCCHN)

In Vivo /Autologous Tumor Cells /Adenovirus /Serotype 5 /p53

Breau, Randall L

In Vivo /Autologous Tumor Cells /Adenovirus /Serotype 5 /p53 cDNA /Cutaneous or Subcutaneous

von Mehren, Margaret

In Vivo /Autologous Tumor Cells /Adenovirus /Serotype 5 /p53 cDNA /Intratumoral Injection

Logothetis, Christopher J

Sponsor: Gencell (Division of Rhone-Poulenc Rorer Pharmaceuticals) 153. Gene Therapy /Phase I /Cancer /Breast /Tumor Suppressor Gene

Phase I /Pilot Study of p53 Intralesional Gene Therapy with Chemotherapy in Breast Cancer

154. Gene Therapy /Phase III /Cancer /Prostate /Tumor Suppressor Gene

A Tolerance and Efficacy Study of Intraprostatic INGN 201 Followed by Pathological Staging and Possible Radical Prostatectomy in Patients with Locally Advanced Prostate Cancer

155. Gene Therapy /Phase I /Cancer /Bladder /Tumor Suppressor Gene

A Phase I Trial of Intravesical Ad-p53 Treatment in Locally Advanced and Metastatic Bladder Cancer

In Vivo /Autologous Tumor Cells /Adenovirus /Serotype 5 /p53 cDNA /Intravesical Administration

Pagliaro, Lance C

156. Gene Therapy /Phase II /Cancer /Non-Small Cell Lung Cancer /Tumor Suppressor Gene (under review)

A Phase II Gene Therapy Study in Patients wigh Non-Small Cell Lung Cancer Using SCH 58500 (rAd /p53) in Combination with Chemotherapy for Multiple Cycles

In Vivo /Autologous Tumor Cells /Adenovirus /Serotype 5 /p53 cDNA /Bronchoscopy or Percutaneous Intratumoral Injection

Dobbs, Tracy W

157. Gene Therapy /Phase I /Other / Coronary Artery Disease (under review)

Phase I Study of Direct Administration of a Replication-Deficient Adenovirus Vector (AdGVVEGF121.10) Containing the VEGF121 cDNA to the Ischemic Myocardium of Individuals with Life Threatening Diffuse Coronary Artery Disease

In Vivo /Ischemic Myocardium /Adenovirus /Serotype 5 /Vascular Endothelial Growth Factor (VEGF) cDNA /Cardiac Administration

Crystal, Ronald G

Sponsor: National Cancer Institue - Cancer Therapy Evaluation Program (NCI-CTEP)

Sponsor: Introgen Therapeutics, Inc.

Sponsor: Schering Plough Research Institute

Sponsor: GenVec, Inc. 158. Naked Plasmid DNA Gene Therapy /Phase I /Colon /Immunotherapy

Phase I Trial of a Polynucleotide Augmented AntiTumor Immunization to Human Carcinoembryonic Antigen in Patients with Metastatic Colorectal Cancer.

In Vivo /Autologous Tumor Cells /Plasmid DNA /Carcinoembryonic Antigen Plasmid Expression Vector /Kanamycin Resistance cDNA /Intratumoral /Direct Injection

Curiel, David

159. Gene Therapy /Phase I /Other /Peripheral Artery Disease

Arterial Gene Transfer for Therapeutic Angiogenesis in Patients with Peripheral Artery Disease.

In Vivo /Vascular Endothelial Cells /Plasmid DNA /Vascular Endothelial Growth Factor cDNA /Intraarterial /Angioplasty Catheter /Hydrogel Coated Balloon

Isner, Jeffrey M. and Walsh, Kenneth

160. Gene Therapy /Phase I /Other /Restenosis

Accelerated Re-endothelialization and Reduced Neointimal Thickening Following Catheter Transfer of phVEGF165.

In Vivo /Vascular Endothelial Cells /Plasmid DNA /Vascular Endothelial Growth Factor cDNA /Intraarterial /Angioplasty Catheter /Hydrogel Coated Balloon

Isner, Jeffrey, M

161. Gene Therapy /Phase I /II /Cancer /NonHodgkin’s B-Cell Lymphoma /Mantle Cell Lymphoma /Immumotherapy

A Phase I /II Study of Vaccine Therapy for B-Cell Lymphoma Utilizing Plasmid DNA Coding for Tumor Idiotype.

In Vivo /Naked Plasmid DNA /Tumor Idiotype /Intramuscular Injection

Levy, Ronald

162. Gene Therapy /Phase I /Cancer /Malignant Glioma /Antisense

A Phase I Study of the Safety of Injecting Malignant Glioma Patients with Irradiated TGF-ß2 Antisense Gene Modified Autologous Tumor Cells.

In Vitro /Autologous Tumor Cells /Lethally Irradiated /Plasmid DNA--Electroporation /TGFß2 /Subcutaneous Injection

Black, Keith L. and Fakhrai, Habib

163. Gene Therapy /Phase I-

Sponsor: Vical, Inc.

Phase I /IB Study of Immunization with Autologous

170

In Vitro /Autologous Tumor Cells /Lethally

Mahvi, David M

Gene Therapy and Molecular Biology Vol 1, page 171 IB /Cancer /Melanoma or Sarcoma /Immunotherapy

Tumor Cells Transfected with the GM-CSF Gene by Particle-Mediated Transfer in Patients with Melanoma or Sarcoma.

Irradiated /Plasmid DNA /Particle Mediated Gene Transfer (Accell速) /Cytokine /GM-CSF cDNA /Subcutaneous Injection

164. Gene Therapy /Phase I /Other /Cubital Tunnel Syndrome

Phase I Single Dose-Ranging Study Of Formulated hIGF-I Plasmid In Subjects With Cubital Tunnel Syndrome.

In Vivo /Autologous Muscle Cells /Plasmid DNA /Polyvinylpyrrolidone (PVP) /Human Insulin-Like Growth Factor-1(hIGF-1) /Intramuscular Injection

Netscher, David

In Vivo /Nasal Epithelial Cells /Respiratory Epithelial Cells /Adeno-Associated Virus /Cystic Fibrosis Transmembrane Conductance Regulator cDNA /Intranasal /Respiratory Tract Administration (Bronchoscope)

Flotte, Terence R and Zeitlin, Pamela L

In Vivo /Maxillary Sinus Epithelial Cells / Adeno-Associated Virus /Cystic Fibrosis Transmembrane Conductance Regulator cDNA /Maxillary Sinus Administration

Gardner, Phyllis

Sponsor: Gene Medicine, Inc. 165. Adeno-Associated Virus Gene Therapy /Phase I /Monogenic Disease / Cystic Fibrosis

A Phase I Study of an Adeno-associated VirusCFTR Gene Vector in Adult CF Patients with Mild Lung Disease.

166. Gene Therapy /Phase II /Monogenic Inherited Disorder /Cystic Fibrosis /Sinusitis /Correction

A Phase I /II Study of tgAAVCF for the Treatment of Chronic Sinusitis With Cystic Fibrosis.

167. IMMUNOTHERAPY or OTHER Gene Therapy /Phase I /Monogenic Disease /Familial Hypercholesterolemia

Ex Vivo Gene Therapy of Familial Hypercholesterolemia.

In Vitro /Low Density Lipoprotein Receptor cDNA /Intrahepatic /Portal Vein Catheter

Wilson, James M

168. Gene Therapy /Phase I /Infectious Disease /Human Immunodeficiency Virus /Replication Inhibition /Antisense

Gene Therapy for AIDS using Retroviral Mediated Gene Transfer to deliver HIV-1 Antisense TAR and Transdominant Rev Protein Genes to Syngeneic Lymphocytes in HIV Infected Identical Twins.

In Vitro /Antisense TAR /Transdominant Rev /Intravenous

Morgan, Richard and Walker, Robert

169. Gene Therapy /Phase I /Cancer /Melanoma /Immunotherapy

Phase I Study of Tumor-Infiltrating Lymphocytes Derived from In Vivo HLA-B7 Gene Modified Tumors in the Adoptive Immunotherapy of Melanoma.

In Vivo /Autologous Tumor Cells /Used to Derive Tumor Infiltrating Lymphocytes /HLAB7 cDNA /Intravenous

Chang, Alfred E. and Nabel, Gary J

170. Gene Therapy /Phase I /Cancer /CEAExpressing Malignancies (type of cancer not specified) /Immunotherapy

A Study of Recombinant ALVAC Virus that Expresses Carcinoembryonic Antigen in Patients with Advanced Cancers.

In Vivo /Autologous Muscle Cells /Canarypox Virus /Carcinoembryonic Antigen cDNA /Intramuscular Injection

Hawkins, Michael J. and Marshall, John L

171. Gene Therapy /Phase I /Cancer /Prostate Adenocarcinoma /Immunotherapy

A Phase I Study of Recombinant Vaccinia that Expresses Prostate Specific Antigen in Adult Patients with Adenocarcinoma of the Prostate.

In Vivo /Vaccination /Vaccinia Virus /Prostate Specific Antigen /Intradermal Injection

Chen, A.P

172. Gene Therapy /Phase I /Cancer /Gastrointestinal Tract, Breast, or Lung Adenocarcinoma (CEA-Expressing Malignancies) /Immunotherapy

Phase I Study of Recombinant CEA Vaccinia Virus Vaccine with Post Vaccination CEA Peptide Challenge.

In Vivo /Vaccination /Vaccinia Virus /Carcinoembryonic Antigen /Intradermal Injection in Combination with Subcutaneous Peptide Challenge

Cole, David J

173. Gene Therapy /Phase I /Cancer /Melanoma /Immunotherapy

Evaluation of Intratumoral Gene Therapy with HLA-B7 /DMRIE /DOPE plus Subcutaneous Low Dose Il-2.

In Vivo /Autologous Tumor Cells /HLA B7 cDNA /Intratumoral /Concurrent Interleukin-2 Therapy

Hersh, Evan M and Sondak, Vernon K.

174. Gene Therapy /Phase I /Cancer /Prostate Adenocarcinoma /Immunotherapy

A Phase I Trial Of Recombinant Vaccina Virus That Expresses PSA In Patients With Adenocarcinoma Of The Prostate.

In Vivo /Vaccination /Vaccinia Virus /Prostate Specific Antigen /Intradermal Injection

Kufe, Donald W., and Eder, Joseph Paul

175. Gene Therapy /Phase I /Cancer /Melanoma /Immunotherapy

Phase I Trial In Patients With Metastatic Melanoma Of Immunization With A Recombinant Fowlpox Virus Encoding The MART-1 Melanoma Antigen.

In Vivo /Fowlpox Virus /MART-1 Melanoma Antigen /Intramuscular Injection

Rosenberg, Steven A

176. Gene Therapy /Phase I /Cancer /Melanoma /Immunotherapy

Phase I Trial In Patients With Metastatic Melanoma Of Immunization With A Recombinant Fowlpox Virus Encoding the GP100 Melanoma Antigen.

In Vivo /Fowlpox Virus /gp100 Melanoma Antigen /Intramuscular Injection

Rosenberg, Steven A

177. Gene Therapy /Phase I /Cancer /Melanoma /Immunotherapy

Phase I Trial In Patients With Metastatic Melanoma Of Immunization With A Recombinant Vaccinia Virus Encoding the MART-1 Melanoma Antigen.

In Vivo /Vaccinia Virus /MART-1 Melanoma Antigen /Intramuscular Injection

Rosenberg, Steven A

178. Gene Therapy /Phase I

A Phase I /II Clinical Trial Evaluating the Safety

Sponsor: Targeted Genetics Corporation

171

In Vivo /Vaccination /Vaccinia Virus /Prostate

Sanda, Martin G

Boulikas: An overview on gene therapy /II /Cancer /Prostate Adenocarcinoma /Immunotherapy

and Biological Acivity of Recombinant Vaccinia-PSA Vaccine in Patients with Serological Recurrence of Prostate Cancer Following Radical Prostatectomy.

Specific Antigen /Intradermal Injection

179. Gene Therapy /Phase I /Cancer /CEAExpressing Malignancies /Immunotherapy

A Phase I Study of Active Immunotherapy With Carcinoembronic Antigen RNA-Pulsed Autologous Human Cultured Dendritic Cells In Patients Wlth Metastatic Malignancies Expressing Carcinoembryonic Antigen.

In Vitro /Autologous Dendritic Cells /RNA Transfer /Carcinoembryonic Antigen /Intravenous

Lyerly, Kim H

180. Gene Therapy /Phase I /Cancer /Melanoma /Immunotherapy

Phase Ib Trial of Intratumoral Injection of a Recombinant Canarypox Virus Encoding the Human Interleukin-12 Gene (ALVAC-hIL-12) in Patients with Surgically Incurable Melanoma

In Vivo /Autologous Melanoma Cell /Canarypox Virus /Cytokine /Interleukin-12 cDNA /Intratumoral Injection

Conry, Robert M

In Vivo /Autologous Muscle Cells /Canarypox Virus /Vaccinia Virus /Carcinoembryonic Antigen cDNA /Intramuscular and Percutaneous Injection

Marshall, John L

In Vivo /Vaccinia Virus /Carcinoembryonic Antigen cDNA /Intradermal and Subcutaneous Injections

Conry, Robert M

InVivo /Autologous Melanoma Cell /Canarypox Virus /B7(CD80) /Interleukin-12 /Cytokine /Intratumoral Injection

Conry, Robert M

In Vitro /Autologous Tumor Cells /Canarypox Virus /B7.1 (CD80) /Intraperitoneal Injection

Freedman, Ralph

In Vivo /Autologous Tumor Cells /Canarypox Virus /Carcinoembryonic Antigen /B7.1 (CD80) /Intradermal Scarification

Kaufman, Howard L

In Vivo /Autologous Tumor Cells /Canarypox Virus /Carcinoembryonic Antigen /B7.1 (CD80) /Intramuscular and Intradermal Injections

von Mehren, Margaret

Sponsor: NCI- Cancer Therapy Evaluation Program 181. Gene Therapy /Phase I /Cancer /Immunotherapy /CEA-Expressing Malignancies

A Pilot Study of Sequential Vaccinations with ALVAC-CEA and Vaccina-CEA with the addition of IL-2 and GM-CSF in Patients with CEA Expressing Tumors

182. Gene Therapy /Phase I /Cancer /Immunotherapy /CEA-Expressing Malignancies

A Phase I Trial of a Recombinant Vaccinia-CEA (180 Kd) Vaccine Delivered by Intradermal Needle Injection Versus Subcutaneous Jet Injection in Patients with Metastatic CEA-Expressing Adenocarcinoma

Sponsor: National Cancer Institute-Cancer Therapy Evaluation Program (NCI-CTEP)

Sponsor: NCI, NIH 183. Gene Therapy /Phase I /Cancer /Melanoma /Immunotherapy

Phase Ib Trial of Intratumoral Injection of a Recombinant Canarypox Virus Encoding Human B7.1 (ALVAC-hB7.1) or a Combination of ALVAChB7.1 and a Recombinant Canarypox Virus Encoding Human Interleukin-12 (ALVAC-hIL-12) in Patients with Surgically Incurable Melanoma Sponsor: National Cancer Institute-Cancer Therapy Evaluation Program (NCI-CTEP)

184. Gene Therapy /Phase I / Cancer /Ovarian /Immunotherapy

Intraperitoneal (IP) Auatologous Therapeutic Tumor Vaccine (AUT-OV-ALVAC-hB7.1) plus IP rIFN-! for Patients with Ovarian Cancer. A Pilot Study Sponsor: NCI Cancer Therapy Evaluation Program (NCI-CTEP)

185. Gene Therapy /Phase I /Cancer /Colorectal /Immunotherapy (under review)

Phase I Clinical Trial of a Recombinant ALVACCEA-B7 Vaccine in the Treatment of Advanced Colorectal Carcinoma.

186. Gene Therapy /Phase I /Cancer /CEAExpressing Malignancies /Immunotherapy (under review)

Phase I /Pilot Study of ALVAC-CEA-B7.1 Immunization in Patients with Advanced Adenocarcinoma Expressing CEA

Sponsor: National Cancer Institute-Cancer Therapy Evaluation Program (NCI-CTEP)

Sponsor: National Cancer Institue - Cancer Therapy Evaluation Program (NCI-CTEP)

172

Gene Therapy and Molecular Biology Vol 1, page 173 Gene Ther Mol Biol Vol 1, 173-214. March, 1998.

The challenge of liposomes in gene therapy Francis Martin 1 and Teni Boulikas 2 1. SEQUUS Pharmaceuticals, Inc., 960 Hamilton Court, Menlo Park, California 94025 2. Institute of Molecular Medical Sciences, 460 Page Mill Road, Palo Alto, California 94306 and Regulon Inc., 249 Matadero Avenue, Palo Alto, CA 94306

__________________________________________________________________________________ Correspondence: Francis Martin, Vice President and Chief Scientist, Tel: (650) 323-9011, Fax: 617-3080, E-mail: FrankM@sequus.com

Summary R e c e n t l y , l i p o s o m e s have gained a special interest as gene delivery systems: over 3 0 human clinical trials for gene delivery using cationic liposomes have been approved; all these delivery methods use intratumoral, subcutaneous and other local delivery but not systemic delivery due to the toxicity of cationic lipids. Stealth liposomes (coated with polyethyleneglycol to camouflage t h e l i p o s o m e a n d e v a d e d e t e c t i o n b y t h e i m m u n e s y s t e m ) h a v e a r e m a r k a b l e l o n g e v i t y i n body fluids, have negligible toxicity with respect to their lipid components, reduce the toxicity of the encapsulated drug, and can deliver efficiently their doxorubicin payload (DOXIL) or cis-platin to tumor l e s i o n s . The mechanism o f stealth liposome accumulation i n tumors i n v o l v e s their extravasation through gaps in the endothelium of tumor vessels. DOXIL can sustain a much higher concentration of Doxorubicin in tumor tissue compared to free drug administration at comparable doses. Liposomes tagged with folate-PEG or with antibodies can target specific t i s s u e s . We propose that “stealth” liposomes, could find future applications to systemically deliver plasmid DNA with therapeutic genes (p53, H S V - t k , angiostatin) to primary tumors and their metastases leading to complete cancer eradication. Abbreviations: AUC, area-under-the-plasma concentration vs time curve CHOL, cholesterol CL, cardiolipin DDAB, dimethyldioctadecylammonium bromide DOGS, dioctadecylamidoglycylspermine DOPE, dioleyl phosphatidylethanolamine

DOSPA , (2,3-dioleyloxy-N-[20({2,5-bis[(3aminopropyl)amino]-1-oxypentyl}amino)ethyl]N,N-dimethyl-2,3-bis(9-octadecenyloxy)-1propanaminium trifluoroacetate DOTMA or lipofectin, N-[1-(2,3-dioleyloxy) propyl]-N, N, N trimethylammonium chloride DOX, doxorubicin DOXIL. “stealth” liposomes loaded with doxorubicin DSPC, distearoyl phosphatidyl choline

DXR, doxorubicin EPC, egg phosphatidyl choline EPG, egg phosphatidyl glycerol HSPE, hydrogenated soy phosphatidyl ethanolamine MPS, mononuclear phagocyte system, PC, phosphatidyl choline PEG, polyethyleneglycol SM, sphingomyelin

caused by drugs and proteins after their encapsulation in liposomes (Gregoriadis and Neerunjun, 1975). Cytotoxic anti-tumor agents were of particular interest as these in general have a narrow therapeutic window, i.e., doselimiting side effects limit their therapeutic utility. (i i i ) Because the liposome uptake by cells warrants entrance of chemicals and other molecules into otherwise inaccessible cells (Segal et al, 1974). (i v ) Because of the tendency of “stealth” liposomes to preferentially accumulate in tumor tissues (Gabizon and Papahadjopoulos, 1988; Gabizon et al, 1994); during tumor growth and vascularization of the cell mass blood vessels are formed from epithelial cells which protrude inside the tumor cell mass at sites digested with collagenases; the new blood vessels need a maturation time to attain the vein/artery-type of wall; during this time

I. Introduction Liposome-mediated drug delivery has clear advantages compared to administration of free drugs: (i ) Because of the slow releasing of drugs, encapsulated into the lumen of their lipid bilayer, into the blood stream of animals including humans. Soon after Alec Bangham and his colleagues first described liposomes in the mid 1960s as closed vesicular structures able to envelop water soluble molecules, pharmacologists recognized their potential value in drug delivery (Bangham and Horne, 1964); the rationale was simple: use liposomes as a safe vehicle for delivering drugs more specifically to sites of disease while limiting exposure of normal tissues. (i i ) Because of the minimization of allergic and other untoward reactions 173

Martin and Boulikas: The challenge of liposomes in gene therapy the vessel wall can be penetrated by liposomes. (v) Because of the possibility of adding various substances on their surface to target particular cell types. Liposomes tagged with folate-PEG (Lee and Low, 1995) or with antibodies (Straubinger et al, 1988; Ahmad et al, 1992, 1993) are promising vehicles for drug and gene delivery.

protein, posttranslational modification of the protein, and in some cases, addition of a signal peptide for export of the protein outside of the cell) would depend on the type of DNA control elements added to the therapeutic gene and not on the liposome. All steps can be experimentally manipulated and improvements in each one of them can enormously enhance the level of expression and therapeutic index of a gene therapy approach.

Liposomes can be prepared by various methods including (i) reverse phase evaporation, (ii) dehydrationrehydration, (iii) detergent dialysis, and (iv) thin film hydration followed by sonication or repeated extrusions through membranes of 400 down to 50-nm diameter pores.

In this article we shall elaborate on the use of stealth liposomes for the delivery of the antineoplastic drug Adriamycin (also called Doxorubicin) to tumors. We will then review the use of cationic liposomes in gene delivery, the ongoing Clinical trials using cationic lipids, and speculate on possible future applications of stealth liposome technology on systemic delivery of plasmid DNA with therapeutic genes for the treatment of primary tumors and their metastases.

Liposomes have found wide applications. As an exotic example, liposomes are also being used for the delivery of water-soluble antibiotics and of the chemotherapeutics trimethoprim and sulfamethoxazole to the larvae of aquatic animals, such as to nauplii of Artemia fransiscana, which are uptaken and concentrated into larvae tissues; these larvae are the main food source of marine fish larvae delivering the drugs to treat infectious diseases in fish cultures (Touraki et al, 1995, 1996).

II. Drug delivery with conventional liposomes

Liposomes can be divided into two major classes: nearly neutral or â&#x20AC;&#x153;trueâ&#x20AC;? liposomes and cationic liposomes or complexes of cationic lipids with plasmid DNA. Cationic liposomes, because of their instant interaction with the strongly anionic DNA, have been used widely in gene delivery. The field of drug and gene delivery using liposomes has been reviewed by Lasic and Martin, 1995; Lasic and Papahadjopoulos, 1995; Ledley, 1995; Boulikas, 1996a, 1998a; Martin, 1997.

A. Doxorubicin as an antineoplastic drug Doxorubicin (DOX, DXR) is one of the most widely used anticancer drugs with the broadest spectrum of antitumor effects. For example, DOX combined with 5fluorouracil has been used for the treatment of D3 stage of prostate cancer causing 50% reduction in PSA in 11 out of 18 patients. However, this treatment, like other cytotoxic chemotherapies (e.g. cyclophosphamide plus granulocytemacrophage colony stimulating factor supplementation), either alone or in combination with endocrine therapy, have shown only marginal survival benefits; hormone refractory prostate cancer is resistant to cytotoxic agents likely via a mechanism involving overexpression of the MDR1 gene by prostate cancer cells in the advanced stage of the disease (reviewed by Hsieh and Simons, 1993).

There is no limit on the size of DNA to be delivered to cells with liposomes compared with the upper limit of 7.5 kb that can be accommodated into viral/retroviral vectors because of packaging limitations. Several key steps can be conceptualized for effective gene transfer to somatic cells using liposomes: (i ) choice of type of lipid, size of liposome, and type of complex with plasmid DNA which will determine the time for its clearance from body fluids, biodistribution in tissues, and efficacy of delivery; (i i ) interaction of the gene-lipid complex with components in the serum or body fluids (plasma proteins, macrophages, immune response cells); (i i i ) targeting to the cell type, organ, tumor, and binding to the cell surface; (i v ) mode of entrance to the cell (poration through the cell membrane, receptor-mediated endocytosis); (v) release from cytoplasmic compartments (endosomes, lysosomes) and release of the plasmid DNA from its lipid complex. The remaining of the steps (nuclear import, maintenance of the plasmid as an extrachromosomal element or integration into the chromosomes of the cell, transcription, splicing and processing of the transcript to mature mRNA, export to the cytoplasm, translation onto polyribosomes into

DOX has also been linked to monoclonal antibodies or proteins in order to reduce its toxicity. The chemistry includes ester bond formation and C-N linkages between 14-bromodaunorubicin and proteins or poly-L-amino acids, and the use of enzyme-sensitive or acid sensitive spacer arms (for references see Nagy et al, 1996). For drug targeting to specific cell types, the 2pyrrolino-doxorubicin, a derivative 500-1000 times more potent than doxorubicin, was coupled to agonistic and antagonistic analogs of luteinizing hormone-releasing hormone (LH-RH); this coupling preserved the binding capacity for rat pituitary LH-RH receptors; the highly cytotoxic 2-pyrrolino-DOX/LH-RH analogs could constitute anticancer drugs for various tumors expressing LH-RH receptors (Nagy et al, 1996). 174

Gene Therapy and Molecular Biology Vol 1, page 175 However the clinical applications of DOX are limited because of its gastrointestinal and cardiac toxicity, suppression to bone marrow cells, and other side effects.

doxorubicin delivered in this fashion retains its activity against systemic tumors (Olsen et al., 1982). The pharmacokinetics and safety of various clinical formulations of liposomal doxorubicin have been reported in the scientific literature (Kumai et al., 1985; Sells, et al., 1987; Delgado et al., 1989; Cowens et al., 1989, 1990, 1993; Rahman et al., 1990; Treat et al., 1990; Creaven et al., 1990; Gabizon et al., 1990, 1991, 1992; Akamo et al., 1991; Owen et al., 1992; Batist et al., 1992; Mazanet et al., 1993; Conley et al., 1993; Embree et al., 1993). Clinical pharmacokinetic measurements confirm that conventional liposome formulations are cleared rapidly from plasma. These data also suggest that a considerable amount of encapsulated doxorubicin is released into plasma prior to MPS uptake (Gabizon et al., 1991; Conley et al., 1993).

B. Encapsulation of antineoplastic drugs into liposomes reduces toxicity Among the dozens of liposome-encapsulated antitumor agents studied in animal models, the anthracycline antibiotics, in particular doxorubicin and daunorubicin, emerged as benefiting substantially from liposome encapsulation (Gabizon et al., 1990). Animals were able to tolerate greater doses of a variety of formulations of liposome-encapsulated doxorubicin compared with the free drug and antitumor activity was, in general, maintained. Clinical trials and animal studies, or studies with cells in culture using liposomes as carriers of DOX show a reduction of complications and side effects, enhanced antitumor activity, and improved therapeutic index (Mayer et al, 1989). These advantages are thought to arise from a sustained release of the liposomal drug into the blood stream (Bally et al, 1990).

D. The liposome-anthracycline family tree

C. Interaction of liposomes with the mononuclear phagocyte system (MPS) Liposomes are rapidly removed from blood by elements of the MPS, fixed macrophages residing in liver, spleen, lung and bone marrow. It is believed that binding of plasma proteins (lipoproteins, immunoglobulins, complement) to the liposome surface triggers such rapid macrophage uptake (Lasic et al., 1991). Despite the lack of true targeting, internalization of liposome-encapsulated anthracyclines by MPS cells was found to diminish exposure of certain tissues to the toxic effects of such drugs. For example, doxorubicin-related nausea/vomiting and cardiomyopathy are believed to be related to the drug’s peak levels in plasma. By using liposome encapsulation to sequester the majority of an injected dose in the MPS, in theory, initial plasma levels of free drug are attenuated and safety improved. The drug is eventually released from MPS organs and distributes to peripheral tissues in free (i.e., unencapsulated) form. In this case, the pharmacokinetic pattern would be intended to mimic that of doxorubicin administered as a prolonged infusion, a regimen known to reduce drug-related side effects (Bielack et al., 1989). Indeed, it has been shown that administration of liposome-encapsulated doxorubicin reduces the drug’s acute and chronic toxicities in preclinical animal models. Moreover, results from animal models indicate that

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Armed with the knowledge that MPS uptake can provide favorable safety advantages for encapsulated doxorubicin, formulation scientists began to optimize liposome carriers for this purpose. As shown in Figure 1, the first major branch of the liposome anthracycline family tree was represented by these “MPS Targeted” formulations. Two alternative formulation approaches (sub-branches) soon emerged. The first, relied upon acidic lipids incorporated into the liposome bilayer (such as cardiolipin (CL) and egg phosphatidyl glycerol (EPG) to bind doxorubicin (which is positively charged at physiological pH) to the membrane itself (Gabizon et al., 1992; Rahman et al., 1990). Formation of such “ionpairs” between the drug and an acidic membrane component provided strong association and robust formulations that were stable in vitro and that could be freeze dried for long-term storage. The second approach, represented by TLC D-99, used true encapsulation of doxorubicin into the aqueous compartment of the liposome and employed a cleaver technique to circumvent the problem of leakage (Cowens et al., 1993). In this case doxorubicin is loaded into the liposomes immediately prior to administration (in a hospital pharmacy) by adding an aqueous solution of doxorubicin (at neutral pH 7.0) to liposomes containing a low pH internal buffer (pH 4.0). The pH gradient thus formed across the liposome membrane leads to mobilization of doxorubicin to the liposomes. Once inside, the low pH environment traps the drug preventing it from leaking out (as long as the pH gradient is maintained).

Martin and Boulikas: The challenge of liposomes in gene therapy

Figure 1: Family Tree illustrating the relationship between formulation strategy and the development of liposomal anthracycline products.

The ion pair formulations have been tested clinically but have not progressed beyond phase 1-2 studies. TLCD99 is in advanced phase 3 trails in metastatic breast cancer.

Europe for the treatment of AIDS-related Kaposi’s sarcoma (see below).

Recognizing that MPS uptake represented the main obstacle to targeting, another branch of liposomes developed were liposomes that resist binding/interaction with plasma proteins (opsonization) with a view toward prolonging liposome blood residence times and targeting potential. Early work suggested that a modest degree of “MPS avoiding” activity could be obtained by formulations composed of high phase transition lipids and cholesterol. Size was also a critical parameter, the smaller the liposome the longer it circulated: 300-nm in diameter liposomes are cleared from the blood approximately three times faster than small 100-nm liposomes (Huang et al, 1992).

A. Polyethylene glycol (PEG)-coated liposomes circulate for long periods in body fluids

III. “Stealth” liposomes

Coating the surface of liposomes with inert materials designed to camouflage the liposome from the body’s host defense systems was shown to increase remarkably the plasma longevity of liposomes. The biological paradigm for this “surface modified” subbranch was the erythrocyte, a cell which is coated with a dense layer of carbohydrate groups, and which manages to evade immune system detection and to circulate for several months (before being removed by the same type of cell responsible for removing liposomes).

This “pure lipid” subbranch arrived at two formulations of small diameter (!50nm), one composed of DSPC/cholesterol (Presant et al, 1990) and the other of sphingomyelin/cholesterol (Webb et al, 1995) both of which showed relatively slow MPS clearance. DaunoXome, a DSPC/cholesterol formulation of daunorubicin, is the only product to emerge from this pure lipid approach. DaunoXome is approved in the US and

The first breakthrough came in 1987 when a glycolipid (the brain tissue-derived ganglioside GM1) was identified which, when incorporated within the lipid matrix, allowed liposomes to circulate for many hours in the blood stream (Allen and Chonn, 1987). A second glycolipid, phosphatidylinositol, was also found to impart long plasma residence times to liposomes and, since it was extracted from soy beans, not brain tissue, was believed to 176

Gene Therapy and Molecular Biology Vol 1, page 177 be a more pharmaceutically acceptable excipient (Gabizon et al, 1989).

subbranch. It, too, is approved in the US and Europe for treatment of Kaposi’s sarcoma.

A major advance in the surface-modified subbranch was the development of polymer-coated liposomes(Allen et al, 1991). Polyethylene glycol (PEG) modification had been used for many years to prolong the half-lives of biological proteins (such as enzymes and growth factors) and to reduce their immunogenicity (e.g. Beauchamp et al, 1983). It was reported in the early 1990s that PEG-coated liposomes circulated for remarkably long times after intravenous administration. Half-lives in the order of 24 h were seen in mice and rats and over 30 hours in dogs. The term “stealth” was applied to these liposomes because of their ability of evade interception by the immune system (in much the same way as the stealth bomber was able to evade radar) (Gabizon and Papahadjopoulos, 1988; Klibanov et al, 1990; Papahadjopoulos et al, 1991; Senior et al, 1991; Huang et al, 1994). The increased hydrophilicity of the liposomes after their coating with the amphipathic PEG5000 leads to a reduction in nonspecific uptake by the reticuloendothelial system.

The mechanism of doxorubicin loading into liposomes is explained in Figure 2 . Liposomes composed of HSPC:CHOL (1:1) and 5% PEG-DSPE of a diameter of 85 nm are prepared in high ammonium sulfate; these are then brought into a solution of high concentration of Doxorubicin in ammonium chloride at a higher pH. Loading is mediated via exchange of ammonia molecules with uncharged doxorubicin; ammonia molecules pass from the inside of the liposomes to the outside, whereas doxorubicin enters liposomes. Loading is driven by the gradient of a chemical potential across the membrane of the liposome, the efflux of ammonia to a larger external volume, the precipitation of doxorubicin inside the liposome, and the pH gradient. The reactions in the outside volume between liposomes involve removal of a proton from the doxorubicin-ammonium chloride complex and its binding to ammonia converting it into ammonium ions; the neutral doxorubicin-ammonia complex crosses the liposome bilayer. The reactions inside the liposome involve protonation of the doxorubicin-ammonia complex from a hydrogen ion removed from the ammonium ion and its precipitation as doxorubicin sulfate (Lasic, 1995).

Whereas the half-life of antimyosin immunoliposomes was 40 min, their coating with PEG increased their halflife to 1000 min after intravenous injection to rabbits (Torchilin et al, 1992).

High resolution cryo-electron microscopy has shown a precipitate of Doxorubicin molecules with sulfate ions inside liposomes into fibrilar colloidal complexes which align into bundles (F i g u r e 3 ). The structure has been confirmed by small angle X-ray scattering where the periodicity of 2.7 nm observed was thought to represent the thickness of the gel fibers (Lasic et al, 1992).

B. Mechanism of loading of doxorubicin into “stealth” liposomes DOXIL, the PEG-coated liposomes packed with doxorubicin, (also called CAELYX in Europe) is the first product to emerge from the surface-modified liposome

Figure 2. The mechanism of loading of DOX into liposomes.

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Martin and Boulikas: The challenge of liposomes in gene therapy

Figure 3. The coffee bean appearance of the precipitated DOX sulfate within liposomes. Courtesy of Dan Lasic.

F i g u r e 4 . Cut-away view of a DOXIL particle.

folate receptor, at a rate 45-fold higher than liposomal DOX although at a rate only 1.6 times higher than that of free DOX (Lee and Low, 1995). The enhanced release of DOX from folate-PEG-liposomes internalized into the acidic endosomal organelles known as caveolae seemed to be responsible for the increased cytotoxicity of DOX to tumor cells (Lee and Low, 1995).

Figure 4 shows schematically a cut-away view of a DOXIL particle. DOX encapsulated into PEG-coated liposomes is cleared 450 times slower than free DOX (Gabizon et al, 1994). For example, uptake of DOX encapsulated into liposomes coated with PEG-folate was found to be uptaken by KB cells in culture, which express high levels of the 178

Gene Therapy and Molecular Biology Vol 1, page 179 growth, (i i ) at effecting cures and/or (i i i ) at prolonging survival times of tumor-bearing animals. Most often, all three endpoints were improved by DOXIL, and in no case was DOXIL less effective than doxorubicin. DOXIL was more active in both solid and dispersed tumors, and was more effective than doxorubicin in preventing spontaneous metastases from intramammary implants of two different mammary tumors in mice. These findings are also supported by studies done with DOXIL in several murine tumor models and human xenograft models (Figure 5).

C. Preclinical antitumor activity of DOXIL The efficacy of DOXIL has been evaluated in a variety of different tumor models, including several human xenograft models (Papahadjopoulos et al, 1991; Huang et al, 1992; Vaage et al, 1992, 1993, 1994; Williams et al, 1993; Siegal et al, 1995; Amantea et al, 1997). In every model examined DOXIL was more effective than the same doses of doxorubicin (i ) at inhibiting or halting tumor

Figure 5: Growth kinetics of human lung cancer xenografts (A) and human prostate cancer (PC-1) xenografts (B ) implanted in SCID mice. Groups of 10 animals were engrafted in the flank with 2 x 106 TL-1 cells at day 0 and treated weekly (via tail vein injection) for 10 weeks starting one week post engraftment with saline (circles), doxorubicin (also called Adriamycin) (3 mg/kg, diamonds) or DOXIL (3 mg/kg, squares). Adapted from Siegal et al, 1995.

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Gene Therapy and Molecular Biology Vol 1, page 180 Figure 6. Electron micrographs showing colloidal gold (arrows) in the intracellular vesicles of a typical mononuclear phagocyte. The particles are often seen within the endosomes (lower insert) and secondary within lysosomes (upper insert) of macrophages at the border of the liver-implanted tumor.

In general, the efficacy of doxorubicin in these models was limited by its toxicity at high doses. Typically, DOXIL could be used at a higher dose, offering an increased therapeutic advantage. Pharmacokinetic and tissue distribution studies suggest that the greater persistence, particularly in tumor tissue, achieved with DOXIL compared to conventional doxorubicin also contributes a therapeutic advantage. The efficacy of DOXIL compared to that of conventional liposomal (non-Stealth) doxorubicin indicated that DOXIL was significantly more effective than conventional liposomal doxorubicin, demonstrating the impact of the long-circulating Stealth liposome. Based on the results of these nonclinical studies, DOXIL appears to be an effective agent for the treatment of both solid and dispersed tumors. Tissue distribution of sterically-stabilized liposomes was studied by Huang et al, (1992). Following tail vein injection the microscopic localization of liposomes labeled with encapsulated colloidal gold was found predominantly in Kupffer cells (macrophages) of the liver (not in hepatocytes) (Figure 6) and within macrophages of the bone marrow. Electron microscopy showed the presence of gold in endosomes and lysosomes of fixed sinusoidal lining macrophages in the liver.

D. Combinations of DOXIL with other anticancer drugs are effective anticancer regiments in preclinical studies Humanized monoclonal antibodies directed against receptors overexpressed on malignant cells such as HER2 or EGFR have been used in the treatment of malignancies but are not active on their own (Chrysogelos et al, 1994; Baselga et al, 1996). Figure 7 shows that the combination of such anticancer regiments with DOXIL results in a synergistic antitumor activity: a combination of the DOXIL with the C225 EGFR antibody had a spectacular effect in inhibiting growth of human breast cancer cells in nude mice.

IV. Clinical trials using DOXIL A. Pharmacokinetics of DOXIL in human patients Population pharmacokinetic analysis has been conducted on a group of 83 patients receiving DOXIL at doses ranging from 10 to 60 mg/m 2 (17 females, 66 males) (Gabizon et al, 1994; Northfelt et al, 1996). At doses ranging from 10 to 40 mg/m 2, DOXIL pharmacokinetics were linear. At dosages above 40 mg/m2,

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Gene Therapy and Molecular Biology Vol 1, page 181 DOXIL displayed nonlinear pharmacokinetics as evidenced by a disproportionate increase in the area-under-the-plasma concentration versus time curve (AUC) with increasing dose amounts. In general, drugs that display nonlinear pharmacokinetics have a potential to accumulate to toxic levels in the plasma if not monitored regularly (e.g., phenytoin). In the case of DOXIL, this is not a concern since the drug is administered a minimum of every three weeks, after which time no drug is detectable in the plasma of patients. Table 1 lists the statistics of selected pharmacokinetic parameters for all 83 patients. There was no evidence of accumulation at dose intervals of " 3 weeks.

time profiles of DOXIL were generated at doses of 10 – 60 mg/m 2 (Figure 8). The nonlinearity of DOXIL pharmacokinetics at higher doses is most evident at doses greater than 40 mg/m2. No correlations were observed between pharmacokinetic parameters and age, weight, body surface area, tumor type, sex, and renal (as determined by serum creatinine) and hepatic function (as determined by total bilirubin levels). DOXIL has also been used as single-agent therapy for advanced breast cancer among elderly patients in a phase clinical trial (Ranson et al, 1997) and as primary therapy for refractory ovarian cancer also in a phase II study

Utilizing the fitted pharmacokinetic parameter results from this analysis, simulated plasma concentration versus Figure 7. Growth kinetics of human xenograft of A431 breast tumor implanted subcutaneously in nude mice. Groups of animals were treated via tail vein injection as indicated in the figure. C225 is a monoclonal antibody against epidermal growth factor receptor EGFR)

Table 1: DOXIL pharmacokinetic parameter estimates n = 83 Statistic

V ss (L/m 2 )

CL i (L/h/m 2 )

Km (mg/L)

AUC 50 ( m g / L ˙ h)

Mean

3.40

0.108

2.01

3260

CV%

18.2

54.2

66.3

54.8

Median

3.42

0.0950

1.85

3018

Minimum

2.20

0.0269

0.428

535

Maximum

5.67

0.393

8.84

9520

Vss – volume of distribution at steady-state, CL i – intrinsic clearance, K m – Michaelis Menton Constant, AUC 50 – area-underthe-curve normalized for a 50 mg/m2 dose of DOXIL

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Gene Therapy and Molecular Biology Vol 1, page 182 from work by Gabizon et al who conducted a pilot pharmacokinetic of DOXIL (Druckmann et al, 1989). In this study the fraction of the liposome-encapsulated and free, non-liposomal drug in circulation after DOXIL administration was quantitated directly using a Dowex column separation method that is able to accurately and reproducibly quantitate _ 7% free drug in the plasma (Speth et al, 1988). Using this method, essentially all the doxorubicin measured in plasma was liposome-associated (Figure 9). These findings suggest that at least 90 to 95% of the doxorubicin measured in plasma, and possibly more, is liposome-encapsulated.

(Muggia et al, 1997). While generally manageable in both type of trials, epithelial cell toxicity manifesting itself as palmar-plantar erythrodysesthesia (PPE, hand-foot syndrome) may limit the amount of DOXIL patients are able to tolerate.

B. Amount of non-liposomal Doxorubicin in plasma Several lines of evidence support the conclusion that the majority of the doxorubicin (between 93% and 99%) in plasma is encapsulated within the liposome after i.v. administration of DOXIL. The most convincing come

Figure 8: Simulated plasma clearance kinetics of doxorubicin after a single 30 minute infusion of DOXIL at doses ranging from 10 to 60 mg/m2 .

Figure 9: Clearance over a one week period of total vs. encapsulated doxorubicin after a single 50 mg/m2 dose of DOXIL in cancer patients. Data points represent mean values Âą standard deviation for 14 patients in the DOXIL group and 4 patients in the doxorubicin group (adapted from Druckmann et al, 1989). The method described in Speth et al, 1988 was used to separate the encapsulated from released drug fractions.

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Gene Therapy and Molecular Biology Vol 1, page 183 The amount of doxorubicin that remains liposomeassociated while circulating in plasma is an important point that deserves further emphasis from a safety perspective. Acute adverse reactions associated with doxorubicin administration including nausea and vomiting and chronic cardiotoxicity are believed to be directly related to peak concentrations of the drug in plasma. As pointed out above, while in the circulation, DOXIL liposomes remain intact, retaining virtually all of the doxorubicin in encapsulated form (Speth et al, 1987a). Although total plasma levels of doxorubicin may be relatively high for several days after DOXIL administration, the majority of the dose is sequestered within the liposome during this period and thus is not bioavailable to distribute (as free drug molecules) to tissues, including the GI tract and myocardium. With respect to level of available drug in plasma, DOXIL resembles more that of a 96 hour continuous infusion of doxorubicin than the usual 30 minute infusion. Prolonged infusion of doxorubicin is known to reduce cardiotoxicity and GI irritation.

C. Comparison of pharmacokinetic parameters: DOXIL vs. Doxorubicin According to literature reports, an i.v. bolus injection of doxorubicin in humans produces high plasma

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concentrations of doxorubicin that decline quickly due to rapid and extensive distribution into tissues (Greene et al, 1982). Apparent volumes of distribution range from 1400 to 3000 L, reflective of the drugâ&#x20AC;&#x2122;s extensive tissue distribution. The doxorubicin plasma concentration-time curve in humans is biphasic, with a distribution half-life of 5 to 10 minutes and terminal phase elimination half-life of 30 hours (Benjamin et al, 1984; Speth et al, 1987a, b). A triphasic curve has also been described with a terminal plasma half-life of approximately 30 hours (Benjamin et al, 1977). Clearance of doxorubicin after doxorubicin administration ranges from 24 to 73 L/hour (Greene et al, 1982). No accumulation in plasma occurs after repeated injections (Benjamin et al, 1984; Speth et al, 1987a, b). The pharmacokinetics of DOXIL are significantly different from those reported for doxorubicin. Administration of DOXIL results in a significantly higher doxorubicin area-under-the-plasma concentration vs time curve (AUC), lower rate of clearance (approximately 0.1 L/hour) and smaller volume of distribution (5 to 7 L) relative to administration of doxorubicin (Figure 10). The first phase of the biexponential plasma concentrationtime curve after DOXIL administration is relatively short (approximately 5 hours), and the second phase, which represents the majority of the AUC, is prolonged (half-life 50 to 55 hours).

Martin and Boulikas: The challenge of liposomes in gene therapy

the end of the infusion, the mean doxorubicinol level following a 20 mg/m2 dose of DOXIL was approximately 22 ng/mL. Using the doxorubicinol:doxorubicin concentration ratio reported in the literature, as described above, predicted free doxorubicin concentration at this time point would be 54 ng/mL in DOXIL-treated patients (the total plasma concentration measured at this time point was 8863 ng/mL). Comparatively, patients in the Northfelt et al study (1996) who received a dose of doxorubicin 20 mg/m 2, had initial plasma concentrations of doxorubicin of approximately 500 ng/mL.

Doxorubicin Cmax after DOXIL administration is 15- to 40-fold higher than after the same dose of doxorubicin, and the ratio quickly increases as doxorubicin is rapidly cleared from circulation. Importantly, the vast majority of the total plasma doxorubicin remains liposome-encapsulated after DOXIL treatment. Because of the high percentage of liposome encapsulation in DOXIL, the amount of free (i.e., â&#x20AC;&#x153;bioavailableâ&#x20AC;?) drug in the plasma appears to be significantly lower than that measured after administration of an equal dose of doxorubicin. This conclusion is supported by the same type of calculations presented above, which derive the apparent concentration of free doxorubicin based on the reported relationship between doxorubicinol and doxorubicin concentrations in plasma. For example, five minutes after

Studies on human cancer xenografts in nude mice demonstrate an increased accumulation of Doxorubicin at the lesion after DOXIL treatment compared to administration of free Adriamycin (F igures 11, 12).

F i g u r e 1 1 . Increased accumulation of Doxorubicin in prostate cancer xenografts (A) and pancreatic carcinoma xenografts (B ) after DOXIL treatment compared to administration of free Adriamycin.

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F i g u r e 1 2 . Increased accumulation of Doxorubicin in C26 tumors in mice after DOXIL treatment compared to administration of free Adriamycin at different time intervals.

D. Doxorubicin levels in KS lesions

Biopsy data 48 hours after DOXIL injection in the 7 patients receiving 20 mg/m 2 are shown in F i g u r e 1 3 . Ninety-six hours after drug treatment, KS lesion doxorubicin levels were 3-fold and 5-fold greater than in normal skin from the same patients in the 10 and 20 mg/m 2 groups, respectively. Median doxorubicin concentration in KS lesions was 4.3 and 3.3 µg/g tissue in 4 patients receiving 10 mg/m 2 and 4 patients receiving 20 mg/m 2 dose, respectively; median concentration in the normal skin was 1.4 µg/g tissue for the 10 mg/m 2 dose group, and 0.7 µg/g tissue in the 20 mg/m2 group.

Biopsies of KS lesion tissue and adjacent normal skin were obtained in 22 patients (T a b l e 2) (Amantea et al, 1997). Doxorubicin levels in KS lesions were higher than the levels in normal skin in 20 of the 22 patients; in 14 patients normal skin levels were below the lower limit of quantitation (0.4 µg/g tissue), whereas all KS lesion levels were quantifiable. Forty-eight hours after DOXIL administration, median doxorubicin levels in biopsies of KS lesions ranged from 3-fold to 16-fold higher than in normal skin from the same patients. The median doxorubicin concentration in KS lesions was 1.3 µg/g tissue in 7 patients receiving 10 mg/m 2 DOXIL and 15.2 µg/g tissue in 7 patients receiving 20 mg/m2 DOXIL; normal skin concentrations were 0.4 and 0.9 µg/g tissue in the 10 and 20 mg/m2 dose groups, respectively.

Although too few time points were studied to allow determination of an AUC for doxorubicin in KS lesions or skin, these data suggest that doxorubicin accumulates in KS lesions after DOXIL treatment. The levels of Doxorubicin were substantially higher in KS lesions after DOXIL compared to free Adriamycin administration (Figure 14).

Table 2: Concentration of doxorubicin in KS lesions and normal skin after DOXIL administration Time after Infusion

48 hr

96 hr

No. of Patients

Dose (mg/m2 )

Doxorubicin Concentration (µg/g tissue) Median (range) KS Lesion

Normal Skin

KS/Normal Skin

1.32 (0.17-22.43)

0.40 (0.26-1.55)

2.43

15.21 (2.98-25.56)

0.92 (0.38-1.74)

20.89

4.26 (1.91-36.44)

1.42 (0.70-2.78)

3.20

3.28 (1.03-4.17)

0.73 (0.55-1.14)

4.94

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F i g u r e 1 3 : Doxorubicin concentration in KS lesion tissue and adjacent normal skin tissue. Seven KS patients were given a 20 mg/m2 dose of DOXIL and, 96 hours later, biopsies were taken of a representative cutaneous KS lesion and normal skin near the lesion. The tissue was homogenized, extracted with solvents and total doxorubicin measured by HPLC.

F i g u r e 1 4 . DOXIL compared to Adriamycin sustains a higher concentration of Doxorubicin in Kaposiâ&#x20AC;&#x2122;s sarcoma lesions in human patients. From Northfelt et al (1996) J Clin Pharmacol 36, 55-63.

E. Combinations of DOXIL with other anticancer drugs in clinical trials The dose-limiting toxicity of Navelbine (vinorelbine tartrate) is granulocytopenia. In combination with doxorubicin, Navelbine produced a 57% overall objective response rate as first-line therapy of advanced breast cancer, 186

however, the incidence of grade 4 granulocytopenia was 83%, with 8% requiring hospitalization due to febrile neutropenia and one septic death (Hochster, 1995). Substitution of doxorubicin with DOXIL in this combination is being explored as a means of maintaining the favorable tumor response of the combination while reducing the incidence of hematological toxicity.

Gene Therapy and Molecular Biology Vol 1, page 187 Navelbine would not be expected to contribute to the skin toxicity seen with DOXIL.

B. Plasma stability and long plasma residence times are critical requirements

The excitement generated by Gianni et al (1995) who reported a greater than 90% objective response rate in metastatic breast for a taxol/doxorubicin combination is tempered by the rather unfavorable side effects profile of this regimen. Severe, febrile neutropenia was common and peripheral neuropathy occurred in one third of the patients. Perhaps more troubling was the development of reversible congestive heart failure (CHF) in 18% of women after a median of 480 mg/m2 doxorubicin, results which raise the specter of taxol-related enhancement of doxorubicin cardiotoxicity.

DOXIL liposomes are intend to carry their payload of doxorubicin directly to tumors. So, any premature release of the drug, while the liposomes are still in route (i.e., in the circulation), would detract from the total amount of encapsulated doxorubicin able to reach the desired target. This requirement highlights the importance of engineering plasma stability into DOXIL liposomes. As mentioned earlier, conventional liposome formulations of doxorubicin have been shown to release a significant proportion of their payload into the bloodstream soon after injection (Gabizon et al, 1991; Conley et al, 1993). Drug release appears to follow protein adsorption/intercalation into the liposome which disrupts the barrier properties of the membrane. Moreover, the liposomes, together with any remaining drug, are removed by cells of the MPS within several minutes to a few hours after injection. As a consequence of this rapid clearance, doxorubicin delivered in conventional liposomes has little opportunity to reach tumors in encapsulated form.

These encouraging preclinical findings are supported by results of a pilot clinical study done by Berry et al (1996). These authors have reported the results of endomyocardial biopsies performed on a series of AIDS-KS patients who received cumulative doses of DOXIL ranging from 469 to 860 mg/m 2. These findings support the rationale for combining taxol (paclitaxel) with DOXIL. Both drugs have demonstrated activity in breast and ovarian cancer. With respect to toxicities, the incidence of severe neutropenia and peripheral neuropathy are lower for DOXIL than taxol. Preclinical and early clinical biopsy results strongly suggest that DOXIL produces less damage to the myocardium relative to comparable cumulative doses of doxorubicin. Thus the cardio-protective effect of DOXIL may translate into a reduced risk of cardiotoxicity relative to the highly active taxol-doxorubicin combination. Moreover, taxol causes relatively little skin toxicity. Based on these considerations, several phase 1 dose-finding trials of DOXIL and taxol have been launched.

By virtue of the PEG groups grafted to their surface, DOXIL liposomes are stable in plasma and release very little drug while in the circulation (see discussion above). Moreover, the PEG coating provides slow clearance; after a single injection, DOXIL can be detected in the circulation for 2-3 weeks. Slow clearance kinetics provide an opportunity for these liposomes to reach sites of disease such a tumors. Measurements made in tumor-bearing animals and cancer patients indicate that uptake of pegylated liposomes by tumors is also slow process. In preclinical tumor models, the peak uptake of DOXIL is reached 24-48 hours after injection (Vaage et al, 1993; Working et al, 1994). In cancer patients given 111Indium encapsulated in pegylated liposomes of the same composition and size as DOXIL, peak uptake in tumors is seen 48-72 hours after injection (Figure 1 6 , Stewart Simon, personal communication, May 20, 1997). Slow uptake in tumors highlights the importance of long circulation times; if liposomes are to have an opportunity to reach and enter tumors in significant numbers, they must circulate for periods of days after injection.

V. Extravasation of â&#x20AC;&#x153;stealthâ&#x20AC;? liposomes into tumors A. Mechanism of enhanced DOXIL accumulation in tumors An understanding of the mechanisms by which liposome-encapsulated doxorubicin accumulates within solid tumors after DOXIL administration, and how this deposition pattern and subsequent slow release of drug improve the antitumor activity of DOXIL relative to treatment with the free drug, is now emerging (refer to Figure 15).

Figures 17 and 18 also show localization of 111Inlabeled Stealth liposomes into a T4 squamous cell carcinoma of the tongue and a squamous cell carcinoma of the lung, respectively. Figure 19 shows complete eradication of a KS lesion after six cycles of treatment with DOXIL.

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F i g u r e 1 5 : Proposed mechanism for DOXIL accumulation in tumors. ! Liposomes containing doxorubicin circulate for 2-3 weeks after injection. During this period virtually all of the drug remains encapsulated. The liposomes pass many times through the blood vessels feeding growing tumors. " Intact liposomes extravasate through defects/gaps present in newly sprouting vessels and enter the tissue

compartment; lodging in the tumor interstitium near the vessel. # Drug molecules are released from the extravasated liposomes. Liposome leakage is believed to be the consequence of conditions present in the interstitial fluid surrounding tumors which lead to physical/chemical breakdown of the liposome membrane (low pH, oxidizing agents, enzymes, uptake by macrophages). $ Free drug

188

molecules penetrate deeply into the tumor and enter tumor cells. % Doxorubicin molecules bind to nucleic acids and kill tumor cells. Note that such a mechanism does not require a close physical encounter between a liposome and target cell, since free drug molecules are able to diffuse through barriers that may intercept liposomes.

Gene Therapy and Molecular Biology Vol 1, page 189

F i g u r e 1 6 : Gamma scintigraphic image of a lung cancer patient 48 and 96 hours after administration of DOXIL liposomes containing 111 Indium. Note that both images are posterior views. Uptake of the radioactive liposomes is seen in certain normal tissues including spleen, liver, bone marrow. The activity visible in the central chest (substernal) and upper abdomen represent liposomes that are still circulating in the heart and major vessels at these time points. The liposomes are taken up by a large tumor in the left upper lung. The density of radioactivity is as high or higher in the tumor than in any normal organ.

F i g u r e 1 7 . Plain anterior view scintigrams of a patient with T4 squamous cell carcinoma of the tongue injected with 111 Inlabeled â&#x20AC;&#x153;stealthâ&#x20AC;? liposomes. The image at 4 h postinjection shows the blood pool, early uptake by the liver reticuloendothelial system, and EDTA-chelated 111 In in the bladder. The tumor is seen clearly (white arrow) 72 hours after injection and is still visible at 10 days. From Stewart and Harrington, 1997 with their kind permission and the permission of Oncology.

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F i g u r e 1 8 . Anterior scintigram of a patient with squamous cell carcinoma of the lung together with SPECT images taken at 72 hours injected with 111 In-labeled â&#x20AC;&#x153;stealthâ&#x20AC;? liposomes. The tumor is seen clearly (arrow) in all images. Prominent 111 In activity can also be seen in the liver, spleen, and bone marrow. From Stewart and Harrington, 1997 with their kind permission and the permission of Oncology.

F i g u r e 1 9 . Therapy of a KS lesion with DOXIL.

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F i g u r e 2 0 . Histological preparation of KS-like lesion nodule. Early lesion and adjacent normal skin in transgenic mice by liposome-encapsulated colloidal gold. A-C: Sections of KS-like lesion nodule were from a 16-month old F2 mouse that had a localized 5-mm spherical erythematous lesion on its back. The sections reveal that the gold particles are localized predominantly in the lesion region. Arrows in C show labeling of spindle cells. D: Section of an early lesion invisible to the naked eye in a 8month-old C4 mouse showing that the gold marker is scattered extravasated erythrocytes in the collagenous dermis. E: Normal skin adjacent to the tumor shown in A. From Huang et al, 1993.

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and other oxidizing agents (Cobbs et al, 1995); or (i i i ) phagocytic cells residing in tumors (Pupa et al, 1996) which are known to engulf liposomes (Huang et al, 1995), may digest the lipid matrix intracellularly and release doxorubicin (or its active metabolites) back into the interstitial fluid (Gabizon et al, 1991). A combination of these possibilities may well be responsible for the observed release of doxorubicin after extravasation of DOXIL liposomes in tumors (Gabizon et al, 1995).

C. Liposomes extravasate through gaps in the endothelium of tumor vessels Stealth liposomes of the same size and lipid composition as DOXIL, but containing entrapped colloidal gold designed to serve as a marker to follow liposome distribution by microscopic techniques, have been shown to enter solid colon tumors implanted in mice (Huang et al, 1992) and KS-like lesions in HIV-transgenic mice (Huang et al, 1993) (Figure 20). In these mouse models, movement of liposomes from the vascular lumen into the tumor interstitium was visualized by light and electron microscopy. Transcytosis of liposomes from the lumen of blood vessels, through endothelial cells, and into the extravascular compartment of KS lesions was seen, as was intracellular uptake of liposomes by some spindle cells within lesions. However, these processes appear to be restricted to a minority of the particles entering the tumor (Huang et al, 1993). The vast majority of the liposomes were seen to enter through gaps in the endothelial cell wall.

The rate of release of doxorubicin within a tumor has yet to be measured directly. In order to do so, it would be necessary to separate encapsulated drug (i.e., drug molecules that have not been released from intact liposomes) from free drug in a solid tissue. Although such a separation is possible in biological fluids (such as plasma; Druckmann et al, 1989) it is technically difficult to conduct in solid tissues such as tumors; the conditions needed for quantitative extraction of doxorubicin lead to liposome disruption. Despite the difficulty of directly measuring release kinetics, indirect methods suggest that the release of doxorubicin from DOXIL liposomes occurs over a period of days to perhaps weeks following administration. In a recent study using a human pancreatic xenograft model in nude mice, Vaage et al showed that tumor levels of doxorubicin peak at 24-48 hours after DOXIL, and fall slowly over a period of a week (Vaage et al, 1997). These results suggest that the liposomes entering the tumor release their drug locally at quite a slow rate.

This finding is consistent with results reported by Yuan, et al who used pegylated liposomes ranging in size from 100-600 nm to probe the cut off size of the gaps present in a human adenocarcinoma xenograft implanted in nude mice (Yuan et al, 1995). This tumor was permeable to liposomes up to 400 nm in diameter, suggesting the cut off size in this tumor is between 400-600 nm. Given their small size (85 nm) and long circulation times, DOXIL liposomes would be expected to extravasate in tumors that exhibit gaps of such dimensions. Gaps/defects are known to be present in solid tumors (Seymour, 1992; Jain, 1989) and KS lesions (Francis et al, 1986; Vogel et al, 1988). Indeed, fluorescent pegylated liposomes of <100 nm in diameter have been visualized by video microscopy extravasating in real time into the interstitium of implanted tumors using window chamber models (Yuan et al, 1994; Huang et al, 1995; Dewhirst and Needam, 1995).

The improved antitumor activity of DOXIL relative to a comparable dose of free doxorubicin can be partially attributed to these slow in situ release kinetics. Consider the distribution kinetics after a dose of free doxorubicin. Drug molecules enter the tumor (and other tissues) quickly, reaching maximal exposure (i.e., peak concentrations) within minutes (Working et al, 1994). During the subsequent 24 hours, tumor doxorubicin concentration drops precipitously to undetectable levels. During this brief â&#x20AC;&#x153;pulseâ&#x20AC;? of doxorubicin, those cells not exposed to a cytotoxic concentration for a sufficient amount of time, or which are not at a sensitive point in the cell cycle, can escape therapy and continue to proliferate. A typical course of doxorubicin is given on a three week cycle. This length of time between injections is needed to allow for recovery from the hematologic toxicity associated with doxorubicin therapy. Following such a schedule, it is quite likely that tumor cells are exposed to cytotoxic levels of drug for only a few hours during the 3 week interval between injections. In the case of DOXIL which is also given in a 2-4 week cycle, not only does more drug reach the tumor, but, by virtue of the slow in situ release kinetics provided by the liposomes, tumor

D. Release of drug following extravasation Encapsulated doxorubicin is released from the DOXIL liposomes after extravasation in tumors (Dewhirst and Needam, 1995). Several possible factors may contribute to liposome breakdown and drug release in tumors: (i ) conditions present in the interstitial fluid surrounding tumors may cause breakdown of the liposomes, such as low pH, (Stubbs et al, 1992) and lipases released from dead or dying tumor cells (Sakayama et al, 1994); (i i ) inflammatory cells (which are often found in tumors (Dvorak et al, 1981) may release factors that lead to liposome destabilization such as enzymes or superoxide 192

Gene Therapy and Molecular Biology Vol 1, page 193 cells are exposed to drug over a period of several days to perhaps a week or more after a single dose. Such a release pattern may contribute to DOXIL’s antitumor response.

toxicity, although significant nausea and vomiting, ototoxicity, peripheral neuropathy and myelotoxicity are also induced by cisplatin administration. Attempts to ameliorate cisplatin-induced toxicity and/or resistance have focused on the development of platinum derivatives that are less toxic and/or more active than the parent compound (Schilder et al, 1994; Kelland and McKeage, 1994). Alternative approaches include altering the pharmacology of the drug by altering the treatment schedule, hydrating patients prior to and during therapy, or administering renal protectant therapy. Encapsulating the drug within liposomes has shown improved therapeutic capacity (Steerenberg et al, 1987; Potkul et al, 1991).

E. Tumor cell penetration and cytotoxicity Given its amphipathic nature, a doxorubicin molecule that is released from a liposome can quickly diffuse through surrounding fluids and connective tissue, enter tumor cells, bind to nucleic acids and inhibit DNA synthesis. Indeed, it is quite likely that drug molecules released from DOXIL can penetrate many cell layers into the tumor, well beyond the point that the liposome itself has reached. Early findings suggest that penetration of “free” drug in this fashion may be essential for DOXIL’s antitumor activity.

SPI-77 is a formulation of cisplatin encapsulated in virtually the same type of liposome as DOXIL. SPI-77 exhibits plasma pharmacokinetics characteristic of sterically stabilized (Stealth) liposomes, with long circulation, high C max and area-under-the-plasma concentration vs time curve (AUC), and low clearance and volume of distribution compared to non-liposomal cisplatin (Figure 21). In vitro leakage studies suggest that plasma levels of platinum primarily or solely represent liposomal cisplatin, i.e., drug that is in liposomes and free or bound to proteins.

As mentioned above, microscopic observations indicate that liposomes extravasate in tumors at particular sites; primarily through vessels forming at the advancing edge of angiogenesis (Yuan et al, 1994). The deposition of extravasated liposomes in these areas is perivascular and focal, occurring primarily at the roots of capillary sprouts where weak spots (possibly defects or gaps) in the endothelium are believed to occur. Given the geometry of the system, liposomes that enter through such gaps may not be able to penetrate deeply into the tumor interstitium. Liposome penetration may be limited by a range of physical obstacles including tight cell-cell junctions (often found in highly differentiated epithelial cell tumors), dense connective tissue stroma, small extracellular volume and high interstitial fluid viscosity (that may be caused by fibrin cross-linking) (Nagy et al, 1995). Ideally all tumor cells, regardless of their proximity to blood vessels or the liposome depots that may from near them, would be exposed to a cytotoxic dose of drug. So, the observation that drug molecules released from focal, perivascular deposits of liposomes are able to penetrate deeply into the tumor mass may be a critical requirement for expression of DOXIL’s antitumor activity.

The therapeutic activity of SPI-77 has been evaluated and compared to non-liposomal cisplatin in various tumor models, including the C26 colon carcinoma in Balb/c mice and a xenograft of the NCI-H82 small cell lung tumor in athymic mice (Figure 22). SPI-77 showed meaningful anti-tumor activity in these tumor models. Cisplatin was only effective in the NCI-H82 xenograft model; carboplatin (Paraplatin) was ineffective in both. SPI-77 only occasionally produced complete tumor responses, but did cause a persistent inhibition of tumor growth during and after treatment. In many animals, tumors grew slowly to intermediate size and then were apparently arrested, with little additional growth evident. Although cisplatin treatment resulted in better inhibition of tumor growth in both trials in the NCI-H82 xenograft models, SPI-77 was more effective in producing a prolonged response to treatment, with persistent inhibition of tumor growth.

VI. Encapsulation of other drugs into stealth liposomes

B. Stealth Vincristine

A. cis-Platinum (SPI-77)

Vincristine is used clinically both as a single agent and in combination regimens, for the treatment of hematological malignancies, head and neck cancer, Kaposi’s sarcoma and lung cancer. Early work with conventional liposomal Vincristine showed no improvement in safety or therapeutic activity relative to the free drug (Layton and Trouet, 1980).

Cisplatin (Platinol) is active alone and or combination chemotherapy regimens against a wide rage of epithelial malignancies including testicular, ovarian, head and neck, lung, bladder, and cervical cancers (Loehler and Einhorn, 1984). Cisplatin chemotherapy is often limited by side effects that prohibit continued treatment. In addition, some tumors are initially resistant or acquire cisplatin resistance with continued exposure. The major doselimiting cisplatin-induced toxicity in humans is renal

Stealth liposome-encapsulated Vincristine (S-Vinc) prolonged the drug’s distribution phase plasma half-life in rats from 0.22 to 10.5 hours. While there was no 193

Martin and Boulikas: The challenge of liposomes in gene therapy significant difference in LD50 between encapsulated and free drug (at doses of #2.5 mg/kg, given by i.v. injection), mice given sublethal doses of S-Vinc experienced

significantly less weight loss compared to animals receiving the same dose of Vincristine. Compared to free

F i g u r e 2 1 : Plasma clearance of cisplatin (circles) or SPI-77 (squares) after single intravenous injection in rabbits.

F i g u r e 2 2 . Tumor growth kinetics of a human small cell lung cancer xenograft (NCI-H82) implanted subcutaneously in athymic mice. Groups of tumor-bearing animals were treated via tail vein injection with 100 mg/kg carboplatin (circles), saline (inverted triangles), 6 mg/kg cisplatin (squares) or 6 mg/kg SPI-77 (triangles).

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F i g u r e 2 3 . Tumor volume in BLAB/c mice given multiple tail vein injections of saline, 1.3 mg/kg Vincristine (Oncovin) or 1.3 mg/kg Stealth liposomal Vincristine (SVINC). Treatment was given on days 10,17 and 24 after implantation of the murine C26 colon carcinoma. (NMT = no measurable tumor)

reticuloendothelial system of the body rapidly removes liposomes from the blood. Gangliosides and sphingomyelin, when included into the lipids of the liposome, act synergistically to diminish the rate of uptake of liposomes by macrophages of the host defense system; this results in extended circulation times of these large unilamellar liposomes (Allen and Chonn, 1987).

drug, S-Vinc was more active against intraperitoneally and subcutaneously implanted tumors. In a subcutaneouslyimplanted murine colon tumor model, multiple doses of free drug did little to retard tumor growth, but S-Vinc slowed tumor growth and improved long-term survival in several dosing regimens (Figure 23; Allen et al, 1995). Stealth liposomes extravasate preferentially to tumors by leaking through new vessels during the process of angiogenesis of the tumor (Papahadjopoulos et al, 1991; Huang et al, 1992, 1993; Gabizon et al, 1994).

Attempts to generate cell-targeting have focused primarily on the addition of monoclonal antibodies to the surface of the liposome. Liposomes tagged on their surface with IgG immunoglobulins directed against a variety of cell membrane proteins and desialylated fetuin which binds to the parenchymal cells of the liver can deliver bleomycin and mediate selective cellular uptake of the entrapped drug (Gregoriadis and Neerunjun, 1975). Apparently the hydrophobic IgG regions penetrate the lipid bilayers whereas the immunologically active portions are facing the exterior of the liposomes and are available for interaction with cells.

Steric hindrance by coating the liposome surface with PEG can inhibit recognition of targeting ligands, such as antibodies, by cell membrane proteins on the targeted cell (Mori et al, 1991; Torchilin et al, 1992). This obstacle can be in part overcome by conjugating a water-soluble drug at the end of the PEG polymer. For example, 66-nm in diameter liposomes can be efficiently targeted to tumor cells that express folate receptors (KB cells) via conjugation of the folate to a PEG spacer of 25 nm in length; shorter PEG spacers were not efficient in mediating binding of the liposomes to KB cells (Lee and Low, 1995). Antibodies attached to long PEG spacers can give stealth liposomes that are effective in target binding and exhibit prolonged circulation times (Papahadjopoulos et al, 1991; Blume et al, 1993).

A. IgG-coated liposomes can target specific cell types

Immunoliposomes tagged with monoclonal antibodies against c-ErbB2 (other names Neu or HER2), product of the protooncogene c-erbB2, a growth factor receptortyrosine kinase, were bound preferentially to breast cancer cells in culture which overexpress this receptor; loading these immunoliposomes with doxorubicin made them more toxic to cell lines overexpressing the c-erbB2 oncogene; furthermore, when this immunoliposome bullet was injected into SCID mice bearing human breast tumor xenografts it was able to deliver the cytotoxic doxorubicin to the tumor cells (Park et al, 1995).

Injected liposomes are localized mainly in the fixed macrophages of the liver and spleen tissue; indeed the

More recently, production of cell-targeting ligands has been achieved by cell-binding peptides specific for different

VII. Cell targeting with liposomes

195

Martin and Boulikas: The challenge of liposomes in gene therapy cell types in culture; these peptides are selected through several rounds of binding to a particular cell type from random peptide-presenting phage libraries (Devlin et al, 1990; Cwirla et al, 1990; Barry et al, 1996).

Cholera toxin trafficking, observed by fluorescence confocal microscopy, might occur via caveolae directed to the Golgi compartment (Bastiaens et al, 1996); cationic amphiphilic drugs (also cationic liposomes?) inhibit the internalization of cholera toxin to the Golgi (Sofer and Futerman, 1995). These studies support a model for internalization of cationic or amphiphilic liposomes via caveolae.

Antibodies have been attached to neutral liposomes (Straubinger et al, 1988; Ahmad et al, 1992, 1993). A disadvantage of using antibodies that are bulky in liposome formulations is the increase in the volume of the liposome: small liposomes extravasate at the site of a tumor more readily than large liposomes and larger liposomes are captured more frequently by macrophages in animal and human studies; thus, keeping the size of the liposome small offers a clear advantage for its use as a delivery system.

2. The GPI anchor The mechanism of GPI anchoring involves covalent attachment of the glycosyl-phosphatidylinositol moiety to the C-terminus of the protein through an ethanolamine linkage. The GPI anchor precursor is synthesized in the endoplasmic reticulum and linked to protein posttranslationally. This occurs soon after the protein synthesis; the GPI anchor is added in the lumen of the endoplasmic reticulum (Takahashi et al, 1996) and might involve either a protease linked to a transferase or a single transpeptidase which breaks a peptide bond at the Cterminus and forms the amide bond to the ethanolamine (Ferguson and Williams, 1988). Synthesis of the GPI anchor involves several steps; the first reaction is transfer of the N-acetyl-glucosamine (GlcNAc) from UDP-GlnNAc to phosphatidylinositol (PI) ; deacetylation of this molecule is then followed by the sequential addition of three mannosyl residues (Man); the last step involves transfer EtN-P to the third mannose from phosphatidylethanolamine. Most of the genes involved in GPI synthesis have been cloned (see Takahashi et al, 1996). The core backbone of the GPI anchor is conserved from yeast to mammals and has the structure: ethanolamine-P6Mana1,2Mana1,6Mana1,4GlcNa1,6myoinositol1-P-lipid (Ferguson and Williams, 1988; Takahashi et al, 1996).

Often antibodies are loaded to preassembled liposomes in order to avoid exposure of the antibody to organic solvents; in other cases antibodies are reacted with preassembled liposomes containing lipids with activated head groups (Heath et al, 1983; Matthay et al, 1989). Antibodies have also been conjugated to N-glutarylphosphatidylethanolamine in aqueous dispersions and have been reassembled with the drug bullet and bilayer lipids by detergent dialysis into targeting liposomes (Maruyama et al, 1990; Lundberg et al, 1993); however, the encapsulation efficiency with hydrophilic drugs is very low.

B. Liposomes tagged with folate receptor and the caveolae vesicle 1. Caveolae The purpose of this approach is to bypass the lysosomal compartment that could modify and degrade foreign DNA during DNA delivery. Caveolae, also known as plasmalemmal vesicles, are cell membrane organelles appearing under transmission electron microscopy as 50-100 nm invaginations of the plasma membrane (Bundgaard et al, 1979; Montesano et al, 1982). Caveolae are abundant in endothelial cells and are rich in glycosyl-phosphatidylinositol (GPI); caveolae concentrate specific proteins that bind to GPI lipids and mediate a unique transcytosis or potocytosis mechanism where the engulfed material is not presented to lysosomes but to Golgi or is emptied to the cytoplasm. Proteins interacting with the GPI lipid components include SRC tyrosine kinases, an anchorage mediated by their palmitoylation (Robbins et al, 1995), the folate receptors $, %, and & , and G protein-coupled receptors, and may thus constitute integral components of the signal transduction from the cell exterior to cytoplasm and the nucleus across the cell membrane (reviewed by Anderson, 1993a,b; Lisanti et al, 1994).

The phosphatidylinositol glycan of complementation class B (PIG-B) is a ER transmembrane protein involved in transferring the third mannose; about 60 aa are to the cytoplasmic site and the large C-terminal portion of 470 aa, that contains the active site, lies within the lumen of the ER (Takahashi et al, 1996). A somatic mutation in the X-linked PIG-A gene involved in the first step in GPI synthesis results in defective GPI anchor and is a somatically acquired genetic disease known as paroxysmal nocturnal hemoglobinuria; the defect arises from afflicted clonal hematopoietic cells (Takeda et al, 1993). The nascent proteins that are to be GPI anchored have a signal peptide sequence at their C-terminus; this Cterminal peptide is cleaved and the new C-terminus is linked to the ethanolamine of the GPI anchor (see Takahashi et al, 1996 for more references). Once attached to the GPI anchor, proteins are transported to the plasma membrane by vesicular transport via the Golgi apparatus; protein molecules with GPI anchors are more mobile in 196

Gene Therapy and Molecular Biology Vol 1, page 197 the lipid bilayer than proteins with a transmembrane domain and such proteins are thought to be localized at specialized regions of the plasma membrane. The importance of the GPI anchor is obvious in the case of the neural acetylcholinesterase, also attached to the membrane via a GPI anchor: rapid destruction of acetylcholine in the region of the synapse triggers neurotransmission. Other protein molecules attached to membranes via a GPI anchor include alkaline phosphatase, 5' nucleotidase, alkaline phosphodiesterase, the lymphoid antigens Thy-1 and RT-6 and others (reviewed by Ferguson and Williams, 1988).

tetrahydrofolate (THF) is composed of the two condensed ring compound 2-amino-4-hydroxy-6-methyltetrahydro pteridine linked to p-aminobenzoic acid which is esterified with the amino group of glutamic acid. N5-methyl-THF is formed by removal of the -CH2OH group from serine and its reduction to -CH3; the methyl group is then donated to homocysteine to form methionine. N10-formyl-THF is a cofactor of the enzyme phosphoribosylaminoimidazolecarboxamide formyltransferase; N5, N10-methylene-THF is a cofactor of the enzyme phosphoribosylglycinamide formyltransferase; both derivatives of THF donate a formyl group to two different intermediates during biosynthesis of inosinic acid, the precursor of adenylic and guanylic acids (AMP and GMP) during the building of the purine ring on D-ribose-5-phosphate. N5, N 10-methylene-THF is also a cofactor of the enzyme thymidylate synthetase which catalyzes methylation of the 5 position of deoxyuridylic acid (dUMP) to deoxythymidylic acid (dTMP); the antifolate drugs aminopterin and amethopterin, used as antineoplastic drugs, are competitive inhibitors of dihydrofolate reductase (DHFR) that converts DHF into THF (Lehninger, 1975).

The GPI anchor can be broken by proteases as in the case of folate receptor (Lacey et al, 1989) or by activation of phospholipase C (PLC) in response to triggering at the cell surface; one of the cleavage products of phosphoinositides by PLC is diacylglycerol which stimulates protein kinase C and another cleavage product is inositol phosphate that triggers the release of Ca++ from intracellular stores. This has led to the idea that breakdown of GPI anchors might be a component of receptor mediated triggering pathway. A number of membrane proteins are known to be associated with caveolae. The protein-tyrosine kinase p59hck is first myristoylated and then palmitoylated at another site, cysteine-3; palmitoylation targets p59hck to caveolae vesicles (Robbins et al, 1995). The FYN tyrosine kinase is also anchored to caveolae membrane via its palmitoylation at cysteine-3 (Shenoy-Scaria et al, 1994). Among the SRC family of tyrosine kinases, myristoylation is a prerequisite for their anchorage to the cell membrane and mutations at the N-terminal glycine where myristoylation takes place results in the exclusive retain of the kinase in the cytoplasm (Resh, 1994). Different types of interactions have been evoked to explain anchorage of the SRC family of tyrosine kinases to the cell membrane including insertion of the myristate moiety into the lipid bilayer, electrostatic protein-lipid interactions, and interactions between the anchor part of the SRC proteins with protein domains already embedded in the cell membrane (Resh and Ling, 1990; Sigal et al, 1994). This suggests that caveolae participate in the transduction of signals across the plasma membrane (Anderson, 1993a,b; Lisanti et al, 1994).

4. Folate receptor (FR) is overexpressed in tumor cells The FR molecule is maximally expressed on the surface of cells cultured in low folate medium and mediates the high affinity accumulation of 5-methyltetrahydrofolate in the cytoplasm of these cells. Because of their increased metabolic rates tumor cells have increased needs for folate and overexpress folate receptor (Matsue et al, 1992; Weitman et al, 1992; Mayor et al, 1994). A special interest for the FR emerged from the finding that its density on the cell membrane is considerably (more than 20-fold) higher in tumor than in normal cells especially ovarian adenocarcinoma and cervical carcinoma cell lines; FR expression, albeit at lower levels, was detected in normal bone marrow, spleen, thymus and ovarian and uterine carcinoma tissue explants (Weitman et al, 1992). FR is also expressed in subsets of breast, lung, and colon cancer, in neuroendocrine carcinomas and rare gliomas (Garin-Chesa et al, 1993). Folate receptor (also called folate-binding protein) was identified by cDNA cloning as the ovarian cancer-associated antigen recognized by the monoclonal antibody MOv18; this monoclonal antibody was used for immunodiagnosis of ovarian cancers. FR was not amplified in 16 out of 16 carcinoma cell lines examined and thus the overexpression of this gene in ovarian cancer involves other mechanisms (Campbell et al, 1991). The overexpression of FR in cancer cells has raised the possibility of targeting tumor cells with folate attached to different ligands such as PEG-liposome-encapsulated doxorubicin (Lee and Low, 1995, see below).

3. Folate as an essential cofactor in purine/pyrimidine biosynthesis Folic acid, broadly distributed in plant leafs, is essential for mammals (vitamin) supporting cell growth; its reduced form, tetrahydrofolate, serves as an intermediate carrier of hydroxymethyl (-CH2OH), formyl (-CHO), or methyl (-CH3) groups in a large number of enzymatic reactions in particular those involved in the intermediary metabolism of purines, pyrimidines, and amino acids; 197

Martin and Boulikas: The challenge of liposomes in gene therapy The folate receptor (FR) is a membrane protein linked to glycosyl-phosphatidylinositol. The anchor to GPI of the protein molecule is a C-terminal 19 aa residue segment: WAAWPFLLSLALMLLWLLS (Lacey et al, 1989; Coney et al, 1991). Thus FR is not a transmembrane protein since it lacks a cytoplasmic tail. The protein is released from the membrane by cleavage of its anchor with phosphatidylinositol phospholipase C, apparently enriched in plasma and responsible for a soluble form of the FR in plasma as well as milk. The cDNA cloning also revealed a signal peptide at the N-terminus responsible for targeting to the lumen of the endoplasmic reticulum: MAQRMTTQLLLLLVWVAVVGEAQT, with the hydrophobic core of the sequence underlined (Lacey et al, 1989). It is noted here that the FR possesses a putative weak nuclear localization signal AKHHKEKPGPEDK; thus, a possible cleavage of the membrane anchor of the molecule inside the cytoplasm, if occurring at all under physiological or pathological conditions, could give a soluble form of the folate receptor similar to that found in human and cow milk, able to enter the nucleus (Boulikas, 1996b).

pass to the lysosomal compartment as do clathrin-coated pits (Rothberg et al, 1990). One additional advantage of the folate-PEG-liposome is that the conjugation of folate-PEG-distearoylphosphatidyl ethanolamine (DSPE) is performed prior to liposome assembly and is thus compatible with the different methods of liposome preparation (Lee and Low, 1995).

VIII. Cationic liposomes in gene delivery A. Principle of cationic liposomemediated gene transfer Cationic liposomes have gained wide recognition as delivery vehicles for plasmid DNA in somatic cell gene transfer often circumventing the shortcomings of the viral and retroviral systems (Lasic and Papahadjopoulos, 1995; Ledley, 1995; Ali単o et al, 1996; Cao et al, 1995); one advantage using cationic liposomes is that there is no limit on the size of DNA to be delivered to cells compared with the upper limit of 7.5 kb that can be accommodated into viral/retroviral vectors. The elimination of therapeutically important cells from the body by the immune system due to expression of viral proteins after ex vivo delivery of genes with recombinant adenovirus seems to be an additional drawback of viral methods (Dai et al, 1995).

The part of the cell membrane with a high density of RFs (clusters of about 750 protein molecules; Rothberg et al, 1990) is potocytosed forming a special type of vesicle known as caveolae; according to a model proposed by Rothberg and coworkers (1990) caveolae, enclosing the RF with the folate bound to it, remain attached to the membrane at the cytoplasmic side of the cell; the pH inside the caveolae vesicle drops by one unit as a result of a proton pump on the vesicle increasing the concentration of H+ inside the vesicle and causing the dissociation of the folate from its receptor; folate then moves across the caveolae membrane via the transporter using the energy generated by the H+ gradient; finally, folate is modified by a chain of glutamic acid residues, a modification entrapping it into the cytoplasm, and the caveolae unseals and presents the receptor to the exterior of the cell for another cycle.

Two approaches have been used for the liposomal delivery of genes: (i ) encapsulation of plasmids (Kaneda et al, 1989) and oligonucleotides (Thierry and Dritschilo, 1992) into true liposomes and (i i ) formation of a complex between liposomes composed of cationic lipids and plasmid DNA (Ali単o et al, 1996; Cao et al, 1995). Use of pH-sensitive liposomes (Wang and Huang, 1987) or liposomes with folate ligands exposed on their surface (Lee and Low, 1995) have been used to circumvent the cumbersome uptake of such complexes into endosomes (lysosomes) by phagocytosis resulting in DNA degradation.

About 600,000 RF molecules per cell have been estimated for MA104 monkey kidney epithelial cells in culture; these are grouped into about 800 clusters per cell each containing 750 RF molecules (Rothberg et al, 1990).

Important parameters affecting cationic liposomemediated transfection efficiency are (i ) the type of lipid, (i i ) the ratio of lipid to DNA, (i i i ) the presence of DNA condensing agents such as spermine, polylysine, histones, (i v ) whether cells in culture or somatic cells in animals in vivo are being targeted, (v) presence of fusogenic peptides in the complex (Wagner et al, 1992), and (v i ) the type of control elements that drive the reporter or therapeutically important gene. The physicochemical properties of such complexes and their interaction with the cell surface are not well understood (Lasic and Papahadjopoulos, 1995).

5. Folate-PEG-liposomes in tumor therapy The folate receptor has been predicted from cDNA molecular cloning and sequencing to be anchored in the membrane via a glycosyl-phosphatidylinositol (GPI) linkage (Lacey et al, 1989). GPI in membranes has a special function (Low and Saltiel, 1988) and molecules internalized into cells via GPI-enriched caveolae do not

The calcium phosphate coprecipitation method and high molecular weight polycations (dextran) are still 198

Gene Therapy and Molecular Biology Vol 1, page 199 extensively used for the introduction of plasmid DNA into cells; however, these methods display a high variability in transfection, are toxic to cells, and result in the introduction of many copies of DNA into a single cell whereas the majority of cells may not be transfected at all. Furthermore, multiple copies of foreign DNA may become integrated into the host's genome; the mechanisms involved have not been fully elucidated.

the heart when injected into the tail vein in mice (Huang, SK, SEQUUS, personal communication). Cationic liposome-plasmid complexes are rapidly taken up by endothelial cells; this explains why the primary tissue target is lung, which has by far the largest surface area of vascular endothelium, followed by liver and heart (Huang, SK and Danilo Lasic, personal communications). The mechanism of internalization of cationicliposome-plasmid complexes by cells in vivo is not thoroughly understood; cationic liposomes could electrostatically bind to the slightly negatively-charged surface of the cells followed by endocytosis leading to the enclosure of the liposome-plasmid complex into endosomes and lysosomes. According to a different model (Danilo Lasic, personal communication) cationic liposome-plasmid complexes enter rapidly the cell like "bullets". It is believed that only a tiny fraction of the plasmid reaches

Compared to viral vectors, liposomes are safer to prepare, their toxicity can be monitored, and the risk of pathogenic and immunological complications is diminished; a great variety of cationic lipids is available for transfection studies (Table 3); liposome compositions can be matched with the appropriate liposome mean diameter which is controlled by ultrasonication or extrusion through membranes of various pore sizes. In vivo studies have shown that cationic liposomesplasmid complexes are cleared rapidly from the blood stream of animals and do not circulate beyond one pulse of

Table 3. Cationic and neutral lipids used in liposome formulations and other polymers for gene transfer Abbreviated name

Full name

Reference

DC-CHOL

3% [N-(N',N'-dimethylaminoethane)carbamoyl]cholesterol

Gao and Huang, 1991; Litzinger et al, 1996; Zuidam and Barenholz, 1997

DDAB

dimethyldioctadecyl ammonium bromide

e.g. Lappalainen et al, 1997

DMRIE

N-[1-(2,3-dimyristyloxy)propyl]-N,N-dimethyl-N-(2hydroxyethyl) ammonium bromide

Felgner et al, 1994

DMTAP

1,2-dimyristoyl-3-trimethylammonium propane

Song et al, 1997; Filion and Phillips, 1997

DODAC

Dioctadecyldimethylammonium chloride

Behr et al, 1989

DOGS

Dioctadecylamidoglycylspermine (Transfectam, Promega)

Behr et al, 1989

DOPC

1,2-dioleoyl-sn-glycero-3-phosphatidylcholine

Zuidam and Barenholz, 1997

DOPE

dioleyl phosphatidylethanolamine (neutral fusogenic lipid)

DOSPA

2,3-dioleoyloxy-N-[2(sperminecarboxamido)ethyl]-N,Ndimethyl -1-propanaminium trifluoroacetate

DOTAP

1,2-dioleyloxypropyl-3-(trimethylammonium)propane

Lappalainen et al, 1997

N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride DOTMA

N-[1-(2,3-dioleyloxy) propyl]-n,n,n-trimethylammonium chloride

Felgner et al, 1987

DOTMA:DOPE 1:1

Lipofectin (GIBCO BRL)

Yoshimura et al, 1992; Zhu et al, 1993; Hyde et al, 1993

DPPES

Dipalmitoyl phosphatidylethanolamidospermine

Behr et al, 1989

DPTAP

1,2- dipalmitoyl-3-trimethylammonium propane

Song et al, 1997; Filion and Phillips, 1997

DSPE

distearoyl phosphatidylethanolamine (neutral lipid)

Felgner et al, 1987

DSTAP

1,2-disteroyl-3-trimethylammonium propane

Song et al, 1997; Filion and Phillips, 1997

ExGen (PEI)

Polyethylenimine

Ferrari et al, 1997

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intact the cytoplasmic compartment and even a smaller fraction is imported into nuclei (Boulikas, 1998b).

Wood et al, 1995); and of the IL-2 gene for prostate cancer therapy (Vieweg et al, 1995).

There is no upper limit in plasmid DNA size to be complexed with cationic liposomes as opposed to adenoviruses, AAV, and retroviruses that can accommodate a maximum of 7.5 kb of foreign DNA because of packaging limitations. This factor is of utmost importance when large genomic regions need to be transferred in order to obtain correct developmental expression after transduction of fetuses or newborn animals.

Cationic liposomes have also been used for arterial gene transfer (Nabel et al, 1990, 1993, Takeshita et al, 1994). Stable cationic lipid/DNA complexes were formed by solubilizing DOSPA:DOPE in 1% octylglucoside, 10 mM Tris pH 7.4 followed by the addition of DNA and exhaustive dialysis of the complex against 10 mM Tris, pH 7.4, 5% dextrose; the lipid-DNA complex had a storage life of up to 3 months after formation with respect to its ability to transfect tissue culture cells and was competent of transfecting cells in the presence of 15% fetal bovine serum (FBS) whereas convenient cationic liposome-DNA complexes are unable to transfect cells in the presence of FBS; a precipitate of the stable cationic lipid-DNA complex formed after 14 days on shelf storage at 40C could be pelleted retaining all the transfection efficiency and displaying lower toxicity because of the removal of the free uncomplexed lipid in the supernatant (Hofland et al, 1996).

Positively-charged liposomes containing distearoyl phosphatidylethanolamine, a lipid which promotes fusion of liposomes with membranes, has been used for the transfer of plasmid DNA into cells (Felgner et al, 1987; Felgner and Ringold, 1989). Liposomes containing head groups able to form discrete complexes with DNA induce wrapping of DNA around unilamellar 80- to 100-nm in diameter vesicles in a way reminiscent of the wrapping of 167 bp of DNA around the 55-nm core histone octamer forming nucleosomes (see Behr et al, 1989). The positively-charged groups of the lipids interact with DNA causing both a condensation of the plasmid by diminishing the negative electrostatic repulsions on DNA as well as electrostatic binding of DNA. The liposomeplasmid DNA complex is then presented to cells in culture or is injected into animals intravenously, intraperitoneally, subcutaneously, intratracheally, or via other routes.

DOTAP DOTAP, a monocationic lipid, has been used to transfer efficiently the lacZ reporter gene and CFTR cDNA into mice without any inflammatory response(McLachlan et al, 1995).

B. Studies on cationic liposomemediated gene transfer

Lipofectin Lipofectin (a 1:1 mixture of the cationic DOTMA with the neutral DOPE) has been shown to deliver reporter genes to the rodent airways after direct intratracheal injection (Yoshimura et al, 1992) or after intravenous administration (Zhu et al, 1993). Lipofectin has been used to alleviate the symptoms of cystic fibrosis in transgenic CF mice after transfer of the CFTR gene (Hyde et al, 1993). Lipofectin has been used for the transfer of the CCK gene to suppress audiogenic epileptic seizures, as well as for the transfer of reporter genes directly injected into mouse brain (Ono et al, 1990; Roessler and Davidson, 1994).

A number of studies in gene therapy have used cationic liposomes as means of delivering DNA. Examples include transfer of prostaglandin G/H synthase to protect lungs in rabbits against endotoxin-induced inflammation and pulmonary hypertension (Conary et al 1994); of $ 1antitrypsin cDNA to protect lungs (Canonico et al, 1994) or $ 1-antitrypsin cDNA encapsulated into negativelycharged liposomes to protect connective tissue from the lytic action of the leukocyte neutrophil elastase (Ali単o et al, 1996); of the human CFTR (cystic fibrosis transmembrane conductance regulator) gene in lungs in CFTR-deficient transgenic mice (Hyde et al, 1993; Alton et al, 1993) or in normal mice (Yoshimura et al, 1992) by tracheal instillation for the transduction of airway epithelial cells for cystic fibrosis; of the MHC class I HLA-B7 heavy chain gene for the treatment of cancer (Lew et al, 1995); of tyrosine hydroxylase (TH) gene in order to alleviate degeneration of dopaminergic nigrostriatal neurons in rat models of Parkinson's disease (Jiao et al, 1993; Cao et al, 1995); of the wild-type p53 gene to treat nude mice inoculated with breast carcinoma cells (Lesoon-

DOGS (Transfectam) DOGS appears to be more efficient than Lipofectin and DOTAP for the transfer of the luciferase gene to polyp and tracheal lung epithelial cells in culture; this may be due to the presence of a secondary amine with a pKa=5.4 in the DOGS molecule which might be able to buffer the acidic endosomes and protect the plasmid DNA from degradation;

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Gene Therapy and Molecular Biology Vol 1, page 201 DOGS, however, was inefficient for gene transfer to submucosal gland cells which are active in producing sticky mucus in CF patients and should be the target cells to be corrected by CFTR gene transfer (Ferrari et al, 1997).

control of CMV promoter to striatal parenchyma and paraventricular brain cells in neonatal mice; however, the expression of the transgene, although significant at early times postinjection diminished over time (Schwartz et al, 1995).

Compaction of plasmid DNA with dipalmitoyl phosphatidylethanolamidospermine (DPPES) and dioctadecylamidoglycylspermine (DOGS), collectively known as lipospermines, gave lipid-coated plasmid DNA rather than liposome-plasmid complexes; lipospermines interact strongly with DNA eventually promoting coating of supercoiled DNA plasmids and promote binding of the complex to the cell membrane (Behr et al, 1989). When dispersed in water (either by sonication or by dilution of an ethanol solution) they form unilamellar vesicles of 80-100 nm, unstable in ionic media, and interacting cooperatively with plasmids because of the strong affinity of the 5 7 -1 spermine group for DNA (10 -10 M ). Small unilamellar vesicles formed between DOGS and egg yolk lecithin were unable to mediate transfection and thus DOGS-mediated transfection is not a liposome-mediated process but a process mediated by cationic lipid-coated plasmid (Behr et al, 1989).

C. Advantages and drawbacks using cationic lipids for gene delivery Cationic lipids may show low efficiency of transfection despite the relatively large amounts of DNA used. Cationic lipid-DNA complexes with a net positive charge may interact with circulating serum proteins or anionic components of the extracellular matrix in the various tissues; this interaction reduces their bioavailability (Schwartz et al, 1995). In addition positivelycharged complexes activate complement and complementdependent phagocytosis by macrophages in the reticuloendothelial system and are, thus, cleared rapidly from body fluids (Plank et al, 1996). Thus, although the optimal lipid-DNA ratio may be to an excess of positive charges for the transfection of cells in culture (mediated by the ability of cationic lipids to interact with the relatively negatively-charged external surface of the cell membrane and higher poration through the membrane) the optimal ratio in vivo is nearly that which gives a neutral complex (Boulikas et al, in preparation, see Boulikas 1998a).

Although the in vitro conditions for transfection seem to favor a ratio of positive charges of lipids to negative phosphate charges on DNA of about 1:8, the in vivo optimal conditions are 1 lipid molecule/30 phosphates using DDAB:DOPE and DDAD:Chol (Boulikas et al, in preparation). Previous studies by Behr and coworkers (1989; reviewed by Behr, 1994) and Schwartz et al (1995) using DOGS have also shown that the in vivo optimal conditions require a lower lipid:DNA charge ratio; the explanation might be that anionic proteins in the blood serum or in the extracellular matrix could interact with cationic lipid particles inhibiting their uptake by the cells in tissues.

D. Entry of liposome-DNA into cells Neutral liposomes are internalized via the endocytic uptake mechanism. However, cationic liposome-plasmid complexes (Capaccioli et al, 1993; Boutorine and Kostina, 1993) as well as Sendai virus-derived liposomes (Compagnon et al, 1992; Morishita et al, 1993) seem to permit a direct passage of the oligonucleotide load through the cell membrane (see also Bongartz et al, 1994).

The in vivo efficacy for transferring episomally replicating plasmids containing the human papovavirus BKV origin of replication/early regulatory region and the large T antigen gene, as well as the luciferase reporter gene under control of RSV promoter were investigated by Thierry and coworkers (1995); dioctadecylamidoglysylspermidine:DOPE liposomes hydrated in the presence of plasmid DNA, injected into the vein of mice, sustained expression in the liver, lung, spleen, heart, and other tissues for up to three months postinjection. These vectors were found to replicate extrachromosomally in the lung tissue; use of nonepisomal vectors under the same conditions gave only transient expression of the luciferase gene which disappeared after about 4-5 days.

E. Enhancement of cationic liposome delivery A number of methods have been invented to augment the efficiency of internalization or release from endosomes of DNA-liposome complexes, or to enhance nuclear import of the plasmid after its release in the cytoplasm. The external cell surface has a net negative charge and thus liposome-DNA particles with a net positive charge can be electrostatically anchored to the external cell membrane. Liposomes are internalized into endosomes which degrade the plasmid; use of folate attached to ligands on the surface of the liposome (Gottschalk et al, 1993) changes the route of internalization and the particles are taken up by the caveolae vesicles rather than endosomes which lack

Cationic liposomes have been used for targeting brain cells after direct intracranial injection of the plasmidliposome complex. DOGS:DOPE liposomes have successfully transferred the luciferase reporter gene under 201

Martin and Boulikas: The challenge of liposomes in gene therapy nucleases and release more readily their content to the interior of the cell compared with endosomes.

The elimination of therapeutically important cells from the body by the immune system due to expression of viral proteins after ex vivo delivery of genes with recombinant adenovirus (Dai et al, 1995) would not apply to liposomal delivery of genes.

Use of pH-sensitive liposomes, such as DOPE: cholesterol: oleic acid liposomes (Wang and Huang, 1987; Huang et al, 1987) seem to induce their break-down at the lower pH of the endosomes releasing the DOPE to the endosome; DOPE then induces endosome membrane breakdown and release of its content to the cytoplasm.

IX. Clinical trials using liposomemediated gene transfer

Use of nuclear proteins in complex with plasmid DNA encapsulated into true (as opposed to cationic) liposomes has been found to increase transfection efficiency; DNA was rapidly transported into the nuclei and its expression reached a maximum within 6-8h after transfection (Kaneda et al, 1989; Kato et al, 1991). According to this procedure Sendai virus was used to fuse DNA-loaded ganglioside liposomes with protein-containing membrane vesicles purified from red blood cells; cointroduction of HMG-1 protein showed rapid uptake of plasmids by nuclei whereas with BSA in the place of HMG the grains of the in situ hybridization were located in the cytoplasm after 6 h reaching the nucleus only after about 24h (Kaneda et al, 1989).

All RAC-approved protocols for gene transfer to human patients with liposomes use cationic lipids (Table 4). Because of the toxicity of cationic lipids none of these protocols employs systemic intravenous injection of the cationic liposome-plasmid complex; instead the type of administration involves (i ) direct intratumoral injection for immunotherapy of melanoma, lymphoma, renal carcinoma and a great variety of metastatic malignancies; (i i ) subcutaneous injection for glioblastoma antisense-IGF therapy, or for immunotherapy of melanoma, colon with hepatic metastases, renal, breast, and small cell lung cancer; (i i i ) intranasal delivery for CFTR and alpha-1antitrypsin deficiency; (i v ) intradermal injection for the immunotherapy of ovarian cancer and advanced or metastatic prostate cancer using IL-2 cDNA; and (v) intraperitoneal and intrapleural delivery of the adenoviral E1A gene to control the regulation of the HER-2/neu oncogene in ovarian and breast cancer T ( able 4).

F . Problems and advantages for liposomal delivery of genes During in vivo delivery foreign DNA can be attacked by macrophages, lymphocytes, or other components of the immune system and the vast majority is cleared from blood, intracellular, or other body fluids before it is given the chance to reach the membrane of the cell target; the half-life of naked plasmids injected intravenously into animals is about 5 min (reviewed by Boulikas, 1998a). On the other hand “stealth” liposomes persist in the body fluids for days as seen in the whole body scintigraphs of empty radioactively-labeled “stealth “ liposomes (Figures 17-19); the encapsulated plasmid DNA would also circulate for long times. However, tumor cells , where the “stealth” liposomes are localized are very often of epithelial origin (brain, colon, breast, head & neck, prostate tumors) and not engaged in the uptake of liposomes or other colloidal particles. Thus, stealth liposomes remain in the extracellular space and slowly releasing their material after lysis over days. One strategy to circumvent this bottleneck would be to include cationic lipids in the lipid bilayer and devise methods for PEG coating to fall off; the cationic lipids are then expected to mediate rapid poration through the cell membrane or uptake, as cationic lipids are known to penetrate rapidly most types of cells.

X. Prospects To-date, only very few conventional (radiation/ chemotherapy) regimens on cancer patients lead to tumor eradication. Most treatments, after cancer is detected at an advanced stage, prolong the life of the patient by few months but reduce its quality because of the adverse effects of antineoplastic treatments. Gene therapy offers strong hopes to millions of desperate people, both patients and relatives. Although "stealth" liposomes extravasate preferentially in solid tumors, they remain in the extracellular space and are not readily internalized by tumor cells; liposomes loaded with doxorubicin concentrate the drug in the solid tumor and release their content over a period of several days. Use of substance P, a peptide known to increase vascular permeability, increased liposome extravasation in tissues that posses receptors for substance P (trachea, esophagus, and urinary bladder); extravasated liposomes remained in the extracellular space beyond the postcapillary venular endothelium at early times. The colloidal gold particles encapsulated into liposomes were localized intracellularly in both endothelial cells and macrophages at 24 h postinjection (Rosenecker et al, 1996).

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Gene Therapy and Molecular Biology Vol 1, page 203

Table 4. RAC-approved human gene therapy protocols using liposomes (For clinical trials using viruses see Appendix 1 in Boulikas, 1998, pages 159-172 of this volume). Protocol number & human disease

Gene and route of administration

Investigators/ Affiliation

Title of protocol & date of RAC/NIH approval

187.

9202-013 (Closed) Gene Therapy /Phase I /Cancer /Melanoma /Adenocarcinoma /Immunotherapy

In Vivo /Autologous Tumor Cells /Cationic Liposome Complex /DCChol /HLA-B7 /Beta-2 Microglobulin cDNA /Intratumoral /Direct Injection /Catheter Delivery to Pulmonary Nodules

Nabel, Gary J.; University of Michigan, Ann Arbor, Michigan

Immunotherapy of Malignancy by In Vivo Gene Transfer into Tumors. RAC Approval: 2-10-92 /NIH Approval: 4-1792 Closed: 11-19-92 (Replaced by Protocol #9306-045)

188.

9306-045 (Open) Gene Therapy /Phase I /Cancer Immunotherapy

In Vivo /Autologous Tumor Cells /Cationic Liposome Complex /HLAB7 /Beta-2 Microglobulin cDNA / Intratumoral /Direct Injection /Catheter Delivery to Pulmonary Nodules

Nabel, Gary J.; University of Michigan Medical Center, Ann Arbor, Michigan

Immunotherapy for Cancer by Direct Gene Transfer into Tumors. RAC Approval: 6-7-93 /NIH Approval: 9-3-93

189.

9306-052 (Open) Gene Therapy /Phase I /Cancer / Glioblastoma /Antisense

In Vitro /Autologous Tumor Cells /Lethally Irradiated / Cationic Liposome Complex /Lipofectin (Gibco BRL) /Insulin-like Growth Factor Antisense / Subcutaneous Injection

Ilan, Joseph; Case Western Reserve University School of Medicine and University Hospitals of Cleveland, Cleveland, Ohio

Gene Therapy for Human Brain Tumors Using Episome-Based Antisense cDNA Transcription of Insulin-Like Growth Factor I. RAC Approval: 6-8-93 /NIH Approval: 12-2-93

190.

9309-053 (Open) Gene Therapy /Phase I /Cancer / Small Cell Lung Cancer / Immunotherapy

In Vitro /Autologous Tumor Cells /Lethally Irradiated / Cationic Liposome Complex /Lipofectin (Gibco BRL) /Cytokine /Interleukin-2 cDNA /Neomycin Phosphotransferase cDNA /Subcutaneous Injection

Cassileth, Peter; Podack, Eckhard R.; Sridhar, Kasi; University of Miami; and Savaraj, Niramol; Miami Veterans Administration Hospital, Miami, Florida

Phase I Study of Transfected Cancer Cells Expressing the Interleukin-2 Gene Product in Limited Stage Small Cell Lung Cancer. RAC Approval: 9-9-93 /NIH Approval: 12-2-93

191.

9312-063 (Open) Gene Therapy /Phase I /Cancer /Melanoma /Immunotherapy

In Vitro /Allogeneic Tumor Cells /Lethally Irradiated /Cationic Liposome Complex /Lipofectin (Gibco BRL) /B7 (CD80) cDNA /Neomycin Phosphotransferase cDNA /Subcutaneous Injection

Sznol, Mario; National Institutes of Health, Frederick, Maryland

A Phase I Trial of B7-Transfected Lethally Irradiated Allogeneic Melanoma Cell Lines to Induce Cell Mediated Immunity Against Tumor-Associated Antigens Presented by HLA-A2 or HLAA1 in Patients with Stage IV Melanoma. RAC Approval: 12-3-93 /NIH Approval: 4-19-94

192.

9312-064 (Closed) Gene Therapy /Phase I /Cancer /Colon /Hepatic Metastases /Immunotherapy

In Vivo /Autologous Tumor Cells /Cationic Liposome Complex /DMRIE-DOPE Vical VCL-1005 /HLA-B7 /Beta-2 Microglobulin cDNA /Intratumoral /Hepatic Injection

Rubin, Joseph; Mayo Clinic, Rochester, Minnesota

Phase I Study of Immunotherapy of Advanced Colorectal Carcinoma by Direct Gene Transfer into Hepatic Metastases.

193.

9312-066 (Open) Gene Therapy /Phase I /Monogenic Disease /Cystic Fibrosis

In Vivo /Nasal Epithelial Cells /Cationic Liposome Complex /DMRIE-DOPE /Cystic Fibrosis Transmembrane Conductance Regulator cDNA /Intranasal

Sorscher, Eric J. and Logan, James L.; University of Alabama, Birmingham, Alabama

Gene Therapy for Cystic Fibrosis Using Cationic Liposome Mediated Gene Transfer: A Phase I Trial of Safety and Efficacy in the Nasal Airway. RAC Approval: 12-3-93 /NIH Approval: 1-4-95

194.

9403-070 (Open) Gene Therapy /Phase I /Monogenic Disease /Alpha-1-Antitrypsin Deficiency

In Vivo /Nasal Epithelial Cells /Respiratory Epithelial Cells /Cationic Liposome Complex /DC-Chol-DOPE /Alpha-1 Antitrypsin cDNA /Intranasal /Respiratory Tract Administration (Bronchoscope)

Brigham, Kenneth; Clinical Research Center at Vanderbilt University Medical Center, Nashville, Tennessee

Expression of an Exogenously Administered Human Alpha-1-Antitrypsin Gene in the Respiratory Tract of Humans.

9403-071 (Closed) Gene Therapy /Phase I /Cancer /Renal Cell /Immunotherapy

In Vivo /Autologous Tumor Cells /Cationic Liposome Complex /DMRIE-DOPE Vical VCL-1005 /HLA-B7 /Beta-2 Microglobulin cDNA /Intratumoral /Direct Injection

Vogelzang, Nicholas; the University of Chicago, Chicago, Illinois

Phase I Study of Immunotherapy for Metastatic Renal Cell Carcinoma by Direct Gene Transfer into Metastatic Lesions.

In Vivo /Autologous Tumor Cells /Cationic Liposome Complex /DMRIE-DOPE Vical VCL-1005 /HLA-B7 /Beta-2 Microglobulin cDNA /Intratumoral /Direct Injection

Hersh, Evan; Arizona Cancer Center, Tucson, Arizona; and Akporiaye; Harris; Stopeck; Unger; and Warneke; University of Arizona, Tucson, Arizona

195.

196.

9403-072 (Closed) Gene Therapy /Phase I /Cancer /Melanoma /Immunotherapy

Sponsor: Vical, Incorporated RAC Approval: 12-3-93 /NIH Approval: 4-1994 Closed: 3-16-95

Sponsor: Gene Medicine, Inc. RAC Approval: 3-3-94 /NIH Approval: 10-2594

Sponsor: Vical, Incorporated RAC Approval: 3-4-94 /NIH Approval: 4-19-94 Closed: 4-5-95

203

Phase I Study of Immunotherapy of Malignant Melanoma by Direct Gene Transfer. Sponsor: Vical, Incorporated RAC Approval: 3-4-94 /NIH Approval: 4-19-94

Martin and Boulikas: The challenge of liposomes in gene therapy

197.

9409-086 (Open) Gene Therapy /Phase I /Cancer /Breast /Immunotherapy

In Vitro /Autologous Tumor Cells /Lethally Irradiated /Cationic Liposome Complex /Avectin™ /Cytokine /Interleukin-2 cDNA /Subcutaneous Injection

Lyerly, H. Kim; Duke University Medical Center, Durham, North Carolina

A Pilot Study of Autologous Human Interleukin-2 Gene Modified Tumor Cells in Patients with Refractory or Recurrent Metastatic Breast Cancer. RAC Approval: 9-12-94 /NIH Approval: 10-25-94

198.

9412-095 (Open) Gene Therapy /Phase I /Solid Tumors /Lymphoma /Immunotherapy

In Vivo /Autologous Tumor Cells /Cationic Liposome Complex /DMRIE-DOPE Vical VCL-1102 /Cytokine /Interleukin-2 cDNA /Intratumoral /Direct Injection

Hersh, Evan; Arizona Cancer Center, Tucson, Arizona; and Rinehart, John; Scott and White Clinic; Temple Texas.

Phase I Trial of Interleukin-2 Plasmid DNA /DMRIE /DOPE Lipid Complex as an Immunotherapeutic Agent in Solid Malignant Tumors or Lymphomas by Direct Gene Transfer. Sponsor: Vical, Incorporated RAC Approval: 12-1-94 /NIH Approval: 3-2-95

199.

9506-108 (Open) Gene Therapy /Phase I /Cancer /Renal Cell /Melanoma /Immunotherapy

In Vitro /Autologous Tumor Cells /Lethally Irradiated /Cationic Liposome Complex /DMRIE-DOPE Vical VCL-1005 /HLA-B7 /Beta-2 Microglobulin cDNA /Subcutaneous Injection

Fox, Bernard A. and Urba, Walter J.; Earle A. Chiles Research Institute, Providence Medical Center, Portland, Oregon

Adoptive Cellular Therapy of Cancer Combining Direct HA-B7 /ß-2 Microglobulin Gene Transfer with Autologous Tumor Vaccination for the Generation of Vaccine-Primed Anti-CD3 Activated Lymphocytes. RAC Approval: 6-9-95 /NIH Approval: 9-30-95

200.

9506-110 (Open) Gene Therapy /Phase I /Cancer /Ovarian /Immunotherapy

In Vitro /Autologous Tumor Cells /Lethally Irradiated /Cationic Liposome Complex /DDAB-DOPE /Cytokine /Interleukin-2 cDNA /Intradermal Injection

Berchuck, Andres and Lyerly, H. Kim; Duke University Medical Center, Durham, North Carolina

A Phase I Study of Autologous Human Interleukin-2 (IL-2) Gene Modified Tumor Cells in Patients with Refractory Metastatic Ovarian Cancer. RAC Approval: 6-10-95 /NIH Approval: 9-3095

201.

9508-115 (Open) Gene Therapy /Phase II /Cancer /Metastatic Malignancies (Breast Adenocarcinoma, Renal Cell Carcinoma, Melanoma, Colorectal Adenocarcinoma, nonHodgkin’s Lymphoma) /Immunotherapy

In Vivo /Autologous Tumor Cells /Cationic Liposome Complex /DMRIE-DOPE Vical VCL 1005 /HLA-B7 /Beta-2 Microglobulin cDNA /Direct Intratumoral Injection

Chang, Alfred E.; Univ of Michigan; Hersh, Evan; Arizona Cancer Center; Vogelzang, Nicholas; University of Chicago; Levy, Ronald; Stanford University; Redman, Bruce; Wayne State University; Figlin, Robert; UCLA; Rubin, Joseph; Mayo Foundation; Rinehart, John J.; Scott and White Hospital, Texas A & M University; Doroshow, James H.; City of Hope; Klasa, Richard; British Columbia Cancer Agency; Sobol, Robert; Sidney Kimmel Cancer Center

Phase II Study of Immunotherapy of Metastatic Cancer by Direct Gene Transfer.

202.

9508-121 (Open) Gene Therapy/Phase I/Cancer/Renal Cell/Immunotherapy

In Vivo/Autologous Tumor Cells/HLA B7 cDNA/ Intratumoral/Concurrent Interleukin-2 Therapy

Figlin, Robert A.; University of California Los Angeles Medical Center, Los Angeles, California

Phase I Study of HLA-B7 Plasmid DNA/DMRIE/DOPE Lipid Complex as an Immunotherapeutic Agent in Renal Cell Carcinoma by Direct Gene Transfer with Concurrent Low Dose Bolus IL-2 Protein Therapy. Sponsor: Vical, Incorporated Sole FDA Review Recommended by NIH/ORDA: 8-14-95

203.

9509-127 (Open) Gene Therapy /Phase I /Monogenic Disease /Cystic Fibrosis

In Vivo /Nasal Epithelial Cells /Cationic Liposome Complex /DOPE /Cystic Fibrosis Transmembrane Conductance Regulator cDNA; Intranasal Administration

Welsh, Michael J. and Zabner, Joseph; Howard Hughes Medical Institute, University of Iowa College of Medicine, Iowa City, Iowa

Cationic Lipid Mediated Gene Transfer of CFTR: Safety of a Single Administration to the Nasal Epithelia.

204.

9510-132 (Open) Gene Therapy /Phase I /Cancer /Locally Advanced or Metastatic Prostate /Immunotherapy

In Vitro /Autologous Tumor Cells /Lethally Irradiated /Cationic Liposome Complex /Cytokine /Interleukin-2 cDNA /Intradermal Injection

Paulson, David; and Lyerly, H. Kim; Duke University Medical Center, Durham, North Carolina

A Phase I Study of Autologous Human Interleukin-2 (IL-2) Gene Modified Tumor Cells in Patients with locally Advanced or Metastatic Prostate Cancer. Sole FDA Review Recommended by NIH /ORDA: 10-19-95

205.

9512-137 (Open) Gene Therapy /Phase I /Cancer /Ovarian,Breast /Oncogene Regulation /HER-2 /neu

In Vivo /Autologous Tumor Cells /Cationic Liposome Complex /DCChol-DOPE /E1A /Intraperitoneal, Intrapleural Administration

Hortobagyi, Gabriel N.; Lopez-Berstein, Gabriel; and Hung, Mien-Chien; MD Anderson Cancer Center, Houston, Texas; Kilbourn, Robert, Rush-Presbyterian /St. Luke’s Medical Center, Chicago, Illinois; Weiden, Paul, Virginia Mason Medical

Phase I Study of E1A Gene Therapy for Patients with Metastatic Breast or Ovarian Cancer that Overexpresses Her-2 /neu.

204

Sponsor: Vical, Incorporated Sole FDA Review Recommended by NIH /ORDA: 8-2-95

Sponsor: Genzyme Corporation Sole FDA Review Recommended by NIH /ORDA: 9-26-95

Sponsor: Targeted Genetics Corporation RAC Approval: 12-4-95 /NIH Approval: 2-2-96

Gene Therapy and Molecular Biology Vol 1, page 205 Center, Seattle, Washington 206.

9512-142 (Open) Gene Therapy /Phase I /Gene Therapy /Cancer /Head and Neck Squamous Cell Carcinoma /Immunotherapy

In Vivo /Autologous Tumor Cells /Cationic Liposome Complex /DMRIE-DOPE Vical VCL 1005 /HLA-B7 /Beta-2 Microglobulin cDNA /Direct Intratumoral Injection

Gluckman, Jack L.; University of Cincinnati Medical Center, Cincinnati, Ohio

Allovectin-7 in the Treatment of Squamous Cell Carcinoma of the Head and Neck. Sole FDA Review Recommended by NIH /ORDA: 12-15-95

207.

9608-156 (Open) Gene Therapy /Phase I /Cancer /Breast /Immunotherapy

InVitro /Allogeneic Tumor Cells /Lethally Irradiated /Cationic Liposome Complex /B7(CD80) cDNA /Subcutaneous Injection

Urba, Walter J., Providence Portland Medical Center, Portland, Oregon

Phase I Trial Using a CD80-Modified Allogeneic Breast Cancer Line to Vaccinate HLA-A2-Positive Women with Breast Cancer . Sole FDA Review Recommended by NIH /ORDA: 8-6-96

208.

9609-161 (Open) Gene Therapy /Phase I /Cancer /Small Cell Lung Cancer /Immunotherapy

In Vitro /Autologous Tumor Cells /Lethally Irradiated /Cationic Liposome Complex /Lipofectin(GibcoBRL) /B7-1(CD80) cDNA / Subcutaneous Injection

Antonia, Scott J., H. Lee Moffitt Cancer Center, Tampa, Florida

Treatment of Small Cell Lung Cancer Patients In Partial Remission Or At Relapse With B7-1 Gene-Modified Autologous Tumor Cells As A Vaccine With Systemic Interferon Gamma. Sole FDA Review Recommended by NIH /ORDA: 10-10-96

209.

9610-162 (Open) Gene Therapy /Phase I /Cancer /Solid Tumors /Oncogene Regulation /HER-2 /neu

In Vivo /Autologous Tumor Cells /Cationic Liposome Complex /DCChol-DOPE /E1A /Intratumoral Injection

LaFollette, Suzanne, Rush /Presbyterian /St. Lukeâ&#x20AC;&#x2122;s Medical Center, Chicago, Illinois; Murray, James L., M.D. Anderson Cancer Center, Houston, Texas; Yoo, George, Wayne State University, Detroit, Michigan

A Phase I Multicenter Study of Intratumoral E1A Gene Therapy for Patients with Unresectable or Metastatic Solid Tumors that Overexpress HER-2 /neu.

210.

211.

212.

Sponsor: Targeted Genetics Corporation Sole FDA Review Recommended by NIH /ORDA: 10-29-96

9611-168 (Open) Gene Therapy /Phase II /Cancer /Melanoma /Immunotherapy

In Vivo /Autologous Tumor Cells /Cationic Liposome Complex /DMRIE-DOPE Vical VCL 1005 /HLA-B7 /Beta-2 Microglobulin cDNA /Direct Intratumoral Injection

Hersh, Evan M., Arizona Cancer Center, Tucson, Arizona; Klasa, Richard, British Columbia Cancer Agency, Vancouver, B.C., Canada; Gonzales, Rene, University of Colorado Cancer Center, Denver, Colorado; Silver, Gary, Northern California Melanoma Clinic, San Francisco, California;Thompson, John A.,U. of Washington Medical Center, Seattle, Washington

Phase II Study of Immunotherapy of Metastatic Melanoma by Direct Gene Transfer.

9611-169 (Open) Gene Therapy /Phase I /II /Cancer /Solid Tumors /Immunotherapy

In Vivo /Autologous Tumor Cells /Cationic Liposome Complex /DMRIE-DOPE Vical VCL 1102 /Cytokine /Interleukin-2 cDNA /Direct Intratumoral Injection

Hersh, Evan, M., Arizona Cancer Center, Tucson, Arizona; Rinehart, John, Scott and White Clinic, Temple, Texas; Rubin, Joseph, Mayo Clinic, Rochester, Minnesota; Sondak, Vernon K., University of Michigan Medical Center, Ann Arbor, Michigan; Gonzales, Rene, University of Colorado Cancer Center, Denver, Colorado; Sobol, Robert E., Sharp HealthCare, San Diego, California; and Forscher, Charles A., CedarsSinai Comprehensive Cancer Center, Los Angeles, California

Phase I /II Trial of Interleukin-2 DNA /DMRIE /DOPE Lipid Complex as an Immunotherapeutic Agent in Cancer by Direct Gene Transfer.

9612-170 (Open) Gene Therapy /Phase I /Monogenic Disease /Cystic Fibrosis

In Vivo /Lung and Nasal Epithelial Cells /Cationic Liposome Complex /DOPE /CFTR cDNA /Aerosol Administration

Sorscher, Eric, University of Alabama, Birmingham, Medical Center

Safety and Efficiency of Gene Transfer of Aerosol Administration of a Single Dose of a Cationic Lipid /DNA Formulation fo the Lungs and Nose of Patients with Cystic Fibrosis.

Sponsor: Vical, Incorporated NIH /ORDA Receipt Date: 11-26-96. Sole FDA Review Recommended by NIH /ORDA: 1-6-97

Sponsor: Vical, Incorporated NIH /ORDA Receipt Date: 11-26-96. Sole FDA Review Recommended by NIH /ORDA: 1-17-97

Sponsor: Genzyme Corporation NIH /ORDA Receipt Date: 12-17-96. Sole FDA Review Recommended by NIH /ORDA: 1-6-97 213.

9703-184 (Open) Gene Therapy /Phase I /Cancer /Prostate Cancer /Immunotherapy

In Vivo /Autologous Tumor Cells /Cationic Liposome Complex /DMRIE-DOPE Vical VCL-1102 /Cytokine /Interleukin-2 cDNA /Intratumoral Injection

Belldegrun, Arie, University of California, Los Angeles, School of Medicine, Los Angeles, California

A Phase I Study Evaluating the Safety and Efficacy of Interleukin-2 Gene Therapy Delivered by Lipid Mediated Gene Transfer (Leuvectin) in Prostate Cancer Patients. Sponsor: Vical, Inc. NIH /ORDA Receipt Date: 3-24-97. Sole FDA Review

205

Martin and Boulikas: The challenge of liposomes in gene therapy Recommended by NIH /ORDA: 5-21-97 214.

9704-186 (Open) Gene Therapy /Phase I /Monogenic Disease /Cystic Fibrosis

In Vivo /Nasal Epithelial Cells /Cystic FibrosisTransmembrane Conductance Regulator cDNA /Cationic Liposome Complex /EDMPC /Intranasal Administration

Noone, Peadar G., Knowles, Michael R., University of North Carolina at Chapel Hill, North Carolina

A Double-Blind, Placebo Controlled, Dose Ranging Study to Evaluate the Safety and Biological Efficacy of the Lipid-DNA Complex GR213487B in the Nasal Epithelium of Adult Patients with CysticFibrosis. Sponsor: Glaxo Wellcome Inc. NIH /ORDA Receipt Date: 4-23-97. Sole FDA Review Recommended by NIH /ORDA: 5-13-97

215.

9705-190 (Open) Gene Therapy /Phase I /Cancer /Squamous Cell Carcinoma of the Head and Neck /Immunotherapy

In Vivo /Autologous Tumor Cells /Cationic Liposome Complex /DOTMA-Cholesterol /Cytokine /Interleukin-2 cDNA /Intratumoral Injection

Oâ&#x20AC;&#x2122;Malley, Bert W., Johns Hopkins Medical Institutions, Baltimore, Maryland

A Double-Blind, Placebo-Controlled, Single Rising-Dose Study of the Safety and Tolerability of Formulated hIL-2 Plasmid in Patients with Squamous Cell Carcinoma of the Head and Neck (SCCHN). Sponser: Gene Medicine, Inc. NIH /ORDA Receipt Date: 5-27-97. Sole FDA Review Recommended by NIH /ORDA: 6-16-97

216.

9706-191 (Open) Gene Therapy /Phase II /Cancer /Head and Neck Squamous Cell Carcinoma /Immunotherapy

In Vivo /Autologous Tumor Cells /Cationic Liposome Complex /DMRIE-DOPE /Vical VCL-1005 /HLA-B7 /Beta-2 Microglobulin cDNA /Direct Intratumoral Injection

Gluckman, Jack L..; Gleich, Lyon L., University of Cincinnati Medical Center, Cincinnati, Ohio; Swinehart, James M., Colorado Medical Research Center, Denver, Colorado; Hanna, Ehab, University of Arkansas for Medical Sciences /Arkansas Cancer Research Center (UAMS), Little Rock, Arkansas; Castro, Dan J., University of California, Los Angeles, Los Angeles, California; Gapany, Markus, Veterans Affairs Medical Center, Minneapolis, Minnesota; Carroll, William, R., University of Alabama at Birmingham, Birmingham, Alabama; and Coltrera, Marc D., University of Wahington Medical Center, Seattle, Washington

Phase II Study of Immunotherapy by Direct Gene Transfer with Allovectin-7 for the Treatment of Recurrent or Metastatic Squamous Cell Carcinoma of the Head and Neck

9709-210 (Open) Gene Therapy /Phase III /Cancer /Melanoma /Immunotherapy

In Vivo /Autologous Tumor Cells /Cationic Liposome Complex /DMRIE-DOPE /Vical VCL-1005 /HLA-B7 /b2-Macroglobulin cDNA /Direct Intratumoral Injection

Gonzales, Rene; University of Colorado Cancer Center, Denver, Colorado and Hersh, Evan; Arizona Cancer Center, Tucson, Arizona

Compassionate Use Protocol for Retreatment with Allovectin-7 Immunotherapy for Metastatic Cancer by Direct Gene Transfer

217.

Sponsor: Vical, Inc. NIH /ORDA Receipt Date: 6-6-97. Sole FDA Review Recommended by NIH /ORDA: 7-7-97

Sponsor: Vical, Inc. NIH /ORDA Receipt Date: 9-8-97. Sole FDA Review Recommended by NIH /ORDA: 9-26-97

218.

9708-211 (under review) Gene Therapy /Phase I /Monogenetic Disease / Canavan Disease

In Vivo /Autologous Brain Cells /Plasmid DNA /Adeno-associated Virus /Poly-L-Lysine /Cationic Liposome Complex /DC-Chol /DOPE /Aspartoacylase cDNA /Intracranial (Ommaya Reservoir) Administration

During, Matthew, J.; University of Auckland, New Zealand; Leone, Paola; and Seashore, Margretta, R.; Yale University, New Haven, Connecticut

Gene Therapy of Canavan Disease: Retreatment of Previously Treated Children NIH /ORDA Receipt Date: 828-97.

219.

9709-212 (Open) Gene Therapy /PhaseI /Cancer /Melanoma /Immunotherapy

In Vivo /Autologous Tumor Cells /Cationic Liposome Complex /DMRIE-DOPE Vical VCL-1005 /HLA-B7 /Beta-2 Microglobulin cDNA /Vical-1102 /Interleukin-2 cDNA /Intratumoral Injection

Gonzales, Rene; University of Colorado Health Sciences Center, Denver, Colorado; and Hersh, Evan M.; Arizona Cancer Center, Tucson, Arizona

Phase I Study of Direct Gene Transfer of HLA-B7 Plasmid DNA /DMRIE /DOPE Lipid Complex (Allovectin-7) with IL-2 Plasmid DNA /DMRIE /DOPE Lipid Complex (Leuvectin) as an Immunotherapeutic Regimen in Patients with Metastatic Melanoma Sponsor: Vical, Inc. NIH /ORDA Receipt Date: 9-18-97. Sole FDA Review Recommended by NIH /ORDA: 10-8-97

220.

9711-222 (under review) Gene Therapy /Phase I /Monogenetic Disease / Canavan Disease

In Vivo /Autologous Brain Cells /Plasmid DNA /Adeno-Associated Virus /Protamine /Cationic Liposome Complex /DC-Cholesterol-DOPE /Aspartoacylase cDNA /Intracranial (Ommaya Reservoir) Administration

Freese, Andrew; Thomas Jefferson University, Philadelphia, Pennsylvania

206

Gene Therapy of Canavan Disease NIH /ORDA Receipt Date: 11-12-97.

Gene Therapy and Molecular Biology Vol 1, page 207 Alton EWFW, Middleton PG, Caplen NJ, Smith SN, Steel DM, Munkonge FM, Jeffery PK, Geddes DM, Hart SL, Williamson R, Fasold KI, Miller AD, Dickinson P, Stevenson BJ, McLachlan G, Dorin JR, Porteous DJ (1 9 9 3 ) Non-invasive liposome-mediated gene delivery can correct the ion transport defect in cystic fibrosis mutant mice. Nature Genet 5, 135-142.

A breakthrough would be the encapsulation of plasmids with therapeutic genes (e.g. antitumor genes such as p53, HSV-tk, angiostatin) into Stealth liposomes with mechanisms inducing PEG fall off after their accumulation into tumors; when a certain amount of cationic liposomes is included into the lipid composition of the Stealth liposome, the PEG-free liposome is expected to be taken rapidly by tumor cells. Fusogenic peptides or other strategies could be combined to release the liposomes from endosomes. These regiments could be combined with nontoxic levels of antineoplastic drugs encapsulated into Stealth liposomes that might act synergistically with the tumor suppressor or tumor killer genes to eradicate cancer. The major advantage, no doubt, would be the systemic delivery of genes with this approach, able to hit, like no other gene therapy regiment currently available, the primary tumor and its metastases.

Amantea, M.A., Forrest, A., Northfelt, D.W. and Mamelok, R. (1 9 9 7 ) Population pharmacokinetics and pharmacodynamics of pegylated liposomal doxorubicin in patients with AIDS-related Kaposiâ&#x20AC;&#x2122;s sarcoma. C l i n . Pharmacol. Therapeutics, 61:301-311. Anderson RGW (1 9 9 3 a ) Caveolae: Where incoming and outgoing messengers meet. P r o c N a t l A c a d S c i U S A 90, 10909-10913. Anderson RGW (1 9 9 3 b ) Plasmalemmal caveolae and GPIanchored membrane proteins. C u r r O p i n C e l l B i o l 5, 647-652. Bally MB, Nayar R, Masin D, Hope MJ, Cullis PR and Mayer LD (1 9 9 0 ) Liposomes with entrapped doxorubicin exhibit extended blood residence times. B i o c h i m . B i o p h y s . A c t a . 1023, 133-139.

Acknowledgements We are indebted to Stewart Simon for providing Figures for this article and to Dan Lasic, Demetrios Papahadjopoulos, Peter Working, Ken Huang, and Joe Vallner for stimulating discussions.

Bangham, A.D. and Horne, R.W. (1 9 6 4 ) Negative staining of phospholipids and their structural modification by surface-active agents as observed in the electron microscope. J . M o l B i o l . 8:660-668. Barry MA, Dower WJ, Johnston SA (1 9 9 6 ) Toward celltargeting gene therapy vectors: Selection of cell-binding peptides from random peptide-presenting phage libraries. Nature Med 2, 299-305.

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Wagner E, Plank C, Zatloukal K, Cotten M, and Birnstiel ML (1 9 9 2 ) Influenza virus hemagglutinin HA-2 N-terminal fusogenic peptides augment gene transfer by transferrinpolylysine-DNA complexes: Toward a synthetic virus-like gene-transfer vehicle. P r o c N a t l A c a d S c i U S A 89, 7934-7938.

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Gene Therapy and Molecular Biology Vol 1, page 215 Gene Ther Mol Biol Vol 1, 215-229. March, 1998.

Gene transfer to the nervous system using HSV vectors M. Karina Soares1, William H. Goins1, Joseph C. Glorioso1,2,3, and David J. Fink1,2 1

Department of Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, E1240 Biomedical Science Tower, Pittsburgh, PA 15261 USA 2 Department of Neurology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261 USA __________________________________________________________________________________________________ 3 Corresponding Author: Joseph C. Glorioso, Tel: (412) 648-8106, Fax: (412) 624-8997, E-mail: joe@hoffman.mgen.pitt.edu

Summary T h e n a t u r a l h i s t o r y o f H e r p e s s i m p l e x v i r u s t y p e 1 ( H S V - 1 ) i n f e c t i o n i n humans suggests its potential for development as a gene transfer vehicle suitable for nervous system applications. HSV-1 has a broad host range, does not require cell division for infection and gene expression and h a s e v o l v e d t o p e r s i s t i n a l i f e - l o n g n o n i n t e g r a t e d l a t e n t s t a t e w i t h o u t t h e e x p r e s s i o n o f viral proteins or evidence of neurodegenerative disease in the immune competent host. The virus also has evolved a unique neuronal-specific promoter system that remains active during latency and fortuitously may be used to express therapeutic proteins without compromising the latent state. The establishment of latency also does not require the expression of viral lytic functions and thus removal of genes required for expression of the viral cascade of expressed products allows for safe vector design without the possibility of reactivation from latency. The HSV-1 genome is 152 Kb in length and of the 84 known genes (Roizman & Sears, 1996), approximately half are dispensable for virus replication in cell culture thereby providing considerable opportunity for introduction of f o r e i g n s e q u e n c e s . I n t h i s r e v i e w , a b r i e f o v e r v i e w o f t h e b i o l o g y o f H S V r e l e v a n t t o vector design and progress in reducing virus cytotoxicity and its relevance to the level and duration of transgene expression is discussed. Methods for the expression of transgenes in the peripheral and central nervous system using the latency active promoters are described and strategies are suggested for potential applications to the treatment of neurodegenerative disease and cancer.

of chronic neurodegenerative conditions (e.g. Alzheimerâ&#x20AC;&#x2122;s disease, Parkinsonâ&#x20AC;&#x2122;s disease) might be ameliorated by the local production of neurotrophic factor(s) that prevent the degeneration of affected cells. Even though there is no evidence that these diseases are caused primarily by trophic factor deficiency, experimental evidence has shown that disease progression in animal models may be prevented by specific neurotrophins (e.g. nerve growth factor, glial derived neurotrophic factor) (Choi-Lundberg et al., 1997; Gash et al., 1996; Kearns and Gash, 1995; Tomac et al., 1995). An ideal vector for this application would constitutively produce low levels of the trophic factor in the region of interest for the life of the host. Intracranial malignancies of neural tissue origin or metastases from other tissues represent a second class of neurologic disease that might be treated by the transient local expression of therapeutic molecules. The therapy of brain tumors could

I. Introduction The nervous system would appear to be a fertile site for the application of gene transfer for the treatment of disease where structural features of this tissue impede traditional pharmacologic therapy. These include the blood-brain-barrier which excludes macromolecules in the blood from entry into brain parenchyma and the cellular and regional specialization within the nervous system which may require the delivery of the therapeutic agent to restricted regions or to particular cells within those regions. Such impediments would be overcome by the direct transfer of a gene whose local expression resulted in the production of a macromolecule required for restoration of normal tissue function. Several different classes of neurologic disease could theoretically be treated by gene transfer. The progression 215

Soares et al: Gene transfer using HSV vectors be enhanced by the expression within tumor cells either of immune modulators that attract tumor killing inflammatory cells and cells capable of differentiating into tumor-specific immune T cells or the transient expression of cytotoxic molecules (e.g. tumor necrosis factor) or enzymes (e.g. thymidine kinase) which locally activate anti-cancer drugs. Activated drugs such an ganciclovir and 5-fluoro-uracil can also kill dividing neighboring cells even without direct infection by cell to cell transmission or uptake of locally released drug. Multiple sclerosis, a relapsing remitting disease caused by immune mediated attack on central nervous system myelin, could be effectively treated by the transient expression of immunomodulatory cytokines that block the autoimmune attack, although prolonged expression of cytokine inhibitors would not likely be beneficial, requiring either repeat dosing of the vector or control of anti-cytokine gene expression by drug manipulation of therapeutic gene expression.

are integrated (Kaplitt et al., 1994). Ex vivo approaches consist of introducing the therapeutic gene into a cell population such as fetal or immortalized neurons, multipotent progenitor neural stem cells, adrenal chromaffin cells, glia, or fibroblasts that can be cultured in vitro and survive at or migrate to the relevant anatomic location upon transplantation back into the host. Given the dividing nature of the target cell population, ex vivo approaches can employ retroviral and adeno-associated viral vectors.

III. HSV vectors HSV is a commonly acquired, naturally neurotropic virus that establishes a life-long, benign association with the human host. The virus replicates within epithelial cells of the cornea or orofacial tissue (Cook and Stevens, 1973; Stevens, 1989), invades local peripheral nerve endings and ascends to the associated sensory ganglion by retrograde axonal transport (F i g . 1 ). HSV is capable of maintaining multiple copies of viral genomes as quiescent episomes within post-mitotic sensory neurons of the peripheral nervous system (Efstathiou et al., 1986; Mellerick and Fraser, 1987; Rock and Fraser, 1985). The HSV genome contains 84 known open reading frames (Roizman and Sears, 1996), approximately half are considered non-essential since they are complemented by host cell proteins or provide accessory functions that influence viral replication and spread in vivo. These can be deleted without affecting the ability of the virus to replicate in culture thus providing the potential to transduce as much as 35 kb of foreign DNA sequence. The current generation of viral vectors exhibit reduced cytotoxicity due to further deletion of viral genes responsible for biochemical and structural alterations in host cell processes that occur in the course of natural infection. These include (i) disaggregation of polyribosomes and degradation of cellular mRNA induced soon after infection by the virion host shut-off protein (vhs) (Kwong et al., 1988; Oroskar and Read, 1989; Read and Frenkel, 1983), (ii) inhibition of RNA splicing by the immediate-early protein ICP27 (Brown et al., 1995; McGregor et al., 1996; Sandri-Goldin and Hibbard, 1996), (iii) fragmentation of host chromosomes, and (iv) destruction of sub-cellular compartments late in infection (Johnson et al., 1992, 1994). Lastly, multiply deleted viral mutants that are incapable of replicating in neurons or any cells other than their stably transformed complementing cell lines have been developed (Marconi et al., 1996; Samaniego et al., 1995; Wu et al., 1996b). These replication-incompetent viral vectors are incapable of reactivating from latency thus providing a relatively safe, gene transfer vector that has a natural propensity for the nervous system.

II. Gene transfer vectors for brain Genes may be introduced into relevant cells of the nervous system directly in vivo or through transplantation of transduced cells (ex vivo approach). A crucial factor in gene replacement therapy is efficient transduction of the therapeutic gene into the target cell population and a wide variety of delivery vehicles including viral vectors, gold particle-DNA conjugate bombardment, direct injection of plasmid DNA, cationic liposomes alone or accompanied by fusion-promoting agents, and receptor-mediated endocytosis have been tested for gene delivery into neurons and glia. Viral vectors are widely and an efficient means of gene delivery. Replication defective herpes simplex virus (HSV), adenovirus (AV), human lentivirus-mouse retrovirus recombinants (HIV-MoMuLV) and adenoassociated viruses (AAV) have all been tested in animals. These viruses differ with respect to tropism, persistence as an episome versus integration into host chromosome, toxicity or antigenicity, longevity and level of gene expression, risk of tumorigenicity, maximum transduction capacity, and the ability to produce high titer viral stocks free of replicating virus contaminants. Retroviruses that require host cell division for their integration and expression can not be employed for in vivo gene transfer to the nervous system (Miller, 1992). On the other hand, viral vectors which either remain extrachromosomal, such as HSV-1 (Fink et al., 1992; Geller and Breakefield, 1988; Geller and Freese, 1990a) and AV (Davidson and Bohn, 1997; Mitani et al., 1995) or HIV recombinants (Naldini et al., 1996; Naldini et al., 1996) which are capable of integrating into the genomes of nondividing cells, are suitable for gene delivery to adult post-mitotic neurons. AAV has been shown to efficiently transduce particular neurons in brain, but it is unclear whether the viral genes 216

Gene Therapy and Molecular Biology Vol 1, page 217 called the tegument which contains numerous important viral proteins, including the virion host shut-off (vhs) protein that mediates the shut down of host cell protein synthesis (Kwong and Frenkel, 1987; Kwong et al., 1988; Oroskar and Read, 1989; Oroskar and Read, 1987; Read and Frenkel, 1983), and VP16 or a-TIF, a protein involved in transactivating the immediate-early class of viral genes (Batterson and Roizman, 1983; Campbell et al., 1984; Gaffney et al., 1985; Kristie and Roizman, 1987; Mackem and Roizman, 1982; McKnight et al., 1987; O'Hare and Goding, 1988; Post et al., 1981; Preston et al., 1988). Infectious virions possess an envelope acquired by budding through the nuclear membrane. The envelope contains at least ten virus-encoded glycoproteins integrated into the bilayer lipid envelope which are instrumental in the attachment, penetration and cell-to-cell spread of HSV in a variety of different cell types (Spear, 1993a; Spear, 1993b; Steven and Spear, 1997).

IV. Overview of relevant HSV biology The HSV virion contains the 152-Kb double stranded DNA genome packaged in the shape of a torus within an eicosadeltahedral capsid (Furlong et al., 1972). The genome consists of two unique segments (UL and US) each flanked by a set of terminal and internal repeats. Surrounding the capsid is an amorphous mass of proteins

A notable feature of the viral lytic cycle is the temporally and sequentially coordinated cascade of viral gene expression (Honess and Roizman, 1974): !transinducing factor (!-TIF), a component of the virus tegument, induces expression of the first kinetic class of viral genes called the immediate-early (IE or !) genes (Batterson and Roizman, 1983; Campbell et al., 1984; Gaffney et al., 1985; Kristie and Roizman, 1987; Mackem and Roizman, 1982; McKnight et al., 1987; O'Hare and Goding, 1988a; Post et al., 1981; Preston et al., 1988). Four of the five ! genes (ICP0, ICP4, ICP22 and ICP27) are involved in transcriptional and post-transcriptional regulation of the next kinetic class of viral genes designated as early or " genes (DeLuca et al., 1985; Dixon and Schaffer, 1980; Preston, 1979b; Preston, 1979a; Rice et al., 1994; Sacks et al., 1985; Sacks and Schaffer, 1987; Sandri-Goldin and Hibbard, 1996; Stow and Stow, 1986; Watson and Clements, 1980). The " genes provide enzymes required for nucleotide metabolism and viral DNA replication. The last kinetic class of viral genes to be expressed, the # genes, provide components of the viral eicosadeltahedral capsid, teguments and envelope glycoproteins.

F i g u r e 1 . Schematic diagram of the viral life cycle in the host. The stages of the life cycle include: (i ) infection of epithelial cells with lytic replication and production of progeny virus particles; (i i ) invasion of local sensory nerve endings and ascension to sensory ganglion via retrograde axonal transport; (i i i ) acute phase of lytic replication in sensory nerve cell body; (i v ) establishment of latency and maintenance of viral genomes as quiescent, nucleosomebound episomes; (v ) resumption of lytic gene expression and production of progeny virions upon reactivation that is induced by stimuli such as stress and UV irradiation; (v i ) anterograde transport back to the periphery with or without manifestation of symptoms such as cold sores or keratitis.

A similar cascade of viral gene expression occurs within nerve cell bodies in the sensory ganglia. Infectious viral particles can be detected up to 7 days following infection. However, unlike most cell types sensory neurons are not lysed during the virus lytic cycle. Viral lytic gene expression is repressed by an unknown mechanism and viral genomes are maintained as nonreplicating, largely quiescent nucleosome-bound episomes within the nucleus of the sensory neuron until induced to reactivate by stimuli such as stress and exposure to UV irradiation. Upon reactivation, viral nucleocapsids are transported back to the periphery where acute replication resumes within epithelial cells. In humans, manifestations 217

Soares et al: Gene transfer using HSV vectors of viral reactivation include characteristic cold sores or herpetic keratitis, depending on the site of recurrence. Thus, the virus oscillates between two states in the host: long periods of dormancy or ‘latency’, interrupted by occasional acute periods of active viral replication.

et al., 1993; Fareed and Spivack, 1994; Hill et al., 1990; Ho and Mocarski, 1989; Javier et al., 1988; Leib et al., 1989; Natarajan et al., 1991; Sedarati et al., 1989; Steiner et al., 1989), but does appear to delay or reduce the ability of the virus to reactivate (Block et al., 1993; Bloom et al., 1996; Devi-Rao et al., 1994; Hill et al., 1990; Leib et al., 1989; Sawtell and Thompson, 1992; Steiner et al., 1989; Trousdale et al., 1991). Because mutations that affect LAT expression have not been shown to prevent the establishment of latency, it should be possible to exploit the LAT promoter-regulatory region to drive latencyspecific expression of a therapeutic gene in place of LAT RNA.

V. HSV latency During latency, a unique set of transcripts is expressed originating from a 10-Kb region located in the internal (IRL) and terminal (TRL) repeats of the viral genome (F i g . 2 ). The predicted 8.3-Kb primary transcript has proven to be difficult to detect by Northern blot analysis and is therefore believed to be highly unstable or in low abundance. 2.0- and 1.5-Kb length RNA transcripts from within that region accumulate in latently infected sensory ganglia and are referred to as the "latency-associated transcripts" (LATs) (Croen et al., 1987; Deatly et al., 1987; Deatly et al., 1988; Gordon et al., 1988; Rock et al., 1987; Spivack and Fraser, 1988; Spivack et al., 1991; Stevens et al., 1987; Wagner et al., 1988). Recent evidence (Zablotony et al., 1997) suggests that the 2.0and 1.5-Kb LATs are introns derived from the 8.3-Kb minor LAT (Farrell et al., 1991), a result that is consistent with the nuclear localization, lack of polyadenylation, and non-linear nature of the LATs (Devi-Rao et al., 1991; Wu et al., 1996a).

VI. Strategies for engineering HSV vectors HSV gene vectors have been developed which are mutated in nonessential and essential genes that compromise virus replication in some or all cell types respectively and replication defective mutants have been used to package plasmid vectors which are largely devoid of viral sequences (F i g . 3 ). Vectors mutated in particular nonessential accessory functions are able to replicate in dividing cells (e.g. tumor cells) but not in post-mitotic cells (e.g. brain neurons). Mutants deleted for genes

The functional role of the LATs is not known. Deletions in the LAT region do not have deleterious effects on the ability of the virus to replicate or to establish latency (Block et al., 1990; Chen et al., 1995; Deshmane

Figure 2. Schematic representation of the LAT loci. Transcription during latency maps to a diploid gene called the LAT locus that maps to the internal and terminal repeats flanking the unique long (UL) segment of the HSV genome. Shown are the location of the two LAT promoter regions, LAP1 and LAP2, relative to the 5’ end of the major latency-associated transcripts that accumulate in latently infected ganglia The dashed line represents the putative 8.7 Kb primary LAT transcript derived from the TATA box-containing LAP1 region. The 2 Kb and 1.5 Kb LATs are co-linear stable introns.

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F i g u r e 3 . HSV Vector Strategies (A) Production of defective full-length HSV-based vectors is carried out in cell lines that are engineered to provide the deleted essential genes in trans. These vectors are incapable of replicating in neurons because of the missing essential genes. (B ) Amplicons are propagated in bacteria (using the bacterial origin of replication), and then transfected into a complementary cell line that is infected with defective “helper” HSV, thus producing particles consisting either of amplicon concatemers (about 150 Kb in length) or defective HSV. (C) Helper virus-free amplicon system doesn’t require either a defective helper virus or a complementing cell line for the amplicon plasmid to be packaged. The amplicon plasmid is transfected into cells along with a five cosmids that contain overlapping fragments that represent the entire HSV genome which encode all the viral proteins necessary to produce infectious particles. In order to insure that only the amplicon plasmid is packaged and not any of the cosmids, the packaging sequence (“a” sequence) was deleted from the cosmid clones.

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Gene Therapy and Molecular Biology Vol 1, page 220 Plasmid or amplicon vectors have been exploited by a number of laboratories for gene transfer (Battleman et al., 1993; Casaccia-Bonnefil et al., 1993; During et al., 1994; Geller and Breakefield, 1988; Geller et al., 1990b; Geschwind et al., 1994; Ho et al., 1993). Amplicon vectors consist of a transgene expression cassette, an HSV origin and packaging recognition sequences. The plasmid is transfected into cells followed by infection with a defective helper virus. Progeny consist of amplicon vector packaged as a concatemer and helper virus particles. Recently this system has been improved by using a cosmid library spanning the entire HSV genome that can not be packaged because they are devoid of packaging signals (Fraefel et al., 1996). While only ampliconcontaining particles are produced, high titer stocks are difficult to prepare since the entire system depends on the efficiency of co-transfection. Ideally a packaging system that does not require transfection would provide the best amplicon system however packaging cell lines will be difficult to engineer since at least 35 viral genes are required for particle production and many of these genes are toxic to cells. Amplicons have also encountered problems in maintenance of transgene expression (During et al., 1994; Fraefel et al., 1996) as have other HSV vectors systems and more research on promoter functions in this context is required.

coding for products involved in DNA synthesis (e.g. thymidine kinase or ribonucleotide reductase) are compromised for growth in brain (Fink et al., 1992; Ramakrishnan et al., 1994) and thus are highly reduced for viral pathogenesis following intracranial virus inoculation. Nevertheless, these mutants can replicate in glioma cells and thus these and other similar mutants have been used as oncolytic vectors for destruction of tumor tissue by the natural cytolytic mechanisms inherent in virus replication (Andreansky et al., 1997; Andreansky et al., 1996; Boviatsis et al., 1994; Chambers et al., 1995; Markert et al., 1993; Martuza et al., 1991; Mineta et al., 1994). Virus replication and spread within tumors may improve the oncolytic property and also will likely be important for effective delivery of genes which induce antitumor immunity or activate anti-cancer drugs locally. There are a number of potential gene knockouts which might allow preferential virus spread in tumor and other tissues many of which have yet to be explored for this purpose. The design of these vectors will require selection of gene deletions which provide the most effective virus spread without compromising vector safety. Such mutant viruses may also prove useful for gene delivery to sensory neurons since inoculation of skin for example will result in amplication of the vector for more efficient virus delivery to peripheral neurons by virus uptake at axon terminals. Here the virus will establish latency by its inherent mechanisms and transgenes could be expressed using the natural latency promoter system of the virus (described below).

Because HSV is a large virus having many nonessential genes, it should be possible to incorporate large amounts of foreign sequences into the vector genome. Most of the right-hand end of the viral genome (approximately 40 kb) of DNA is nonessential and the two essential genes in this region have been introduced successfully into the genome of cell lines for propagation of highly defective mutants potentially lacking these sequences. All of these sequences have been removed individually or as a large block of genes (Laquerre et al., 1997; Meignier et al., 1988; Rasty et al., 1997; Weber et al., 1987) and experiments are in progress to remove the entire region for replacement with foreign DNA.

Replication defective virus mutants will be the preferred gene transfer vehicles for applications involving transgene delivery to brain or other tissues where long term expression is required. The safest and most efficient mutant would be deleted for the viral immediate early genes that are required for activation of early and late viral functions. Removal of these genes will substantially reduce viral cytotoxicity and prevent viral antigen production, a problem in the immune competent host where immunologic memory will activate effector T cells that could readily eliminate vector containing cells. There are five IE genes most of which have been shown to be cytotoxic to cells (Johnson et al., 1992, 1994) and mutants deleted for these genes are capable of transducing cells without causing cell death at least at multiplicities of infection of 10 or less (Krisky et al., 1997; Marconi et al., 1996; Samaniego et al., 1995; Wu et al., 1996b). These vectors will require the use of promoter systems which are active in a quiescent viral genome and may require the use of cellular promoters which are active in particular tissues. Thus far little research has been carried out along these lines since such highly defective mutant viruses have only recently become available.

VII. Control of transgene expression in HSV vectors A variety of promoters have been employed to drive reporter/therapeutic gene expression from first generation HSV gene transfer vectors. Candidate promoters included neuronal-specific promoters such as the neuron-specific enolase promoter, the neurofilament promoter and tyrosine hydroxylase promoter, as well as viral promoters such as the HSV thymidine kinase promoter and the constitutively strong HCMV immediate early promoter. Although high levels of reporter gene expression from the HSV lytic cycle or the HCMV IE promoter were detected in rat brain soon after stereotactic injection of the replication-defective

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Figure 4. Putative promoter elements within the LAT loci. Schematic representation of the predicted transactivation factor binding sites in the two latency associated promoter regions, LAP1 and LAP2. LAP1 is the TATA box containing promoter located 600 bases upstream of the 5’ end of the major LATs within a 203 bp PstI fragment that contains binding sites for several eucaryotic transcription factors including Sp1, USF, CRE, Egr-1 and a member of the POU domain family of transcription factors. LAP2 is a TATA less promoter that resembles several housekeeping gene promoters. LAP2 encompasses several unique sequence motifs such as a GC box, a polyT which binds to a HMGI(Y) and C/T-rich region that binds to Sp1 and a family of factors (PuF, NSEP1, 2F87) that bind to a similar element in the C-myc promoter .

YY1 and Brn sites. In addition, there is an enhancer region at the start of transcription that plays a role in upregulating LAP1 basal activity (Soares et al., 1996) and also contains an ICP4 binding site that down-regulates LAT expression (Batchelor et al., 1994; Farrell et al., 1994; Rivera-Gonzalez et al., 1994). We have recently shown that the TATA box, USF1, CRE and the putative Brn site all contribute to LAP1 activity in vivo (Soares et al., 1996, and Soares and Glorioso, unpublished data).

vector, this expression soon waned and could not be detected beyond 7 to 10 days (Fink et al., 1996; Glorioso et al., 1995; Glorioso et al., 1992). At best, expression from the HSV ICP0 promoter and the HCMV IE promoter could be extended out to 4 weeks in the context of a mutant vector background deleted for the IE genes ICP4, ICP27 and ICP22 (Ramakrishman et al., unpublished), suggesting that one of the immediate early gene products deleted in this vector caused the attenuation of reporter gene expression from previous generation single IE deletion viral vectors.

LAP2 resembles housekeeping promoters in that it is TATAless, has a high G + C content, and in transient assays is 5- to 10-fold weaker than LAP1 (Goins et al., 1994). Although LAP2 is also not the predominant promoter during natural HSV latency (Chen et al., 1995), LAP2 can independently drive long term reporter gene expression in the PNS (Goins et al., 1994) (as long as 10 months) and, albeit more weakly, in the CNS (Chen et al., unpublished). Binding of HMG I(Y) protein to a polyT stretch within LAP2 promotes the recruitment of Sp1 and perturbs the local DNA conformation (French et al., 1996).

In our hands the native HSV latency-associated promoter-regulatory region has proven to be the only promoter system that supports sustained gene expression from recombinant HSV-1 vectors in the nervous system (Chen et al., unpublished). The functional contribution of specific cis-acting elements to latency-associated gene expression is currently being assessed in an attempt to build an optimized promoter system using the native LAT promoter-regulatory region as a foundation. We now know that there are two latency active promoters (LAPs), LAP1 and LAP2, upstream of the LAT coding sequence (Batchelor and O'Hare, 1990, 1992; Dobson et al, 1989; 1995; Goins et al, 1994; Nicosia et al, 1993; Wang et al, 1995; Zwaagstra et al, 1989, 1990) (F i g . 4). LAP1 contains a TATA box (Ackland-Berglund et al., 1995; Soares et al., 1996; Rader et al., 1993) with upstream control elements such as CAAT (Batchelor and O'Hare, 1992) USF1 (Zwaagstra et al., 1991) CRE (AcklandBerglund et al., 1995; Kenny et al., 1994; Leib et al., 1991; Rader et al., 1993; Soares et al., 1996) and Sp1,

The ability of LAP1 and LAP2 to drive expression of foreign genes from the virus genome during latency was a natural outcome of the studies characterizing these promoters and the specific cis-elements therein (summarized in T a b l e 1). LAP1 was first shown to provide long-term expression of ß-globin in mouse PNS in a virus where the ß-globin genomic clone was inserted immediately downstream from LAP1 (Dobson et al., 1989), however expression waned dramatically over time (Margolis et al., 1993). A similar virus with the !-

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Soares et al: Gene transfer using HSV vectors interferon (!-IFN) cDNA at the identical position downstream from LAP1 failed to express !-IFN during latency (Mester et al., 1995), suggesting that sequences within the ß-globin first intron of the genomic clone may have played a role in expression of that particular transgene from the latent viral genome. Another recombinant in which the rat ß-glucuronidase cDNA was inserted downstream from LAP1 in a virus that was deleted for part of LAP2 was highly active in expression of the transgene acutely in mouse trigeminal ganglia and brainstem and also expressed ß-glucuronidase during latency (Wolfe et al., 1992). However, like the ß-globin recombinant, the number of neurons expressing the transgene and the level of expression decreased with time. In other studies LAP1 failed to provide long-term transgene expression either in the native LAT loci (Margolis et al., 1993) or in an ectopic site within the genome (Lokensgard et al., 1994) such as glycoprotein C (gC). Fusion of the Moloney murine leukemia virus (MoMLV) LTR to LAP1 did result in long-term foreign gene expression from the virus vector in neurons of the murine PNS (Lokensgard et al., 1994). Insertion of a MoMLV LTR-lacZ expression cassette 800bp upstream from the 5’ end of the 8.3-Kb LAT in the opposite orientation to LAT led to long-term transgene expression whereas the neurofilament promoter was not active (Carpenter and Stevens, 1996). Insertion of the transgene immediately downstream of LAP2 in the native LAT loci (Chen et al., 1997; Ho and Mocarski, 1989) or downstream of LAP2 alone in the gC ectopic site (Goins et al., 1994) resulted in long-term activity in both mouse PNS and rat CNS neurons, although the level of transgene expression in brain was reduced compared to that observed in sensory neurons of the PNS. We have also shown that LAP2 is capable of long-term expression of nerve growth factor in trigeminal and dorsal root ganglia neurons in either the tk or gC ectopic loci (Goins et al., unpublished). These results suggest that some element(s) present in the LAP2 region is responsible for mediating expression during latency and that the MoMLV LTR can substitute for that activity. Also, further modification of these sequences will be required to achieve physiologic levels of therapeutic gene expression in brain.

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In a recent report, the encephalomyocarditis virus internal ribosome entry site (IRES) was juxtaposed to a reporter gene cassette that was introduced downstream from the LAPs in the native LAT loci, to examine its affect on transgene expression from the LAPs (Lachmann and Efstathiou, 1997). These recombinants yielded long-term expression of ß-galactosidase in murine sensory and motor neurons, although the level and site of expression varied within the population of latently infected cells. Thus, insertion of the IRES may allow for efficient transport of the message to the cytoplasm and thereby increase the level of foreign gene expression.

VIII. Regulated transgene expression in HSV vectors Our first attempt at engineering a regulatable viral vector created an autoregulatory loop that consisted of a promoter with five tandem copies of the 17-bp Gal4 DNA recognition element to enable transactivation by vectorencoded chimeric Gal4/VP16 protein. This strategy was based on the ability of the Gal4 to transactivate promoters containing this site (Carey et al., 1990; Chasman et al., 1989; Sadowski et al., 1988) despite the repressive presence of nucleosomes (Axelrod et al., 1993; Xu et al., 1993). Completion of the autoregulatory loop achieved enhanced, albeit transient expression of the transgene in the CNS, thus serving as an encouraging proof-ofprinciple experiment (Oligino et al., 1996). We have since modified this system to achieve an inducible promoter system: the transactivator is a chimeric molecule consisting of the hormone binding domain of the progesterone receptor fused to the previously used transactivation domain of VP16 and DNA binding domain of Gal4 (Vegeto et al., 1992; Wang et al., 1994). In presence of the progesterone analog RU486, the inactive chimeric transactivator assumes a conformation that can bind to and transactivate the Gal4 recognition sitecontaining promoter driving transgene expression. We have been able to induce high levels of viral vector-derived transgene expression in rat brain upon intraveinous administration of the inducing agent RU486 demonstrating the feasibility of the drug-inducible viral vector delivery system (Oligino et al., unpublished).

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Table 1. Transgene expression from recombinant HSV vectors in the nervous system. This table summarizes the location and expression profiles of reporter genes inserted into the LAT locus or driven by LAT promoter-regulatory regions in an ectopic genomic locus.

quiescent genome in order to produce either constitutive long-term expression of transgenes in neurons, or regulatable transient expression as required for specific applications. In the peripheral nervous system the virus is well adapted for vector genome persistence and long term gene expression using the native viral constituents and mechanisms for gene expression. Here it remains to be demonstrated that the virus will be effective in treating peripheral nervous system disease in animal model systems. While the virus readily establishes latency in brain, the latency promoter is much less active and will

IX. Future directions for HSV vector development. Herpes simplex virus has many features which make it a promising platform for the creation of vectors for gene transfer to the nervous system. The wild-type virus is capable of naturally establishing long-term persistence in neurons of the brain and peripheral nervous system. Work in our laboratories has focused on modifying the virus to reduce cytotoxicity of the initial infection, and understanding the regulation of gene expression from the 223

Soares et al: Gene transfer using HSV vectors Battleman, D., Geller, A., and Chao, M. (1 9 9 3 ). HSV-1 vector-mediated gene transfer of the human nerve growth factor receptor p75hNGFR defines high-affinity NGF binding. J . N e u r o s c i . 13, 941-951.

almost certainly require manipulation to improve promoter function. Possibilities include amplification systems in which the latency promoter is used to express artificially transactivators or the engineering of cis- acting elements into the promoter which are responsive to brain derived transcription factors. Moreover it may be possible to introduce large cellular promoter elements which will be active in the vector genome. Applications involving conditionally replication competent vectors for cancer therapy or spread in vivo will require considerably more research to define a suitable genetic background. These vectors might be improved considerably if virus infection were targeted to particular cell types by modifying the envelope glycoproteins in a manner to eliminate the natural receptor binding ligands with replacement with novel binding ligands that recognize a specific cell type in vivo.

Block, T. M., Deshmane, S., Masonis, J., Maggioncalda, J., Valyi-Nagi, T., and Fraser, N. W. (1 9 9 3 ). An HSV LAT null mutant reactivates slowly from latent infection and makes small plaques on CV-1 monolayers. V i r o l o g y 192, 618-630. Block, T. M., Spivack, J. G., Steiner, I., Deshmane, S., MacIntosh, M. T., Lirette, R. P., and Fraser, N. W. (1 9 9 0 ). A herpes simplex virus type 1 latency-associated transcript mutant reactivates with normal kinetics from latent infection. J . V i r o l . 64, 3417-3426. Bloom, D., Hill, J., Devi-Rao, G., Wagner, E., Feldman, L., and Stevens, J. (1 9 9 6 ). A 348-base-pair region in the latency-associated transcript facilitates hereps simplex virus type 1 reactivation. J . V i r o l . 70, 2449-2459. Boviatsis, E., Chase, M., Wei, M., Tamiya, T., Hurford, R., Kowall, N., Tepper, R., Breakfield, X., and Chiocca, E. (1 9 9 4 ). Gene transfer into experimental brain tumors mediated by adenovirus, herpes simplex virus, and retrovirus vectors. Hum. Gene Ther. 5, 183-191.

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Soares, M. K., Hwang, D.-Y., Schmidt, M. C., Fink, D. J., and Glorioso, J. C. (1 9 9 6 ). Cis-acting elements involved in transcriptional regulation of the herpes simplex virus type-1 latency-associated promoter 1 (LAP1) in vitro and in vivo. J . V i r o l . 70, 5384-5394.

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Gene Therapy and Molecular Biology Vol 1, page 229 Stevens, J. G., Wagner, E. K., Devi-Rao, G. B., Cook, M. L., and Feldman, L. T. (1 9 8 7 ). RNA complementary to a herpesviruses a gene mRNA is prominent in latently infected neurons. S c i e n c e 255, 1056-1059.

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Gene Therapy and Molecular Biology Vol 1, page 231 Gene Ther Mol Biol Vol 1, 231-239. March, 1998.

The baculovirus vector system for gene delivery into hepatocytes Christian Hofmann1, Wolfgang Lehnert 1 and Michael Strauss2,3 1

HepaVec AG für Gentherapie, Robert-Rössle-Str. 10, D-13122 Berlin-Buch, Germany

Humboldt University Berlin, Max Delbrück Center for Molecular Medicine, Robert-Rössle-Str. 10, D-13122 Berlin-Buch, Germany 3

Danish Cancer Society, Institute of Cancer Biology, Strandboulevarden 49, DK-2100 Copenhagen, Denmark

________________________________________________________________________________________________ Correspondence to: Prof. Michael Strauss, Humboldt University Berlin, Max Delbrück Center for Molecular Medicine, RobertRössle-Str. 10, D-13122 Berlin-Buch, Germany. Tel: +49/30/94063307, Fax: +49/30/94063306, E-mail: strauss@mpg.mdh4.mdc-berlin.de

Summary Gene therapy in the liver requires powerful vectors capable of mediating sufficient gene delivery and expression in affected hepatocytes. Viral vectors are amongst the most efficient tools for gene delivery, and the search for tissue-specific infecting viruses is important for the development of in v i v o gene therapy strategies. We have recently shown for the first time that a genetically modified baculovirus Autographa californica c a n efficiently and specifically transfer genes into cultured l i v e r c e l l s f r o m various origin. The efficiency o f baculovirus-mediated gene transduction into hepatocytes was determined to approach 100% using appropriate virus titers. Apart from these features, potential advantages of baculovirus vectors are the nearly unlimited capacity for insertion of foreign DNA, a supposed restriction of viral promoters to the arthropod host and the ease of generating high vector titers. Uptake of the virus occurs via the endosomal pathway, most likely via a receptor that i s currently under investigation. Baculovirus-mediated gene expression i s t r a n s i e n t i n d i v i d i n g c e l l s , but prolonged expression can be achieved i n non-dividing primary hepatocytes. Baculovirus-mediated gene transfer i s feasible into e x v i v o perfused human liver tissue. Systemic application of baculovirus vectors i n v i v o i s h a m p e r e d b y t h e c o m p l e m e n t ( C ) system. Current attempts to facilitate baculovirus-mediated gene transfer i n v i v o include strategies for both, blocking or avoiding the C system and generation of new baculovirus vectors that are not affected by the C system. Alternatively, direct injection of baculovirus vectors was successful into normal mouse liver and into induced human hepatocarcinomas i n nude mice. The potential of baculovirus vectors i n v i t r o and the feasibility of vector application i n v i v o provide the basis for gene therapy strategies for metabolic diseases and tumors of the liver.

tremendous efforts are made to develop potent gene transfer vectors for in vivo application. Viral vectors, such as retroviruses and adenoviruses, are generally considered superior to non-viral vectors with regard to gene transfer efficiency. However, retroviruses for example are not able to integrate their genome into non-dividing cells so that hepatic gene transfer by retroviral vectors requires stimulation of liver cell division (Ferry et al., 1991; Cardoso et al., 1993; Rettinger et al., 1993). Adenoviruses can deliver genes at a high frequency into the liver (Li et

I. Introduction Gene therapy in the liver is a promising approach for the treatment of various inherited and malignant diseases affecting this organ. In order do realize this concept, powerful tools capable of transferring therapeutic genes into affected hepatocytes at sufficient efficacy are required. The principle of an ex vivo approach for liver gene therapy does presently not allow for sufficient rates of genetically corrected hepatocytes (Grossman et al., 1995). Therefore, 231

Hofmann et al: Baculovirus for gene delivery into hepatocytes al., 1993), but induce a strong immunological response in vivo (Yang et al., 1994). An important drawback of all existing viral vectors is, in addition, the lack of liver cell specific targeting. Since the mainly used viral gene transfer vectors are derived from mammalian species, general problems have to be considered, such as emergence of replication competent vectors, preexisting or induced immune response and undesired gene expression from the viral backbone. Baculoviruses comprise a large group of viral pathogens of arthropods particularly of insects. The best studied member of this family, Autographa californica nuclear polyhedrosis virus (AcNPV), is a large, enveloped virus with a double-stranded, circular, completely sequenced DNA genome of about 130 kilobase pairs (Ayres et al., 1994). Baculoviruses are normally used for the overproduction of recombinant proteins under control of strong baculoviral promoters in insect cells (Luckow and Summers, 1988; Fraser et al., 1992; Kidd and Emery, 1993; Miller, 1993) or as biopesticides (Cory et al., 1994). Although the ability of AcNPV to infect mammalian cells was studied in the past (Doller et al., 1983; Tjia et al., 1983; Carbonell and Miller, 1987; Hartig et al., 1992), neither gene expression nor DNA replication could be observed. Since these studies did not include hepatocytes, a block of infection of mammalian cells was assumed.

II. Baculovirus-mediated gene transfer in vitro We have recently shown that the baculovirus, AcNPV, can efficiently deliver genes into hepatocytes (Hofmann et al., 1995). This unexpected property of baculovirus was confirmed by others (Boyce and Bucher, 1996). Further applications of baculovirus vectors were recently presented for recovery of an infectious virus from cDNA by means of a hybrid baculovirus-T7 RNA polymerase system (Yap et al., 1997). This study highlighted the lack of replication and toxicity after baculovirus-mediated gene transfer into mammalian cells in contrast to the vaccinia-T7 polymerase system, which is widely used for that purpose. The major prerequisit for the expression of a baculovirally transferred gene in either application is its control by a functionally active promoter in mammalian cells. Recombinant baculoviruses are generated in insect cells via homologous recombination after cotransfection of a linearized AcNPVgenome and a baculovirus transfer vector bearing the mammalian expresssion unit. The expandability of the capsid structure of baculoviruses allows for packaging and expression of very large genes with an until now not challenged upper size limit.

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A. Cell-type specificity of baculovirus In order to investigate the cell-type tropism of baculovirus vectors, we constructed recombinant baculoviruses bearing the luciferase reporter gene under control of the immediate early promoter of cytomegalovirus. After incubation of this virus with a large panel of cells, high levels of gene expression could be detected in hepatocytes, including primary cultures derived from various species (T able 1). In contrast, no or very low levels of gene expression could be detected in more than 40 tested non-hepatic cell lines. Relative gene transfer efficiencies among hepatocytes seem to decline in a species specific manner by a maximal factor of 40 from human > rabbit > to mouse. Therefore, a human nonhepatic cell line (T47-D) was just as susceptible to baculovirus infection as primary mouse hepatocytes. In other studies using baculovirus vectors, high levels of gene expression were also achieved only in hepatocytes (Boyce and Bucher, 1996). Thus, the present in vitro data show baculovirus to be liver cell specific, which would be a highly advantageous feature of the vector, if it could be confirmed in vivo.

B. Efficiency of gene transfer by baculovirus In the first report on baculovirus-mediated gene transfer into hepatocytes, we described a baculovirus vector coding for a C-terminally truncated simian virus 40 large tumor antigen under control of the cytomegalovirus (CMV) immediate early promoter. With this vector, we demonstrated the ability of baculovirus vectors to approach a transduction efficiency of 100% in human hepatocytes (Hofmann et al., 1995). A dose-response analysis was performed in the hepatocarcinoma cell line Huh7 by using a baculoviral vector (AcNPV-ß-gal) with a nuclear localised ß-galactosidase gene under control of the RousSarcoma-Virus long terminal repeat (RSV-LTR). The gene transfer efficiency increased gradually with the respective multiplicity of infection (moi). After infection at a moi of 750, almost all cells were positive as determined by histochemical staining for ß-galactosidase (Sandig et al., 1996). However, the histochemical ß-gal staining method often underestimates the percentage of actually transduced cells and does not allow for an analysis of gene transfer events on living cells. Therefore, we constructed a baculovirus vector bearing the green fluorescent protein under control of the human CMV-promoter (AcNPVGFP). We found that all Huh7 cells were successfully transduced after infection at a moi of only 100 (Figure 1). Analogous to previous reports, no signs of cell toxicity were observed even if very high doses were applied.

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F i g u r e 1 . Baculovirus-mediated expression of the green fluorescent protein in Huh7 cells. Human hepatocarcima cells (Huh7) were infected with recombinant baculoviruses bearing the green fluorescent protein (GFP) under control of the CMV immediate early promoter (AcNPV-GFP) at a moi of 100. (A) Expression of GFP was detected 42 hours after infection by direct immunofluorescence of living cells. (C) Corresponding phase-contrast micrograph. (B ) Immunofluorescence of non-infected Huh7 cells and corresponding phase-contrast micrograph (D).

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Gene Therapy and Molecular Biology Vol 1, page 234 Baculovirus-mediated gene transfer is most likely independent of the cycling status of the cell, since nondividing primary hepatocytes from different origin could be efficiently transduced. Boyce and Bucher reported a gene transfer efficieny > 70% in primary cultures of rat hepatocytes using a moi of 430 (Boyce and Bucher, 1996).

C. Mode of hepatocytes

baculovirus

uptake

A obvious assumption as to the striking preference of baculovirus-mediated gene transfer for hepatocytes would be the existence of a specific receptor on hepatocytes. Although the desialiated baculoviral envelope proteins represent putative ligands for the hepatocyte-specific asialoglycoprotein receptor, various experiments excluded this initially postulated candidate for baculoviral entry into hepatocytes (Hofmann et al., 1995). Indications for a receptor on hepatocytes became apparent, however, within the same study by both, competition experiments and a clear dose-response curve of baculovirus-mediated gene transfer into hepatocytes. We started investigating the mechanism of baculovirus uptake by hepatocytes by following data available from its natural arthropod host. Baculovirus enters insect cells by adsorptive endocytosis (Volkman and Goldsmith, 1985). A receptor on insect cells has not yet been identified, but it was proven by the use of neutralizating monoclonal antibodies (mAb) that the main baculoviral envelope protein gp64 is responsible for entry of baculovirus into insect cells (Volkman et al., 1984). Therefore, we treated luciferase expressing baculovirus with mAbs against gp64 prior to infection of hepatocytes and compared subsequently measured luciferase levels with those obtained with untreated vector. No influence on baculoviral gene transduction was observed after virus preincubation with mAb, AcV5 or AcN9. In contrast, AcV1 completely blocked baculovirus-mediated gene expression in hepatocytes (Table 2). These data reflect exactly the ability of these mAb to block baculovirus infection of insect cells (Hohmann and Faulkner, 1983; Withford et al., 1989). In order to determine if the AcV1-mediated block of baculovirus infection of hepatocytes is due to a block of receptor binding or due to later fusion events during endocytosis, we investigated the ability of AcV1-treated baculovirus to bind to hepatocytes. We observed that binding of baculovirus to hepatocytes is not affected by the neutralizing mAb, AcV1 (Figure 2). This result indicates that AcV1 blocks baculovirus penetration into hepatocytes or plays a role in low pH-dependent fusion. The necessity of endosomal maturation for the transport of baculovirus was demonstrated for both, insect cells (Volkman and Goldsmith, 1985; Charlton and Volkman, 1993) and

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hepatocytes (Hofmann et al., 1995; Boyce and Bucher, 1996).

D. Kinetics of baculoviral gene expression The stability of gene expression is an important aspect of the use of a vector for treatment of disorders, which require permanent provision of a missing gene product. Retroviruses and adeno-associated viruses are able to integrate their genome into the target cell which should allow for long term gene expression. However, integration of an expression cassette into the target cell does not preclude that other events, such as a promoter shut-off (Lรถser et al., in press) or elimination of the transduced cell by the immune system, prevent from stable gene expression. We compared the duration of gene expression after baculovirus-mediated gene transfer into the hepatic cell line Huh7 and into non-dividing primary mouse hepatocytes using a luciferase expressing virus (Sandig et al., 1996). The instability of luciferase RNA and protein allowed to draw conclusions as to vector stability from expression data obtained with this reporter gene. We observed transient gene expression in the dividing hepatocarcinoma cell line Huh7, peaking at 42 hours and decreasing continously over four orders of magnitude within 19 days. Baculovirus shares short-term expression of genes transferred into dividing cells with other non-replicating and also with non-integrating vector systems due to the manifold arthropod-specific requirements for replication (Pearson et al., 1992; Kool et al., 1994; Lu and Miller, 1995) and due to the lack of an integration machinery. The liver consists, however, of cells with low regenerative activity. Therefore, baculovirus-mediated gene expression in primary cultures of hepatocytes declines more slowly and the kinectic is almost equal to that recorded from a stably transducing retroviral vector (Sandig, et al., 1996). These results support the idea that the baculoviral genome

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F i g u r e 2 . Role of AcV1 mAb in inhibition of baculovirus uptake by Huh7 cells. (A , B ) Baculovirus and (C) baculovirus, pretreated with infectivity neutralizing amounts of AcV 1 mAb were allowed to adsorb onto Huh7 cells for 1 h at 4o C. (D) Huh7 cells were preincubated with AcV1 without baculovirus as control. After washing, cells were fixed and (A) incubated with AcV1 mAb. Bound virus (exemplary marked by arrowheads) was visualized using a fluoresceinconjugated goat anti-mouse antibody (A-D).

the observation that baculoviral gene transduction into hepatocytes is dramatically reduced by heat-labile serum components.

may persist in hepatocytes in vivo for some time leading to longer periods of expression as has also been observed with adenoviral vectors in immunodeficient animals (Dai et al., 1995). In contrast to first generation adenovirus vectors (Yang et al., 1994), an advantageous feature of baculovirus could be the evasion of a cellular immunitiy to viral antigens because of the strong restriction of baculoviral promoters even within different arthropod species (Morris and Miller, 1992; Bilimoria et al., 1993).

A. Inactivation of baculovirus by serum Incubation of baculovirus with native serum from different species prior to infection, causes a marked decrease in its ability to mediate gene expression in hepatocytes. In contrast, complete survival of baculovirus vectors was observed upon preincubation with the corresonding heat-treated sera. Since most of the components of the complement (C) cascades are heatlabile, we used sera deficient in different C components and determined baculovirus survival. The C component C4 is involved in triggering the classical complement cascade, whereas C3 is a component of both, the classical and the alternative pathway. Neither C3-deficient nor C4-deficient guinea-pig serum had a significant influence on baculovirus survival (T a b l e 3). These data indicate that

III. Baculovirus-mediated gene transfer in vivo We have undertaken a number of attempts for systemical and intraportal application of baculovirus vectors in rodents. The absence of a significant number of positively transduced cells in these in vivo experiments indicated that the virus is somehow inhibited in transferring genes to the liver. Clues as to the reasons for the inefficiency of baculovirus vectors in vivo derived from 235

Hofmann et al: Baculovirus for gene delivery into hepatocytes activation of the classical pathway of the C system has an impact on baculovirus survival in vivo. Triggering of the C cascade is also a major cause for the inactivation of a variety of currently used gene delivery vectors and contributes to inefficient gene transfer after in vivo application. C activation has been shown for liposomes (Szebeni et al., 1994), for various synthetic DNA complexes (Plank et al., 1996) based on polylysins, dendrimers or polyethyleneimine and for murine retrovirus vectors in primate serum (Takeuchi et al., 1996). However, inactivation of baculovirus in the presence of C can be prevented by treatment with complement blocking agents, such as cobra venom factor (CVF) or anti C5 antibodies (Hofmann and Strauss, 1998). The usefulness of CVF or anti-C mAb has already been demonstrated to protect murine retroviruses from C inactivation (Rother et al., 1995).

Another possibility to circumvent complementmediated neutralization of baculovirus seems to be likely by in situ perfusion methods, which have already been used for retrovirus-mediated gene transfer into the liver (Ferry et al., 1991; Cardoso et al., 1993; Rettinger et al., 1993). We established an ex vivo perfusion model of human liver segments (F i g u r e 3 ). Human liver tissue was chosen because of the high levels of baculovirusmediated gene expression obtained in human hepatocytes in vitro (Table 1). The liver segments were perfused with culture medium

B. Gene transfer into ex vivo perfused liver tissue

F i g u r e 3 . Ex vivo perfusion model of human liver tissue. Human liver segments were perfused with conditioned culture medium through a main vessel. After introduction of luciferase expressing baculoviruses into this system, perfusion was maintained for an additional period of time (22-42h), following analysis of gene expression. Each experiment using baculovirus vectors within this model system resulted in substantial gene expression distributed in all perfused parts of the liver segments.

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F i g u r e 4 . Baculovirus-mediated gene transfer in vivo. Baculoviruses bearing the lacZ gene under control of the Rous sarcoma virus long terminal repeat (AcNPV-ß-gal, 108 plaque forming units) were directly injected into (A) the big liver lobe of AKR-mice or into (B ) Huh7 cell derived human hepatocarcinomas generated in nude mice. Histochemical staining for ß-galactosidase of the injection sites was performed 48 hours after infection. The number of successfully transduced cells decreases with increasing distance to the injection sites. Uninfected liver or tumor stained negative (data not shown).

through a main vessel immediately after resection from patients with liver metastases of colon carcinoma. After application of a luciferase expressing baculovirus vector to this model system and subsequent analysis of small regions of the liver segments for gene expression, we found varying levels of luciferase activity distributed in all perfused parts of the liver segments (Sandig et al., 1996). These experiments demonstrated on the one hand that baculovirus-mediated gene transfer is not restricted to cells in culture and on the other hand that in situ perfusion methods represent an attractive means to facilitate gene transfer into the liver in vivo using baculovirus vectors.

C. Gene transfer into normal liver tissue of mice Based on the knowledge that the complement system poses so far a major hurdle for the success of baculovirus vectors in vivo, we evaluated the ability of baculovirus vectors to transfer genes into the livers of C-deficient mice (Lynch and Kay, 1995). In these pilot experiments, we injected a ß-galactosidase expressing baculovirus directly into the liver parenchyma of AKR-mice (C5-deficient). After histochemical staining for ß-galactosidase, we could detect a convincing amount of successfully transduced hepatocytes around the injection site (Figure 4A). The number of positive staining cells decreased with increasing distance to the injection site. These results demonstrate for the first time that baculovirus-mediated gene transfer in the liver is feasible in vivo. Just as important is the

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availability of a model, which is useful to evaluate important requirements on baculovirus vectors in vivo, such as duration of gene expression and interactions of the cellular immune system with the successfully transduced hepatocytes. These aspects of baculovirus-mediated gene transfer are currently under investigation with respect to the treatment of liver diseases, where already expression of low levels of the therapeutic gene product results in a therapeutic effect (Wilson´s diseases and heamophilias).

D. Gene transfer into liver tumors in vivo The treatment of liver tumors by gene transfer is highly dependent on the quality of the vector as well as on the gene-therapeutic concept. Although, the development of baculoviral vectors is not nearly ready, the usefulness of this vector system for the treatment of liver tumors is conceivable. In preliminary experiments, we generated human liver cell tumors derived from the cell line Huh7 in nude mice and injected the ß-galactosidase expressing baculovirus vector into the tumors. Even though nude mice possess an intact complement system, the ß-gal staining of the tumor revealed a successful gene transfer using this intratumoral vector application (Figure 4B). A definite answer for the usefulness of baculovirus vectors for the treatment of liver tumors will result from an experiment that combines the features of this new vector with an established concept for treating tumors with complementing tumorsuppressor genes (Sandig et al., 1997; Strauss et al., 1997).

Hofmann et al: Baculovirus for gene delivery into hepatocytes

IV. Future vector improvements and prospects The investigation of the baculovirus vector system for gene transfer into hepatocytes has, since its discovery, revealed a variety of advantegeous features of the vector, but there are still hurdles to overcome. Even if evasion or inactivation of the C system in vivo seems to be feasible, the ultimate goal will be generation of C-resistant viruses. We are currently approaching this goal by screening of baculovirus vector mutants as well as by insertion of C regulating molecules, such as decay accelerating factor (Lublin and Atkinson, 1989), into the viral envelope. Preclinical experiments using existing and improved baculovirus vectors have to be carried out for the treatment of inherited and malignant diseases of the liver. The outcome of those studies will provide clues as to the most promising application of baculovirus vectors in the field of liver gene therapy.

References

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Boyce, F.M., and Bucher, N.L.R. (1 9 9 6 ). Baculovirusmediated gene transfer into mammalian cells. P r o c . N a t l . A c a d . S c i . U . S . A . 93 , 2358-2352. Carbonell, L.F. and Miller, L.K. (1 9 8 7 ). Baculovirus interaction with nontarget organisms: a virus-borne reporter gene is not expressed in two mammalian cell lines. A p p l . E n v i r o n . M i c r o b i o l . 53 , 1412-1417. Cardoso, J.E., Branchereau, S., Jeyaraj, P.R., Houssin, D., Danos, O., and Heard, J.M. (1 9 9 3 ). In situ retrovirusmediated gene transfer into dog liver. Hum. Gene Ther. 4, 411-418. Charlton, C.A., and Volkman, L.E. (1 9 9 3 ). Penetration of Autographa californica nuclear polyhedrosis virus nucleocapsides into IPLB Sf 21 cells induces actin cable formation. V i r o l . 154, 245-254. Cory, J.S., Hirst, M.L., Hails, R.S., Goulson, D., Green, B.M., Carty, T.M., Possee, R.D., Cayley, P.J., and Bishop, D.H.L. (1 9 9 4 ). Field trial of a genetically improved baculovirus insecticide. Nature 370, 138-140. Dai, Y., Schwarz, E.M., Gu, D., Zhang, W.W., Sarvetnick, N., and Verma, I.M. (1 9 9 5 ). Cellular and humoral immune responses to adenoviral vectors containing factor IX gene: Tolerization of factor IX and vector antigens allow for long-term expression. P r o c . N a t l . A c a d . S c i . U . S . A . 92 , 1401-1405.

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Kidd, I.M. and Emery, V.C. (1 9 9 3 ). The use of baculoviruses as expression vectors. A p p l . B i o c h e m . B i o t e c h n o l . 42 , 137-159. Kool, M., Ahrens, C.H., Goldbach, R.W., Rohrmann, G.F., and Vlak, J.M. (1 9 9 4 ). Identification of genes involved in DNA replication of the Autographa californica baculovirus. P r o c . N a t l . A c a d . S c i . U . S . A . 91 , 11212-11216. Li, Q., Kay, M.A., Finegold, M., Stratford Perricaudet, L.D., and Woo, S.L. (1 9 9 3 ). Assessment of recombinant adenoviral vectors for hepatic gene therapy. Hum. Gene Ther. 4, 403-409. Lรถser, P., Jennings, G., Strauss, M., and Sandig, V. (1 9 9 8 ). Reactivation of the previously silenced cytomegalovirus major immediate early promoter in mouse liver: involvement of NF!B. J . V i r o l . In press. Lu, A. and Miller, L.K. (1 9 9 5 ). The roles of eighteen baculovirus late expression factor genes in transcription and DNA replication. J . V i r o l . 69 , 975-982. Lublin D.M., and Atkinson J.P. (1 9 8 9 ). Decay-accelerating factor: Biochemistry, molecular biology, and function. A n n . R e v . I m m u n o l . 7, 35-38.

Gene Therapy and Molecular Biology Vol 1, page 239 Luckow, V.A. and Summers, M.D. (1 9 8 8 ). Trends in the development of baculovirus expression vectors. B i o / T e c h n o l o g y 6, 47-55.

cell death. In Concepts in Gene Therapy, M. Strauss and J.A. Barranger, eds. (Berlin; New York: deGruyter), 521537.

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Szebeni, J., Wassef, N.M., Spielberg, H., Rudolph, A.S., and Alving, C.R. (1 9 9 4 ). Complement activation in rats by liposomes and liposome-encapsulated hemoglobin: evidence for anti-lipid antibodies and alternative pathway activation. B i o c h e m . B i o p h y s . R e s . Commun. 205, 255-263.

Miller, L.K. (1 9 9 3 ). Baculoviruses: high-level expression in insect cells. C u r r . O p i n . i n G e n . a n d D e v . 3, 97101. Morris, T.D., and Miller, L.K. (1 9 9 2 ). Promoter influence on baculovirus-mediated gene expression in permissive and nonpermissive insect cell lines. J . V i r o l . 66 , 73977405.

Takeuchi, Y., Porter, C.D., Strahan, K.M., Preece, A.F., Gustafsson, K., Cosset, F.-L., Weiss, R.A., and Collins, M.K.L. (1 9 9 6 ). Sensitization of cells and retroviruses to human serum by (a1-3) galatosyltransferase. Nature 379, 85-88.

Pearson, M., Bjornson, R., Pearson, G., and Rohrmann, G. (1 9 9 2 ). The Autographa californica baculovirus genome: evidence for multiple replication origins. S c i e n c e 257, 1382-1384.

Tjia, S.T., zu Altenschildesche, G.M., and Doerfler, W. (1 9 8 3 ). Autographa californica nuclear polyhedrosis virus (AcNPV) DNA does not persist in mass cultures of mammalian cells. V i r o l o g y 125, 107-117.

Plank C., Mechtler K., Szoka F.C., Jr., and (1 9 9 6 ). Activation of the complement synthetic DNA complexes: a potential intravenous gene delivery. Hum. G e n e . 1437-1446.

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Volkman, L.E., Goldsmith, P.A., Hess, R.T., and Faulkner, P. (1 9 8 4 ). Neutralization of budded Autographa californica nuclear polyhedrosis virus by a monoclonal antibody: identification of the target antigen. V i r o l . 133, 354362.

Rettinger, S.D., Ponder, K.P., Saylors, R.L., Kennedy, S.C., Hafenrichter, D.G., and Flye, M.W. (1 9 9 3 ). In vivo hepatocyte transduction with retrovirus during in-flow occlusion. J . S u r g . R e s . 54 , 418-425.

Volkman, L.E. and Goldsmith, P.A. (1 9 8 5 ). Mechanism of neutralization of budded Autographa californica nuclear polyhedrosis virus by a monoclonal antibody: inhibition of entry by adsorptive endocytosis. V i r o l . 143, 185195.

Wagner system barrier Ther.;

Rother, R.P., Squinto, S.P., Mason, J.M., and Rollins, S.A. (1 9 9 5 ). Protection of retroviral vector particles in human blood through complement inhibition. Hum. Gene Ther. 6, 429-435. Sandig, V., Hofmann, C., Steinert, S., Jennings, G., Schlag, P., and Strauss, M. (1 9 9 6 ). Gene transfer into hepatocytes and human liver tissue by baculovirus vectors. Hum. Gene Ther. 7, 1937-1945. Sandig, V., Brand, K., Herwig, S., Lukas, J., Bartek, J., and Strauss, M. (1 9 9 7 ). Adenovirally transferred p16(INK4/CDKN2) and p53 genes cooperate to induce apoptotic tumor cell death. Nature Medicine , 3, 313319. Strauss, M., Brand, K., and Sandig, V. (1 9 9 7 ). Tumor suppressor gene therapy - growth arrest and programmed

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Whitford, M., Stewart, S., Kuzio, J., and Faulkner, P. (1 9 8 9 ). Identification and sequence analysis of a gene encoding gp67, an abundant envelope glycoprotein of the baculovirus autographa californica nuclear polyhedrosis virus. J . V i r o l . 63 , 1393-1399. Yang, Y.P., Nunes, F.A., Berencsi, K., Furth, E.E., Gonczol, E., and Wilson, J.M. (1 9 9 4 ). Cellular immunity to viral antigens limits E1-deleted adenoviruses for gene therapy. P r o c . N a t l . A c a d . S c i . U . S . A . 91 , 4407-4411. Yap, C., Ishii, K., Aoki, Y., Aizaki, H., Hideki, T., Shimizu, H., Ueno, Y., Miyamura, T., and Matsuura, Y. (1 9 9 7 ). A hybrid baculovirus-T7 RNA polymerase system for recovery of an infectious virus from cDNA. V i r o l . 231, 192-200.

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Gene Ther Mol Biol Vol 1, 241-251. March, 1998.

Delivery systems for the MDR1 gene Caroline G. L. Lee1 , Wilfred D. Vieira1 , Ira Pastan2 and Michael M. Gottesman1 . 1Laboratory of Cell Biology, 2Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health

_____________________________________________________________________________________ Corresponding Author: Michael M. Gottesman, Laboratory of Cell Biology, Bldg 37, Room 1A09, National Cancer Institute, National Institutes of Health, 37 Convent Drive MSC 4255, Bethesda, Maryland 20895-4255, Tel: (301)-496-1530, Fax: (301)-402-0450, Email: mgottesman@nih.gov

Summary The acquired resistance of cancer cells to a wide variety of structurally unrelated anti-cancer drugs as w e l l a s t h e i n c r e a s e d s e n s i t i v i t y o f c e l l s o f the hematopoietic system t o these same drugs have contributed to the limited success o f l o n g term cancer chemotherapy. The overexpression o f the multidrug resistance (M D R 1) gene was found to be associated with this acquired resistance and has been exploited to protect normal bone marrow cells from myelosuppression that may result in life threatening leukopenia and thrombocytopenia following high dose chemotherapy. Thus far, retroviral transfer of the M D R 1 g e n e h a s b e e n t h e m a i n r o u t e o f d e l i v e r y i n t o b o n e m a r r o w c e l l s . A l t h o u g h widely used as one o f the most efficient vehicles for gene delivery, safety and other concerns associated with viruses cannot be ignored. We have explored other means of introducing the M D R 1 gene into recipient cells. A variety of vectors can be introduced into cultured cells and bone marrow c e l l s i n v i t r o using lipofection. One new system under development uses lipofection to introduce an Epstein Barr Virus (EBV)-based episomal vector carrying the M D R 1 cDNA. High efficiency transfection of cultured cells has been achieved with this system.

I. Drug resistance in cancer Metastatic and disseminated cancers that are not amenable to surgical removal or radiation can be treated by chemotherapy. Although many different malignances have been successfully treated by various antineoplastic drugs, the majority of solid tumors are either refractory to treatment or become non-responsive after an initial effect. Dose escalation to improve the efficacy of these chemotherapeutic agents is severely limited by their toxic side effects since normal tissues, especially the bone marrow, are particularly sensitive to anti-cancer agents. The mechanism by which cancers evade chemotherapy has been intensively pursued. This has led to the elucidation of various cellular and genetic changes that confer increased drug resistance to cancer cells. These include decreased influx or increased extrusion of anti-cancer drugs; metabolic and cell-cycle changes in response to these drugs; augmented repair of drug-induced damage; to name a few (Gottesman et al., 1994). T a b l e 1 illustrates examples of gene products implicated in mediating resistance to anti-cancer drugs. Of these, the multidrug transporter, MDR1, has generated

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significant interest since it confers resistance to a wide variety of structurally unrelated drugs and has been found to be expressed at levels likely to contribute to drug resistance in 50% of metastatic and disseminated cancers (Gottesman and Pastan, 1997).

II. Structure of the multidrug transporter The human MDR1 gene encodes a 170 kDa cell-surface phospho-glycoprotein known as P-glycoprotein (â&#x20AC;&#x153;Pâ&#x20AC;? also stands for permeability or pump). P-glycoprotein is an ATPdependent efflux pump belonging to a superfamily of ATPbinding cassette (ABC) transporters. It is composed of two homologous halves, each spanning the plasma membrane six times and containing an ATP utilization site (F i g . 1 ). ATP binding and hydrolysis is important for substrate transport (Azzaria et al., 1989). Mutational analysis revealed three Nlinked glycosylation sites present at the amino-terminal half in the first extracytoplasmic loop (Schinkel et al., 1993). These glycosylation sites may contribute to the correct

Gene Therapy and Molecular Biology Vol 1, page 253 Gene Ther Mol Biol Vol 1, 253-263. March, 1998.

Tumor killing using the HSV-tk suicide gene Rajagopal Ramesh 1, Aizen J. Marrogi 1 and Scott M. Freeman2,3 1

Department of Surgery and Gene Therapy Program, LSU School of Medicine, New Orleans, Louisiana, USA.

Department of Pathology, Tulane University School of Medicine, New Orleans, Louisiana, USA.

____________________________________________________________________ 3

Corresponding author. Dr. Scott M. Freeman. Associate Professor. Department of Pathology, Tulane University School of Medicine, 1430 Tulane Avenue, New Orleans, Louisiana 70112, USA. Tel: (504) 585-4089, Fax : (504) 587-7389, E-mail: sfreema@tmc.tulane.edu

Summary The Herpes Simplex virus thymidine kinase gene (HSV-tk) has been widely used as a suicide gene in cancer gene therapy. Since the first demonstration of the ability of HSV-tk gene modified tumor cells to generate a bystander effect, a number of clinical trials have been initiated to treat human cancers. However, the mechanism of the HSV-tk mediated bystander tumor killing has remained controversial and i s under intense investigation. The present report discusses the various mechanisms proposed by which the HSV-tk mediated bystander tumor killing occurs and highlights the importance of the host immune system in mediating the tumor killing. In addition, the present report also demonstrates that the initial tumor killing results in an inflammatory response leading to a cytokine cascade. This subsequently leads to an immune response resulting in an influx of macrophages and tumor infiltrating lymphocytes. Finally, strategies t o augment the HSV-tk mediated bystander tumor killing by immunization are discussed and conclude with potential pitfalls of using HSV-tk/GCV system in a clinical setting.

I. Gene therapy

During the course of developing the suicide genes, it was realized that if the suicide gene can be delivered directly to a tumor, they can be used for cancer therapy. This concept forms the basis for suicide gene therapy. The most common strategy utilized in suicide gene therapy involves the delivery of a gene encoding an enzyme that will metabolize a nontoxic prodrug into a toxic metabolite, leading to killing of the cells expressing the gene. The activated prodrug interferes with the replication of the transfected cells, while not affecting the non transfected cells. Therefore, systemic toxicity is minimal making this approach attractive for tumor gene therapy or as a safety device in the use of live tumor cell vaccines. The two most commonly used suicide genes, which have progressed into clinical trials, are the herpes simplex virus thymidine kinase (HSV-tk) gene coupled with the pro-drug ganciclovir (GCV) and the cytosine deaminase (CD) gene coupled with the pro-drug 5' fluorouracil (5-FU) (Freeman et al., 1992a; Mullen et al., 1992; Huber et al., 1994). Other candidate suicide genes which are being tested include the xanthine guanine phosphoribosyl transferase (XGPRT) and purine nucleoside phosphorylase (Besnard et al., 1987, Mroz and Moolten., 1993).

Gene therapy has been defined as the alteration of the genetic material of a cell with resultant benefit to a patient. Gene transfer has two broad categories : one in which a therapeutic gene is delivered to the cells with the aim of treating a disease; and second where a marker gene is delivered to label a cell type to determine the fate of a cell or the marker gene. Gene therapy is now becoming a rapidly developing therapeutic modality for experimental treatment of some cancers and diseases that have no alternative treatment (Anderson, 1992; Friedman and Roblin, 1972).

II. Suicide genes and suicide gene therapy Definition o f suicide gene: Suicide gene was originally developed as a safety measure to control the expression of a foreign gene introduced into a cell such that the gene modified cell can be eliminated if gene expression is no longer desired or if the gene modified cells become transformed. (Blaese, 1992).

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Ramesh et al: Tumor Killing Using the HSV-tk Suicide Gene methylation) resulting in loss of expression of the recombinant protein (i v ) incomplete tumor killing. Thus modifications of the existing approach is required before suicide gene therapy can be applied as a prophylaxis for cancer. Since it is difficult to genetically modify all tumor cells within an individual, killing of unmodified tumor cells needs to occur in order for this approach to be therapeutically effective. Using the HSV-tk/GCV system, Freeman and colleagues (1992a; 1992b; 1993) demonstrated that HSV-tk gene-modified tumor cells are toxic to nearby unmodified tumor cells when the mixed tumor population is exposed to GCV. This phenomenon where untransduced tumor cells, not expressing the HSV-tk enzyme, are killed has been termed the "bystander effect". The effectiveness of the "bystander effect" to kill tumor cells has been shown both in vitro and in vivo and occurs even when only a fraction (10%) of the tumor mass contains the HSV-tk gene-modified tumor cells (Freeman et al., 1993). In addition the bystander effect has been demonstrated when syngeneic or xenogeneic HSV-tk gene modified tumor cells were used, indicating that irrespective of cell type, the gene modified cells need to be in close proximity to the unmodified tumor cells for the antitumor effect (Freeman et al, 1995a). Several other investigators have subsequently demonstrated the occurrence of the bystander effect using different tumor cells lines expressing the HSV-tk gene (Culver et al., 1992; Vile et al., 1993; Barba et al., 1993; Ram et al., 1993). The demonstration of the bystander effect has important implications in cancer therapy since it removes the burden of the need for delivery of the gene to 100% of the tumor cell population. The use of HSV-tk/GCV system in the treatment of cancer offers several advantages : (i) rapidly replicating tumor cells are more susceptible to impairment of DNA synthesis (ii) chemotherapy resistant tumors can be made sensitive when genetically modified with the HSV-tk gene and (iii) HSV-tk/GCV-treated tumor cells have the ability to kill neighboring tumor cells through the bystander effect. Such a strategy has been tried to treat various experimental tumors (Culver et al., 1992; Ezzedine et al., 1991; Takamiya et al., 1992). After some encouraging results from experimental animal studies, many clinical trials have been approved worldwide (Freeman et al., 1995b; Clinical Protocols 1993; Clinical Protocols 1994a; 1994b). Although clinical protocols have been initiated, the precise mechanism of the bystander effect is unclear and is currently under intense investigation (Kolberg, 1994; Seachrist, 1994). Several hypothesis have been proposed for the mechanism of bystander effect which includes : apoptosis, endocytosis of toxic cell debris, blood vessel destruction and the involvement of the host immune system. In addition, reports from several groups indicate that the bystander killing varies depending upon the type of tumor cell used. Whatever the mechanism is, the generation of the bystander effect explains at least in part, the success of the delivery experiments in vivo that have successfully eradicated growing tumors despite the improbability of having delivered HSV-tk to every tumor cell. The observation and

III. HSV/tk GCV system and the bystander effect The herpes simplex virus thymidine kinase (HSV-tk) gene is the most commonly used suicide gene. Initially, Moolten et al., (1990) demonstrated that tumors cells expressing the suicide gene (HSV-tk) can be specifically killed both in vitro and in vivo when exposed to the antiviral drug, ganciclovir (GCV). The HSV-tk gene specifically monophosphorylates the guanosine analogue ganciclovir (GCV) which is subsequently converted into the toxic GCV-triphosphate form by endogenous mammalian kinases. The GCV-triphosphate is incorporated into replicating DNA by cellular DNA polymerase, thereby arresting DNA replication and causing cell death (Elion, 1980). The HSV-tk enzyme is almost 1000 fold more efficient at monophosphorylating GCV than the cellular thymidine kinase (Elion et al., 1977). Therefore, GCV is highly toxic to cells that express HSV-tk but are minimally toxic to unmodified or uninfected cells at therapeutic concentrations of the drug (1-10mmol/L). However, neutropenia can be a clinical manifestation as result of GCV (Shepp et al., 1985; Elion, 1980; Freeman et al., 1996). The phosphorylation of GCV curtails its movement across cell membrane resulting in a longer half life (t1/2=18-24 hrs) within the cells than unmodified GCV (Elion, 1980). The increased half life of GCV is an important feature in the anti-tumor effects of HSV-tk gene modified tumors. Based on the evidence that most cancers are clonal in origin, and that HSV-tk gene modified tumor cells are sensitive to GCV, initial strategy was to generate a mosaicism within an individual such that cells become HSV-tk positive randomly (Moolten et al., 1986; Moolten et al., 1990a). Any tumor arising later from one of the HSV-tk sensitized cells, then all the tumor cells will carry the sensitivity gene as a clonal property and thereby can be treated with GCV to eliminate the tumor (Moolten et al., 1990b). Additional drug sensitivities can be achieved by using a combination of suicide genes (e.g.: CD and XGPRT) such that a complete mosaicism can be obtained. In such a situation, cells expressing three different kinds of suicide genes would exist within an organ. If a cancer developed later from a cell carrying any one of these genes, then those cells can be selectively eliminated by using the appropriate drug treatment. Thus, the normal nonmalignant cells will be spared with very minimal damage and thereby can repopulate. Although the mosaic theory for cancer therapy using suicide genes is an attractive approach, due to current limitations in the available technology it may not be immediately applicable in the clinic. The various difficulties currently faced include (i ) inefficient gene transfer into cells of an organ in particular when retroviral vectors are used as a result of which only a small portion of an organ can be modified (i i ) transient gene expression when adenoviral vectors are used as a result of which, gene expression is lost in a rapidly dividing cell population (e.g.; malignant growth) (i i i ) silencing of the gene (e.g.; 254

Gene Therapy and Molecular Biology Vol 1, page 255 results from our laboratory and others, studying the mechanism of the bystander effect, will be discussed in detail in the following sections.

bystander effect in vitro, their role in the in vivo bystander tumor killing has not been tested.

V. In vivo mechanism of bystander tumor killing

IV. In vitro HSV-tk mediated bystander effect Since the initial findings by Freeman et al., (1992a, 1992b, 1993) demonstrating the occurrence of a bystander effect, the mechanism of bystander tumor killing has been controversial and has been the subject of intensive investigation. Initial in vitro studies suggested that toxic metabolites of GCV from HSV-tk gene modified tumor cells contained in apoptotic vesicles were transferred to the adjacent unmodified tumor cells by phagocytosis (Freeman et al., 1993). This was based upon the observation that HSV-tk gene modified tumor cells when exposed to GCV undergo apoptotic cell death as evidenced by cytoplasmic shrinkage, chromatin condensation and nuclear DNA fragmentation. Additional in vitro studies demonstrated that the bystander tumor killing resulted from the transfer of toxic GCV metabolites through apoptotic vesicles to nearby unmodified tumor cells (Samejima et al., 1995; Colombo et al., 1995). However, subsequent studies by Bi et al., (1993) using radiolabeled GCV demonstrated that the anti-cancer effect occurs in vitro by the transfer of toxic GCV metabolites from the dying HSV-tk tumor cells to the adjacent unmodified tumor cells through gap junctions. Similar results demonstrating the role of gap junctions in HSV-tk mediated bystander killing have been reported by other investigators (Fick et al., 1995; Elshami et al., 1996). Like other nucleotides, phosphorylated GCV cannot pass through the plasma membranes except when traversing to neighboring cells by gap junctions. Gap junctions are intercellular communicating channels that connect adjacent cells and which are in dynamic equilibrium exchanging ions and proteins between cells. These channels are permeable to molecules smaller than Mr 1000, such as cyclic AMP, calcium, and inositol triphosphate, but do not allow the transfer of proteins and nucleic acids. Gap junction channels are formed by proteins called connexins. The family of connexin proteins include at least 13 members in rodents. The role of connexins, in particular connexin 26 (Cx26) in gap junctional mediated bystander killing in vitro was demonstrated by Mesnil et al., (1996). More recently, connexin 46 (Cx 46), a tumor suppressor gene, has also been demonstrated to mediate the bystander tumor killing (Mesnil et al., 1997). Tumor cells when cotransfected with Cx23 or Cx46 along with HSV-tk gene showed enhanced bystander killing when exposed to GCV. In contrast, tumor cells transfected with HSV-tk alone showed decreased cell death while cells transfected with Cx23 or Cx46 alone showed no cell death upon exposure to GCV. Although gap junctions probably play a key role in the mechanism of

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Although the mechanism of HSV-tk bystander tumor cell killing in vitro has been demonstrated to occur between cells in close proximity through gap junctions, the in vivo mechanism of tumor killing remains unresolved. This is partly due to the conflicting reports that have been generated using different tumor models. However, results are now emerging from several laboratories including ours suggesting that additional mechanism may be operational in vivo, namely the host immune system. The observation that the host immune system participates in mediating the bystander effect in vivo stems from the initial findings demonstrating severely diminished or abrogated bystander tumor killing in animals that lacked an intact immune system, particularly T-cells (Freeman et al., 1992a, 1992b; Freeman et al., 1993; Freeman et al., 1994; Vile et al., 1994; Whartenby et al., 1995; Colombo et al., 1995; Ramesh et al., 1996a). Furthermore, HSV-tk gene-modified tumor cells and GCV can prolong animal survival when injected intraperitoneally (i.p.) into i.p. tumor bearing mice (Freeman et al., 1992; Freeman et al., 1993). In vivo autopsy results showed a rapid centralized hemorrhagic tumor necrosis which occurs within 24 hours after injection of HSV-tk gene modified tumor cells and GCV (Figure 1) suggesting that a more rapid mechanism of tumor killing was occurring (Freeman et al., 1994; Whartenby et al., 1995; Ramesh et al., 1996a). This is in contrast to the in vitro tumor cell death which is mediated by apoptosis that occurs over a period of 48-72 hours. The rapid occurrence of hemorrhagic tumor necrosis following the initial killing of the injected HSV-tk modified tumor cells after exposure to GCV indicated that soluble factors (cytokines and chemokines) which are capable of causing necrosis are released (Carswell et al., 1975; Schall and Bacon, 1994). This is due to the fact that tumor necrosis occurred inside, from the center of the tumor, rather than from the outside of the tumor on the periphery (Figure 1). The occurrence of tumor necrosis was also reported by Ram et al., (1994) using HSV-tk vector producer cells. However, the observed tumor necrosis was attributed to be due to the transfer of retroviral particles carrying the HSVtk gene to the endothelial cells lining the tumors blood vessel, which were destroyed when exposed to GCV. Although, the HSV-tk delivery system used were different ( Freeman et al., 1993; Ram et al., 1994), the occurrence of tumor necrosis was observed to be a common phenomenon in both the studies. Several other investigators have also subsequently documented the occurrence of tumor necrosis following HSV-tk/GCV treatment (Barba et al., 1994; Bovistias et al., 1994).

Ramesh et al: Tumor Killing Using the HSV-tk Suicide Gene

F i g u r e 1 : H e m o r r h a g i c T u m o r N e c r o s i s . BALB/c mice with intraperitoneal murine tumors were injected with HSV-tk gene modified tumor cells with or without GCV. Tumors were harvested 24 hours later and examined microscopically by hematoxylin and eosin staining (H&E). A. Absence of necrosis in tumors not receiving GCV. B . Necrosis observed in tumors from animals receiving HSV-tk and GCV treatment.

speculate that the observed tumor necrosis is caused by TNF-" and IL-1 (Carswell et al., 1975; Dinarello, 1996). To this extent, i.p. tumor bearing animals were treated with HSV-tk gene modified tumor cells and analyzed for cytokine production. Expression of TNF, IL-1 and IL-6 mRNA was observed within 24 hours following HSVtk/GCV treatment which coincides with the observed centralized tumor necrosis (Freeman et al., 1994; Freeman et al., 1995a; Ramesh et al., 1996a). In addition, an increase in the message for TNF" was observed (Ramesh et al., 1996a). Since TNF" and IL-1" are known potent activators of host anti-tumor cells such as macrophages, NK cells, and cytotoxic lymphocytes, and can serve to potentiate the proliferative response of T lymphocytes (Urban et al.,

Further evidence demonstrating that soluble factors are responsible for centralized tumor necrosis comes from the observation that when fluorescein labeled HSV-tk tumor cells are injected intraperitoneally into an i.p. tumor bearing animal, these cells preferentially homed to actively growing tumor in-situ (Figure 2). The argument for the role of soluble factors is based on the fact that if apoptotic cell death or gap junctional mediated bystander tumor killing was to occur following GCV treatment, then cell death would occur at the periphery of the tumor rather than on the inside (Shastri et al., unpublished data). Furthermore, apoptotic cell death leads to the upregulation of IL-1! converting enzyme (ICE) which causes IL-1 secretion (Hogquist et al., 1991). Based on the known functions of some of the soluble factors such as tumor necrosis factor-" (TNF-"), and interleukin-1 (IL-1) which can cause rapid necrosis, one can

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Figure 2: Homing of Fluorescein Labeled HSV-tk gene modified tumor cells. Fluorescein labeled (experimental) or unlabeled (control) HSV-tk gene modified tumor cells were injected intraperitoneally (i.p.) into i.p. tumor bearing mice and analyzed for their fate. The tumors were isolated 24 hours post injection and analyzed by light microscopy (a & b ) and fluorescent microscopy (c & d). The HSV-tk tumor cells home onto actively growing in-situ tumor and adhere to the outer surface of the tumor as seen by the fluorescence in experimental animals (d). Unlabeled cells when injected do not fluoresce and were used as a control (c ).

mononuclear and T cells within the tumor after injection of the HSV-tk gene modified tumor cells could be due in part to the TNFa induced hemorrhagic necrosis as well as increased up-regulation of adhesion molecules caused by inflammatory cytokines such as IL-1". Similarly, the infiltration of effector T cells and macrophages into the tumor following HSV-tk/GCV treatment, may be a direct consequence of the induction of localized cytokine expression. Although PCR can detect mRNA expression within the tumor it does not necessarily reflect protein expression, even though we can demonstrate an increase in TNF mRNA in the HSV-tk treated animals compared to the untreated

1986; Palladino et al., 1987; Nakano et al., 1989; Harada et al., 1994) immunohistochemical studies were performed on tumor tissues from control and experimental animals for infiltrating macrophages and T-cells. It was found that in the tumors from animals receiving HSV-tk and GCV treatment there was a significant increase in the macrophages and T cells as compared to the control animals (Ramesh et al., 1996a). Studies supporting these findings have been further documented by other investigators demonstrating infiltration of immune cells (macrophages, CD4+ , CD8 + ) following HSV-tk/GCV treatment (Caruso et al., 1993; Barba et al., 1994; Vile et al., 1994; Pope et al., 1996; Gagandeep et al.; 1996). Increase in 257

Ramesh et al: Tumor Killing Using the HSV-tk Suicide Gene animals. Immunohistochemical studies showed that mononuclear cells infiltrating the tumor were expressing TNF and IL-1 suggesting that cytokines are generated in the process of development of an antitumor response (Freeman et al., 1994; Ramesh et al., 1996a). More recently, Vile et al., (1997a) using B16 melanoma tumor cells demonstrated the production of cytokines along with pronounced intratumoral infiltration of macrophages and lymphocytes following HSV-tk/GCV treatment in vivo. The ability to induce cytokine production in vivo has also been demonstrated for other agents such as the E. coli lipopolysaccharide (LPS). However, we found that LPS injections stimulated a different cytokine repertoire within the tumor than the HSV-tk tumor cells and did not prolong survival of tumor bearing mice indicating that in addition to cytokine production, the events that set the cascade appear to be critical (Freeman et al., 1996; Shastri et al., unpublished data). Based on these findings, we hypothesized that the HSV-tk gene-modified tumor cells alter the tumor's microenvironment from one that suppresses an anti-tumor immune response to a stimulatory one (Freeman et al., 1994; Ramesh et al., 1996a). Failure of a tumor-specific T cell response in tumor bearing mice might in part result due to lack of tumor antigen expression or from the inability or inadequate expression of adhesion molecules (ICAM-1) and other cell surface molecules such as the costimulatory molecules (B7) by the tumor cells. Among the different accessory molecules expressed on antigen presenting cells (APC), cytokines can upregulate expression of co-stimulatory molecules like B7 and ICAM which have been suggested to play a major role in T cell activation (Freedman et al., 1987; Chang et al., 1994). B7 might be preferentially involved in stimulation of antigen primed T cells whereas ICAM-1 which is constitutively expressed on all APCs would be most efficient in co-stimulation of resting cells. The ability of pro-inflammatory cytokines (TNF, IL-1) to upregulate B7 (B7-1 and B7-2) expression has been demonstrated (Chang et al., 1995). Furthermore, B7 expression modulates the differentiation of T cells into Th1 or Th2 (Kuchroo et al., 1995). Since HSV-tk/GCV treatment results in production of cytokines (TNF, IL-1, IFN-# ) in vivo, we investigated whether HSV-tk/GCV treatment could also elicit the expression of co-stimulatory molecules, B7-1, B7-2 and ICAM which are critical for the induction of anti-tumor immunity. Analysis for the expression of these cell surface immune regulatory molecules in-vivo after treatment of tumor bearing mice (i.p.) with HSV-tk gene-modified tumor cells and GCV demonstrated i.p. tumor bearing control mice do not express B7-1, while low levels of B7-2 and ICAM-1 are expressed (Freeman et al., 1995c; Ramesh et al., 1996b). Interestingly, it has been reported that low levels of B7-2 are expressed on the cell surface of naive leukocytes. Only upon activation does B7-1 become expressed. In mice with an i.p. tumor, inoculation of HSV-tk gene modified tumor cells with GCV led to upregulation of B7-1, B7-2 and ICAM-1 thus indicating a state of activation within the tumor (Freeman et al., 1995c;

Ramesh et al., 1996b). Furthermore, T-cells isolated from the spleen of tumor bearing mice treated with HSV-tk genemodified tumor cells and GCV showed a proliferative response in-vitro to parental syngeneic tumor cells and released IL-2 which is often associated with an activated state. The proliferative response thus observed appeared to be specific since murine mastocytoma cells did not stimulate T-lymphocytic proliferation (Ramesh et al., 1996b). This type of response suggests that cells become activated after treatment with HSV-tk gene modified tumor cells and GCV with the generation of a tumor specific immune response in-vivo. The alteration in the tumor microenvironment following HSV-tk/GCV treatment has also been suggested by Vile et al., (1997a).

VI. Augmenting the HSV-tk mediated bystander killing by immunization Based on the importance of the immune system in the generation of the "bystander effect", we examined whether enhancement of the immune system could augment the "bystander effect". This would be extremely important since it may not only be possible to treat local tumors but also metastatic tumors which are life threatening to the patient. Potential means for enhancement of the bystander tumor killing include (i ) using biological response modifiers (BRM), such as cytokines to augment the immune/inflammatory response generated by the HSV-tk gene modified tumor cells and ganciclovir and (i i ) immunization to a known tumor antigen before treatment with HSV-tk gene modified cells and ganciclovir. Although active (tumor vaccination) and adoptive (TIL, LAK) immunotherapy has been extensively studied over the past decade, there has been only marginal clinical benefit to these approaches. One potential problem is that the peripheral blood "activated" immune effector cells generated by these approaches may become inactivated upon entering the immunosuppressive tumor environment. Thus, unless the tumor microenvironment can be altered, immunotherapeutic approaches may continue to have problems generating effective anti-tumor responses. The "activated" immune stimulatory environment developed by the inflammatory response to the HSV-tk gene-modified cells allows for the development of an immune response. But more importantly, it provides an environment for the efficient functioning of immune effector cells which exist within the host's peripheral blood. This latter issue relates to the enhanced anti-tumor response which we can demonstrate when combining immunization with HSV-tk gene-modified since immune effector cells in the peripheral blood generated by immunization can traffic to the tumor after treatment with HSV-tk gene-modified cells and GCV (Ramesh et al., 1997). However, the cell type used for immunization was found to be critical. To further understand how this enhancement of the "bystander effect" occurs, we began to evaluate how the tumor environment is altered after immunization and treatment with HSV-tk gene-modified cells. Since we have previously demonstrated the occurrence of a centralized 258

Gene Therapy and Molecular Biology Vol 1, page 259 hemorrhagic necrosis in vivo with release of soluble factors, in unimmunized mice treated with HSV-tk gene, cytokine mRNA expression was analyzed in intraperitoneal tumors by RT-PCR initially. We detected mRNA to all three cytokines (TNF, IL-1 and IL-6) in the tumors of mice within 24 hours in both the immunized untreated group and immunized treated group of mice, but could not detect the message in untreated mice. This observation is somewhat surprising since although it appears that the tumor environment can be altered by immunization, animal survival is unchanged by immunization alone. More importantly, subsequent analysis for other cytokines demonstrated IL-2 expression only in mice immunized with syngeneic tumor and treated with HSV-tk gene-modified tumor cells and GCV. IL-2 was detected at 48 hours post injection of the HSV-tk gene-modified cells and GCV. None of the other cytokines tested (IL-10, GMCSF, IL-4 and IFN-# ) were detectable in mice from any of the immunized groups, although results from our earlier studies demonstrate mice which were unimmunized and treated with HSV-tk gene-modified cells showed expression of GM-CSF and IFN-# mRNA. The production of IL-2 only in mice receiving HSV-tk gene-modified tumor cells and GCV is intriguing since in our previous study using unimmunized mice receiving HSV-tk and GCV, IL-2 was not observed. The demonstration of IL-2 mRNA after treatment may have significant implications in the enhancement of the "bystander effect" since it may activate T cells which in turn might be triggering and amplifying the antitumor effector response (Vieweg and Gilboa, 1995). Although, the subset of T lymphocytes (CD4/CD8) infiltrating the tumor was not characterized, T cells from immunized mice receiving HSV-tk gene-modified cells and GCV demonstrated an increased proliferation to syngeneic tumor than either mice immunized only or mice treated with HSVtk/GCV only. In addition, an increase in the number of tumor infiltrating lymphocytes was observed. This is probably because of the hemorrhagic tumor necrosis which develops after treatment and the release of cytokines which allows the immune effector cells to enter the tumor and provides an immune stimulatory environment for them to function. Since the HSV-tk modified tumor cells and GCV can alter the tumor microenvironment to one that is immunostimulatory through the release of cytokines, as evidenced by increased expression of immune regulatory molecules such as ICAM-1 and B7, immune effector T cells already present secondary to immunization can function in the "activated" tumor microenvironment and kill the tumor. This situation, unlike that observed in unimmunized mice treated with HSV-tk tumor cells where only proinflammatory cytokines are produced, thus provides a potential new therapeutic cancer approach. Attempts are currently being made to develop vectors for the delivery of HSV-tk gene along with other immunostimulatory genes that can be expressed at the same time as the cells are killed and can enhance the antitumor immune response. However, studies from various laboratories have met with varying degrees of success. For

instance, when IL-2 secreting tumor cells were injected in conjunction with HSV-tk gene modified tumor cells, no enhanced bystander tumor killing was observed (Ram et al., 1994). Similar results were reported by Chen et al., (1995). However, combining HSV-tk gene modified tumor cells with IL-2 resulted in an increased long term tumor immunity in the surviving animals. In contrast, when interferon alpha (IFN-") was combined with HSV-tk gene modified tumor cells, an enhanced bystander tumor killing was observed (Santodonato et al., 1996). Interleukin-12 (IL-12) which is a potent activator of T-cells is also being evaluated along with HSV-tk to enhance tumor cell elimination in vivo (Vile et al., 1997b). Thus in-vivo therapy with cytokines capable of inducing either tumor cells or host immune cells to express molecules important in immunogenicity may be efficacious either independently or as an adjunctive therapy with HSV-tk.

VII. Proposed hypothesis Based on the findings mentioned above, we would like to advance the following mechanism(s) to explain the bystander killing following HSV-tk/GCV treatment where the tumor microenvironment is altered from an immunosuppressed to an immunostimulated environment (F i g u r e 3 ). Injection of HSV-tk gene modified tumor cells home to actively growing in-situ tumor through adhesion molecules. Primary killing of these HSV-tk tumor cells occurs with exposure to GCV resulting in an inflammatory response against the dying tumor cells which subsequently leads to an immune response. The inflammatory response generated by the dying HSV-tk gene modified tumor cells resembles the inflammatory response to microbial pathogens. This is partly because the HSV-tk gene modified cells die through apoptosis, which is facilitated by the transfer of toxic metabolites, releasing soluble factors such as TNF-" and IL-1". This process then leads to hemorrhagic tumor necrosis with the simultaneous activation of leukocytes/lymphocytes (Th), by costimulatory signals (B7) and adhesion molecules (ICAM, VCAM) within the tumor resulting in the increased production of cytokines. The cytokines released within the tumor microenvironment may improve indirect tumor presentation by host cells and influence the type of immune mechanism(s) resulting in either a Th1 or Th2 like response. Furthermore, the chemotactic factors and cytokines produced regulate the influx of natural killer cells (NK), neutrophils, eosinophils and monocyte/ macrophages (Mac) into the site of inflammation or tumor deposit and thereby affect the tumor microenvironment. The initial inflammatory response generated is usually too weak to eliminate the entire tumor mass, allowing the tumor to grow to a size that is too large to be killed when anti-tumor immunity develops several weeks later. However, in immunized mice, the "activated" immune effector T cells (CD4+ , CD8+ ) which are already present in the host's peripheral circulation possess strong anti-tumor activity which can function in the immune stimulatory tumor 259

Ramesh et al: Tumor Killing Using the HSV-tk Suicide Gene environment generated by treatment with HSV-tk and GCV. Thus, this anti-tumor effect mediated by HSV-tk suicide gene therapy can be enhanced to be effectiveclinically.

For example, neither the role of natural killer cells (NK) nor the role of macrophages in bystander tumor killing has been investigated. Till date, experimental studies have used T-cell deficient mice. However, these animals are neither deficient in NK cells nor in macrophages. Therefore, studies are required using animals which are completely lacking the immune system (NK and macrophages), for

VIII. Conclusions Although, based on animal experimental studies a role for the host immune system in the bystander tumor killing in vivo has been demonstrated further studies are warranted.

F i g u r e 3 : P r o p o s e d m e c h a n i s m o f t h e i n v i v o b y s t a n d e r e f f e c t . The injected HSV-tk gene modified tumor cells (TK) home to the actively growing in situ tumor. Treatment with GCV results in the killing of the HSV-tk gene modified tumor cells and the transfer of toxic metabolites to the adjacent bystander tumor cells resulting in hemorrhagic necrosis. The dying tumor cells (inflammatory response) release soluble factors (cytokines and chemokines) and shed tumor proteins. The resident macrophages (Mac) act as antigen presenting cells (APC's) resulting in the presentation of tumor antigens to the T-cells (Th). During this process, the cytokines (TNF, IL-1) upregulate the expression of costimulatory (B7) and adhesion molecules (ICAM, VCAM) on the lymphocytic infiltrates resulting in their activation. The activated lymphocytes produce more cytokines resulting in an influx of macrophages and T-cells (cytotoxic) which recognize the tumor antigens and kill the residual tumor (1o immune response). Upon rechallenge the T-cells specifically recognize the tumor antigens (specific immunity) and kill any tumor cell present (2o immune response) .

expression in adoptively transferred gene modified cells. However, these strategies can fail completely depending on the appropriate target cell. In particular, in a clinical setting, in vitro testing of the suicide effect may be useful to predict whether this approach has the potential to

instance by using immune-deficient animals such as SCID or SCID-beige mice. In conclusion, suicide gene strategies using the HSV-tk/GCV system can be highly effective for treatment of local tumor growth or to switch off gene

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Gene Therapy and Molecular Biology Vol 1, page 261 eliminate an individual tumor. Further understanding of the "bystander effect" will lead to improved uses for suicide gene therapy.

Clinical Protocol: Gene transfer for the treatment of cancer. ( 1 9 9 3 ) . Hum. Gene Ther. 4, 361.

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Gene Therapy and Molecular Biology Vol 1, page 263 Samejima, Y., and Meruelo, D. ( 1 9 9 5 ) . "Bystander killing" induces apoptosis and is inhibited by forskolin. Gene Ther. 2, 50-58. Santodonato, L., Ferrantini, M., Gabriel, L., et al. ( 1 9 9 6 ) . Cure of mice with established metastatic Friend leukemia cell tumors by a combined therapy with tumor cells expressing both interferon-1" and herpes simplex thymidine kinase followed by ganciclovir. Hum. Gene Ther. 7, 1-10. Schall, T.J., and Bacon, K.B. ( 1 9 9 4 ) . Chemokines, leukocyte trafficking, and inflammation. C u r r . O p i n . Immunol. 6, 865-873. Seachrist, L. ( 1 9 9 4 ) . Successful gene therapy has researchers looking for the bystander effect. J N a t l . C a n c e r I n s t . 86, 82. Shepp, D.H., Dandliker, P., DeMiranda, P., Burnette, T.C., Cederberg, D.M., Kirk, L.E., and Meyer, J.D. ( 1 9 8 5 ) . Activity of 9-[2-hydroxymethyl) ethoxymethyl] guanine in the treatment of cytomegalovirus pneumonia. A n n . Intern Med. 103, 368-373. Takamiya, Y., Short, M.P., Ezzeddine, Z.D. et al. ( 1 9 9 2 ) . Gene therapy of malignant brain tumors : A rat glioma line bearing the herpes simplex virus type 1- thymidine kinase gene and wild type retrovirus kills other tumor cells. J . N e u r o s c i . R e s . 33, 493-503. Urban, J.L., Shepard, H.M., Rothstein, J.L., Sugarman, B.J., and Schreiberm H. ( 1 9 8 6 ) . Tumor necrosis factor: a potent effector molecule or tumor cell killing by activated macrophages. P r o c . N a t l . Acad. S c i . USA. 83, 5233-5237. Vieweg, J., and Gilboa, E. ( 1 9 9 5 ) . Considerations for the use of cytokine secreting tumor cell preparations for cancer treatment. Cancer Invest. 13,193- 201. Vile, R.G., and Hart, I.R. ( 1 9 9 3 ) . Use of tissue-specific expression of the herpes simplex virus thymidine kinase gene to inhibit growth of established murine melanomas following direct intratumoral injection of DNA. Cancer R e s . 53, 3860-3864. Vile, R.G., Nelson, J.A., Castleden, S., Chong, H., and Hart, I.R. ( 1 9 9 4 ) . Systemic gene therapy of murine melanoma using tissue specific expression of the HSV-TK gene involves an immune component. Cancer Res . 54, 62286234. Vile, R.G., Castleden, S.C., Marshall, J., Comle-John, R., Upton, C., and Chong, H. ( 1 9 9 7 a ) . Generation of a Th1 like immune response in an nonimmunogenic tumor : HSV-tk killing in vivo stimulates a mononuclear cell infiltrate and a Th1-like profile of intratumoral cytokine expression. Int. J. Cancer. 71, 267-274 Vile, R.G., Diaz, R.M., Castleden, S., and Chong, H. ( 1 9 9 7 b ) . Targeted gene therapy for cancer : herpes simplex virus thymidine kinase gene-mediated cell killing leads to anti-tumor immunity that can be augmented by coexpression of cytokines in the tumor cells. B i o c h e m So c . T r a ns. 25, 717-722. Whartenby, K.A., Abboud, C.N., Marrogi, A.J., Ramesh, R., and Freeman, S.M. ( 1 9 9 5 ) . The biology of cancer gene therapy. L a b . I n v e s t . 72 , 131-145.

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Gene Therapy and Molecular Biology Vol 1, page 265 Gene Ther Mol Biol Vol 1, 265-277. March, 1998.

Neurotrophic factor gene therapy for neurodegenerative diseases Martha C. Bohn and Derek L. Choi-Lundberg1 Dept. of Pediatrics, Northwestern Univ. Med. Sch., Chicago, IL 60614

__________________________________________________________________________________ Correspondence: Martha C. Bohn, Ph.D., Medical Research Institute Council Professor, Children’s Memorial Institute For Education and Research, Northwestern University Medical School, Department of Pediatrics, 2300 Children’s Plaza, #209, Chicago, IL 60614, Tel: 773-868-8052, Fax: 773-868-8066, E-mail: m-bohn@nwu.edu 1

Current address: Department of Neurosciences, Genentech, Inc., DNA Way, South San Francisco, CA 94080, Tel: 415-225-6237

Key words: retrovirus, adenovirus, herpes simplex virus, adenoassociated virus, neurotrophic factors, GDNF, CNTF, BDNF, neurotrophins, adrenal, tyrosine hydroxylase, dopamine, substantia nigra, neurodegeneration, neural transplantation

Summary The delivery of genes to the central nervous system (CNS) whose products protect specific types o f nerve c e l l s from dying or stimulate their growth i s applicable t o a wide range of neurodegenerative diseases and injuries t o the nervous s y s t e m . This review focuses on Parkinson’s disease as a prototype for studying adenoviral mediated gene therapy. The dopaminergic (DA) neurotrophic factor, glial cell line-derived neurotrophic factor (GDNF), is a potent DA factor that ameliorates the behavioral and histological consequences of lesioning DA neurons in mice, rats and monkeys following injection of large amounts of recombinant GDNF protein into the brain. Adenoviral mediated gene therapy with GDNF was studied in a rat model of Parkinson’s disease in which the DA neurons die slowly following injection of the neurotoxin, 6-hydroxydopamine (6-OHDA), near their terminals. The survival of DA neurons was markedly enhanced in rats injected with Ad GDNF, but not with control vectors (approximately 80% in Ad GDNF treated rats vs 30% in control rats). Moreover, this degree of neuronal protection was observed at levels of biosynthesized GDNF in the nanogram range which persisted in the injection site for at least 7 weeks. These studies suggest that neurotrophic factor gene therapy may be valuable to treat Parkinson’s disease in humans. A review of related studies of neurotrophic factor gene therapy suggests that the potential applications o f neurotrophic factor gene therapy are extensive, once advances are made in vector delivery, persistence, safety and testing in non-human primate brain.

replacement for hereditary gene defects and delivery of killer genes for brain tumors are similar to approaches taken in other tissues, gene therapies aimed at brain repair address rather unique processes in which neurons fail to develop or degenerate later in life. Neurodegenerative diseases, such as Parkinson’s, Alzheimer’s, and Lou Gehrig’s diseases, have devastating consequences and are incapacitating to the individual, as well as of high cost to society. In addition, there are limited pharmacological means of intervening against the neuronal degenerations

I. Introduction Gene therapy applied to the nervous system is unique in many ways compared to most tissues. The cellular heterogeneity and complexity of the nervous system present exceptional challenges for directing genes to specific cells that are beyond the blood brain barrier and not accessible by ordinary routes of vector administration. In the brain, most cells are postmitotic, making approaches requiring gene integration into host chromatin not particularly effective. In addition, while gene 265

Bohn and Choi-Lundberg: Neurotrophic Factor Gene Therapy that occur in these diseases, as well as those accompanying stroke or trauma, such as in spinal cord and head injuries.

hydroxyl radical, hydrogen peroxide and superoxide anion, molecules that damage lipids, proteins and DNA and lead to cell death. Injection of 6-OHDA into the rat striatum produces a progressive loss of DA neurons over several weeks (Przedborski et al., 1995; Sauer and Oertel, 1994), whereas injection into the medial forebrain bundle or substantia nigra produces a rapid loss of DA neurons. MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) causes parkinsonian symptoms in humans, non-human primates and mice following its oxidation to MPP+ (1methyl-4-phenyl pyridinium) by monamine oxidase type B (MAO-B). MPP+ enters DA neurons by high affinity uptake through the DA transporter and interferes with ATP production by inhibiting complex I of the mitochondrial electron transport chain.

There are interesting parallels in the neurodegenerations that occur in disease and injuries to the nervous system. Regardless of the type of insult and the neuronal cell type involved, most neurodegeneration appears to proceed through apoptosis, an active process requiring new gene expression. This presents a window of opportunity for intervention by delivering genes whose products block or slow the progress of apoptosis. Another approach is to provide genes that replace the neurotransmitter systems that are lost following degeneration of specific classes of neurons. For example, studies in animal models of Parkinson’s disease suggest that gene replacement of tyrosine hydroxylase (TH), the rate limiting enzyme in dopamine (DA) synthesis, increases DA levels in the brain and in some studies has been shown to improve parkinsonian behavior (Cao et al., 1995; During et al., 1994; Kaplitt et al., 1994; Lundberg et al., 1996; Wolff et al., 1989). While this approach may ameliorate disease symptoms, an approach that prevents neurons from dying or that stimulates regrowth in damaged neurons has the potential of permanently restoring brain function and of reversing or at least slowing the progression of neurodegenerative diseases.

Since a unilateral lesion of the nigrostriatal pathway produces an imbalance in the levels of DA and DA receptors in the target regions of the DA nerve terminals (the striatum), behavioral tests of this asymmetry have been useful for assessing the efficacy of therapeutic approaches. For example, DA stores are depleted and striatal postsynaptic DA receptors are upregulated on the lesioned side. Animals exhibit rotational behavior in response to amphetamine and DA agonists such as apomorphine that can be easily quantified (Ungerstedt, 1971). In addition, unilaterally lesioned animals exhibit deficits in contralateral limb use in several spontaneous behaviors (Olsson et al., 1995; Schallert, 1995). MPTP lesioned non-human primates exhibit symptoms similar to humans with Parkinson’s disease, including bradykinesia or akinesia, resting tremor and rigidity.

This chapter will review studies applying gene therapy to prevent neurodegeneration or stimulate regeneration, focusing on animal models of Parkinson’s disease in which DA neurons degenerate.

II. Animal models of Parkinson’s disease

III. Neurotrophic factors for dopaminergic neurons

Parkinson’s disease (PD) affects approximately 1% of people over the age of 50, with nearly 500,000 patients in the United States. The cardinal symptoms of PD include bradykinesia or akinesia, rigidity, resting tremor and postural instability. The hallmark of PD is the slow degeneration of DA neurons in the substantia nigra region of the midbrain. Due to this discrete, well-defined lesion and the availability of neurotoxins that selectively kill DA neurons, several well-characterized animal models of Parkinson’s disease have been developed (Bankiewicz et al., 1993, review). Transfection of the medial forebrain bundle, which contains nigrostriatal and mesolimbic DA axons, causes a reduction of DA phenotypic markers and cell death in the substantia nigra and ventral tegmental area. Injection of the neurotoxin 6-hydroxydopamine (6OHDA) into the striatum, medial forebrain bundle or substantia nigra results in a selective loss of DA neurons. 6-OHDA, an analog of DA, is concentrated in DA neurons through uptake by the high affinity DA transporter. 6-OHDA undergoes auto-oxidation, generating

The discovery and characterization of neurotrophic factors that promote the survival, neurite outgrowth and phenotypic differentiation of DA neurons has been an area of intense research in recent years. Early studies showed that glia from different brain regions and glial cell lines produce factors that influence the survival and differentiation of embryonic DA neurons in culture (Engele et al., 1991; Rousselet et al., 1988). Previously identified growth factors, such as fibroblast growth factors 1 and 2, were found to enhance survival of DA neurons in vitro, but their effects were shown to be indirectly mediated through glial cells (Engele and Bohn, 1991). The first neurotrophic factor shown to act directly on DA neurons was brain-derived neurotrophic factor (BDNF) (Hyman et al., 1991). Subsequently, glial cell line-derived neurotrophic factor (GDNF) was purified from the glial cell line, B49, and shown to be a very potent DA factor that enhances survival and neurite outgrowth of embryonic 266

Gene Therapy and Molecular Biology Vol 1, page 267 DA neurons in vitro (Lin et al., 1993). A second member of the GDNF family, neurturin, was recently identified and also shown to be a potent DA factor (Kotzbauer et al., 1996). The receptors for GDNF and neurturin are widely expressed in the CNS and periphery in distinct, but overlapping, patterns, and many neuronal populations in addition to DA neurons respond to these factors (Baloh et al., 1997; Buj-Bello et al., 1997; Creedon et al., 1997; Henderson et al., 1994; Klein et al., 1997; Nosrat et al., 1996; Olson, 1997; Williams et al., 1996). Consequently, both GDNF and neurturin are excellent candidates for gene therapy for Parkinsonâ&#x20AC;&#x2122;s disease. A summary of growth factors with DA neurotrophic activity is shown in Table I.

diseases as reviewed previously (Arenas, 1996; Eide et al., 1993; Lindsay et al., 1993; Mizuno et al., 1994), see Table II. However, the initial identification of neurotrophic factors has for the most part relied on in vitro assays using cultured embryonic neurons from mouse or rat. Consequently, studies in animals are necessary to determine if damaged or aged neurons respond to these factors in the same manner in vivo.

Table II. Potential applications of neurotrophic factor gene therapy Enhance cell survival Prolong life of diseased neurons. Decrease vulnerability of neurons to damage in the nervous system. Enhance survival and/or differentiation of grafted neurons or neuronal stem cells.

Table I: Neurotrophic factors for dopaminergic neurons (selected references)

Stimulate Regeneration or Sprouting Stimulate regeneration of damaged, diseased neurons Stimulate sprouting in healthy, neighboring neurons Block inhibitory growth molecules

TGF- Superfamily GDNF (Lin et al., 1993) Neurturin (Kotzbauer et al., 1996) GDF-5 (Krieglstein et al., 1995b) TGF-!1 (Krieglstein et al., 1995a) TGF-!2, TGF-!3 (Krieglstein et al., 1995a; Poulsen et al., 1994) Activin A (Krieglstein et al., 1995a)

E nhanc e ne ur onal func tion Upregulate neurotransmitter expression or metabolism Affect neuronal activity

Neurotrophins BDNF (Hyman et al., 1991) NT-3 (Hyman et al., 1994) NT-4/5 (Hyman et al., 1994)

In the case of GDNF, there is much in vivo evidence showing that GDNF protects neurons in the adult brain from neurotoxin and axotomy induced degeneration (Arenas et al., 1995; Beck et al., 1995; Choi-Lundberg et al., 1997a,b; Emerich et al., 1996; Gash et al., 1995; Henderson et al., 1994; Houenou et al., 1996; Matheson et al., 1997; Munson and McMahon, 1997; Oppenheim et al., 1995; Sagot et al., 1996; Shults et al., 1996; Tomac et al., 1995; Tseng et al., 1997; Yan et al., 1995). GDNF also may stimulate sprouting or regeneration from damaged DA neurons in the adult brain as in one study of polymer-encapsulated GDNF-secreting BHK cells where DA fibers growing into the capsule were observed (Lindner et al., 1995). However in our studies, we have not observed a GDNF stimulated increase in the density of DA fibers in the striatum (Choi-Lundberg and Bohn, unpublished observations). Similar findings for BDNF have been reported in the 6-OHDA lesioned rat in which BDNF improved DA dependent behavior and increased striatal DA levels in the absence of an effect on the density of DA fibers in the striatum (Altar et al., 1994; Altar et al., 1992; Yoshimoto et al., 1995). Thus, studies of embryonic neurons in culture may not be completely

Cytokines Cardiotrophin-1 (Pennica et al., 1995) CNTF (Hagg and Varon, 1993; Magal et al., 1993) Il-1! (Akaneya et al., 1995) Il-6 (Hama et al., 1991; von Coelln et al., 1995) Il-7 (von Coelln et al., 1995)

Mitogenic Growth Factors aFGF, bFGF (Beck et al., 1993; Engele and Bohn, 1991; Ferrari et al., 1989; Otto and Unsicker, 1993) EGF (Casper et al., 1991) Insulin (Knusel et al., 1990) IGF-I (Beck et al., 1993) IGF-II (Liu and Lauder, 1992) Midkine (Kikuchi et al., 1993) x (Othberg et al., 1995) TGF- (Alexi and Hefti, 1993)

The identification of factors that affect either survival and/or differentiated properties of specific classes of neurons, such as neurite outgrowth, neurite branching, or neurotransmitter expression, led to the concept of using these factors as therapeutic agents for neurodegenerative 267

Bohn and Choi-Lundberg: Neurotrophic Factor Gene Therapy predictive of the effects of neurotrophic factors on neurons in the adult brain. It remains entirely unknown to what extent the effects of neurotrophic factors on neurons in animal models of disease, in which neurons are either physically or chemically damaged, will predict their actions on diseased neurons in the human brain, and only clinical studies will reveal this.

As a first step in studying the potential of neurotrophic factor gene therapy for Parkinson’s disease, adenoviral vectors of the serotype Ad5, deleted in E1a and E3, were made containing the DNA sequences for wild type human GDNF preproprotein and a mutant human GDNF with a 12 amino acid deletion (Ad- GDNF and Ad mGDNF, respectively). An Ad vector harboring the ß-galactosidase gene with the SV40 large T nuclear localization sequence was also used as an additional control (Ad LacZnl). All vectors used in our studies to date have contained the Rous sarcoma virus (RSV) long terminal repeat promoter (ChoiLundberg et al, 1997a,b).

There is abundant evidence supporting an application of GDNF to Parkinson’s disease. As discussed above, administration of recombinant GDNF in mouse, rat and non-human primate models of Parkinson’s disease protects DA neurons against the effects of at least two neurotoxins acting through different mechanisms and against physical lesions. This had led to clinical trials, newly undertaken by Amgen, which involve repeated intracerebral administration of GDNF. Although the outcome of this trial remains unknown, several issues must be addressed in the development of neurotrophic factor therapies for neurodegenerative diseases. Long-term trophic support for the diseased neurons will be required as neurodegenerative diseases are slowly progressive. Since neurotrophic factors are labile substances that are unable to cross the bloodbrain barrier, it will be necessary to develop methods for delivering these substances in a controlled manner to specific types of neurons. These methods need to be minimally invasive, safe and sophisticated enough to avoid general effects on other types of neurons that might lead to intolerable side effects. Repeated injections of recombinant neurotrophic factors into the human brain are unlikely to be practical and are likely to elicit deleterious side-effects over the long term. In this respect, clinical trials in which neurotrophic factors were administered in large amounts in the periphery were stopped due to unanticipated, intolerable side-effects (ALS CNTF Treatment Study Group, 1996; Miller et al., 1996). In addition, one patient with Alzheimer’s disease experienced the side effects of weight loss, pain and sleep disturbances following intraventricular infusion of NGF (Olson et al., 1994; Olson et al., 1992). Gene therapies for delivering neurotrophic factors to the CNS have the potential to circumvent or even eliminate the drawbacks of recombinant protein therapies. By transferring neurotrophic factor genes to the CNS, there is the potential to produce these substances in a continuous, regulatable manner and even to direct synthesis of these substances only in selected cells through the use of a cell specific promoter. This approach is applicable not only to neurotrophic factor genes, but also to other growth promoting genes and anti-apoptotic genes.

To determine whether these vectors conferred to cells the ability to synthesize the transgene, and in the case of GDNF to secrete bioactive protein, several assay systems were used. PC12 cells, a pheochromocytoma clonal cell line, were used to test infectivity and degree of cytotoxicity. Counts of viable cells four days after infection with vector stocks with different titers and total particles showed that the degree of cytotoxicity correlated with particle ratio rather than infectious titer (F i g u r e 1 ; Choi-Lundberg, 1997a,b). Consequently, in vivo studies (discussed below) were performed with the same titer of vector stocks with similar particle ratios in order to control for both the degree of cytotoxicity and the multiplicity of infection in experimental groups. Since medium conditioned by PC12 cells does not contain neurotrophic activity for DA neurons (Engele et al., 1991), PC12 cells were also used to determine whether bioactive GDNF was secreted following infection with the Ad GDNF vector. Media from PC12 cells infected with Ad GDNF or Ad mGDNF at 10 pfu/cell were collected and tested for presence of DA neurotrophic factor activity. Cultures of dissociated embryonic mesencephalon containing DA neurons were used as the assay system. As shown in Figure 2, the survival of DA neurons as assessed by counting neurons positive for tyrosine hydroxylase (TH), the rate limiting enzyme in DA synthesis, was significantly increased in cultures grown in the presence of medium conditioned by Ad GDNF infected PC12 cells, but not by control medium (Choi-Lundberg, 1997; Choi-Lundberg et al., 1997b). A further demonstration of the bioactivity conferred by the vectors was shown by directly infecting cultures of embryonic mesencephalon at 1 or 10 pfu/cell . The survival of DA neurons also was significantly increased only by the Ad GDNF vector (Choi-Lundberg, 1997; Choi-Lundberg et al., 1997b); Figure 2.

IV. GDNF adenoviruses: Construction and testing 268

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Figure 2 .

Bioactive GDNF is produced in vitro after infection with Ad GDNF. The number of TH immunoreactive (TH-IR) neurons was counted in E14.5 cultures of dissociated rat ventral mesencephalon maintained on 50% PC12 CM or directly infected with Ad GDNF or Ad mGDNF at 10 pfu/cell, or mockinfected (mean ± SEM, n=6). Ad GDNF increased survival versus Ad mGDNF and no virus in both groups (analysis of variance (ANOVA), F=10.49, P<0.001; symbol indicates Tukey’s post hoc pairwise comparisons at a family error rate of 0.02). Reprinted with permission from Choi-Lundberg et al., 1997b. Copyright 1997 American Association for the Advancement of Science.

mately 1 x 10 7, 1 x 108, or 1 x 109 total particles, respectively. All vector stocks had a particle ratio between 20 and 26. Rats were sacrificed at 4, 30 or 60 days and the number of blue nuclei in the injection site counted. At 4 days little tissue damage was noted around the needle tract in rats injected with the 2 lower titers, whereas significant tissue damage extending up to 0.5mm from the site of injection was observed at the highest titer. At 30 and 60 days, many blue cells persisted in rats injected with the two highest titers; however, macrophages were present in the needle tracts of rats injected with each titer.

F i g u r e 1 . Cytotoxicity of Ad vectors in vitro correlates more closely with total particle number per cells (A) than with infectious plaque forming units per cell (B ). PC12 cells were infected for two hours with 100 to 100,000 particles per cell of Ad vectors with particle ratios 20, 65 or 190, or mock infected. Four days later, cells were trypsinized, centrifuged, resuspended and counted in a hemacytometer. Cell counts were plotted versus total particle number per cell (A) and infectious plaque forming units (pfu) per cell (B).

Cell count data (Figure 3; Choi-Lundberg, 1997) revealed that a high level of persistent gene expression could be obtained with titers in the range of 4 x 106 to 4 x 107 pfu with infected cells extending 0.6 mm or greater from the injection site. Notably, there were no significant

Prior to testing Ad vectors in rodents models of Parkinson’s disease in vivo, it was deemed important to optimize in vivo injection paradigms to obtain a high level of transgene expression that persisted for at least two months, but which resulted in minimal tissue damage. Fischer 344 rats were injected in the striatum with 4 x 105, 4 x 10 6, or 4 x 107 pfu of Ad LacZnl containing approxi-

differences in the numbers of blue nuclei at 4, 30 or 60 days at any of the titers, suggesting transgene expression from Ad vectors is stable in the Fischer 344 rat brain. Retrograde transport of either the Ad LacZnl vector or ß-

269

Bohn and Choi-Lundberg: Neurotrophic Factor Gene Therapy OHDA lesion suggested that neurons that had supposedly degenerated had actually lost TH-immunoreactivity and seemed to “reappear” after GDNF administration (Bowenkamp et al., 1996; Hoffer et al., 1994; Kearns and Gash, 1995). Although our cell counts were made of FG labeled cells, we also noted that virtually all of the FG labeled cells were also positive for TH prior to lesioning (unpublished observations). One week following the vector and FG injections, the subpopulation of DA neurons on one side of the brain was lesioned by injection of 6-OHDA into the striatum in the vicinity of their terminals. The DA neurons on the other side of the brain were labeled with FG, but remained unlesioned and untreated as an internal control in each animal. Seven weeks after vector injection, rats were assessed for the degree of DA cell survival and the persistence of transgene expression.

Figure 3 . Expression of ß-galactosidase transgene protein in the striatum at 4, 30 and 60 days after injection of 4 x 105 , 4 x 106 , or 4 x 107 pfu.

DA neurons undergo a slow, progressive degeneration following injection of 6-OHDA near their terminals so that by 2 months, approximately 75% of the neurons have died (Sauer and Oertel, 1994). This degeneration was almost completely prevented by the Ad GDNF vector. As shown in F i g u r e 4 , Ad-GDNF protected an average of about 80% of the FG labeled DA neurons with two of the rats displaying nearly complete protection (Choi-Lundberg et al., 1997b). In contrast, only an average of about 30% of the neurons remained in the 3 control groups. In lesioned rats that received no treatment or injection of Ad LacZnl or Ad mGDNF, there were few large FG labeled DA neurons remaining and many small FG labeled cells. Many of the small cells had the characteristic morphology of microglia, which presumably had phagocytosed dying DA neurons, or alternatively were shrunken DA neurons in the process of dying. These observations clearly demonstrated that increasing levels of synthesis of a neurotrophic factor in the brain can have remarkable effects on protecting neurons from chemically induced cell death. It remains to be determined whether this approach will also protect diseased neurons from cell death.

Tissue sections of striatum at 160 µm intervals were stained with X-gal histochemistry and the number of blue nuclei in the striatum counted.

galactosidase protein also occurred since numerous DA neurons in the substantia nigra with blue nuclei were observed. However, these labeled neurons were observed only in rats injected with the highest titer of vector (ChoiLundberg, 1997).

V. Ad GDNF gene therapy directed to DA cell bodies Injection of microgram amounts of recombinant GDNF protein into the striatum or substantia nigra has been shown to rescue DA neurons from cell death following chemical lesions from 6-hydroxydopamine (6OHDA) and MPTP, as well as that caused by physical lesions (Beck et al., 1995; Bowenkamp et al., 1995; Gash et al., 1995; Gash et al., 1996; Kearns and Gash, 1995; Shults et al., 1996; Tomac et al., 1995). To determine whether low levels of GDNF biologically synthesized near the DA neurons would also protect against cell death, Ad GDNF, Ad-mGDNF, or Ad LacZnl were injected stereotaxically immediately above the substantia nigra in the ventral midbrain. In the same surgery as the vector injection, a subpopulation of DA neurons that projected to a specific site in the striatum were labeled with the retrograde tracer fluorogold (FG). This labeling permitted a quantitative assessment of the degree of cell survival without relying on any DA neuronal phenotypic marker.

To determine the amount and stability of GDNF transgene expression in the brain following injection of Ad GDNF, a time course of vector expression was undertaken. A comparison was made of levels of transgene protein, transgene mRNA and vector DNA for both GDNF and LacZnl at 1, 4 and 7 weeks after injection of vector into brain. The levels of human GDNF protein at 1, 4 and 7 weeks in the ventral midbrain were found to be 13, 5 and 2 nanograms, respectively, while those of ß-

This was considered important since previous studies in which rats were injected with GDNF following a 6270

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Figure 4 . In vivo gene therapy with Ad GDNF prevents progressive degeneration of fluorogold-prelabeled DA neurons. Six weeks following intrastriatal 6-OHDA, many large, FG positive cells (DA neurons, arrows) were observed in the SN on the unlesioned side (A) and on the lesioned side of a rat treated with Ad GDNF (B). In contrast, fewer large FG+ cells, but many small, secondarily labeled FG+ cells (microglia and other non-neuronal cells, arrowheads) were noted in rats treated with Ad mGDNF (C) or untreated (D). Scale bars in A-D are 100 Âľm and 50 Âľm for the insets. Reprinted with permission from Choi-Lundberg et al., 1997b. Copyright 1997 American Association for the Advancement of Science.

Lundberg et al., 1997b). This observation also suggests that the use of different promoters, e.g. cellular promoters, may increase the stability of transgene expression from adenoviral vectors.

galactosidase protein were 57, 20 and 17 nanograms (ChoiLundberg, 1997b; Choi-Lundberg et al., 1997). Although the levels of transgene declined over time, nanogram levels of GDNF are high with respect to the K d for components of the GDNF receptor system that are in the picomolar range (Durbec et al., 1996; Jing et al., 1996; Trupp et al., 1996). The levels of transgene mRNAs also declined. In contrast, the levels of vector DNA did not decline between 1 and 7 weeks, suggesting that host responses to the vectors repressed transgene expression, but did not result in the loss of infected cells (Choi-Lundberg, 1997; Choi-

The observation that neurotrophic factor gene therapy near the cell bodies of the DA neurons provided neuroprotection against the effects of 6-OHDA raises several issues. One issue is whether GDNF gene therapy directed to the terminals of DA neurons will also offer neuroprotection. This question is important since a clinical application of this therapy would require injection 271

Bohn and Choi-Lundberg: Neurotrophic Factor Gene Therapy into striatum, a brain region relatively accessible and safe compared to injection into the midbrain. Another issue is whether the GDNF gene therapy results in a functional improvement. Studies addressing these questions were undertaken in the same progressive degeneration rat model of Parkinson’s disease as above, but in which vectors were injected into the striatum rather than into the mesencephalon. Preliminary results from these studies show that Ad-GDNF injected near the terminals of DA neurons also protected against 6-OHDA induced cell death (Choi-Lundberg, 1997a; Choi-Lundberg et al., 1997). Furthermore, DA dependent behaviors were also improved in the Ad GDNF treated rats (Choi-Lundberg, 1997; ChoiLundberg et al., 1997a).

vectors harboring BDNF or GDNF (Gimenez y Ribotta et al., 1997). Retinal degeneration is also a potential target of neurotrophic factor gene therapy. In this respect, injection of an Ad vector harboring ciliary neurotrophic factor (CNTF) intravitreally in rd transgenic mice rescued photoreceptors from apoptosis (Cayouette and Gravel, 1997). Although not yet studied in vivo, transduction of explant cultures of rat spiral ganglia with an HSV amplicon vector harboring BDNF elicited neuritic outgrowth, suggesting that neurotrophic factor gene therapy may also be applicable to deafness (Geschwind et al., 1996). Ex vivo gene therapy approaches with neurotrophic factors have used a variety of cell types, including fibroblasts, myoblasts, astrocytes, stem cells and a variety of cell types encapsulated in polymers. Ex vivo neurotrophic factor gene therapies have shown protective effects or behavioral improvement in animals whose cholinergic, dopaminergic or striatal systems have been damaged; however, these studies are too numerous to be reviewed here (for reviews, see (Choi-Lundberg and Bohn, 1997; Gage et al., 1987; Jinnah et al., 1993). In the case of Lou Gehrig’s disease, ex vivo neurotrophic factor gene therapy has progressed to the clinical trial stage where encapsulated cells secreting CNTF are being implanted intrathecally (Aebischer et al., 1996a, b). In animal models of spinal cord injury, implanted fibroblasts secreting NGF or BDNF increased sprouting from sensory, motor and noradrenergic fibers and reduced the area of spinal tissue injury and locomotor deficits (Kim et al., 1996; Tuszynski et al., 1996). In aged rats, implantation of GDNF-secreting fibrobasts increased locomotor activity and bar pressing, as well as increased the density of TH staining in the striatum suggesting an effect of GDNF on DA neurons in aged rats (Emerich et al., 1997). An interesting recent study showed that implantation of mouse stem cells responsive to the proliferative effects of epidermal growth factor (EGF) and generated from mice in which NGF is under control of the astrocyte specific promoter, glial fibrillary acidic protein (GFAP), not only protected striatal neurons from excitotoxin-induced cell death, but also resulted in the integration of grafted cells as astrocytes into host tissue (Kordower et al., 1997). The grafting of neuronal precursors carrying transgenes, including neurotrophic factor genes, in which such cells integrate into host tissue is beginning to be explored (Martinez-Serrano et al., 1995; Snyder and Fisher, 1996; Snyder et al., 1995), and has significant potential for both cell replacement and gene therapies.

VI. Other directions for neurotrophic factor based gene therapies. The idea of using vectors to deliver genes that may slow or even reverse the progression of neurodegenerative diseases is exciting as there are presently no cures for diseases such as Parkinson’s, Huntington’s, Alzheimer’s or Lou Gehrig’s. However, this research area is in its infancy and new vectors need to be developed that are safe and offer stable gene expression in the CNS, most specifically in the primate CNS. Vectors that target genes to specific cell types in the nervous system, as well as those that contain promoters that can be regulated by peripheral drug administration, may offer distinct advantages over those presently in use. Both ex vivo and in vivo gene therapy studies employing neurotrophic factors have generated promising results in animal models of disease and aging. Other in vivo gene therapy studies in rat models of Parkinson’s disease using adenoviral and adeno-associated viral vectors harboring GDNF have demonstrated protective effects similar to those observed in our studies (Bilang-Bleuel et al., 1997; Horellou et al., 1997; Mandel et al., 1997). Injection of an Ad vector harboring nerve growth factor (NGF) into aged rats increased the size of cholinergic neurons (Castel-Barthe et al., 1996). Another study using herpes simplex virus harboring NGF prevented the decline in TH activity in sympathetic neurons that occurs after axotomy (Federoff et al., 1992). In a rat model of stroke, injection of an adenoviral vector harboring a cDNA for interleukin receptor antagonist decreased the amount of tissue damage in the area of focal ischemia (Betz et al., 1995). Adenoviral vectors for ciliary neurotrophic factor (CNTF) and neurotrophin 3 (NT-3) improved indices of motoneuron function and increased life span of pmn mice, a mouse strain characterized by motoneuron nerve terminal and axonal degeneration (Haase et al., 1997). Motoneuron cell death following facial nerve lesions in newborn rats was reduced by muscular injection of Ad

In conclusion, the merging of gene therapy with recent discoveries in the field of developmental neurobiology, such as novel neurotrophic factors, molecules involved in neuronal death and differentation, and techniques for 272

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generating neuronal precursor cells, presents exciting, relatively unexplored horizons for developing therapies for neurodegenerative diseases and trauma to the nervous system. Yet, advances in vectors that will elicit safe, stable gene expression in the human brain are pivotal to bringing these potentials to practical use. As a first step, it is crucial that new generation vectors be developed that elicit persistent transgene expression in specific cell types and that these be demonstrated to be effective and safe in the non-human primate CNS.

Arenas, E. (1 9 9 6 ). GDNF, a multispecific neurotrophic factor with potential therapeutic application in neurodegenerative disorders. Molecular P s y c h i a t r y 1, 179-82. Arenas, E., Trupp, M., Akerud, P., and Ibanez, C. F. (1 9 9 5 ). GDNF prevents degeneration and promotes the phenotype of brain noradrenergic neurons in vivo. Neuron 15 , 1465-1473. Baloh, R., Tansey, M., Golden, J., Creedon, D., Heuckeroth, R., Keck, C., Zimonjic, D., Popescu, N., Johnson Jr., E., and Milbrandt, J. (1 9 9 7 ). TrnR2, a novel receptor that mediates neurturin and GDNF signaling through Ret. Neuron 18 , 793-802.

Acknowledgment This review was supported by NIH grant NS31957 and the Medical Research Institute Council of Childrenâ&#x20AC;&#x2122;s Memorial Hospital. The secretarial assistance of Ms. Gabrielle Pearlman is appreciated.

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Gene Therapy and Molecular Biology Vol 1, page 279 Gene Ther Mol Biol Vol 1, 279-292. March, 1998.

Hemophilia A: current treatment and future gene therapy Sheila Connelly and Michael Kaleko Genetic Therapy, Inc., 938 Clopper Road, Gaithersburg, MD 20878, USA ________________________________________________________________________________________________ Corresponding author: Sheila Connelly, Phone: (301) 258-4812, Fax: (301) 590-2638, E-mail: Sheila.Connelly@pharma.novartis.com

Summary The last two decades has seen significant progress i n the treatment o f hemophilia A . The development of highly purified and recombinant FVIII pharmaceutical products has dramatically increased the life expectancy and quality of life for many hemophiliacs. However, the high cost and short supply of these replacement products has resulted in their availability limited to less than 10% of the worldâ&#x20AC;&#x2122;s hemophiliac population. Gene therapy for hemophilia A would provide prophylactic expression of FVIII and correction of the coagulation defect. A gene therapy protocol allowing s i m p l e , infrequent vector administration may extend hemophilia treatment t o remote locations worldwide that currently lack access to FVIII replacement therapy. While progress has been made with each of the gene therapy vector systems described below, each still faces obstacles to its clinically utility. However, with the efforts that are currently directed toward overcoming these limitations, gene therapy for hemophilia A will ultimately become a reality.

I. Introduction Hemophilia A is the most common hereditary coagulation disorder and is caused by a deficiency or abnormality in blood clotting factor VIII (FVIII). This Xlinked disease affects 1 in 5-10,000 males in all populations with approximately one third of the occurrences due to spontaneous genetic mutations (Sadler and Davie, 1987). Hemophiliacs suffer from uncontrolled bleeding into the joints, muscles, and internal organs and repeated joint bleeding frequently leads to a disabling arthropathy (Sadler and Davie, 1987). Hemophilia A is categorized into severe, moderate, or mild forms, with over half of the patients manifesting the severe disease (Sadler and Davie, 1987). The severity of the bleeding disorder is related to the nature of the underlying mutation of the FVIII gene (Antonarakis et al., 1995). Current treatment involves replacing the missing clotting factor with plasma-derived or recombinant FVIII protein infusions in response to bleeding crises. While prophylactic treatment of hemophilia A has been shown to reduce the frequency and severity of bleeding, such therapy is limited by the availability and high cost of purified FVIII, the short half-life of FVIII in vivo, and the

difficulties associated with frequent intravenous administrations (Rosendaal et al., 1991; DiMichele, 1996). Prior to the development of recombinant FVIII and advanced viral screening and inactivation techniques, FVIII cryoprecipitates derived from pooled human plasma resulted in transmission of several human viruses, including HIV and hepatitis, to over 50% of the hemophiliac population (DiMichele, 1996). A major complication to the treatment of hemophilia A is the development of inhibitory antibodies against the infused FVIII protein. While the majority of hemophiliac patients are immunologically unresponsive to FVIII infusions, over 20% of severe hemophiliacs develop a FVIII-specific antibody response that can become strong enough to render further FVIII administrations ineffective. Although hemophilia A therapy has progressed considerably, present treatments remain suboptimal. Somatic cell gene therapy, which would provide constant blood levels of FVIII, would be a significant treatment improvement. Hemophilia A has been discussed widely as a candidate disease for gene therapy for several reasons (Lozier and Brinkhous, 1994; Fallaux et al., 1995; Hoeben et al., 1995; Smith, 1995; Connelly and Kaleko,

Connelly and Kaleko: Hemophilia A gene therapy 1997). The human FVIII gene has been cloned (Wood et al., 1984; Toole et al., 1984) and the protein well characterized (Kaufman, 1992; Vehar et al., 1991; Hoyer et al., 1994). Human physiological levels of FVIII are low, 100-200 ng/ml (by definition, 1U/ml), and a therapeutic benefit can be achieved with as little as 5% of normal levels (Vehar et al., 1991; Hoyer et al., 1994). As a secreted protein, FVIII expression need not be regulated or limited to a specific target organ or tissue, provided that FVIII is biologically active and absorbed into the blood. However, features of FVIII biology such as the size of the FVIII cDNA, the inhibited accumulation of the mRNA (Kaufman et al., 1989; Lynch et al., 1993; Hoeben et al., 1995; Koeberl et al., 1995) and the instability of the protein (Kaufman, 1992) have hindered efforts to develop FVIII gene transfer vectors. Despite these obstacles, significant progress has been made on the development of gene therapy for hemophilia A. Recent advances in gene transfer technology have enabled the expression of therapeutic to physiological levels of human FVIII in normal animals as well as hemophiliac mice and dogs. However, current gene transfer vectors each face limitations to their clinical utility. Hemophilia A gene therapy research is focused on improving gene transfer vehicles and delivery methods to enable sustained clotting factor expression, treatment readministration, and circumvention of the host immune response to treatment.

II. Historical perspective and current treatment of hemophilia A Hemophilia was documented in the Talmud over 1,700 years ago, where the death of several infant boys from uncontrolled bleeding following circumcision was described (Rosner, 1969). By the early 1800s, hemophilia was characterized as a sex-linked disorder (Otto, 1803), and by 1840, whole blood transfusion was found to halt a hemophilia bleeding episode (Lane, 1840). The presence of FVIII in blood was demonstrated in 1911 by the ability of normal plasma to shorten the clotting time of hemophilic blood (Addis, 1911). In 1937, the critical role of FVIII in hemostasis was recognized, and the missing factor designated â&#x20AC;&#x153;antihemophilic globulinâ&#x20AC;? (Patek and Taylor, 1937). By 1962, the blood coagulation protein was renamed factor VIII by the International Committee on Thrombosis and Haemostasis (Wright, 1962). However, a detailed biochemical and structural characterization of FVIII has been achieved only within the last 20 years. A significant advance in the understanding of FVIII biology has resulted from the isolation and expression of the FVIII gene (Gitschier et al., 1984; Toole et al., 1984; Vehar et al., 1984; Wood et al., 1984). The clinical severity of hemophilia is related to the degree of FVIII deficiency. The three degrees of hemophilia

were first noted by Legg (1872) and are designated severe, moderate, and mild. Severe hemophiliacs are defined as having <1% of normal FVIII levels and experience frequent, spontaneous bleeding into the joints and soft tissues. Patients with 2 to 5% of normal FVIII levels are considered to have moderate hemophilia. Spontaneous hemarthroses are rare, and severe bleeding into joints and tissues usually result from trauma. Mild hemophilia is defined as 6 to 50% of normal FVIII levels, and often goes undiagnosed for many years. The hemostatic defect becomes apparent only after severe trauma or surgical procedures. Historical treatments for hemophilia included methods that are in use today, such as cautery, the application of ice, and splinting, in addition to the advice to avoid any procedure or activity that could produce trauma. However, hemophiliac mortality rates were 90% by the age of 21 years until the first half of the 20 th century (DiMichele, 1996). Although the first transfusion to treat hemophilic bleeding was attributed to Lane (1840), transfusion therapy was not firmly established until 100 years later (Macfarlane, 1938). In the 1950s, fresh frozen plasma and early FVIII concentrates were developed and employed (Kekurck and Wolf, 1957). The development of a simple method for FVIII purification from human plasma by cryoprecipitation represented a milestone in hemophilia therapy (Pool and Shannon, 1965), and in the 1970s lyophilized intermediate purity FVIII concentrates were employed, each lot manufactured from more than 2000 donors. The average life expectancy and quality of life improved dramatically, for even severe hemophiliacs, from 11 years in 1921 to 60 years in 1980 in one report (Larsson, 1985). In the 1980s, the transmission of hepatitis C from these concentrates was recognized in 60 to 95% of hemophiliacs (DiMichele, 1996). Additionally, from 1979 through 1985, 55% of the hemophiliac population was infected with HIV-1 (DiMichele, 1996). Therefore, highly purified replacement products that were free from viral contamination were actively pursued in the 1980s, and the development of affinity chromatography purification, mandatory screening of all donor plasma, and improved viral inactivation methods, resulted in high purity FVIII concentrates (DiMichele, 1996). Consequently, no cases of HIV-1 transmission from FVIII products have been documented since 1986. The 1990s saw the advent of recombinant FVIII products (Lusher et al., 1993; Bray et al., 1994), and with it the hope of an end to viral transmission through replacement therapy. While the safety and purity of FVIII products has been improved dramatically, current purification techniques are not infallible as parvovirus has been reported recently in plasma-derived and recombinant, albumin-containing FVIII products (Laurian et al., 1994; Eis-Hubinger et al., 1996). In addition, the transmission of prions, the proposed

Gene Therapy and Molecular Biology Vol 1, page 281 causative agents of Creutzfeldt-Jakob disease and bovine spongiform encephalopathy, through plasma-derived FVIII or bovine products employed in the production of the recombinant protein, has been debated vigorously (Arnold, 1995). Frequently, the major obstacle encountered in the late 1990s that restricts the implementation of optimal therapy for hemophilia A is the limited availability and high cost of FVIII pharmaceutical products. The most common current treatment of hemophilia A involves infusion of plasma-derived or recombinant FVIII in response to bleeding crises. Early treatment, at the first onset of symptoms, limits both the amount of the bleeding and the extent of the ensuing tissue damage (DiMichele, 1996). However, in many cases, such therapy is not sufficient to prevent inflammation of the synovial membrane, and subsequent joint damage. Based on observations that moderate hemophiliacs rarely develop chronic arthropathy, it was theorized that adequate prevention would be accomplished by maintaining FVIII at levels of 1-5% of normal. Prophylactic treatment has been performed in Europe over the last 20 years, and, in most cases, involves protein infusion three times weekly to maintain FVIII at therapeutic levels. In a Swedish study, patients who initiated therapy at 1-3 years of age, before significant orthopedic damage occurred, had fewer than one bleeding crisis per year and normal joints over a ten year period (Nilsson et al., 1992). However, such treatment resulted in a significant increase (3-4 fold) in FVIII usage, and required the placement of a central venous catheter in young children. Prophylaxis has not been widely adopted in the United States, the major deterrent being the current lack of cost-effectiveness data regarding such therapy (DiMichele, 1996). One of the major complications of hemophilia treatment is development of antibodies (inhibitors) against the infused FVIII (reviewed by White and Roberts, 1996). Inhibitor incidence is as great as 20% of severe hemophiliacs, and when the titer of these antibodies becomes sufficiently elevated, treatment with FVIII, even in tremendous doses, is completely ineffective. FVIIIspecific antibodies may function by two general mechanisms: the inhibition of FVIII function or the clearing of FVIII from the blood. Most FVIII antibodies characterized clinically inhibit FVIII function. Several therapeutic approaches are currently available for the treatment of inhibitor patients. These include the induction of immune tolerance by infusion of large or moderate amounts of FVIII protein twice daily until inhibitor titer declines (Brackmann, 1984; Ewing et al., 1988; Mauser-Bunschoten et al., 1991). A second protocol involves the reduction of antibody titer by plasmapheresis, suppression of de novo antibody synthesis by the administration of cytotoxic drugs, daily infusion of FVIII, and intravenous IgG administration (Nilsson et al.,

1993). An effective treatment for patients with autoantibodies against FVIII is the infusion of intravenous gamma globulin (Sultan et al., 1994). For inhibitor patients who must be treated acutely, FVIII bypassing agents, or porcine FVIII, which may not cross react with the human inhibitor, have shown successful application (White and Roberts, 1996). For the future, it may be possible to prepare and administer synthetic peptides that mimic the FVIII epitopes recognized by the inhibitor (White and Roberts, 1996). The use of chimeric FVIII molecules, such as human/porcine hybrids, not recognized by the inhibitor, has also been investigated (Lollar, 1997).

III. The role of factor VIII in blood coagulation Normal blood coagulation requires the rapid activation of a series of sequential enzymatic reactions in which plasma proteins and proteins released from damaged cells have essential roles (reviewed by Davie, 1995). The lack or deficiency of any of the proteins involved in this cascade blocks the propagation of the initial stimulus. Blood clotting begins with injury to a blood vessel. The damaged vessel wall causes adherence and accumulation of platelets, which, in turn, activate the plasma proteases in the intrinsic pathway of coagulation leading to the localized generation of thrombin and the conversion of fibrinogen to fibrin. The deposit of insoluble fibrin stabilizes the platelet plug and impedes blood flow through the damaged vessel. Thrombin generation requires the interaction of proteases, cofactors, and substrate zymogens, which assemble on a phospholipid surface (reviewed by Kaufman, 1992). FVIII functions in the blood coagulation cascade as a cofactor accelerating the activation of factor X by activated factor IX (factor IXa). FVIII, in turn, is activated by factor Xa and thrombin cleavage. The initial activation of FVIII may be caused by a trace amount of factor Xa generated by the tissue factor-factor VIIa complex (Hoyer, 1994). The formation of factor Xa by this mechanism is rapidly restrained by tissue factor pathway inhibitor, however. Therefore, to sustain hemostasis, the activation of factor X by factor IXa, accelerated though thrombin activation of FVIII, is required (Hoyer, 1994). Subsequently, factor Xa acts in the presence of activated factor V, negatively charged phospholipids, and calcium to convert prothrombin to thrombin. The mechanism by which FVIII functions in the factor Xa-generating complex remains poorly understood. FVIII circulates in the plasma in a noncovalent complex with von Willebrand factor (vWF) and has binding sites for factor IXa, factor X, calcium, phospholipid, and vWF (Kaufman, 1992). vWF is an adhesive glycoprotein that is essential for platelet aggregation and adhesion to the vessel wall in response to

Connelly and Kaleko: Hemophilia A gene therapy vascular injury (Kaufman, 1992). Major functions of vWF are to protect FVIII from proteolysis and to concentrate FVIII at the sites of active hemostasis (Kaufman, 1992). The association of FVIII with vWF was misunderstood for many years, and the distinction between the two coagulation factors was not realized until the mid-1970s (Hoyer, 1994).

IV. Structure and function of factor VIII The isolation of the FVIII gene in 1984 (Gitschier et al., 1984; Toole et al., 1984; Vehar et al., 1984; Wood et al., 1984) represented a significant advance in the understanding of FVIII biology and structure (Figure 1). The human FVIII gene maps to the most distal band of the long arm of the X chromosome, Xq28. The genomic DNA is 186 kb and contains 26 exons, making it one of the largest human genes identified to date. The exon lengths vary considerably, from 69 to 262 base pairs (bps) with the exception of the 3106 bp exon 14 (encoding the B-domain, see below), and the 1958 bp exon 26 (reviewed by Antonarakis et al., 1995). The FVIII mRNA is approximately 9 kb, 7053 nts of which is the coding region (F i g u r e 1 ). Interestingly, two additional RNA transcripts, initiated in intron 22, have been identified (Levinson et al., 1990; 1992). One transcript, the 1.8 kb F8A, is transcribed in the opposite orientation from that of FVIII (Levinson et al., 1990). The F8B transcript is 2.5 kb and is transcribed in the same direction as FVIII (Levinson et al., 1992). The function of F8A and F8B mRNAs and their potential protein products are unknown. Notably, the F8A gene and several kb of surrounding sequence are duplicated elsewhere on the X chromosome (Levinson et al., 1990; Freije and Schlessinger, 1992). FVIII mRNA is expressed in the liver, spleen, kidney, and lymph nodes, but not in peripheral blood lymphocytes or endothelial cells (Wion et al., 1985). Within the liver, the hepatocyte is the cell type that synthesizes FVIII (Wion et al., 1985; Zelechowska et al., 1985). The FVIII protein is synthesized as a 2351 amino acid (aa), single-chain precursor having the domain structure A1-A2-B-A3-C1-C2 (Figure 1; Toole et al., 1984; Vehar et al., 1984). The 19 aa signal peptide is removed upon translocation to the endoplasmic reticulum. Upon transit to the Golgi, FVIII is cleaved specifically within the Bdomain to generate the heavy chain, composed of domains A1-A2-B, and the light chain, composed of domains A3C1-C2 (Kaufman, 1992). The large B domain has no detectable homology to any known genes. The A domains share homology with ceruloplasmin and factor V, while the C domains are homologous to factor V, and discoidin I, a phospholipid-binding protein (Antonarakis et al., 1995).

In plasma, FVIII consists of a heterodimer composed of a heterogenously sized heavy chain polypeptide extending up to 200 kDa in a metal ion complex with the 80 kDa light chain (F i g u r e 1 ). FVIII circulates, in a complex with vWF, as an inactive cofactor. On exposure to thrombin or factor Xa, the heterogenous heavy chain is first cleaved into a 92 kDa fragment, followed by further cleavage into 54 and 44 kDa fragments, both of which are required for procoagulant activity (Hoyer, 1994). Concurrently, a small fragment is cleaved from the light chain to disassociate vWF. These processing steps generate the activated FVIII heterotrimer, FVIIIa. FVIIIa is an unstable molecule that rapidly loses cofactor function, due to subunit dissociation (Hoyer, 1994).

V. Molecular etiology of hemophilia A Since the identification of the FVIII gene, the DNA of hemophilia A patients has been examined extensively for molecular defects (reviewed by Antonarakis et al., 1995), and a data base of FVIII mutations has been established (Tuddenham et al., 1991). As expected, a variety of FVIII mutations have been identified, although their characterization has been impeded by the large size of the FVIII gene (see above). Initial studies were performed by restriction analysis of patient DNA, and revealed that most families carried distinctive mutations, and that approximately one third of hemophilia A cases are the result of new mutations (Anatonarakis et al., 1995). Approximately 5% of severe hemophiliacs have large deletions of the FVIII gene, and 24% have point mutations resulting in missense or nonsense mutations. Many of these base changes were identified by the alteration of TaqI restriction sites (TCGA) within the FVIII gene. TaqI sites are established hot spots for the occurrence of point mutations because they contain CpG dinucleotides in which cytosine can be methylated and subsequently deaminated to thymine (Youssoufian et al., 1986). More than 80 different missense mutations have been identified. These base changes usually involve single aa substitutions at sites critical for FVIII function and are associated with normal or reduced levels of FVIII antigen and the production of a dysfunctional FVIII molecule. However, until 1993, the mutation causing severe hemophilia A in approximately 50% of patients remained elusive. Lakich et al. (1993) and Naylor et al. (1993) discovered that these patients have a partial inversion of the FVIII gene caused by homologous recombination between the region within intron 22 encoding the F8A gene, and one of the two other homologous regions located elsewhere on the X chromosome. These inversions originate almost exclusively in male meiosis (Rossiter et al., 1994), suggesting that nearly all mothers of hemophiliacs with inversions are carriers.

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F i g u r e 1 . Schematic representation of the structure of the factor VIII gene, mRNA and protein. A) Factor VIII (FVIII) gene structure. The double horizontal line depicts the human FVIII gene, with exons represented by vertical lines or solid boxes. The scale is drawn in kilobases (kb). The human FVIII gene is 186 kb and contains 26 exons (Gitschier et al., 1984; Toole et al., 1984; Vehar et al., 1984; Wood et al., 1984). B) FVIII mRNA structure. The single horizontal line represents the FVIII mRNA coding region. The exon boundaries are depicted as vertical lines. The human FVIII mRNA is approximately 9 kb, 7053 nts of which is the coding region. C) FVIII protein structure. The 19 amino acid secretary leader peptide, the three A domains, A1, A2, and A3, the B domain, and the two C domains, C1 and C2 are represented by open boxes. The 2351 amino acid single-chain precursor is displayed. The leader peptide is removed upon translocation to the endoplasmic reticulum (ER). In the Golgi, FVIII is cleaved specifically within the B-domain to generate the heavy chain, A1-A2-B, and the light chain A3-C1-C2. In plasma, FVIII circulates as a heavy chain and light chain heterodimer in a complex with von Willebrands factor (vWF). Activation of FVIII upon exposure to thrombin or activated factor X (FXa), results in cleavage of the heavy chain into a 92 kd fragment, followed by further cleavage into 50 and 43 kd fragments. The light chain is concurrently cleaved into a 73 kd fragment, resulting in the release of vWF. Activated FVIII (FVIIIa) functions as a cofactor in the intrinsic blood coagulation cascade. FVIIIa is rapidly inactivated by subunit dissociation (Hoyer, 1994).

VI. Gene therapy for hemophilia A Gene therapy for hemophilia A, the transfer and expression of a functional FVIII cDNA or gene to hemophiliac patients, remains a viable treatment option for the future. Gene therapy would provide a significant treatment benefit by providing constant, prophylactic blood levels of FVIII and correction of the coagulation

defect. Two basic gene therapy strategies, ex vivo and in vivo, have been employed to date. Ex vivo gene transfer involves the isolation of host cells, expansion and genetic modification of the cells in culture, and reimplantation of the transduced cells into the host. Alternatively, the in vivo approach involves the direct delivery of the gene transfer vehicle, in most cases, a viral vector, to the patient.

Connelly and Kaleko: Hemophilia A gene therapy The success of an ex vivo gene therapy strategy would require that FVIII-transduced cells exhibit prolong survival, sustained FVIII expression, and allow efficient entry of FVIII into the blood. To date, most ex vivo gene transfer strategies have employed retroviral vectors derived from murine retroviruses. Retroviral vectors can infect a broad spectrum of cell types and stably integrate into the genome allowing long-term persistence of the transgene and transfer to all progeny cells. A disadvantage of retroviral vectors is that host cell division is necessary for vector transduction and integration (Miller et al., 1990), thus limiting retroviral-mediated gene therapy to actively dividing host cells. Until recently, the development of hemophilia A gene therapy was focused almost exclusively on ex vivo strategies utilizing retroviral vectors for FVIII gene transfer and expression (Dwarki et al., 1995; Lynch et al., 1993; Chuah et al., 1995; Hoeben et al., 1990; 1992; 1993; Israel and Kaufman, 1990). For use in the development of FVIII-encoding retroviral vectors, the FVIII cDNA was modified by deletion of the B-domain. Removal of the Bdomain reduces the FVIII cDNA from >7 kb, too large to be effectively packaged into most viral vectors for gene transfer, to 4.5 kb (Eaton et al., 1986; Toole et al., 1986). Removal of the B-domain coding region from the FVIII cDNA has no effect on FVIII function, activity, or immunogenicity (Eaton et al., 1986; Toole et al., 1986; Pittman et al., 1993). However, the inclusion of the FVIII cDNA into retroviral vectors was demonstrated to dramatically decrease vector titer (Lynch et al., 1993; Chuah et al., 1995; Israel and Kaufman, 1990). The identification of RNA accumulation inhibitory sequences within the FVIII cDNA (Lynch et al., 1993; Koeberl et al., 1995; Chuah et al., 1995), reported to function as a transcriptional silencer (Hoeben et al., 1995) or as a block to transcriptional elongation (Koeberl et al., 1995), were cited as the cause of the decreased vector titer. Furthermore, conservative mutagenesis of the entire 1.2 kb inhibitory region described by Lynch et al. (1993) failed to increase FVIII expression or retroviral vector titer (Chuah et al., 1995). Despite these difficulties, the development of retroviral vectors encoding the human B-domain deleted FVIII cDNA demonstrated the feasibility of retrovirus-mediated transfer and expression of human FVIII (Hoeben et al., 1990; Israel and Kaufman, 1990). Transduction of mouse 3T3 fibroblasts resulted in secretion of biologically active FVIII at peak levels of 56 mU/ml (Israel and Kaufman, 1990). Similarly, transduction of murine fibroblasts and primary human skin fibroblasts resulted in expression of FVIII at levels of 120 mU/ml/10 6 cells/day and 25 mU/ml/10 6 cells/day, respectively (Hoeben et al., 1990). However, subcutaneous implantation of collagen matrices containing transduced rodent fibroblasts or primary human

fibroblasts into immune-deficient nude mice did not result in expression of detectable levels of human FVIII (Hoeben et al., 1993). The genetically modified cells persisted in vivo, and cells capable of secreting FVIII could be rescued from the implants for up to two months (Hoeben et al., 1993). Furthermore, the transplantation of transduced murine bone marrow into lethally irradiated mice also did not result in FVIII expression in the plasma although the vector was detected in individual hemopoietic progenitor cell-derived spleen colonies (Hoeben et al., 1992). A significant advance in retroviral titer and FVIII expression was achieved by the addition of an intron into vectors encoding the B-domain deleted cDNA (Chuah et al., 1995; Dwarki et al., 1995). These vectors were based on the MFG vector system comprised of the Moloney murine leukemia virus (MMLV) splice donor and acceptor sites incorporated upstream of the transgene cDNA (Krall et al., 1996). Inclusion of the intron was demonstrated to significantly increase vector titer and FVIII expression up to 40-fold (Chuah et al., 1995). Dwarki et al. (1995) constructed a similar retroviral vector which mediated expression of high levels of FVIII (peak of 2000 ng/ml/10 6 cells/24 hrs) in transduced primary human fibroblasts. Intraperitoneal implantation of the vectortransduced cells on neo-organs consisting of polytetrafluoroethylene coated with collagen into SCID (severed combined immunodeficiency) mice resulted in high level expression of FVIII (100 ng/ml) in the mouse plasma at two days (Dwarki et al., 1995). However, by day 13, FVIII expression levels had declined to background. Limited survival of the transduced cells within the neo-organ implants and transcriptional inactivation of the FVIII expression cassette may have contributed to the cessation of FVIII expression. Notably, a similar strategy using a transduced myoblast cell line and muscle implantation was not successful suggesting that the secreted FVIII was poorly absorbed into the circulation (Dwarki et al., 1995). As an alternative approach for hemophilia A gene therapy, a non-viral transfection strategy for delivery and expression of human FVIII has been described (Zatloukal et al., 1994). Using receptor-mediated, adenovirusaugmented gene delivery, primary mouse fibroblasts were transfected with a B-domain deleted FVIII expression plasmid and then surgically implanted into mouse spleens. Low-level FVIII expression (peak of 17 ng/ml) was detected one day after intrasplenic administration, but expression persisted less than 48 hrs. Recently, in vivo gene therapy approaches to hemophilia A treatment have been described. A facile, intravenous administration of a FVIII-encoding vector would provide a more benign and cost-effective treatment than ex vivo protocols involving surgical procedures. Currently, adenoviral vectors represent the most efficient

Gene Therapy and Molecular Biology Vol 1, page 285 means to transfer an exogenous gene to target cells in vivo. Most adenoviral vectors are derived from human adenovirus serotype 5 and rendered replication-deficient by removal of critical viral regulatory elements (Berkner, 1988; Trapnell and Gorziglia, 1994). Adenoviral vectors can transduce a broad spectrum of cell types and, unlike retroviral vectors, do not require target cell proliferation for gene transfer and expression. In addition, the adenovirus chromosome remains episomal in the transduced cell, thus avoiding the possibility of insertional mutagenesis (reviewed by Gingsberg, 1984; Horwitz, 1990). The main disadvantage of adenoviral vectors is that the host immune response, in general, appears to limit the duration of transgene expression and the ability to readminister the vector. Considerable progress has been made recently in the development of adenoviral-mediated gene therapy of hemophilia A (Connelly et al., 1995, 1996a, 1996b, 1996c, 1998). Adenoviral vectors are an efficient system for in vivo FVIII gene delivery since a peripheral vein injection in mice (Smith et al., 1993; Kozarsky and Wilson, 1993; Connelly et al., 1995) and dogs (Connelly et al., 1996c) results in efficient transduction of hepatocytes, cells capable of secreting FVIII directly into the blood (Kaufman, 1992). The transduction of human hepatoma cells with an adenoviral vector in which a liverspecific, albumin promoter directed expression of a human B-domain deleted FVIII cDNA resulted in secretion of high levels of biologically active human FVIII, >2,400 mU/106 cells/24 hrs (Connelly et al., 1995). Intravenous administration of the vector to normal C57BL/6 mice, via the tail vein, resulted in expression of human FVIII in the mouse plasma at levels averaging 300 ng/ml one week postinjection. Therapeutic plasma levels of FVIII were sustained for several weeks and the human FVIII expressed in the mice was biologically active (Connelly et al., 1995). The inclusion of an untranslated exon and intron from the human apolipoprotein 1 gene (Swanson et al., 1992) upstream of the FVIII cDNA in a second, more potent FVIII vector, boosted in vivo FVIII expression approximately 10-fold (Connelly et al., 1996b). Administration of low, non-toxic doses of this vector to normal, adult mice resulted in expression of FVIII at levels 4-fold above the human therapeutic range sustained for at least five months (Connelly et al., 1996a). In contrast, when high, hepatotoxic doses of the vector were administered, FVIII expression declined rapidly to background levels suggesting that dose-dependent vector toxicity limited vector persistence (Connelly et al., 1996a). Similarly, using a human ! 1-antitrypsin-encoding adenoviral vector, it was observed that high viral doses limit the duration of transgene expression (Morral et al., 1997). Furthermore, FVIII expression, directed by the albumin promoter, was demonstrated to be liver-specific

(Connelly et al., 1996a and 1996c) thus providing a potential margin of safety for the use of adenoviral vectors to treat hemophilia. Although no problems are anticipated from ectopic expression of FVIII, the consequences of expression in organs other than the liver are presently unknown. The achievement of phenotypic correction in FVIIIdeficient dogs, a large, clinically relevant animal model of hemophilia A demonstrated the potential utility of adenoviral vectors for the treatment of hemophilia A (Connelly et al., 1996c). Peripheral vein administration of a FVIII adenoviral vector resulted in normalization of the clinical clotting parameters and expression of human FVIII in the canine plasma at levels well above therapeutic (peak levels of 8000 mU/ml). However, phenotypic correction in the treated dogs was transient, as the animals developed a strong antibody response directed to the human protein (Connelly et al., 1996c). In contrast to human FVIII, the canine FVIII protein is less immunogenic in hemophiliac dogs (Tinlin et al., 1993). Therefore, the establishment of sustained phenotypic correction in hemophiliac dogs may require the development of vectors that encode the canine cDNA. The recent generation of FVIII-deficient mice, by gene disruption techniques, provides the first small animal model of hemophilia A (Figure 2; Bi et al., 1995). Affected mice have FVIII activity levels that are <1% of normal and display lethal bleeding after the trauma of tail biopsy (Bi et al., 1995). The mice frequently are anemic, exhibit prolonged bleeding after routine procedures such as ear tagging, and occasionally develop joint bleeds (S.C. unpublished data). Therefore, the murine phenotype is similar to that of human hemophiliacs (Sadler and Davie, 1987). Treatment of the hemophiliac mice with a FVIII adenoviral vector resulted in expression of biologically active human FVIII sustained at levels well above the human therapeutic range for over nine months (Connelly et al., 1998). Furthermore, a tail-clip survival study demonstrated that FVIII vector-treated mice readily survived tail clipping with minimal blood loss, while mice that received a similar dose of a Ă&#x;-galactosidase-encoding vector and untreated hemophiliac mice suffered 70-95% mortality. These data directly demonstrate sustained phenotypic correction of murine hemophilia A by in vivo gene therapy (Connelly et al., 1998). Notably, human B-domain deleted FVIII expressed endogenously in the vector-treated mice was not immunogenic, while hemophiliac mice injected intravenously with human full-length FVIII protein rapidly develop a potent anti-FVIII antibody response (Qian et al., 1996). These observations represent preliminary evidence to suggest that constant level, endogenous expression of human FVIII may be less immunogenic than intermittent, intravenous protein administration.

Gene Therapy and Molecular Biology Vol 1, page 286

F i g u r e 2 . Hemophiliac mice. The factor VIII (FVIII)-deficient hemophiliac mice bleed severely from scratches and routine procedures such as ear tagging. Application of topical thrombin controls the bleeding. Two genotypes of factor VIII (FVIII)deficient hemophiliac mice were generated by disruption of exon 16 or exon 17 of the murine FVIII gene (Bi et al., 1995). Affected mice of both genotypes have FVIII levels <1% of normal and display lethal bleeding after trauma. The hemophiliac mice suffer from joint bleeds, subcutaneous bleeding, and spontaneous death indicating a similarity to the pathophysiology of human hemophilia A. Notably, hemophiliac females of both genotypes survive pregnancy and birth (Bi et al., 1996; Connelly et al., 1998).

The treatment of human patients with an adenoviral vector will require that the vector efficiently transduce and express in human hepatocytes. Cultured primary human hepatocytes were exposed to low doses (10, 100 and 1000 particles/cell) of vectors encoding Ă&#x;-galactosidase or a Bdomain deleted human FVIII. Hepatocyte transduction efficiency was high, 50%, 90% and 100%, respectively, and FVIII was secreted into the tissue culture media at levels of 300, 2500, and 3000 mU/ml per 106 cells per 60 hrs, respectively (S.C., manuscript in preparation). Additionally, the cultured primary human hepatocytes were used to test the potency of a recently generated adenoviral vector that encodes the full-length FVIII cDNA. Transduction with this vector yielded biologically active FVIII at levels 10-fold lower than those obtained with the B-domain deleted FVIII vector (S.C., manuscript in preparation). These data are consistent with previous studies with transfected COS cells in which the B-domain deleted FVIII was expressed at a higher level than the fulllength protein (Toole et al., 1985). Processing and secretion of the B-domain deleted FVIII protein may be more efficient than that of the full-length protein as they follow different secretary pathways (Dorner et al., 1987). Although sustained expression of FVIII has been achieved in mice, the clinical utility of adenoviral vectors

may be limited as expression is not likely to be life-long and an antibody response directed to the viral capsid prevents repeated administration. The duration of expression is limited by at least two aspects of vector biology. The vector remains episomal and may be lost as the hepatocytes slowly proliferate. In addition, a cytotoxic T lymphocyte (CTL) response, directed against vector backbone gene products, may result in elimination of the transduced hepatocytes (Yang et al., 1994a, 1994b). Current efforts are directed towards removing viral backbone genes to diminish the host immune response and to increase the duration of gene expression (Armentano et al., 1995; Wang et al., 1995; Yeh et al., 1996; Gao et al., 1996; Gorziglia et al., 1996; Kochanek et al., 1996; Clemens et al., 1996; Lieber et al., 1996; Haecker et al., 1996; Kumar-Singh and Chamberlain, 1996; Fisher et al., 1996; Zhou et al., 1996; Hardy et al., 1997; Morral et al., 1997).

Gene Therapy and Molecular Biology Vol 1, page 287 A major limitation in the application of adenoviral vectors to the treatment of hemophilia is the block to repeated administration. Immunosuppressive strategies designed to prevent the formation of antibodies to the viral capsid have been successful in mice (Smith et al., 1996; Yang et al., 1995; 1996). Smith et al. (1996) have demonstrated that the immune response to a systemically administered adenoviral vector is dose-dependent and can be modulated by transient immunosuppression with cyclophosphamide or deoxyspergualin (DSG) at the time of initial vector injection to allow effective repeated treatment. More recently, using low dose combination immunotherapy, at least three efficacious adenoviral vector treatments were achieved (TAG Smith, personal communication). However, an immunosuppressive protocol that is clinically relevant to the treatment of human disease will require a means of further diminishing vector immunogenicity either through tolerization or by capsid modification. Several other viral vector systems are currently under development which may be applicable to the treatment of hemophilia A. Among the most promising are recombinant adeno-associated viral vectors (AAV). AAV is a nonpathogenic, defective parvovirus that establishes a latent infection by integrating into the host genome (Kotin, 1994). Vectors derived from AAV have been shown to transduce several tissues in vivo including muscle (Xiao et al., 1996), brain (McCown et al., 1996), lung (Halbert et al., 1997), and liver (Snyder et al., 1997). Following portal vein infusion of a purified human factor IX (FIX)-encoding AAV vector into normal mice, human FIX was detected in mouse plasma for at least 36 weeks in one animal (Synder et al., 1997). An AAV vector encoding the B-domain deleted human FVIII cDNA has been described (Gnatenki et al., 1996), although in vivo expression data has not yet been reported. The use of MMLV retroviral vectors for in vivo gene delivery has been described recently. The development of complement resistant vectors, in addition to improved methods of vector concentration and purification have allowed in vivo hepatocyte delivery (Bosch et al., 1996). High dose intravenous infusion of a purified retroviral vector to juvenile animals resulted in transduction of 1% of hepatocytes (Greengard et al., 1997). Peripheral vein administration of a high dose of a vector encoding a human B-domain deleted FVIII cDNA to rabbits resulted in expression of therapeutic levels of FVIII in 50% of the animals, sustained for at least one year (Greengard et al., 1997). Similar treatment of two normal dogs resulted in FVIII expression in one animal, sustained for at least two months (Greengard et al., 1997). A novel retroviral vector system derived from lentiviruses has emerged recently (Naldini et al., 1996a;

1996b) and may be well suited for the treatment of hemophilia A. The lentivirus life cycle, the prototype for which is HIV, is distinguished from that of murine retroviruses in that the capsid is readily transported into the nucleus thus enabling the efficient transduction of nondividing cells (Naldini et al., 1996a). An HIV-derived vector pseudotyped with VSV G protein demonstrated localized, efficacious transduction of rat neurons in vivo and transgene expression sustained for at least three months (Naldini et al., 1996a, 1996b). Present efforts are aimed at the generation of stable vector packaging cell lines (Corbeau et al., 1996) and safe, clinically acceptable vectors. Finally, nonviral or synthetic vectors are receiving increasing attention as gene transfer vehicles. Synthetic vectors have the potential to be less immunogenic than viral vectors, and can be assembled in cell-free systems from well defined components. The elimination of viral components from the vector system may diminish patient anxiety in a population ravaged by viral illnesses. Currently, the most efficient synthetic system for gene transfer to hepatocytes following intravenous injection is composed of DNA/polylysine/asialoglycoprotein conjugates which utilize hepatocyte receptors for targeting and gene delivery (Wu and Wu, 1988; Perales et al., 1994). Using an optimized human FVIII expression cassette, this gene transfer strategy yielded therapeutic plasma levels of FVIII in mice sustained for at least 30 days (Ill et al., 1997). Current challenges faced with this approach include achieving a consistent formulation, demonstrating reproducibility of transgene expression and delivery, and developing nonimmunogenic conjugates suitable for repeated treatments.

Acknowledgements We than Drs. Soumitra Roy and Alan McClelland for critical review of the manuscript, Dr. Theodore A. G. Smith for communication of data prior to publication, and Ian Springer for assistance with the graphics.

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Gene therapy for haemophilia Rob C. Hoeben Applied-Virology group, Laboratory of Molecular Carcinogenesis, Dept. of Molecular Cell Biology, Leiden University, Wassenaarseweg 72, 2333 AL Leiden, The Netherlands. ________________________________________________________________________________________________ Corresondence: Rob C. Hoeben, Ph.D., Tel: (+31) 71 527 6119, Fax: (+31) 71 527 6284, E-mail: Hoeben@Rullf2.MedFac.LeidenUniv.NL

Summary Gene therapy is an appealing prospect for the treatment of human diseases. In this chapter, I will describe the hopes that gene therapy has brought for hemophilia patients, as well as the hurdles that the researchers have encountered on the route that shall lead to the development of a clinically applicable protocol.

I. Introduction

II. Basic strategies for hemophilia gene therapy

Haemophilia is a congenital coagulation disorder characterized by uncontrolled haemorrhagic episodes that are crippling and potentially life-threatening. Haemophilia A results from subnormal levels of an essential cofactor protein, factor VIII (F.VIII), and affects 1 in every 10,000 males; haemophilia B is associated with a lack of an essential protease, factor IX (F.IX), and occurs in 1 out of 50,000 males. Due to the absence of these key intermediates in the clotting cascade, haemorrhage is the most frequent cause of death in untreated haemophiliacs.

Two strategies are being pursued for haemophilia gene therapy. In the ex-vivo gene-transfer approach, cells are isolated from the patient, cultured and genetically modified in the laboratory. The treated cells, that now should synthesize the factor VIII protein, are reimplanted into the patient in order to bring about a continued production of the desired clotting factor. For this approach, skin fibroblasts, keratinocytes, endothelial cells, hepatocytes, hematopoietic progenitor cells, and myoblasts have been considered. Alternatively, the in-vivo approach aims at genetic modification of some of the patients cells in-situ. In this strategy, gene-transfer vehicles are administered to the patient and should deliver the genes to the tissue of interest. This approach concentrates on genetic modification of the liver, the main site of factor VIII synthesis in healthy individuals (see Fallaux and Hoeben (1996) for a more extensive review).

To date protein-replacement therapy is the treatment of choice. This treatment essentially normalized both the life expectancy and the quality-of-life. Notwithstanding its tremendous achievements, this therapy has several drawbacks. The treated patient is still prone to spontaneous haemorrhages with the associated risk of chronic joint damage. In addition, therapy with plasma-derived F.VIII has resulted in transmission of several human viruses, such as HIV and hepatitis viruses. The risk of exposure to blood-borne pathogens has been virtually eliminated by improved manufacturing procedures and, more recently, by application of recombinant-DNA-derived F.VIII. Nevertheless, the ideal therapy would be independent of blood-derived products (Peake et al., 1993) and would provide a sustained therapeutic effect. Gene therapy may hold the promise of such a treatment of haemophilia and could, in theory, completely cure the disease.

III. Gene Therapy for Hemophilia A A. Relevant properties of factor VIII The F.VIII protein is a large multimeric glycoprotein (300 kDa) that circulates in plasma in low concentrations. The protein is synthesized mainly in the liver as a singlechain polypeptide, which by intracellular processing, is converted in a two-chain dimer of 80-kDa and 200-kDa subunits. Before the actual activation of the F.VIII protein, a large segment of the 200 kDa subunit (the B-domain), is 293

Hoeben: Gene therapy for haemophilia removed, resulting in a 90-kDa heavy chain complexed to the 80-kDa light chain. Further proteolytic cleavage activates the F.VIII protein (Pittman and Kaufman, 1989; Pittman et al., 1994). The F.VIII protein is translated from an mRNA of approx. 9000 nt, of which 7053 nt are coding. The F.VIII gene, located on the X-chromosome, is about 186.000 bp in size. Production of recombinant DNA-derived F.VIII using the human F.VIII cDNA has been difficult. Firstly, the F.VIII cDNA has been found to contain sequences that repress its expression, resulting in low levels of F.VIIIspecific mRNA (Lynch et al., 1993; Hoeben et al., 1995). Secondly, the majority of the F.VIII protein is transported inefficiently from the endoplasmatic reticulum to the Golgi system due to retention of the protein in the ER (Pittman and Kaufman, 1989; Pittman et al., 1994). Thirdly, the protein is extremely sensitive to proteolytic degradation and needs to be stabilized by the von Willebrand factor. In addition, the protein undergoes extensive post-translational modification and needs to be proteolytically cleaved for its functional activation (Pittman and Kaufman, 1989).

B. Ex-vivo gene therapy: problems with retroviral vectors. Many studies focused on the development of retroviral vectors for transfer of a F.VIII gene. In all studies published so far, F.VIII cDNA clones were used in which the non-essential B-domain was removed. The sequences coding for the 90-kDa heavy chain were fused in-frame to the 80-kDa light chain codons. Removal of the B-domain does not significantly affect any known function of the protein; the complete and the B-domain-deleted F.VIII variants are virtually identical in functional assays (Pittman et al., 1993). These B-domain-deleted cDNA clones have a size of approx. 4,500 base pairs, and therefore can be inserted in retroviral vectors without exceeding the packaging capacity of the virus. Retrovirusmediated transfer of the B-domain-deleted F.VIII cDNA has been achieved into various cell types, e.g., skin fibroblasts (Israel and Kaufman, 1990; Hoeben et al., 1990; Lynch et al., 1993), endothelial cells (Chuah et al., 1995; Dwarki et al., 1995), myoblasts (Zatloukal et al., 1994), and haematopoietic progenitor cells (Hoeben et al., 1992). The F.VIII secreted by these cells was functional, illustrating that also cells of non-hepatic origin have the capacity for proper post-translational modification of the F.VIII protein. This illustrates the idea that gene therapy for haemophilia in not necessarily restricted to genetic modification of the hepatic cells that normally produce F.VIII. In general, synthesis of F.VIII by genetically modified cells in culture has been quite low. Both the titre of the 294

retroviral vectors, and the amounts of F.VIII secreted by the transduced cells are reduced about 100-fold in comparison to FIX and other cDNAs (Lynch et al., 1993; Hoeben et al., 1995) . The low titres and the reduced amounts of F.VIII produced are caused, at least in part, by the very low amounts of F.VIII-specific transcripts that accumulate in the transduced cells (Lynch et al., 1993). There is now ample evidence that the inhibition of expression is caused by sequences in the F.VIII cDNA itself and that repression occurs at the level of transcription (Lynch et al., 1993; Hoeben et al., 1995; Koeberl et al., 1995). Lynch et al. (1993) located a 1.2-kb stretch of the F.VIII cDNA ('INS') that reduces the titre of the F.VIII retroviral vectors. These sequences inhibit the F.VIII mRNA accumulation in the cytoplasm. In independent experiments we identified a 305-bp region in the F.VIII cDNA that is involved in the repression phenomenon (Hoeben et al., 1995). Intriguingly, the latter fragment is located near the 'INS' region. In the 305-bp region, sequences were found that resemble the Autonomously Replicating Sequence-consensus (ARSc) sequences of yeast, and the A/T rich sequences found in mammalian Matrix-Attachment Regions (MAR). It has been shown that multimerization of the F.VIII cDNA-derived ARSc/MAR-like sequences could functionally mimic the repression phenomenon when linked to a heterologous reporter gene. Also, de-repression of expression by sodium butyrate could be mimicked using multimers of the F.VIIIderived sequences. This suggests that such ARSc/MARlike sequences, dispersed throughout the F.VIII cDNA, may alter the chromosomal context of the F.VIIIexpression vector (e.g. by associating to the nuclear matrix), resulting in repression of expression (Fallaux et al., 1996). In the F.VIII cDNA the presence of a number of multiple elements in the F.VIII cDNA could form a functional MAR. Such model can explain the difficulties in pinpointing the sequences involved in the repression. So far there is no evidence to support any physiological relevance for the presence of the repressor sequences in the F.VIII cDNA. To improve the expression, Chuah and co-workers (1995) used a conservative mutagenesis strategy to introduce the maximum number of nucleotide changes in the 1200-bp 'INS' region. Despite their impressive efforts, this neither increased the virus titre nor F.VIII expression. However, the insertion of an intron in their retroviral vector increased F.VIII expression up to 20-fold, and boosted virus titres up to 40-fold. This correlated with an increase in mRNA accumulation, which suggests that the inclusion of an intron in the retroviral backbone relieved the transcriptional repression (Chuah et al., 1995). Although the problematic expression has been found to occur with many retroviral vectors, some appear to be less prone to the inhibition. Dwarki and colleagues (1995)

Gene Therapy and Molecular Biology Vol 1, page 295 reported F.VIII expression levels and vector titres that are at least 10- to 100-fold higher than those reported by others. In this vector, based on the MFG retroviral vector, the F.VIII cDNA is located at the exact position of the retrovirus env gene. Thus, the F.VIII message is translated from the spliced sub-genomic mRNA. Although its efficiency is not easily understood considering the repression that has been reported by others, it is the first F.VIII vector that meets the requirements with respect to efficiency of a clinically applicable retroviral vector.

recombinant adenoviral vector, Av1ALH81, in which the F.VIII cDNA is driven by a liver-specific mouse albumin promoter. The use of this vector circumvented many of the problems associated with retroviral vectors in ex-vivo gene transfer strategies. HepG2 hepatoma cells transduced with Av1ALH81 secreted high levels of biologically active human F.VIII (>240 ng/106 cells/24h). Administration of Av1ALH81 to mice resulted in an efficient transduction of the liver (the systemically administrated adenovirus exhibits a strong hepatotrophism). The resulting F.VIII levels in the recipients plasma peaked at 300 ng/ml. These levels are even more impressive if one considers the short half-life of the human protein in mice. Normal F.VIII levels in humans are 100-200 ng/ml, and levels as low as 10 ng/ml are therapeutic. Thus, the mice were producing human F.VIII at levels that exceeded those in normal human plasma. In the recipient mice F.VIII levels in plasma peaked at day 7, and decreased slowly to background levels 7 weeks after treatment. The decline in plasma F.VIII levels correlated with the loss of vector DNA from the liver. This is caused by elimination of the transduced hepatocytes by the hosts' immune system (Yang et al., 1994; Engelhardt et al., 1994). An optimized F.VIII adenoviral vector, Av1ALAPH81, was generated that carries an intron in the F.VIII expression cassette (Connelly et al., 1996b). The F.VIII plasma levels (up to 2.000 ng/ml) in mice that received this vector exceeded those obtained with Av1ALH81. This allowed the administration of lower, less toxic vector doses while maintaining sufficient levels of human F.VIII in the plasma of the recipient mice. F.VIII levels in plasma in the therapeutic range persisted for at least 22 weeks after a single administration of the vector (Connelly et al., 1996a) in mice. In hemophiliac dogs the bleeding tendency could be completely, although transiently, corrected (Connelly et al., 1996c). This provided the much awaited proof-ofconcept of gene therapy for hemophilia A in a large animal model for hemophilia A. It remains to be established whether also in the large animal models for haemophilia A (e.g. haemophiliac dogs) and, ultimately, in humans, vector virus-doses can be found that combine adequate and persistent F.VIII levels in plasma with the absence of apparent hepatotoxicity.

C. Implantation of retrovirallytransduced cells Several cell types can be considered as targets for genetic modification in a protocol for gene therapy for haemophilia. Diploid skin fibroblasts are attractive targets. These cells can easily be harvested from patients, can be grown to large numbers in tissue culture and can be transduced with retroviral vectors with relative ease. In initial experiments F.VIII-secreting fibroblasts of murine or human origin, embedded in an artificial collagen matrix, were implanted subcutaneously on the midbacks of nude mice. In the case of human fibroblasts, cells isolated from the grafts 8 weeks after implantation still had the capacity to secrete F.VIII when regrown in culture. These results demonstrate the persistence of the transplanted cells in a metabolically active state (Hoeben et al., 1993). Unfortunately, no human F.VIII could be detected in the recipients' plasma that might have been secreted by the implanted cells. This was be attributed to the short halflife of the human F.VIII protein in mice. Dwarki and colleagues (1995) observed circulating F.VIII after intravenous and intra-peritoneal injection of recombinant F.VIII protein. In parallel experiments these authors could not detect human F.VIII following intra-muscular or subcutaneous injection. This can be due to the susceptibility of the protein to proteolysis, resulting in degradation of F.VIII before it can reach the circulation. After intraperitoneal implantation of F.VIII-secreting fibroblasts into immunodeficient mice circulating human F.VIII could be detected (maximally 100 ng/ml) in their plasma for up to 10 days (Dwarki et al., 1995). The capacity of transduced cells to deliver the F.VIII into the circulation was dependent on the site of implantation. These data convincingly demonstrate the feasibility of this approach, although the persistence of expression obviously needs to be increased.

IV.Gene therapy for haemophilia B A. Relevant properties of Factor IX The F.IX protein is much smaller in size (55 kDa), and 500 times more abundant on weight basis than F.VIII. Its gene is located on the X chromosome and is 33.000 bp in size. Whereas the F.VIII has no intrinsic enzymatic activity, the activated F.IX functions as a serine protease. It is secreted as an inactive precursor protein that can be activated by proteolytic cleavage. The F.IX protein is

D. In-vivo gene therapy: encouraging results with adenoviral vectors. Conceptually protocols involving in-vivo gene transfer are more straight forward than the ex-vivo approaches. Connelly et al. (1995) studied this approach using a 295

Hoeben: Gene therapy for haemophilia modified extensively. The first 12 glutamic-acid residues of the Gla domain are gamma-carboxylated posttranslationally. This modification is essential for Ca2+ binding and F.IX function (reviewed by Roberts (1993)).

cells (Chen et al., 1997) and in vivo (Koeberl et al., 1997). With these vectors significant levels of F.IX protein could be observed in the recipient mice up to 5 months post-infection. Although the expression is still low, the AAV-derived vectors capacity to infect nonmitotic cells makes it an important alternative for the retroviral vectors, especially for in-vivo liver-directed gene transfer.

B. Status of hemophilia-B gene therapy The developments in the field of gene therapy for haemophilia B paralleled, and often preceded, those for haemophilia A. Starting in 1987 (Anson et al., 1987), a variety of cultured cells have been transduced with retroviral F.IX vectors (reviewed by Fallaux and Hoeben, 1996). In general, functional F.IX was found to be secreted in significant amounts. However, transplantation of the transduced fibroblasts into mice, resulted in transient F.IX plasma levels that were lower than would be expected on the bases of the F.IX secretion in-vitro (Scharfmann et al., 1991; Axelrod et al., 1990; Palmer et al., 1989; Palmer et al., 1991; St.Louis and Verma, 1988). In some of the recipients the formation of F.IX inhibitors could be established, explaining the disappearance of circulating F.IX (St.Louis and Verma, 1988). In addition, the retroviral LTR-promoter that drives expression of the gene of interest was found to be inactivated in fibroblasts in vivo (Axelrod et al., 1990; Palmer et al., 1989; Palmer et al., 1991). Although the latter problem can be overcome by using a cellular promoter (St.Louis and Verma, 1988), such promoters are generally not very strong. Despite these problems, in 1993, Lu and colleagues initiated a phase-I gene-therapy trial with retrovirus-transduced autologous skin fibroblasts (Lu et al., 1993). Two brothers with haemophilia B were treated. It has been reported that in one patient F.IX-clotting activity increased significantly (from 2.9% to 6.3%), and persistently (over 6 months), but not in the other individual. Although encouraging, this trial is still a matter of debate (Thompson, 1995).

The efficacy of in-vivo gene therapy for haemophilia has been demonstrated by Kay and collaborators (Kay et al., 1993). They infused F.IX retroviruses in haemophiliac dogs (Beagles) that had previously undergone partial hepatectomy to stimulate the remaining hepatocytes to divide. Despite the low amounts of F.IX produced (ca. 0.1 % of normal), the average clotting-time was reduced by approximately 60%. The production of the clotting factor persisted for over 9 months (Kay et al., 1993). These results are very promising, although a further 10-100 fold increase in production is required to reach a clinically beneficial range. Also adenoviral vectors have been used for the gene transfer of a human F.IX gene into mice. After a single intra-venous dose into the tail vein, amounts of 400 ng/ml human F.IX could be detected in the recipient mice (Smith et al., 1993). However, the levels slowly decreased to baseline within the course of 10 weeks. A second administration of the virus did not re-establish human F.IX plasma levels. This was due to high amounts of circulating antibodies that were generated and neutralized the vector viruses upon re-challenge (Smith et al., 1993). Similar results have been obtained in F.IX-deficient dogs (Kay et al., 1994). After a single dose of the virus (administered into the portal vein) the bleeding tendency of these dogs was transiently corrected with an increase in F.IX levels from 0 to 300% of the level present in normal dogs. Although therapeutic levels could be maintained for 1-2 months, the F.IX levels decreased significantly in time.

In parallel, many other cell types have been efficiently transduced with F.IX retroviral vectors, including myoblasts (Hortelano et al., 1996; Yao and Kurachi, 1992; Dai et al., 1992; Yao et al., 1994; Baru et al., 1995; Wang et al., 1996), endothelial cells (Axelrod et al., 1990; Yao et al., 1991), hepatocytes (Kay et al., 1993; Kay et al., 1994), keratinocytes (Gerrard et al., 1993; Gerrard et al., 1996; Fenjves et al., 1996), and haematopoietic cells (Hao et al., 1995). Although in laboratory animals circulating F.IX protein has been detected after transplantation of the genetically modified cells, in many cases the synthesis is low and transient, similar to the fibroblast-transplantation experiments. However, it can be anticipated that improvement in the vector technology and transplantation procedures may increase the F.IX levels considerably. Recently, also vectors derived from the adeno-associated virus have been used for the expression of F.IX in cultured

To prolong the expression of the transduced F.IX gene, the administration of the adenovirus vector was combined with immuno-suppression by cyclosporin A, which allowed expression to persist up to 6 months (Fang et al., 1995). However, neutralizing antibodies were formed, making subsequent administrations of the vector ineffective. The occurrence of neutralizing antibodies could be reduced by transient immuno-suppression with deoxyspergualin or cyclophosphamide, allowing repeated administrations of the vector (Dai et al., 1995; Smith et al., 1996). It has been also been reported that, in mice, tolerance could be induced if the adenovirus was administered neonatally (Walter et al., 1996), allowing repeated administrations of the vector. However, given the differences in the development of their respective immune 296

Gene Therapy and Molecular Biology Vol 1, page 297 systems, this procedure can not be translated directly to dogs or humans.

although harmless in normal individuals, may become pathogenic in severely immune-compromised hosts. Even the C-group adenoviruses that we use as vectors, may become pathogenic if the immune system is compromised, e.g. after a bone-marrow transplantation (Hierholzer, 1992; Landry et al., 1987; Bertheau et al., 1996). Thus, we should adapt the vector to the patient, and not vice versa.

In order to prolong the expression of F.IX without the need of immune suppression, vectors have been generated and tested in which the adenovirus E2A gene carries the ts125 mutation which makes the protein product of the E2A gene, the single-stranded DNA Binding Protein (DBP), temperature sensitive. At the body temperature of mice and dogs, the ts125 DBP is non-functional, resulting in a reduced level of adenovirus late-gene expression, and consequently, in reduced immuno-genicity. However, the ts125 did not increase the persistence of expression neither in mice, nor in haemophilic dogs (Fang et al., 1996). An approach that appears more successful is to maintain the E3 region in the adenoviral vector. The protein products of the E3 region can suppress host immune reactions by interference with the expression of MHC class I molecules and by other mechanisms. Side-by-side comparison of !E1/!E3 F.IX adenoviral vectors with !E1 F.IX adenoviral vectors demonstrated a longer persistence of the expression with the former type (Poller et al., 1996). This strongly argues for use of vectors that have a wild-type E3 region. However, deletion of the E3 is often required to generate the space required for the insertion of the gene of interest, especially with larger genes (e.g. the F.VIII cDNA).

These issues above are not unique for haemophilia, but are imperative for all gene-therapy approaches for the treatment of congenital disorders. A concern that is more prominent in the case of haemophilia than in other disorders, is the potential humoral response against the transgene product (viz. F.VIII or F.IX). Such inhibitors, that also are formed in a minority of patients upon regular treatment, inhibit not only the genetic therapy but also the conventional replacement therapy. It needs to be established at what frequency inhibitors (F.VIII or F.IXantibodies) occur after the gene therapy. To determine such frequencies, studies must employ the homologous cDNA. The cloning of the canine F.IX cDNA (Evans et al., 1989b) and the murine F.VIII cDNA (Elder et al., 1993) permits to evaluate the gene-therapy procedures in the established canine (Mauser et al., 1996; Evans et al., 1989) and murine (Bi et al., 1995) models for haemophilia. This will allow a detailed comparison of the current and the future methods for haemophilia management with respect to safety and efficacy.

V. The future Some of the hurdles on the road to gene therapy for haemophilia have been taken. The results obtained so far have demonstrated the potential efficacy and provided the conceptual 'proof-of-principle'. However, several aspects need to be improved before clinical application can be considered for the treatment of haemophilia. In the ex-vivo approaches the techniques for cell isolation, gene transfer and cell transplantation need further improvement. Also the persistence of expression and the level of expression need to be enhanced. On the in-vivo route it will be essential to efficiently target the gene-transfer vector to the desired tissue to ensure specific delivery of the curative gene into the cell type of choice. Ways must be found around the immune problems that restrict the applicability in vivo of the current adenovirus vectors. It will be essential to limit the cellular immune response directed against the transduced cells. Also the rapid humoral response which generates neutralizing antibodies that inhibit subsequent virus-mediated gene transfer, reduces the applicability. Although the results obtained with transient immuno-suppression of the recipients are promising, strategies in which the immunogenicity of the vector is reduced by removing all the viral protein-coding regions are preferable (Kochanek et al., 1996; Haecker et al., 1996; Chen et al., 1997). We should not forget that viruses 297

Notwithstanding the promising results, we should realize that gene therapy has only recently emerged as an approach for the treatment of various diseases. With the input from academic institutions and (biotech)-industry steadily growing, the number of potential applications, too, is increasing. Applications are found for the treatment of e.g. AIDS, cancer, arthritis, Parkinson's disease and many hereditary diseases. Some of these applications have already reached the stage of phase-I clinical trials. With the increased input also the range of available tools is expanding. New viral-vector systems are being developed with improved applicability, yield and safety features. In addition, novel very efficient non-viral genetransfer methods have been described that eventually may match and even surpass the efficiency of the viral vector systems. In this respect it is worthwhile to note how the viral and non-viral systems converge. On one hand the safety of viral gene-transfer systems is further increased by reducing the content of virus (-derived) products in the vector. On the other hand the non-viral vectors mimic the viral functions as much as possible using synthetic ingredients, resulting in artificial 'viroid-particles'. In this respect the pioneering work of Birnstiel and colleagues (Zatloukal et al., 1994), and others (Lozier et al., 1994; Ferkol et al., 1993) is exemplary and has already been used for the expression of clotting-F.VIII and IX in rodents. It is, therefore, reasonable to anticipate that the future will

Hoeben: Gene therapy for haemophilia hold promise of vector systems that can be administered systemically and that will target the gene-of-choice to a predetermined target tissue in a very efficient and highly specific manner.

Chen, H.H., Mack, L.M., Kelly, R., Ontell, M., Kochanek, S., and Clemens, P.R. ( 1 9 9 7 ) . Persistence in muscle of an adenoviral vector that lacks all viral genes. P r o c . N a t l . A c a d . S c i . U . S . A . 94, 1645-1650.

In addition to these "scientific" aspects we will need considerable efforts at the level of the production of the vectors. The type of therapeutics that is being considered for clinical application differs in several aspects from the more "conventional" drugs. Hence at the production side, considerable investments need to be made in order to acquire the technology to produce 'clinical-grade' vectors in sufficient quantities.

Chen, L., Perlick, H., and Morgan, R.A. ( 1 9 9 7 ) . Comparison of retroviral and adeno-associated viral vectors designed to express human clotting factor IX. Hum. Gene Ther. 8, 125-135. Chuah, M.K., Vandendriessche, T., and Morgan, R.A. ( 1 9 9 5 ) . Development and analysis of retroviral vectors expressing human factor VIII as a potential gene therapy for hemophilia A. Hum. Gene Ther. 6, 1363-1377.

Gene-therapy research thus requires the concerted action of scientists from many disciplines, e.g. from fundamental research in virology, genetics and process technology to (pre-)clinical research in the fields of haematology, pediatrics and surgery. Once we have been able to solve the 'scientific' and the 'technical' problems and only if we have unequivocally demonstrated the long-term safety and efficacy of this new technology, gene therapy can become a significant alternative for the current treatment of haemophilia.

Acknowledgments

Connelly, S., Smith, T.A., Dhir, G., Gardner, J.M., Mehaffey, M.G., Zaret, K.S., McClelland, A., and Kaleko, M. ( 1 9 9 5 ) . In vivo gene delivery and expression of physiological levels of functional human factor VIII in mice. Hum. Gene Ther. 6, 185-193. Connelly, S., Gardner, J.M., Lyons, R.M., McClelland, A., and Kaleko, M. ( 1 9 9 6 a ) . Sustained expression of therapeutic levels of human factor VIII in mice. B l o o d 87, 4671-4677. Connelly, S., Gardner, J.M., McClelland, A., and Kaleko, M. ( 1 9 9 6 b ) . High-level tissue-specific expression of functional human factor VIII in mice. Hum. Gene Ther. 7, 183-195. Connelly, S., Mount, J., Mauser, A., Gardner, J.M., Kaleko, M., McClelland, A., and Lothrop, C.D., Jr. ( 1 9 9 6 c ) . Complete short-term correction of canine hemophilia A by in vivo gene therapy. B l o o d 88, 3846-3853.

I thank the members of the Applied-Virology group for their constructive criticism on the manuscript. The haemophilia gene-therapy programme is supported by The Netherlands Organization for Scientific Research (NWO) and by the Dutch Praeventie Fonds.

Dai, Y., Roman, M., Naviaux, R.K., and Verma, I.M. ( 1 9 9 2 ) . Gene therapy via primary myoblasts: long-term expression of factor IX protein following transplantation in vivo. P r o c . N a t l . Acad. S c i . U . S . A . 89, 10892-10895.

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Lozier, J.N., Thompson, A.R., Hu, P.C., Read, M., Brinkhous, K.M., High, K.A., and Curiel, D.T. ( 1 9 9 4 ) . Efficient transfection of primary cells in a canine hemophilia B model using adenovirus-polylysine-DNA complexes. Hum. Gene Ther. 5, 313-322.

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Gene Therapy and Molecular Biology Vol 1, page 301 Gene Ther Mol Biol Vol 1, 301-308. March, 1998.

Kallikrein gene therapy in hypertension, cardiovascular and renal diseases J Chao and L Chao Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, South Carolina, USA __________________________________________________________________________________________________ Co r r e spo nde nc e to: Julie Chao, Ph.D., Department of Biochemistry and Molecular Biology, Medical University of South Carolina, 171 Ashley Avenue, Charleston, South Carolina 29425-2211, USA, Phone: (803) 792-4321, Fax: (803) 792-1627, Email: Chaoj@musc.edu Keywords: kallikrein; gene therapy; hypertension; renal damage; cardiac hypertrophy

Summary Somatic gene delivery approaches have received wide attention in recent years as a new technique f o r s t u d y i n g g e n e e x p r e s s i o n a n d a s a p o t e n t i a l t h e r a p e u t i c t o o l i n t r e a t i n g both inherited and infectious diseases. Hypertension, which is a polygenic disease influenced by environmental and dietary factors, shows abnormality of the tissue kallikrein-kinin system in its pathogenesis. To demonstrate potential therapeutic effects of gene delivery in treating hypertension, we introduced the human tissue kallikrein gene i n the form o f naked DNA or an adenoviral vector into h y p e r t e n s i v e r a t s . A s i n g l e i n j e c t i o n o f t h e k a l l i k r e i n g e n e c a u s e d a s u s t a i n e d blood pressure reduction for several weeks in spontaneously hypertensive rats (SHR), two kidney-one clip (2K1C) Goldblatt hypertensive rats, and Dahl salt-sensitive (Dahl-SS) rats. The expression o f human tissue kallikrein in rats receiving gene delivery was identified in tissues relevant to cardiovascular function including the kidney, heart, aorta, lung and liver. Adenovirus-mediated kallikrein gene delivery attenuated cardiac hypertrophy and renal damage in 2K1C and Dahl-SS rats fed on a high salt diet. Kallikrein gene delivery also caused significant increases i n renal blood f l o w , glomerular filtration rate and urine f l o w as w e l l as i n water intake, urine excretion, urinary electrolyte output, kinin, nitrite/nitrate (NOx) and cGMP levels. These findings are consistent with the mechanisms of blood pressure reduction and enhanced renal function mediated via kinin through a NO-cGMP dependent signal transduction pathway f o l l ow i ng kallikrein gene delivery. The ability of kallikrein gene delivery to produce a wide spectrum of beneficial effects makes it an excellent candidate in treating hypertensive, cardiovascular and renal diseases.

I. Tissue kallikrein and hypertension Essential hypertension is a polygenic disease which is governed by the combined action of several genes and results in an increase in blood pressure. Hypertensive subjects are more likely to develop other cardiovascular diseases such as coronary heart disease, congestive heart failure and peripheral vascular and renal diseases. There is ample evidence documenting the role of the tissue kallikrein-kinin system in the pathogenesis of hypertension (Katori and Majima, 1996; Margolius, 1995). Extensive epidemiological studies showed that urinary kallikrein levels are inversely correlated with blood pressure (Zinner et al., 1978; Margolius et al., 1974). Furthermore, a large family pedigree study has shown that

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a dominant allele expressed as high urinary kallikrein excretion may be associated with a decreased risk of essential hypertension (Berry et al., 1989). Since renal kallikrein originates from the kidney, these studies suggest that renal kallikrein defects may contribute to the development of human hypertensive diseases. In addition, reduced urinary kallikrein levels have been observed in a number of genetically hypertensive rats (Ader et al., 1985; Margolius et al., 1972). Several restriction fragment length polymorphisms (RFLP)s have been mapped in the tissue kallikrein gene and their regulatory regions in spontaneously hypertensive rats (SHR) (Woodley-Miller et al., 1989). These findings indicate a possible difference in the tissue kallikrein gene locus between SHR and normotensive Wistar-Kyoto (WKY) rats. Furthermore, a

Chao and Chao: Kallikrein gene therapy in cardiovascular and renal diseases tissue kallikrein RFLP has been shown to cosegregate with high blood pressure in the F2 offspring of SHR and normotensive Brown Norway crosses suggesting a close linkage between the kallikrein gene locus and the hypertensive phenotype of SHR (Pravenec et al., 1991). These findings combine to suggest that low renal kallikrein levels may contribute to hypertension and that high urinary kallikrein may offer a protective effect against the development of high blood pressure and renal diseases.

II. Vasodilating kallikrein-kinin system counter-balances vasoconstricting reninangiotensin system Tissue kallikrein (E.C. 3.4.21.35) belongs to a subgroup of serine proteinases which process kininogen substrates and release vasoactive kinin peptides (Figure 1). The well recognized function of tissue kallikrein is mediated by kinins. Kinins are cleaved by kinin degrading enzymes to produce a number of kinin metabolites or inactive fragments. Intact kinins bind to B2 receptors while kinin metabolites, such as Des-Arg9-bradykinin or Des-Arg10-Lys-bradykinin, bind to B1 receptors. The binding of kinins to the B2 receptor activates second messengers which trigger a broad spectrum of biological effects such as vasodilation, smooth muscle contraction and relaxation, inflammation, pain and cell proliferation

(Bhoola et al., 1992). Activation of the B1 receptor may induce biological effects such as inflammation, cell proliferation and vasoconstriction or vasodilation (Marceau, 1995). The vasodilating kallikrein-kinin system is linked to the vasoconstricting renin-angiotensin system by angiotensin converting enzyme (ACE), also known as kininase II, a kinin degrading enzyme. Renin converts angiotensinogen to angiotensin I which is then cleaved by ACE to produce the potent vasoconstrictor, angiotensin II. Administration of ACE inhibitors causes inhibition of angiotensin II production as well as accumulation of kinin. Therefore, the anti-hypertensive effect of ACE inhibition could be attributed, in part, to increased kinin levels (Figure 1). The renin-angiotensin system is well known for its important role in the development and maintenance of hypertension in both essential hypertensive patients and in animal models of hypertension (Rosenthal, 1993; Fyhrquist et al., 1995). Interruption of the reninangiotensin system by pharmacological manipulations can control high blood pressure and other cardiovascular complications (Nicholls et al., 1994; Linz et al., 1995). Hypertension could result from either an excess of vasoconstrictive substances or a deficiency of vasodilating substances. Therefore, pharmacological and/or genetic manipulation of the vasodilating kallikrein-kinin system could potentially counter-balance the vasopressor reninangiotensin system in blood pressure regulation.

F i g u r e 1 . Mechanisms of tissue kallikrein in blood pressure regulation.

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Gene Therapy and Molecular Biology Vol 1, page 303 suggest that hypotension in kallikrein transgenic mice is mediated by binding of kinin to bradykinin B2 receptors. This notion is further supported by the finding that heterozygous transgenic mouse lines expressing human bradykinin B2 receptor under the control of Rous sarcoma 3'-LTR are hypotensive (Wang et al., 1997a). Together, these results provide direct molecular evidence linking the physiological function of the tissue kallikrein-kinin system in blood pressure regulation. Since it is not possible to introduce the human tissue kallikrein gene into hypertensive patients by the transgenic approach, we explored the potential of gene therapy in treating hypertension by introducing the human tissue kallikrein gene into hypertensive animal models by somatic gene delivery.

III. Kallikrein protein therapy in hypertension Intravenous infusion of tissue kallikrein or kinin results in a transient reduction of blood pressure which lasts only 1-2 min (Chao and Chao, 1997; Schachter, 1969). The short duration of tissue kallikrein/kinin in the circulation is due to the presence of tissue kallikrein inhibitors in the circulation as well as kinin degrading enzymes in the vasculature. Oral administration of purified pig pancreatic kallikrein has been used to temporarily lower both the supine and upright blood pressures of hypertensive patients (Overlack et al., 1981; Ogawa et al., 1985). However, continuous oral kallikrein intake three times daily was required to maintain the blood pressure-lowering effect. The benefit of kallikrein-induced blood pressure reduction disappeared quickly upon the termination of oral kallikrein intake. Therefore, protein therapy is not considered a practical approach for antihypertensive therapy. Gene delivery would be the only alternative designed to circumvent these difficulties. To evaluate the role of the tissue kallikrein-kinin system and potential therapeutic effects in hypertensive and cardiovascular diseases, we employed molecular genetic approaches by manipulating the expression of the genes encoding tissue kallikrein-kinin system components in intact animals.

V. Systemic delivery of the naked human tissue kallikrein gene reduces blood pressure in spontaneously hypertensive rats To evaluate potential therapeutic effects of tissue kallikrein in hypertension, SHR were subjected to somatic gene therapy. The human tissue kallikrein gene or cDNA constructs were created under the promoter control of MRE, albumin, cytomegalovirus or Rous sarcoma virus 3'-LTR (Wang et al., 1994; Xiong et al., 1995; Chao et al., 1996). The human tissue kallikrein gene in the form of naked plasmid DNA was introduced into SHR via intravenous, intraportal vein or intraperitoneal injections. A single injection of the naked human kallikrein plasmid DNA caused a significant delay in blood pressure increase in SHR for more than 6 weeks, as compared to control SHR injected with the vector DNA (Xiong et al., 1995; Chao et al., 1996; Wang et al., 1995). The extent of blood pressure reduction was dependent on the dose of DNA injected, time post injection, gender of the animals, the promoter directing kallikrein expression and the route of injection (Chao et al., 1996; Chao et al., 1997a). Although intravenous delivery of the kallikrein gene into young adult or adult male SHR consistently produced a delay in blood pressure increase in SHR, kallikrein gene delivery did not have significant effects on the blood pressure reduction of adult female SHR (Chao et al., 1997a). The gender difference in response to kallikrein gene therapy in SHR was not expected and it may be attributed to a higher basal expression level of tissue kallikrein in female rats than in male rats. This notion is supported by the observation that tissue kallikrein mRNA levels are significantly higher in the kidney of adult female rats than in male rats (Gerald et al., 1986). Ovariectomized rats showed a significant reduction in tissue kallikrein mRNA levels and in immunoreactive tissue kallikrein content in the kidney which can be corrected by estrogen and progesterone replacement (Gerald et al., 1986). Furthermore, tissue kallikrein levels in humans are apparently regulated by sex hormones, as urinary kallikrein

IV. Transgenic mice expressing human tissue kallikrein or bradykinin B2 receptor are hypotensive Transgenic technologies were employed for the development of transgenic mouse lines expressing the human tissue kallikrein or the human bradykinin B2 receptor gene under the control of various promoters (Wang et al., 1994; Song et al., 1996; Wang et al., 1997a). The human tissue kallikrein gene under the control of the mouse metallothionein promoter, a metalresponsive element (MRE), was first introduced into mouse embryos via microinjection, and transgenic mouse lines expressing human tissue kallikrein were established (Wang et al., 1994). The transgenic mice overexpressing human tissue kallikrein were permanently hypotensive throughout their lifetime, compared to their control littermates (Chao and Chao, 1996). In order to determine the role of circulating tissue kallikrein in blood pressure regulation, transgenic mice with liver-targeted expression of human tissue kallikrein under the control of a mouse albumin enhancer and promoter were developed (Song et al., 1996). Three lines of independently established transgenic mice were hypotensive. The blood pressure of these transgenic mice expressing human tissue kallikrein can be restored by aprotinin, a tissue kallikrein inhibitor, or by incatibant (Hoe 140), a specific bradykinin B2 receptor antagonist (Song et al., 1996). Since human tissue kallikrein is capable of processing mouse kininogen to produce kinins (Wang et al., 1994), these results

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Chao and Chao: Kallikrein gene therapy in cardiovascular and renal diseases levels in women are higher than in men, decrease with age, and peak at the progestinic phase of the menstrual cycle (Albano et al., 1994). The regulation of human tissue kallikrein by sex hormones is consistent with the identification of potential estrogen response elements in the promoter region of the human tissue kallikrein gene (Murray et al., 1990; Madeddu et al., 1991). Therefore, a lack of response to kallikrein gene therapy in female SHR may be attributed to high expression of tissue kallikrein at the transcriptional level in females. Moreover, sex dimorphism of bradykinin B2 receptor mRNA and differential cardiovascular responses to early blockade of bradykinin receptors in male vs. female rats indicates a gender difference in the regulation of cardiovascular function (Madeddu et al., 1996). Collectively, these results suggest that higher expression of tissue kallikreinkinin system components in females than in males may be a contributing factor to vascular function and responsiveness in hypertensive animal models.

VI. Adenovirus-mediated kallikrein gene delivery reduces blood pressure in genetically and experimentally-induced hypertensive rats Although somatic delivery of the human tissue kallikrein gene in the form of plasmid DNA produces a prolonged delay in blood pressure increase, the efficiency of cellular uptake of the naked DNA and the expression of the gene product are limited. To improve the efficiency of foreign gene expression in animal models following somatic gene delivery, we constructed an adenovirus vector carrying the human tissue kallikrein gene under the control of cytomegalovirus or Rous sarcoma virus 3'-LTR. Adenovirus-mediated gene delivery results in high efficiency expression of human tissue kallikrein. A profound and rapid blood pressure reduction was observed 1 to 2 days post gene delivery. The delay in blood pressure increase lasted for 4-6 weeks in spontaneously hypertensive rats, (SHR), two kidney-one clip (2K1C) Goldblatt hypertensive rats and Dahl-SS rats fed on a high salt diet (Yayama et al., 1997; Chao et al., 1997b; Jin et al., 1997). When the same amounts of the adenovirus carrying the human tissue kallikrein gene or the control

adenovirus carrying the LacZ gene under the cytomegalovirus (CMV) promoter control were injected into normotensive WKY rats, the blood pressure remained normotensive during 7 weeks post gene delivery in both the experimental and control groups (Jin et al., 1997). Human tissue kallikrein levels in sera and urine of WKY rats were similar to those of SHR following kallikrein gene delivery. Therefore, the differential effects of gene delivery on blood pressures between hypertensive and normotensive rats may be attributed to their different sensitivities to the exogenous kallikrein in the vasculature. Table 1 summarizes the comparison of kallikrein gene delivery based on naked DNA or adenovirus vector in blood pressure reduction.

VII. Local delivery of the tissue kallikrein gene reduces high blood pressure in hypertensive rats Similar to systemic delivery, local delivery of the human tissue kallikrein gene causes a sustained blood pressure reduction in SHR (Xiong et al., 1995). For example, intramuscular delivery of the naked DNA into SHR produced a prolonged reduction of blood pressure which lasted for more than 8 weeks (Xiong et al., 1995). Central administration of the human tissue kallikrein gene via intracerebroventricular (ICV) injection caused a delay in blood pressure increase in SHR, as compared to control rats receiving the vector DNA or injected with adenovirus containing the LacZ gene (Wang et al., 1997b). Adenovirus-mediated delivery of the human tissue kallikrein or kallistatin gene into rat salivary gland via direct intracapsular injection results in expression of human kallikrein or kallistatin in the salivary gland (Wang et al., 1997c; Xiong et al., 1997). Human tissue kallikrein can also be detected in sera and saliva after direct gene delivery into salivary glands, demonstrating that locally synthesized kallikrein in the salivary gland can be secreted into both the vascular compartment and saliva. Therefore, local delivery of the kallikrein gene into salivary glands may provide a unique opportunity for studying the role of the kallikrein-kinin system in the salivary gland.

Table 1. Kallikrein gene delivery based on naked DNA or adenovirus vector in blood pressure reduction Vector Onset Duration Repeated Administration Expression Efficiency Serum Kallikrein Levels Site of Expression Immune Response

Naked DNA CMV-cHK 1-2 weeks 6-8 weeks yes low n.d. liver, kidney, heart, lung n.d.

n.d.: not detectable.

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Adenovirus Ad.CMV-cHK 1-2 days 4-6 weeks no high up to 500 ng/ml liver>>kidney>aorta>heart yes

Gene Therapy and Molecular Biology Vol 1, page 305 Table 2. Kallikrein gene delivery attenuates hypertension, cardiac hypertrophy, renal injury and stenosis Rat Models Reference Blood Cardiac Renal Injury Stenosis Pressure Hypertrophy SHR Xiong et al, ! 1995 Dahl-SS ! ! ! Chao et al, 1997b 2K1C Goldblatt ! ! Yayama et al, 1997 Nephrotoxicity ! ! Murakami et al, 1997 Angioplasty ! SHR: spontaneously hypertensive rats; Dahl-SS: Dahl salt sensitive rats; 2K1C Goldblatt: two kidney, one clip Goldblatt hypertensive rats. "-" not observed.

VIII. Adenovirus-mediated kallikrein gene delivery protects cardiovascular and renal function Long-term infusion of purified rat tissue kallikrein via a minipump has been shown to attenuate glomerular sclerosis without affecting the blood pressure of Dahl-SS rats fed on a high salt diet (Uehara et al., 1997). This finding indicates that a continuous supply of tissue kallikrein might provide protective effects on salt-induced renal injury. Our recent studies showed that adenovirusmediated kallikrein gene delivery not only caused a prolonged blood pressure reduction but also reduced left ventricular weight and cardiomyocyte size as well as attenuated glomerular and tubular damage in Dahl-SS rats fed on a high salt diet (Chao et al., 1997b). Moreover, kallikrein gene delivery into 2K1C Goldblatt hypertensive rats significantly attenuated cardiac hypertrophy and improved renal function by increasing glomerular filtration rate, renal blood flow and urine flow (Yayama et al., 1997). The protective effects of kallikrein gene delivery in renal tubular injury was also observed morphologically in rats with gentamycin-induced nephrotoxicity (Murakami et al., 1997). Moreover, kallikrein gene delivery inhibited neointimal thickening in balloon-injured rat artery. Collectively, these results lend strong support to an important role of tissue kallikrein in cardiovascular and renal function. Table 2 summarizes the beneficial effects of kallikrein gene delivery on hypertension, cardiac hypertrophy, renal injury and stenosis.

IX. Expression and localization of human tissue kallikrein in rats post gene delivery Expression of human tissue kallikrein mRNA in rats following injection of naked plasmid DNA was identified by reverse transcription-polymerase chain reaction (RTPCR) followed by Southern blot analysis using specific oligonucleotide probes for human tissue kallikrein.

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Human tissue kallikrein mRNA can be identified in heart, aorta, kidney, adrenal gland, lung and liver of rats injected with the kallikrein gene but not in the corresponding tissues of rats injected with the control DNA. Low levels of immunoreactive human tissue kallikrein can be detected in rat tissues following kallikrein gene delivery by a specific enzyme-linked immunosorbent assay (ELISA) (Chao et al., 1996; Wang et al., 1995). Expression of adenovirus-mediated gene delivery via intravenous injection was readily detectable in the hepatocytes 3 days post delivery of the LacZ gene by blue staining with demonstrated "-galactosidase activity. The expression of recombinant human tissue kallikrein in rat liver or kidney was rapidly secreted into the circulation and urine. Although an accurate evaluation of the level of adenovirusmediated transduction can not be determined, following the time-course of human tissue kallikrein levels in the serum could well serve as an indicator of efficiency in the expression of the foreign gene. We observed that human tissue kallikrein levels in the serum reached a peak at days 3-5 and declined gradually following adenovirus-mediated kallikrein gene delivery into SHR. Adenovirus-mediated gene delivery also produced similar serum profiles of human tissue kallikrein levels in hypertensive Dahl-SS rats and Goldblatt hypertensive rats. The reason for the decline of human tissue kallikrein levels 1 week after kallikrein gene delivery is not clear at this time, but two likely causes are inactivation or clearance of the adenovirus by the immune system. Immunoreactive human tissue kallikrein was not detected in control rats injected with the control adenovirus containing the lacZ gene. These results show that the human tissue kallikrein gene is expressed in tissues relevant to cardiovascular and renal function post systemic gene delivery.

X. Mechanisms of kallikrein gene therapy on blood pressure reduction Tissue kallikrein cleaves LMW kininogen substrate to release the kinin product by limited proteolysis. Kinins are capable of binding to B2 receptors, activating second

Chao and Chao: Kallikrein gene therapy in cardiovascular and renal diseases messengers in target tissues via a G-protein coupled signal transduction pathway and triggering biological effects such as vasodilation or vasoconstriction (Bhoola et al., 1992; Linz et al., 1995). There are multiple steps to inhibit or block the tissue kallikrein-kinin system, such as aprotinin, a potent tissue kallikrein inhibitor or incatibant (Hoe 140), a bradykinin B2 receptor antagonist. Kallikrein gene delivery caused a prolonged delay in the blood pressure increase in SHR and the hypotensive effect of kallikrein gene delivery was abolished by aprotinin, a potent tissue kallikrein inhibitor. This suggests that the hypotensive effect of kallikrein gene delivery is due to the expression of functional kallikrein (Wang et al., 1995). The antihypertensive effect in SHR post kallikrein gene delivery was reversed by incatibant (Hoe 140), a specific bradykinin B2 receptor antagonist, suggesting that the blood pressure-lowering effect following somatic gene delivery of human tissue kallikrein is mediated by a bradykinin B2 receptor-mediated signal transduction pathway (Xiong et al., 1995). Adenovirus-mediated kallikrein gene delivery into various hypertensive rat models led to significant increases of urinary kinin, nitrite/nitrate (NOx) and cGMP levels, suggesting that blood pressure reduction was mediated via kinin through a NO-cGMP dependent signal transduction pathway (F igure 1).

XI. Antisense inhibition of the tissue kallikrein-kinin system An antisense inhibition strategy, based on interference of information flow from genes to proteins, was used to determine the role of the tissue kallikrein-kinin system in blood pressure regulation. Acute intracerebroventricular (ICV) injection of antisense oligonucleotides which block rat kininogen mRNA or bradykinin B2-receptor mRNA translation caused a significant blood pressure increase in SHR which returned to basal levels within 24 hours (Madeddu et al., 1996). Prolonged vasopressor effects were observed after repeated injections of antisense oligonucleotides. Mean arterial blood pressure was not altered by intravenous injection of antisense oligonucleotides or by central injection of sense or scrambled oligonucleotides. Uptake of the antisense oligonucleotides of rat kininogen mRNA or bradykinin B2-receptor mRNA was detected in the hippocampus, thalamus and hypothalamus periventricularis one hour after the central injection of fluorescein isothiocyanateconjugated antisense oligonucleotides. Kininogen levels were significantly lower in the brain of SHR injected with antisense kininogen oligonucleotides via the ICV delivery compared with controls injected with the sense oligonucleotides. These results indicate that the brain kallikrein-kinin system plays a role in the central regulation of blood pressure and suggest that this system may exert a protective action against further elevation of blood pressure in SHR. In contrast, ICV injection of the antisense oligonucleotides targeted to rat B1 receptor 306

mRNA induced a profound blood pressure reduction in SHR while similar administration of sense or scrambled oligonucleotides had no effect on their blood pressure (Emanueli et al., 1997). The fact that B1 receptor blockade can decrease blood pressure in SHR suggests that activation of B1 receptors by brain kinin metabolites exerts a vasoconstrictor activity. These findings suggest that bradykinin B1 and B 2 receptors play different roles in the central regulation of blood pressure.

XII. Concluding remarks We showed that kallikrein gene delivery into various rat models exhibits protection such as reduction of high blood pressure, attenuation of cardiac hypertrophy, inhibition of renal damage and restenosis (Table 1). The ability of kallikrein gene transfer to produce such a wide spectrum of beneficial effects makes it an excellent candidate for treating salt-related hypertension as well as cardiovascular and renal diseases. Somatic gene therapy has several unique advantages over traditional pharmaceuticals. For example, gene therapy produces long-lasting effects. It is inexpensive and simple to administer. Gene therapy has the potential to offer permanent gene replacement or to treat diseases not treatable by drugs. Several important factors should be taken into consideration in gene delivery using animal models. These factors include animal age, gender, species, strains, and the choice of local or systemic delivery. Under certain conditions, it might be necessary to deliver genes into specific organs or tissues. Catalytic vs. stoichiometric actions of the therapeutic proteins and availability of the substrate in vivo for an enzyme should be considered. Attention should also be directed to potential immune responses to DNA, gene products, and vectors. Also, in order for the therapeutic proteins or peptides to exhibit their function in vivo, the half-life of these gene products should be sufficiently long and they should not have any significant side-effect. Information obtained from kallikrein gene delivery studies in genetically and experimentally hypertensive animal models is crucial for future clinical applications in treating hypertensive, cardiovascular and renal diseases by gene therapy.

Acknowledgements This work was supported by National Institutes of Health grants HL 29397 and HL 56686.

References Ader JL, Pollock DM, Butterfield MI, Arendshorst WJ. (1 9 8 5 ) Abnormalities in kallikrein excretion in spontaneously hypertensive rats. A m J P h y s i o l 248: F396-F403. Albano JD, Campbell SK, Farrer A, Millar JG. (1 9 9 4 ) Gender differences in urinary kallikrein excretion in man:

Gene Therapy and Molecular Biology Vol 1, page 307 variation throughout the menstrual cycle. C l i n S c i 86: 227-231. Berry TD et al. (1 9 8 9 ) A gene for high urinary kallikrein may protect against hypertension in Utah kindreds. H y p e r t e n s i o n 17: 242-246. Bhoola KD, Figueroa CD, Worthy K. (1 9 9 2 ) Bioregulation of kinins, kallikreins, kininogens, and kininases. Pharmacol Rev 44, 1-80. Chao J, Chao L. (1 9 9 7 ) Experimental approaches using kallikrein gene therapy for hypertension. In, Gene Transfer and Cardiovascular Biology, Experimental Approaches and Therapeutic I m p l i c a t i o n s . ed., K.L. March, pps. 449-473.

Margolius HS. (1 9 9 5 ) Kallikreins and kinins. Some unanswered questions about system characteristics and roles in human disease. H y p e r t e n s i o n 26, 221-229. Margolius HS, Geller R, De Jong W, Pisano JJ. (1 9 7 2 ) Altered urinary kallikrein excretion in rats with hypertension. Circ Res 30, 358-362. Margolius HS, Horwitz D, Pisano JJ, Keiser HR. (1 9 7 4 ) Urinary kallikrein excretion in hypertensive man. Relationships to sodium intake and sodium-retaining steroids. Circ Res 35, 820-825. Murakami H, Chao L, Chao J (1 9 9 7 ) Human kallikrein gene delivery protects gentamycin-induced nephrotoxicity in rats. Kidney Int. In press.

Chao J et al. (1 9 9 7 a ) Kallikrein Gene Therapy in Newborn and Adult Hypertensive Rats. Canadian J P h y s i o l Pharmacol. 75, 750-756.

Murray SR, Chao J, Lin FK, Chao L. (1 9 9 0 ) Kallikrein multigene families and the regulation of their expression. J Cardiovasc Pharmacol 15, S7-S16.

Chao J, Zhang J, Lin KF, Chao L. (1 9 9 7 b ) Adenovirusmediated kallikrein gene delivery attenuates hypertension, cardiac hypertrophy and renal injury in Dahl-salt sensitive rats. Human Gene Ther. In press.

Nicholls MG et al. (1 9 9 4 ) Blockade of the renin-angiotensin system. J Hypertens 12, S95-103.

Chao J, Chao L. (1 9 9 6 ) Functional analysis of human Tissue kallikrein in transgenic mouse models. H y p e r t e n s i o n 27, 491-494. Chao J et al. (1 9 9 6 ) Systemic and portal vein delivery of human kallistatin gene reduces blood pressure in hypertensive rats. Human Gene Ther 7, 901-911. Emanueli C et al. (1 9 9 7 ) Role of the brain bradykinin B 1 receptor in the central regulation of blood pressure in rats. H y p e r t e n s i o n . submitted. Fyhrquist F, Metsarinne K, Tikkanen I. (1 9 9 5 ) Role of angiotensin II in blood pressure regulation and in the pathophysiology of cardiovascular disorders. J Human Hypertens 9, S19-S24. Gerald WL, Chao J, Chao L. (1 9 8 6 ) Sex dimorphism and hormonal regulation of rat tissue kallikrein mRNA. B i o c h i m B i o p h y s A c t a 867, 16-23. Jin L, Zhang J, Chao L, Chao J. (1 9 9 7 ) Gene therapy in hypertension, Adenovirus-mediated kallikrein gene therapy in hypertensive rats. Human Gene Ther. 8 , 1753-1761. Katori M, Majima M. (1 9 9 6 ) Pivotal role of renal kallikreinkinin system in the development of hypertension and approaches to new drugs based on this relationship. Jpn J Pharmacol 70, 95-128. Linz W et al. (1 9 9 5 ) Contribution of kinins to the cardiovascular actions of angiotensin-converting enzyme inhibitors. Pharmacol Rev 47, 25-49. Madeddu P et al.( 1 9 9 6 ) Sexual dimorphism of cardiovascular responses to early blockade of bradykinin receptors. H y p e r t e n s i o n 27, 746-751. Madeddu P et al. (1 9 9 6 ) Antisense inhibition of the brain kallikrein-kinin system. H y p e r t e n s i o n 28, 980-987. Madeddu P et al. (1 9 9 1 ) Regulation of rat renal kallikrein by estrogen and progesterone. J Hyperten 9, S244-S245. Marceau F. (1 9 9 5 ) Kinin B1 receptors, Immunopharmacology 30, 1-26.

review.

Ogawa KT et al. (1 9 8 5 ) Effects of orally administered glandular kallikrein on urinary kallikrein and prostaglandin excretion, plasma immunoreactive prostanoids and platelet aggregation in essential hypertension. K l i n W o c h e n s c h r 63, 332-336. Overlack A et al. (1 9 8 1 ) Antihypertensive effect of orally administered glandular kallikrein in essential hypertension. Results of double blind study. H y p e r t e n s i o n 3, I18-I21. Pravenec M et al. (1 9 9 1 ) Cosegregation of blood pressure with a kallikrein gene family polymorphism. H y p e r t e n s i o n 17, 242-246. Rosenthal J. (1 9 9 3 ) Role of renal and extrarenal reninangiotensin system in the mechanism of arterial hypertension and its sequelae. S t e r o i d s 58, 566-572. Schachter M. (1 9 6 9 ) Kallikreins and kinins. P h y s i o l R e v 49, 509-547. Song Q, Chao J, Chao L. (1 9 9 6 ) Liver-targeted expression of human tissue kallikrein induces hypotension in transgenic mice. C l i n E x p H y p e r t e n s 18, 975-993. Uehara Y et al. (19 9 7 ) Long-term infusion of kallikrein attenuates renal injury in Dahl salt-sensitive rats. Am J Hypertens 10, S83-S88. Wang DZ, Chao L, Chao J. (1 9 9 7 a ) Hypotension in transgenic mice overexpressing human bradykinin B2 receptor. H y p e r t e n s i o n 29, 488-493. Wang C, Chao C, Chao L, Chao J. (1 9 9 7 b ) Central delivery of human tissue kallikrein gene reduces blood pressure in hypertensive rats. Gene Ther. Under revision. Wang C, Chao C, Chao L, Chao J. (1 9 9 7 c ) Expression of human tissue kallikrein in rat salivary glands and its secretion into circulation following adenovirus-mediated gene transfer. Immunopharmacology 36, 221-227. Wang C, Chao L, Chao J. (1 9 9 5 ) Direct gene delivery of human tissue kallikrein reduces blood pressure in spontaneously hypertensive rats. J C l i n I n v e s t 95, 1710-1716. Wang J et al. (1 9 9 4 ) Human tissue kallikrein induces hypotension in transgenic mice. H y p e r t e n s i o n 23, 236-243.

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Chao and Chao: Kallikrein gene therapy in cardiovascular and renal diseases Woodley-Miller C, Chao J, Chao L. (1 9 8 9 ) Restriction fragment length polymorphisms mapped in spontaneously hypertensive rats using kallikrein probes. J Hypertens 7, 865-871. Xiong W, Chao J, Chao L. (1 9 9 7 ) Expression and localization of human kallistatin in rat submandibular gland after intracapsular gene injection. B i o c h e m B i o p h y s R e s Commun 231, 494-498. Xiong W, Chao J, Chao L. (1 9 9 5 ) Muscle delivery of human tissue kallikrein gene reduces blood pressure in spontaneously hypertensive rats. H y p e r t e n s i o n 2 5 , 715-719. Yayama K, Wang C, Chao L, Chao J. (1 9 9 7 ) Human tissue kallikrein gene delivery attenuates hypertension, cardiac hypertrophy and enhances renal function in Goldblatt hypertensive rats. H y p e r t e n s i o n . Under revision. Zinner SH, Margolius HS, Rosner B, Kass EH. (1 9 7 8 ) Stability of blood pressure rank and urinary kallikrein concentration in childhood, an eight follow-up. C i r c u l a t i o n 58, 908-915.

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Gene Therapy and Molecular Biology Vol 1, page 309 Gene Ther Mol Biol Vol 1, 309-321. March, 1998.

Chemically defined, cell-free cancer vaccines: use of tumor antigen-derived peptides or polyepitope proteins for vaccination Michael Buschle1, Walter Schmidt1, Manfred Berger1, Gotthold Schaffner1, Robert Kurzbauer1, Iris Killisch1, Julia-Kristina Tiedemann 1, Barbara Trska1, Helen Kirlappos 1, Karl Mechtler1, Franz Schilcher2, Cornelia Gabler3, and Max L. Birnstiel1 1

Research Institute of Molecular Pathology, Vienna Biocenter, Dr. Bohrgasse 7, 1030 Vienna, Austria Institute of Pathology and Forensic Veterinary Medicine, University of Veterinary Medicine Vienna, Josef Baumanngasse 1, 1210 Vienna, Austria 2

Institute of Nutrition, University of Veterinary Medicine Vienna, Josef Baumanngasse 1, 1210 Vienna, Austria

__________________________________________________________________________________ Correspondence: Max L. Birnstiel, Tel: (+43-1) 797 30-474, Fax: (+43-1) 797 30-490, E-mail: birnstiel@nt.imp.univie.ac.at

Summary With the advent of gene therapy there has been a revival of immunotherapy of cancer. Preclinical studies with the so called tumor vaccines – syngeneic, irradiated tumor cells secreting cytokines – are at present entering clinical trials and hold much promise for efficacious treatment, maybe even cures, of cancer. However, autologous whole cell vaccines which are cytokine gene-modified are expensive and difficult to standardize. In addition, autologous tumor cell cultures, and hence tumor vaccines, cannot be prepared for all patients. For these reasons we have investigated alternative schemes for vaccination against cancer. We have developed vaccines on the basis of tumor antigenderived peptides using a method, we called ”transloading”, t o transfer peptides efficiently into c e l l s . Such vaccines are chemically w e l l defined and inexpensive t o produce. We show i n a preclinical mouse model system for mastocytoma, and to a somewhat lesser extent for melanoma, that peptide vaccines give excellent protection against tumor take, provided tumor antigen peptides are injected subcutaneously in conjunction with polycations like polyarginine. Immunohistological investigations on thin sections show that three days after injection, the vaccine bolus is heavily infiltrated by antigen presenting c e l l s which take up peptides. We posit that such antigen presenting cells then migrate into draining lymph nodes where they activate naive T c e l l s to become anti-tumor cytotoxic lymphocytes (CTL). The consequences o f strong dependency of peptide sequence on the HLA-type of patients and possible remedies are discussed. One avenue to b e t e s t e d i n m o u s e m o d e l s i s t h e i n j e c t i o n o f m i x t u r e s o f p e p t i d e s c o v e r i n g a multiplicity of MHC-specific peptides. Another i s the production o f recombinant proteins as vaccines whose application is considerably less dependent on HLA-types of patients to be treated. In addition, such proteins would be expected to contain both class I and class II restricted peptides. Alternatively, artificial proteins incorporating many CTL epitopes of known tumor antigens, including members of the MAGE family, thus yielding a SUPERMAGE vaccine with utility for several human HLAtypes could be designed. The prophylactic application o f peptide/polyepitope vaccines can be envisaged. have attempted to harness the exquisite specificity, sophistication and power of immunology to combat cancer, but the results hitherto have been rather disappointing and have led to a rather pessimistic assessment of immunotherapy of cancer. Indeed, at certain periods the postulate emerged that cancers were not

I. Tumor-specific and tumor-associated antigens After the great successes of vaccination against bacterial and viral infections, physicians and scientists

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Buschle et al: Chemically defined, cell-free cancer vaccines immunogenic. Recent molecular and immunological investigations, however, have revealed the existence of tumor antigens in several tumor types (Boon et al., 1994; Boon and van der Bruggen, 1996; Rosenberg, 1997). This has led to the conclusion, that most, and perhaps all tumors are indeed immunogenic, but that the response of the immune system to tumor antigens is inadequate and in need of enhancement if immunotherapy of cancer is to be attempted. Peptides derived from tumor antigens, mostly presented by cancer cells in the MHC class I context (Boon et al., 1994; Boon and van der Bruggen, 1996; Robbins and Kawakami, 1996; Rosenberg, 1997) can be classified as having been derived from tumor specific (TSAs) or tumor associated antigens (TAAs). They have been identified mostly by means of CTL clones obtained from cancer patients and by gene cloning or by peptide elution followed by tandem mass spectrometry analysis (Boon et al., 1994; Slingluff et al., 1994; Wang and Rosenberg, 1996). As shown recently by Kinzler and Vogelstein’s lab, cancerous transformation leads to extensive reprogramming of the cancer cell in that as many as 500 mRNA types are either up or down regulated (Zhang et al., 1997). As a consequence, cancer cells can express tumor associated antigens which are unconnected with the gene expression programme of the parental cell type from which the cancer cell arose. Thus it is known for melanoma, that malignant cells express, amongst other TSA and TAA, the MAGE, BAGE and GAGE family of genes which are normally expressed in male germline cells and placenta (Boon and van der Bruggen, 1996) and thus are clearly self-proteins. Because germ line cells are immunologically privileged and do not express MHC surface receptors, clonal selection presumably cannot act to eliminate MAGE, BAGE and GAGE specific CTLs under normal circumstances. The frequent appearance of these related genes in tumors different from melanoma such as head and neck cancer, lung cancer or bladder carcinoma may be a consequence of random demethylation of genes in cancer cells and activation of their promoters by demethylation, as has been shown for the MAGE-1 promoter (De Smet et al., 1996). Another interesting example of TAAs in melanoma cells are the so called differentiation antigens tyrosinase, tyrosinase related proteins 1 and 2 (TRP-1, TRP-2) and other proteins normally encountered during melanocyte differentiation (see Table I and Boon et al., 1997; Rosenberg, 1997). Interestingly, the immune system treats these proteins, dysregulated and expressed in the ”wrong” context of tumor cells, as foreign antigens. Despite potential earlier clonal selection by the presumed interaction of the immune system with melanocytes, cytotoxic T cells which recognise peptides derived from these proteins are generated and hence are reactive against melanoma cells. The encoded proteins of oncoviral genes, mutated oncogenes and tumor suppressor genes or mutated cellular genes, are tumor specific (Table I). Examples for the first are the oncoproteins of the Epstein-Barr virus (Rickinson 310

and Moss, 1997) or E6 and E7 of HPV16 (Tindle, 1996), for the second and third the mutated Ras or p53 proteins (Disis and Cheever, 1996) and for the last are connexin (Mandelboim et al., 1994; Mandelboim et al., 1995), Her 2/neu (Disis and Cheever, 1997), MUC 1 (Finn et al., 1995), CDK4 (Wolfel et al., 1995) and ß-catenin (Robbins et al., 1996). Being tumor specific, any of these have the potential of eliciting anti-tumor immunity when used as a vaccine.

II. TSA and TAA peptide vaccines Previous attempts at creating tumor vaccines have concentrated on the stimulation of the immune response by cytokines. The great advantage of using cytokinesecreting autologous tumor cells as vaccines is that in this strategy, unidentified and unknown TSAs and TAAs are presented to the immune system in a milieu of high local cytokine concentration (Pardoll, 1995; Zatloukal et al., 1995). The most widely used cytokine is IL-2, the level of which is highly critical for the outcome of the vaccination as indicated in preclinical studies using the M3 melanoma model (Figure 1a and Schmidt et al., 1995). We further determined cytokine optima in the B16/F10 model where the number of metastases were scored. The IL-2 optimum for B16/F10 appears to be higher (3,300 units IL2/vaccine; Figure 1b) as compared to 1,500 units IL2/vaccine for the M3 model (Figure 1a and Schmidt et al., 1995). As shown previously for the M3 system, GMCSF shows no dosis maximum, but both in the M3 and B16/F10 model, protection increases with cytokine dosage (Figure 1c). In the case of IFN-! it appears that a dose of 165 ng/vaccine yields best protection (Figure 1c). It has been the experience of different groups that despite the great potential that autologous cancer vaccines have, the procedure is cumbersome and expensive, and of limited utility since tissue cultures can only be prepared in roughly 2/3 of the cases for malignant melanoma (E. Wagner, Vienna, unpubl. results). These circumstances call for alternative strategies. Syngeneic dendritic cells pulsed with tumor antigen peptides matched to the MHC type of the mice strains have repeatedly been shown to protect animals against tumor take (Celluzzi et al., 1996; Mayordomo et al., 1996; Mayordomo et al., 1995; Paglia et al., 1996; Porgador et al., 1996). This approach has also been used in a clinical setting: lymphoma patients were treated with dendritic cells pulsed with autologous idiotype protein (Hsu et al., 1996; Hsu et al., 1997). The results of these studies are promising. In a seminal investigation, Boon and coworkers vaccinated fifteen terminally-ill melanoma patients with a HLA-A1 matched immunogenic MAGE-3 peptide and detected clinical benefits in five of them in that some tumors regressed (Marchand et al., 1995). This result is even more remarkable, because no adjuvants of any kind were used. Curiously, despite these clinical effects, no

Gene Therapy and Molecular Biology Vol 1, page 311

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Gene Therapy and Molecular Biology Vol 1, page 312 b l e I : (previous page) Human tumor specific and tumor associated antigens recognized by T cells known to date. After Boon et al., 1997; Robbins and Kawakami, 1996; Rosenberg, 1997; Wang et al., 1995 with adaptions. Figure 1: Cytokine dosage effects in experimental murine melanoma systems. Figure 1a: IL-2 dosage curve as determined in DBA/2 mice (8-10 animals/group) with prophylactic treatment against M3 melanoma tumor challenge (Schmidt et al., 1995). Mice were vaccinated twice at weekly intervals (105 irradiated cells/vaccination/animal) and challenged one week after the second vaccination (3 x 105 viable M-3 melanoma cells). Reproduction of a previously published Figure (Schmidt et al., 1995). IL-2 (Figure 1b), GM-CSF and IFN-! (Figure 1c) dosage effects in the treatment of experimental B16-F10 lung metastases in C57BL/6J mice (eight animals/group). Animals were vaccinated three times (105 irradiated cells/vaccination/animal). Lung metastases were established by intraveneous injection of 104 B16-F10 cells three days before the first vaccination. After 28 days lung metastases were scored and the number obtained for all eight animals of the control group receiving mock-transfected B16-F10 was set to 100 % (n=215).

peptide specific CTLs could be detected in vaccinated patients. However, as reported at this conference, no clinical effects were detected in a repeat experiment by the Parmiani group (Milano). Following in the footsteps of Boon et al. (Marchand et al., 1995) we have carried out a preclinical study in mice for the efficacious vaccination against tumor take in both the murine M3 melanoma and the P815 mastocytoma model system using small TAA-peptides matched to the MHC-type of the experimental animals (Schmidt et al., 1997a). Since no known tumor antigens are available for the M3 system, it was ascertained by means of RT-PCR that M3 cells expressed tyrosinase and tyrosinase related protein-1 (TRP-1) which are known as TAAs in human melanomas. Much progress has been made in establishing the binding rules for small peptides to MHC class I molecules (Rammensee et al., 1995), therefore, potential tumor antigen peptides can be predicted up to a certain extent by computer analysis. Six potential H-2Kd binding peptides are found in tyrosinase (Table II, see also (Schmidt et al., 1997a) and four in TRP-1 (Table II). Two each of these two classes of peptides were arbitrarily selected (Table II) and combined for subcutaneous injection into mice. In the case of the P815 system it was known that a â&#x20AC;?selfâ&#x20AC;?-peptide SYFPEITHI derived from the JAK1 tyrosine kinase protein occupied an unusually high proportion of 5% of all MHC class I molecules on P815 cancer cells (Harpur et al., 1993; Rammensee et al., 1995) and this peptide was therefore judged to represent a useful target for immunological rejection. Immunohistological investigations of the vaccination Ta

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Table II: Putative peptide antigens used in the M3 melanoma model. The sequences of mouse tyrosinase and tyrosinase related potein 1 (TRP-1) were searched for peptides fulfilling criteria necessary for binding to H-2Kd molecules (?Y??????L/I) (Rammensee et al., 1995).

Figure 2: Peptide immunization prevents tumor growth. Figure 2a: P815 mastocytoma model. DBA/2 mice were immunized with the JAK-1 kinase derived peptide SYFPEITHI. Mice (8 animals per group) were vaccinated three times at weekly intervals and challenged one week after the last immunisation as described in (Schmidt et al., 1997a). Tumor cells stably transfected with a GM-CSF gene (P815 GM-CSF) served as gold standard for vaccine efficiency. Peptides were injected subcutaneously in PBS, emulsified in incomplete Freundâ&#x20AC;&#x2122;s adjuvant (IFA) or as a mixture of peptide and polyarginine (pArg). Figure 2b: Model as in Figure 2a using the novel sorbitol containing injection medium.

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Figure 2c: M3 melanoma model. Experimental metastases (104 parental M3 cells) were set 5 days before the first vaccination. Animals were vaccinated subcutaneously 3 times at weekly intervals with a mixture of tyrosinase and TRP-1 derived peptides (Table II). A cellular vaccine consisting of IL-2 gene transfected, irradiated M3 was used to assess the relative efficiency of the peptide vaccine.

site (Bannerji et al., 1994; Maass et al., 1995; Schmidt et al., 1997b) as well as complementary studies of others (Huang et al., 1994) have identified antigen presenting cells (APCs), rather than T cells, to play a pivotal role in initiating the cascade leading to the generation of tumor specific T cells. Our choice of the subcutaneous route for injection of the peptides was the logical consequence of the above cytological investigations in that it was our intention to elicit a cellular response based on APCs rather than a humoral immune reaction. APCs are present in skin at high frequency, with Langerhans constituting as much as 3-4% of the cell mass (Stingl, 1990). We surmised that these APCs could be engaged and thus initiate the immunological events which would lead to the production of anti-peptide (hence anti-tumor) CTLs capable of attacking the tumor cell challenge or pre-existing experimental metastases placed counterlaterally on the back of the mouse. A trivial finding is that in physiological saline solutions tumor antigen peptides are often quite insoluble and are possibly inactivated by precipitation in the injection medium. This can be circumvented by dissolving the peptide at low ionic strength and making the solution isotonic by addition of sorbitol (unpublished result). The JAK 1 peptide by itself as well as peptide solutions to which incomplete Feundâ&#x20AC;&#x2122;s adjuvant (IFA) had been added did not elicit any protection against tumor take in the P815 test system (Fig 2a, see also Schmidt et al., 1997a). When peptide was dissolved in the novel sorbitol medium tumors developed somewhat slower, however, 314

only administration of peptide in conjunction with polyarginine did result in protection levels comparable to a cellular vaccine secreting GM-CSF (Figure 2b). We hypothesize that the failure of peptides to elicit protection against tumor take may be caused by a lack of peptide uptake into APCs. Such a hypothesis is supported by the finding that at least in vitro APC do not take up small peptides efficiently, but do so, if polycations such as polylysine and polyarginine are included in the peptide medium (Buschle et al., 1997). Such an enhancement of peptide uptake can also be demonstrated to occurin vivo: Sectioning and immuno-staining the injection site after application of a fluorescently labelled small peptide clearly reveals importation of the small peptides into APCs when co-applied with polycations, but not, when these compounds are eliminated (F igure 3 and unpubl. results). As shown in Figure 2b, the JAK 1 peptide administered in the sorbitol medium together with polyarginine was as good as a GM-CSF secreting cellular vaccine and better than peptide injected in isotonic saline (Figure 2a). Hence, under appropriate conditions, the potency of peptide vaccines can equal that of a cytokine secreting cellular vaccine in the P 815 model system. The M3 melanoma yields similar answers, although the first generation results show the peptides from tyrosinase and tyrosinase related protein to be less potent (Figure 2c and Schmidt et al., 1997a). The less efficient protection against tumor take seen in the M3 system may be explained by a poor immunodominance of the peptides arbitrarily selected for this experiment. Experiments with

Gene Therapy and Molecular Biology Vol 1, page 315 the novel sorbitol medium are in progress. When comparing the peptide vaccines with cytokine secreting cellular vaccines, which have become the gold standard of immunotherapy of cancer, it may be concluded from the P815 experiments that peptide vaccines can be equally efficacious. The situation with the M3 experiment can only be judged once we have identified potent, immunodominant tumor antigen peptides. Also, as demonstrated by Boonâ&#x20AC;&#x2122;s group with a tumor antigen peptide (Marchand et al., 1995), humans may be less refractory to peptide vaccination so that even the incomplete protection seen in the M3 system may already be relevant for the human situation. However, the success of peptide vaccines will always depend on a cogent selection of peptides.

From the above we wish to conclude: l . Peptide vaccines can be made to work as efficiently as cytokine secreting cellular vaccines. 2. By contrast with cellular vaccines, peptide vaccines are chemically defined and can be easily standardised. 3. They are inexpensive and are available for vaccination without delay and have a long shelf-life. 4. Although todayâ&#x20AC;&#x2122;s focus is on developing vaccines to cure cancer or at least minimal residual disease of cancer, the availability of peptide vaccines has the potential to vaccinate prophylacticly to prevent the occurrence of cancer.

Figure 3: Immunohistological analysis of the injection site after application of peptide vaccines in the presence (A , B , C ) or absence (D, E) of polyarginine. Fluorescence labeled peptides were injected subcutaneously alone (D,E) or as a mixture of peptide with polyarginine (A,B,C). A , D: Fluorescence photomicrographs of injection sites 3 days after vaccination. Cryosections were stained with an antibody recognizing MHC class II followed by detection with a Texas Red tagged secondary antibody. Nuclei were counterstained with 4,6diamidino-phenylindole (DAPI). B , C , E : Confocal microscopy of injection sites 7 days after vaccination. Sections were processed as above except that DAPI was omitted.

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III. Cancer peptide vaccines and HLA heterogeneity

IV. Polyepitope proteins as vaccines Melief et al. (Melief et al., 1996) have recently suggested a general utility of polyepitope proteins to vaccinate against cancer. We wish to point out that when a TAA protein is degraded and some of its peptides ”presented”, much of the protein sequence is dead-weight because peptides derived from most of the TAA sequences will not fit a given MHC molecule. Now, it has been demonstrated (An and Whitton, 1997; Thomson et al., 1996; Thomson et al., 1995; Whitton et al., 1993) that polyepitope proteins consisting entirely of contiguous short CTL epitopes are correctly processed by the immune system to yield CTLs against each of the epitopes. Assuming that this is a general principle we have constructed a gene encoding an artificial protein, “SUPERMAGE”, which consists entirely of known short peptides derived from known melanoma TAA and TSA capable of generating CTLs in different HLA contexts (Figure 4). Thus, it would be expected on the basis of the artificial SUPERMAGE sequence that such a protein could be used as a melanoma vaccine, in an HLA setting of A1, A2, A24, A31, B44, and Cw6. Thus a majority of melanoma patients, could be vaccinated with this antigen. It is not surprising that at this stage HLA-A2 peptides are over-represented in the SUPERMAGE protein, reflecting the present day state of the art in CTL analysis. The corresponding gene sequence shown inFigure 4 contains restriction sites within the coding sequence for easy modification of the protein sequence, as new epitopes become known. Large quantities of the SUPERMAGE protein could rather easily be produced using standard recombinant technologies including expression of SUPERMAGE in insect cells. Our investigations of the injection sites following peptide vaccination revealed that invasion of the bolus of injected peptides requires several days F ( igure 3a, b and c). By contrast, peptides injected without adjuvants rapidly diffuse from the injection site and are undetectable at time points beyond 24 hours (Figure 3d, e and unpublished results). At day 7 numerous APCs are loaded with flourescence labelled peptides, but only when polyarginine is coinjected (Figures 3 b, c and e). Possibly, application of a larger polyepitope protein rather than peptide mixtures may enhance the efficiency of the vaccine by displaying decreased diffusion rates at least when injected subcutaneously. These ideas are now being tested in murine model systems. However, before such strategies including the generation of SUPERMAGE and related proteins of different kinds can be routinely implemented in man it will be necessary to establish the potency of individual epitopes (e.g. in form of peptides) and to determine what minimal number of each epitope in particular is necessary to elicit clinical benefits in different HLA contexts.

We are encouraged by these many positive aspects of peptide vaccines, but a discussion of this field would be incomplete without considering the downside and possible remedies thereof. The single most important impediment for the wide application of such vaccines is the dependence of the TSA and TAA peptide sequence on the HLA-type of the patient. Another type of heterogeneity one has to content with is the apparently diverse appearance of TAAs even within one class of tumors such as melanoma. Another issue which impinges on practicality of these vaccines, is whether different types of cancer share common antigens. The situation here is in flux and no final assessment is as yet possible. Each cancer type may ultimately require its own vaccine composition. A possible solution to some of these problems, for instance TSA and TAA heterogeneity within one tumor type, is to work with peptide mixtures where it is assumed that mismatched peptides are simply inert. The same approach may be useful when dealing with HLAheterogeneity although here the complexities are immense and vexing. One positive aspect is that certain HLA-types occur more frequently than others, as is the case for HLAA2 which in Caucasians is encountered in nearly half of the population. It can be calculated that by combining HLA-A2 with other relatively frequent HLA-types like A1, A3 and A24 approximately 80% of the Caucasian population will be covered on statistical grounds. This proportion can be increased by adding rarer types, but with diminishing returns and, in practice, 100% will never be attained. TSAs and TAAs, as is the case for cellular proteins, are degraded in the cytoplasm by the proteasome and the resulting peptides transported into the endoplasmatic internal space by the transporter associated with antigen processing (TAP) (York and Rock, 1996). Association of the peptides binding tightly to the groove of the MHC class I molecule occurs in small cellular vesicles which then fuse with the cell membrane, thus exposing the peptide-charged MHC molecule to the extracellular environment. The consequence of such a MHC dependent selection of peptides is that HLA-dependence in peptide vaccines can be largely circumvented by using the entire TSA or TAA for vaccination. The task then becomes that of developing efficient procedures to import the unprocessed (recombinant) TSAs and TAAs into APCs. We have ascertained that the polycations also enhance the entry of large proteins into APCs (unpublished results) and hence this approach should be applicable to protein vaccination. One major advantage of such protein vaccines is that class I as well as MHC class II restricted epitopes are contained within the sequence of a given protein and thus can be presented following entry into APCs (Germain and Margulies, 1993; Kovacsovics Bankowski and Rock, 1995; Watts, 1997; York and Rock, 1996).

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A. DNA sequence ggaattcgccgccATGGAAGCTGACCCGACAGGTCACAGTTACGAGGTAGATCCCATCGGGCATTTGTACTTTTTGTG GGGACCCAGGGCTTTGGTATACCGTCCCCGACCGCGACGTTACGTTCTCCCAGATGTGTTCATACGTTGTGCG TATGGCTTAGATTTTTACATATTAGCAGCCGGGATTGGAATCTTGACGGTAATCCTGACAGTAATCCTCGGTGT TTTAAAAACTTGGGGCCAATATTGGCAGGTAATCACTGACCAAGTTCCCTTTAGTGTTTACCTAGAACCGGGG CCCGTAACCGCGCTACTGGATGGTACGGCGACGCTACGGTTGGTACTCTATCGCTATGGAAGTTTTAGCGTAG TCTACTTCTTTCTACCAGACCACCTA GTCGACATGTTACTCGCTGTACTGTATTGTTTGTATATGAATGGCACT ATGTCACAAGTGAGTGAAATCTGGAGAGATATTGACTTTGCCTTCCTTCCCTGGCATAGACTATTCATGAGTTT ACAAAGGCAGTTCCTGCGCCTATTGCCGGGTGGGAGACCCTATCGTtaatctagatt

B. Amino acid sequence EADPTGHSY EVDPIGHLY FLWGPRALV YRPRPRRY VLPDVFIRC AYGLDFYIL AAGIGILTV ILTVILGVL KTWGQYWQV ITDQVPFSV YLEPGPVTA LLDGTATLRL VLYRYGSFSV VYFFLPDHL-VD-MLLAVLYCL YMNGTMSQV SEIWRDIDF AFLPWHRLF MSLQRQFLR LLPGGRPYR-Stop

C peptide EADPTGHSY EVDPIGHLY FLWGPRALV YRPRPRRY VLPDVFIRC AYGLDFYIL AAGIGILTV ILTVILGVL KTWGQYWQV ITDQVPFSV YLEPGPVTA LLDGTATLRL VLYRYGSFSV VYFFLPDHL MLLAVLYCL YMNGTMSQV SEIWRDIDF AFLPWHRLF MSLQRQFLR LLPGGRPYR

protein MAGE-1 MAGE-3 MAGE-3 GAGE-1 N-acetylglucosaminyl-transferase-V p15 Mart-1/Melan A Mart-1/Melan A gploo/pmell7 gploo/pmell7 gploo/pmell7 gploo/pmell7 gploo/pmell7 gploo/pmell7 tyrosinase tyrosinase tyrosinase tyrosinase TRP-1 TRP-2

MHC HLA-A1 HLA-A1 HLA-A2 HLA-Cw6 HLA-A2 HLA-A24 HLA-A2 HLA-A2 HLA-A2 HLA-A2 HLA-A2 HLA-A2 HLA-A2 HLA-A24 HLA-A2 HLA-A2 HLA-B44 HLA-A24 HLA-A31 HLA-A31

Figure 4: Artificial, polyepitopic SUPERMAGE antigen for the treatment of human melanoma of the HLA haplotypes A1, A2, A24, A31, B44 and Cw6. A) DNA sequence of the SUPERMAGE polypeptide. 5’ Eco R I and 3’ Xba I sequences were added for subcloning (underlined). A Sal I site was included for later modification of the molecule (underlined). The ATG start codon (bold) is preceeded by the sequence gccgcc to allowing efficient translation (Kozak, 1984). B ) Amino acid sequence of the SUPERMAGE antigen (single letter amino acid code). C) Peptides used in the SUPERMAGE antigen. For references see Table II.

peptides or multiepitope proteins are available, patients would be vaccinated with these defined vaccines. Because of the HLA heterogeneity, in practice probably around 80% of the patients (see above) can be anticipated to qualify for such a protocol, if the vaccine is tailored to the four most frequent HLA-types. Alternatively, patients could be treated with recombinant TSA, TAA proteins. If neither of the above are available, either because of a rare HLA genotype or lack of known antigens, the patients would proceed to treatment with either autologous or allogeneic cellular tumor vaccines, if either of these can be shown to be effective in the ongoing clinical trials. If there is no reoccurrence of metastases after peptide/protein vaccination, no further steps would be required. If there is

V. A schedule for curing of cancers ? Looking far ahead and assuming that both cellular and at least one form of peptide/protein vaccination can be shown to be efficacious in patients, the following scheme shown in Figure 5 may ultimately apply. For immunotherapy of cancer, tumors would be resected or, if indicated, patients would undergo chemotherapy or radiation therapy in order to remove the cancerous tissue. In this way cancer would be reduced to a minimal residual disease. Blood cells or excised tumor tissue could be used to determine the HLA-type of the patient and the tumor tissue would be screened for any known TSA and TAA by RT-PCR or immunological techniques. If the appropriate

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Figure 5: Potential treatment schedule for human cancers

reocurrence of tumor metastases, either because progression of tumors renders the vaccine ineffectual or the peptide epitopes prove insufficiently immunogenic, the patients would undergo remission-inducing conventional therapy followed by treatment with cellular vaccines at a later stage. It is hoped that by applying the above scheme at least select patient populations will benefit from immunotherapy of cancer.

Acknowledgements We would like to thank Drs. Cotten and Bello-Fernandez for critical reading of the manuscript and Ivan Botto for preparation of antibodies. This work was supported in part by the Vienna Business Promotion Fund (WWFF).

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Thomson, S. A., Khanna, R., Gardner, J., Burrows, S. R., Coupar, B., Moss, D. J., and Suhrbier, A. (1 9 9 5 ). Minimal epitopes expressed in a recombinant polyepitope protein are processed and presented to CD8+ cytotoxic T cells: implications for vaccine design. Proc Natl Acad Sci U S A 92 , 5845-5849.

Watts, C. (1 9 9 7 ). Capture and processing of exogenous antigens for presentation on MHC molecules. Annu Rev Immunol 15 , 821-850.

Tindle, R. W. (1 9 9 6 ). Human papillomavirus vaccines for cervical cancer. Curr Opin Immunol 8, 643-650.

Whitton, J. L., Sheng, N., Oldstone, M. B., and McKee, T. A. (1 9 9 3 ). A "string-of-beads" vaccine, comprising linked minigenes, confers protection from lethal-dose virus challenge. J Virol 67 , 348-352.

Topalian, S. L., Gonzales, M. I., Parkhurst, M., Li, Y. F., Southwood, S., Sette, A., Rosenberg, S. A., and Robbins, P. F. ( 1 9 9 6 ). Melanoma-specific CD4+T cells recognize nonmutated HLA-DR-restricted tyrosinase epitopes. J Exp Med 183, 1965-1971.

Wolfel, T., Hauer, M., Schneider, J., Serrano, M., Wolfel, C., Klehmann-Hieb, E., De Plaen, E., Hankeln, T., Meyer zum Buschenfelde, K.-H., and Beach, D. (1 9 9 5 ). A p16INK4ainsensitive CDK4 mutant targeted by cytolytic T lymphocytes in a human melanoma. S c i e n c e 269, 12811284.

Topalian, S. L., Rivoltini, L., Mancini, M., Markus, N. R., Robbins, P. F., Kawakami, Y., and Rosenberg, S. A. (1 9 9 4 ). Human CD4+ T cells specifically recognize a shared melanoma-associated antigen encoded by the tyrosinase gene. Proc Natl Acad Sci U S A 91 , 94619465.

Wolfel, T., Schneider, J., Meyer zum Buschenfelde, K. H., Rammensee, H. G., Rotzschke, O., and Falk, K. (1 9 9 4 ). Isolation of naturally processed peptides recognized by cytolytic T lymphocytes (CTL) on human melanoma cells in association with HLA-A2.1. Int J Cancer 57 , 413418.

Traversari, C., van der Bruggen, P., Luescher, I. F., Lurquin, C., Chomez, P., Van Pel, A., De Plaen, E., Amar Costesec, A., and Boon, T. (1 9 9 2 ). A nonapeptide encoded by human gene MAGE-1 is recognized on HLA-A1 by cytolytic T lymphocytes directed against tumor antigen MZ2-E. J Exp Med 176, 1453-1457.

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Buschle et al: Chemically defined, cell-free cancer vaccines B., Meyer zum Buschenfelde, K. H., and Boon, T. (1 9 9 4 ). Two tyrosinase nonapeptides recognized on HLA-A2 melanomas by autologous cytolytic T lymphocytes. Eur J Immunol 24 , 759-764. York, I. A., and Rock, K. L. (1 9 9 6 ). Antigen processing and presentation by the class I major histocompatibility complex. Annu Rev Immunol 14 , 369-396. Zatloukal, K., Schneeberger, A., Berger, M., Schmidt, W., Koszik, F., Kutil, R., Cotten, M., Wagner, E., Buschle,

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Gene Therapy and Molecular Biology Vol 1, page 323 Gene Ther Mol Biol Vol 1, 323-332. March, 1998.

Somatic transgenesis by immunoglobulin genes Sidong Xiong, Mara Gerloni and Maurizio Zanetti The Department of Medicine and Cancer Center, University of California, San Diego, 9500 Gilman Drive, La Jolla CA 92093-0063 _______________________________________________________________________________________________ _ Correspondence: Maurizio Zanetti Tel: (619) 534-7217 Fax: (619) 534-5792 Email: mzanetti@UCSD.edu

Summary In this chapter we describe and discuss somatic transgenesis produced in adult immunocompetent mice using plasmid DNA containing immunoglobulin genes under control o f tissue-specific regulatory elements. We review our experience to date and discuss the findings in relation to the known rules for intracellular usage of immunoglobulin genes i n activated and differentiated B c e l l s . B e c a u s e i m m u n o g l o b u l i n g e n e s a r e controlled by B lymphocytes specific promoter and enhancer elements, somatic transgenesis i s a new approach t o selective targeting o f B lymphocytes i n v i v o for transcription and long-term expression o f exogenous immunoglobulin genes. Owing to the fact that transgenic immunoglobulins synthesized and secreted i n v i v o are immunogenic for the host and that immunoglobulin genes can be engineered t o code for heterologous epitopes, ligands or receptors, somatic transgenesis offers unique features for the development of new strategies of DNA-based immunization and gene therapy.

I. Introduction Advances in molecular medicine are based on the possibility to efficiently deliver genes into specialized tissues. Two essentially independent disciplines are predicated on this technological approach, gene therapy (Mulligan, 1993) and DNA vaccination (Cohen, 1993; Donnelly et al., 1997). In both instances success is determined by a series of factors all of which depend on the efficiency of gene delivery and gene expression in vivo. Strategies have been developed to realize gene delivery via receptor mediated pathways (Ferkol et al., 1995; Ferkol et al., 1996; Wu et al., 1989) exploiting specific structures on somatic cells and their mechanisms to internalize and transport macromolecules. Targeted delivery of DNA also needs to be gauged through the specificity of regulatory elements, i.e., promoters and enhancers, which allow the transgene to be transcribed and translated only in selected tissues. The efficiency of these processes constitutes the rate limiting factor for in vivo efficacy of both gene therapy and DNA vaccination. The use of polynucleic acid for vaccination is complicated by the necessity to achieve sufficient secretion of the transgene product in an immunogenic form. An ideal DNA vaccine should yield molecules with sufficient conformational resemblance to the native antigen to elicit immunity with the greatest cross-reactive potential. In 323

addition, the process of DNA vaccination should also be tailored to include in the process the participation of antigen-presenting cells to heighten both humoral and cellular immune responses. It is based on this reasoning that we developed a new, rational method of immunization using DNA molecules, somatic transgene immunization (STI), to combine selective targeting of B lymphocytes and their antigenpresenting capacity with expression of conformationally constrained antigen molecules (Gerloni et al., 1997). We directed our considerations to immunoglobulin (Ig) genes as â&#x20AC;&#x153;immunogensâ&#x20AC;? in DNA vaccination. In this chapter we will review what known about the principles of immunogenicity by DNA and subsequently describe our findings on somatic transgenesis as they relate to in vivo targeting of B lymphocytes.

II. DNA and immunogenicity DNA itself is scarcely immunogenic as it has proven extremely difficult to induce a response against DNA (Madaio et al., 1984) even though there exist clinical situations in which autoantibodies to double and single stranded DNA are produced (Koffler et al., 1969; Pincus et al., 1969; Tan et al., 1966). The immunogenicity of DNA per se depends on its origin, i.e., eukaryotic or prokaryotic. For instance it was shown that mice

Xiong et al: Somatic Transgenesis by Immunoglobulin Genes immunized with Escherichia coli DNA complexed with methylated BSA in adjuvant produce significantly greater amounts of antibodies than mice similarly immunized with calf thymus DNA (Gilkeson et al., 1989; Gilkeson et al., 1989). This indicated that DNA molecules differ in their immunogenic potential, a characteristic likely due to unique sequences or structures present in bacterial DNA but rarely in mammalian DNA (Gilkeson et al., 1989; Gilkeson et al., 1989). Bacterial DNA possess immunostimulatory properties (Tokunaga et al., 1984), is mitogenic for B cells and induces polyclonal antibody production (Messina et al., 1993), and enhances the lytic activity of natural killer cells with production of IFN-! (Tokunaga et al., 1984; Yamamoto et al., 1992). This stimulatory properties are linked to a six-base nucleotide motif consisting of an unmethylated CpG dinucleotide (Krieg et al., 1995) expressed nearly twenty times more frequently in bacterial than in vertebrate DNA (Cardon et al., 1994). In 1992 Tang and coworkers reported that inoculation of plasmid DNA induces specific immunity in vivo (Tang et al., 1992). There is a fundamental difference between Tangâ&#x20AC;&#x2122;s accomplishment and previous attempts to immunize against DNA as it showed that it was possible to use functional genes to generate immunity against a specific gene product. Implicit in this discovery was the fact that factors regulating gene expression would also regulate immunogenicity in vivo. Many reports have appeared since demonstrating the use of plasmid DNA in eliciting immunity against viruses (Davis et al., 1993; Raz et al., 1994; Ulmer et al., 1993; Wang et al., 1993), bacteria (Huygen et al., 1996; Tascon et al., 1996), parasites (Doolan et al., 1996; Sedegah et al., 1994; Xu and Liew, 1995), tumor antigens (Conry et al., 1994), self antigens (Gilkeson et al., 1996; Waisman et al., 1996) and allergens (Hsu et al., 1996; Raz et al., 1996). The rapidly raising hope to develop simple and cost effective methods of vaccination was followed by the demonstration that plasmid DNA could be used to induce antibody responses (Cox et al., 1993; Davis et al., 1994; Fynan et al., 1993; Raz et al., 1994; Robinson et al., 1993; Sedegah et al., 1994; Ulmer et al., 1993; Wang et al., 1993) as well as cell-mediated responses of helper (Xiang et al., 1994) or cytotoxic T-cell type (Raz et al., 1994; Sedegah et al., 1994; Ulmer et al., 1993; Wang et al., 1993).

III. The development of somatic transgene immunization Immunization via DNA inoculation relies on in vivo transfection, production and possibly secretion of the transgene product, and antigen presentation by specialized cells. However, in most studies neither the in vivo transfected cells nor the antigen presenting cells involved in the process have been identified. Most experiments use foreign DNA under the control of viral promoters which have limited tissue specificity. Therefore, no tissue-

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specific control of expression is possible other than the site of DNA inoculation. Since the quality of nucleic acids and gene expression are intimately connected with the vaccination process we began studies considering together three factors that, alone or in combination, could affect the success of DNA immunization: (i ) the efficiency of in vivo transfection including DNA uptake by the host cells, (i i ) the efficiency with which transfected cells can utilize the DNA and synthesize the transgene product, and (i i i ) the ability of in vivo transfected cells to serve as antigen-presenting cells (APCs). Most reports still indicate that expression of the transgene, synthesis of the corresponding gene product, its presentation to immunocompetent cells and induction of immunity occur after repeated inoculations of plasmid DNA (Whalen and Davis, 1995). The genetic organization and the molecular events leading to expression of immunoglobulin genes are well, albeit not completely, understood and consist in a cascade of tissue-specific genetic events occurring during B-cell differentiation and resulting in the synthesis of immunoglobulin molecules (Alt et al., 1987; Sleckman et al., 1996). Studies in vitro have shown that transfection of B cell lymphomas with rearranged Ig gene is followed by a prompt utilization of the transgene and by secretion of immunoglobulins encoded by the transgene (Morrison, 1985). A variety of rearranged functional Ig genes have also been introduced in the germline to create mice with homogeneous antigen receptor expression on B cells and secretion of immunoglobulins with identical chemical and immunologic characteristics (Storb, 1987). It is then obvious that DNA immunization with Ig genes, e.g., heavy (H) chain genes under the control of their own tissue-specific promoter and enhancer elements (Banerji et al., 1983; Gillies et al., 1983; Grosschedl and Baltimore, 1985; Mason et al., 1985) could be a way to target B lymphocytes and satisfy basic requirement for immunogenicity such as synthesis and secretion of the transgene product, and antigen presentation. In our first experiments we inoculated adult C57Bl/6 mice with a plasmid DNA containing a chimeric (mouse/human) immunoglobulin H chain gene with tissue-specific promoter and enhancer elements. We immediately observed that a single intraspleen inoculation was followed by (i) uptake and persistence of the transgene in B lymphocytes for 3-4 months, (ii) secretion of transgenic immunoglobulins (transgene H chain + endogenous L chain) in amounts ranging between 15 and 30 ng/ml, and (iii) production of IgM anti-Ig antibodies. The circulating H chain transgene product was found to be associated prevalently with " light (L) chains a fact per se not surprising since " L chains represent 95% of the pool of genes utilized in vivo in the mouse. A booster immunization with Ig coded by the same transgene at various times after priming with DNA showed the generation of a typical secondary immune response with IgG1 and IgG2b antibodies (Gerloni et al., 1997). Thus, a single inoculation of an Ig H chain gene targeted to spleen lymphocytes was sufficient to initiate immunity

Gene Therapy and Molecular Biology Vol 1, page 325 and establish immunologic memory. We termed this process somatic transgene immunization (STI) to reflect the fact that a foreign gene is transported inside somatic cells and initiates immunity (Gerloni et al., 1997). It became evident that from this point on the physiological machinery provided by STI could be exploited to program oneâ&#x20AC;&#x2122;s individual immune response in a rational way.

IV. Investigations on the transgene Intraspleen inoculation of the H chain transgene resulted in immunity and established immunologic memory. To explore the basis for this phenomenon we undertook a systematic analysis of (i ) the tissue

These studies were based on an Ig H chain gene purposely engineered in the CDR3 of the VH domain to have a 36 base-pair exogenic molecular marker F ( igure 1).

A. Tissue distribution Genomic DNA extracted from various lymphoid (i.e., spleen, lymph-nodes and bone-marrow) and non-lymphoid (i.e., liver, kidney, lung and muscle) tissues explanted at different times, was analyzed for specific amplification of the transgene VDJ by PCR and Southern blot hybridization. An amplification product was readily visible in splenic genomic DNA. No specific amplification occurred in any of the other tissues (Table 1). This did not vary at any of the time points analyzed. To control for specificity and increase the sensitivity of the reaction, two additional PCR assays were performed using primers designed to anneal sites within the VDJ region. One set of primers (pSE/pNAD) specifically amplified the molecular maker; another (inner primers: pNEL/pNED) served for nested PCR (Figure 2). The results confirmed those obtained with VDJ amplification. Southern blot analysis using a probe specific for the molecular marker further confirmed the PCR results (Table 1). Thus, after intraspleen inoculation the transgene H chain persists in vivo for a period of 3 months in the organ in which it was inoculated.

B. Fate of the transgene

F i g u r e 1 . Schematic representation of plasmid !1NANP. The plasmid contains the Ig H chain construction which is the product of the fusion between the productively rearranged murine VH62 gene and the human !1 C region gene present in genomic configuration in plasmid vector pNeo!1 (Sollazzo et al., 1989). The VH gene was modified by insertion in the CDR3 of 36 bp heterologous sequence coding for three repeats of the amino acid sequence Asn-Ala-Asn-Pro (NANP). The plasmid DNA carries the regulatory elements, promoter (Pr) r and enhancer (En) needed for tissue-specific expression. Neo r = neomycin and Amp = ampicillin, are the resistance genes. CDR = complementarity-determining region. FR = framework region.

distribution of the transgene, (i i ) its fate in vivo over time, (i i i ) the cell type involved in targeting, and (iv) the potential for somatic mutation of the transgene in vivo.

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PCR and Southern blot hybridization were also used to monitor the kinetics of the presence of the transgene in vivo in mice analyzed at various times after DNA inoculation (Xiong et al., 1997). Amplification of the transgene VDJ region was visible up to 12 weeks after a single DNA inoculation. No amplification was seen at subsequent time points (16, 24, 36 and 52 weeks) (Table 1). Southern blot hybridization with the marker-specific probe further confirmed the PCR results (Table 1).

C. The transgene is harbored in B lymphocytes As discussed above mice undergoing STI produce transgene Ig (15-30 ng/ml) for a protracted period of time in vivo. Coupled with the demonstration that the transgene could only be found in the spleen (the organ of inoculation) and consistent with the use of a gene under control of promoter and enhancer elements specific for B lymphoid cells, it became obvious that B lymphocytes could be the cell population accounting for the initiation of STI (i.e., transgene uptake, the persistence of the transgene, its transcription and secretion of transgenic Ig).

Xiong et al: Somatic Transgenesis by Immunoglobulin Genes

F i g u r e 2 . Schematic representation of the VH gene contained in plasmid !1NANP DNA. The annealing sites of the primers, the predicted amplification fragments and their molecular size, are identified. VDJ refers to a fragment inclusive of the coding region for the rearranged V-D-J gene segments; (NANP)3 refers to a 384 bp fragment containing the coding sequence for three NANP repeats in the CDR3 of the VH region between nucleotides 304-340; NESTED refers to a 198 bp fragment inclusive of the coding region for FR3 and the CDR3. Any other position in the gene is numbered in reference to nucleotide +1 (the first nucleotide in the coding region of FR1).

Table 1. PCR amplification and Southern blot hybridization of the transgene i n v i v o Time (weeks) 0* 4 12 16 24 52

Spleen +/+ +/+ -

Lymph nodes -

Bone Marrow -

Liver

Kidney

Lung

Muscle

* Time zero refers to results generated with tissues extracted from a naive mouse (negative control). PCR amplification of the murine #-actin gene served as an internal control. Plus signs correspond to positive PCR amplification (+/ ) and positive Southern blot hybridization ( /+)

To formally demonstrate this assumption we analyzed populations of spleen B and T cells (Xiong et al., 1997). Splenic B and T lymphocytes were isolated to a high degree of purity (97-99%) by FACS sorting and their respective genomic DNA was amplified by PCR. Twentyeight days after DNA inoculation distinct amplification products were detected in B lymphocytes (Figure 3). Southern blot hybridization confirmed the specificity of the amplification products. It appears, therefore, that B lymphocytes in the spleen are the cell population targeted by the Ig H chain gene. The transgene could not be amplified from peripheral blood lymphocytes.

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Questions yet to be answered are “How is the transgene internalized in B cells?” and “How many B cells are part of this process in vivo?” As to the first question several possibilities are considered such as uptake of plasmid DNA by B cells may have occurred either through surface Ig specific for DNA (Glotz et al., 1988; Holmberg et al., 1986) or through the non-Ig receptor for DNA described in murine and human lymphocytes (Bennett et al., 1985). As to the second question we have no definitive answer yet. Suffice it to say that the phenomenon of STI is supported by a limited number of B cells transfected in vivo. We estimated these cells to be fewer than 5x104/spleen or 0.07% of total cells.

Gene Therapy and Molecular Biology Vol 1, page 327

D. Integration in the host genome Protracted secretion of transgenic Ig led us to consider the possibility of integration of the H chain transgene in the host genome. Two approaches were pursued. In the first one (Figure 4), genomic DNA extracted from splenic tissue harvested 17 days after DNA inoculation was analyzed using a multiplex PCR approach (Chen et al., 1994; Daniel et al., 1995; Donaldson et al., 1993) with ELONGASE, a mix of DNA polymerases with improved proofreading activity for amplification of large (<20 Kb) DNA fragments, and seven pairs of specific primers to amplify seven different fragments of plasmid ! 1WT DNA. Primers were designed to facilitate the detection of

F i g u r e 3 . Isolation of splenic B and T lymphocytes and detection of the H chain transgene in the purified lymphocyte populations. B and T lymphocytes from the spleen of DNAinoculated mice were sorted and purified on a fluorescenceactivated cell sorter (FACS) 28 days after DNA inoculation. From left to right the lanes are as follows: lane 1 - fragment amplified with the primers pCL/pCD (VDJ); lane 2 - fragment amplified with the primers pSE/pNAD [(NANP) 3 ]; lane 3 fragment amplified with the primers pNEL/pNED (NESTED); lane 4 - fragment amplified with the primers pbA1/pbA2 specific for the murine #-actin gene (internal control).

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fragmented plasmid and integration break-points in the plasmid, if they occurred (i.e., failure to amplify one or several DNA segments in the genomic DNA would demonstrate integration). In experiments repeated under a wide range of MgCl 2 molar concentrations as well as different annealing temperatures two fragments consistently failed to amplify. This pattern indicated a break-point localized in or around the neomycin resistance gene (Gerloni et al., 1997). In the second approach (Figure 5) genomic DNA from splenic tissue was first digested with Xba I and subsequently ligated and amplified (Xiong et al., 1997). We reasoned that a pattern of multiple molecular size products (smear) would suggest integration of the transgene whereas a single band of m.w. ~ 15 kb would suggest persistence of the transgene in episomal form. Both genomic DNA extracted from the spleen 4 and 12 weeks after plasmid DNA inoculation gave rise to amplified products of multiple molecular sizes (a smear). No such a pattern was observed in the genomic DNA extracted 36 weeks after inoculation, consistent with the kinetics of transgene detection. The presence of a nonintegrated (episomal) form of the transgene (in addition to the integrated one) was sought by PCR amplification in which again an ELONGASE mix was used to ensure amplification of large ($ 20 kb) DNA fragments. We reasoned that if plasmid DNA exists in episomal form, a

F i g u r e 4 . Schematic representation of multiplex PCR analysis to determine integration of the immunoglobulin H chain transgene into chromosomal DNA. Plasmid !1WT-TAC (to scale) and localization of plasmid fragments (A through G) used to analyze integration. P1 through P7 refer to specific primers and their topographical site of annealing. Neor = neomycin and Ampr = ampicillin are the resistance genes. Plasmid !1WT-TAC is in every respect identical to plasmid !1NANP with exception of the NANP-coding sequence which has been deleted.

Xiong et al: Somatic Transgenesis by Immunoglobulin Genes sharp band of molecular size corresponding to the reference plasmid DNA would be seen. No sharp band corresponding to the reference plasmid DNA was observed. We concluded that only the integrated form is present. By Southern blot the hybridization pattern was similar (smear) to the amplified PCR products.

E. Lack of somatic mutation The antigenic and immunogenic potential of a transgene-encoded product relies on the fact that no sense somatic mutation will affect the nucleotide sequence of the transgene while this is harbored in vivo. Hypermutation occurs frequently in the VDJ region of Ig, mainly in the CDRs, in agreement with the notion that hypermutation takes place during antigen selection and affinity maturation of the antibody response (Griffiths et al., 1984). Although

Figure 5 . Schematic representation of the approach and rationale used to demonstrate integration of the transgene. Genomic DNA is digested with Xba I which will cut (X) at multiple sites in the chromosomal DNA and in two sites in the transgene H chain. The digested DNA fragments are then recircularized with T4 DNA ligase and subsequently amplified by PCR using a set of primers (p62L/p62U) designed to anneal and extend in opposite directions (see ). If genomic DNA contains the H chain transgene (integration), PCR amplification will give origin to a multitude of DNA products within an extended range of molecular sizes. This will be reflected in a pattern of diffuse gel migration (smear). Symbols are as follows: ( ) = splenic genomic DNA; ( ) = backbone of plasmid DNA g1NANP; (X) = Xba I site; VH = variable region of the transgene H chain; C(g1) = constant region of the IgG1 subclass of the transgene H chain.

F i g u r e 6 . Schematic representation of the four main events relevant to somatic transgenesis by immunoglobulin genes.

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Gene Therapy and Molecular Biology Vol 1, page 329 Table 2. Lack of transgene mutations in PCR-generated clones from splenic genomic DNA TIME (Weeks)

No. of Clones Sequenced

No. of Clones Mutated

No. of Nucleotides Mutated

Rate of Mutation* (%)

1/6

1**

0/3

2.9x10 -4

* Number of mutations /total number of base pairs sequenced. ** A silent (C ->T) mutation in FR3.

the Ig H chain gene used lacks a transmembrane domain, rendering cell surface anchoring unlikely, experiments were performed to assess accumulation of mutations as a result of protracted in vivo persistence in integrated form. The transgene VDJ region was amplified from splenic genomic DNA, subcloned and sequenced by the dideoxy termination method. No evidence of hypermutation was found in the VDJ region of the transgene in vivo even after 3 months (Table 2).

V. Concluding remarks The studies presented in this chapter indicate that the biological phenomenon of somatic transgenesis relies on series of cellular events which we begin to understand and which seem to fulfill most of the conditions posed as requirements for a new, rational approach to DNA-based immunization. For sake of brevity our comments will concentrate on four points essential in our opinion to understand the phenomenon and the future potential of our work (Figure 6). First, is targeting of B lymphocytes in vivo. As mentioned above two main considerations guided our experiments in this direction: One consideration is that B lymphocytes have receptors with specificity for DNA. This makes it possible that DNA, which sticks to the surface of a cell due to its negative charge, is subsequently internalized by receptor-mediated endocytosis. As pointed out both Ig and non-Ig receptors for DNA exist on normal B lymphocytes (Bennett et al., 1985; Glotz et al., 1988; Holmberg et al., 1986). Presumably, after internalization only a fraction of the DNA resists degradation in the endosomes and reaches the cytosol. For instance, 24 hours after in vivo receptor mediated gene transfer there exists only 1 copy of DNA per cell compared to 100 copies at four hours (Wilson et al., 1992). To analyze conditions of binding and internalization new experiments are in progress to determine whether the same effect can be obtained in vitro. The other consideration is that tissue specificity is provided by the Ig gene regulatory elements (see below).

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Second, is integration. Studies in mice transgenic for Ig genes have sufficiently shown that transgenes are generally integrated in multiple tandem copies at one or a few sites in the host genome. Importantly, integration does not occur into the homologous H or L locus (Storb, 1987). Similarly, somatic cell hybridomas and nonsecreting B-cell lymphomas transfected with Ig genes both harbor integrated foreign gene(s) (Morrison, 1985) randomly. On the other hand, site specific integration can be achieved using suitably modified expression vectors such as replacement (Kardinal et al., 1996) or integration (Lang and Mocikat, 1994) vectors. From the foregoing, it does not surprise that during somatic transgenesis integration occurs randomly. It is quite likely that the transgene enters the nucleus during cell division (Figure 6). An aspect intimately connected with integration of the transgene and its expression in B cells is its relation with the endogenous Ig gene since, as a rule, a single B cell expresses only one H chain together with one L chain, allelic exclusion (Pernis et al., 1965). Although at this stage we have no data in favor or against allelic exclusion during somatic transgenesis, it is of note that germ-line transgenic mice have variably shown some leakage and that the endogenous Ig gene is expressed together with the transgene product (Storb, 1995). Apart from the cell biological relevance, even partial lack of allelic exclusion in somatic transgenic cells would lead to secretion of mixed antibody molecules. Further studies will need to address this point and their potential implication for immunization. Third, is the process of transcription and translation. Possibly this is the step that confers the highest degree of selectivity to the entire process. It is well known that Ig gene expression is restricted to lymphoid cells and among them to cells of the B-cell lineage (Banerji et al., 1983; Gillies et al., 1983; Grosschedl and Baltimore, 1985; Mason et al., 1985). Therefore, the use of B cell specific promoter and enhancer elements introduces a stringent control mechanism on tissue specificity and utilization of the transgene in vivo. One essential aspect to a full understanding of the events starting somatic transgenesis is

Xiong et al: Somatic Transgenesis by Immunoglobulin Genes â&#x20AC;&#x153;How does the first B cell become activated and how does this B cell begin to produce transgenic Ig molecules?â&#x20AC;? A plausible explanation is that bacterial DNA of the plasmid backbone possesses immunostimulatory properties for B cells (Messina et al., 1993; Tokunaga et al., 1984), an activity mediated by a six-base, unmethylated CpG dinucleotide (Krieg et al., 1995). Thus, activation of B cells by plasmid DNA may be crucial to initiate transcription and translation, and ultimately to set in motion the immunogenic process. Fourth, is somatic mutation. It was appreciated early on that Ig genes isolated from myelomas and hybridomas are mutated in the V region compared with the corresponding germ-line (Crews et al., 1981). Hypermutation occurs frequently in the VDJ region, mainly in the CDRs. This is commonly explained with hypermutation taking place during antigen selection and affinity maturation of the antibody response, and is an important means to increase antibody diversity (Griffiths et al., 1984). Hypermutation arises through one of two mechanisms: antigen selection or intrinsic mutational bias independent of selection. In the first case it is required that the Ig is expressed at the surface of B lymphocytes for antigen to exert selective pressure. As indicated in Figure 1 the transgene used in our studies lacks a transmembrane domain. This renders cell surface anchoring unlikely with no possibility for somatic mutation to occur via this mechanism. Another consideration is that we used a H chain transgene coding for an already rearranged V region segment, hence ruling out the possibility of mutations introduced during rearrangement. Others have shown that transgenic mice engineered with already rearranged " chain genes do not mutate unless hyperimmunization is in place (O'Brien et al., 1987). In the second case, i.e., transcriptional error or transcription-driven hypermutation, there appears to be a dependence on the physical distance between the exon and the promoter element (Peters and Storb, 1996). This does not seem to apply to our model of somatic transgenesis as no mutation was found among the clones examined, albeit only a limited number of clones at selected time points were sequenced (Table 2). Understanding lack of transcription-driven hypermutation during somatic transgenesis will need to be addressed with further studies. In conclusion, we have shown and discussed the use of H chain Ig genes in somatic transgenesis as an in vivo step of targeted transgene expression which preceding the phase of immunogenicity which is central to our attempts to develop a new rational approach to immunization, somatic transgene immunization. Others have used a similar rationale to target B lymphocytes in vivo using retroviral vectors for stable transgene expression (Sutkowski et al., 1994). The considerations made in this paper are relevant to better understand the nature of somatic transgenesis and to its future applications for DNA vaccination and gene therapy.

Acknowledgments This work was supported by NIH grant AI36467. During the performance of the experiments reported in this article S.X. was on leave of absence from Shanghai Medical University of the Peopleâ&#x20AC;&#x2122;s Republic of China.

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Gene Therapy and Molecular Biology Vol 1, page 333 Gene Ther Mol Biol Vol 1, 333-344. March, 1998.

Applications of gene therapy in transplantation Mary E. White-Scharf1, Papia Banerjee 1, David H. Sachs 2, and Christian LeGuern2 (1) BioTransplant, Inc., Charlestown, MA and (2) Transplantation Biology Research Center, Massachusetts General Hospital/Harvard Medical School, Boston, MA. _________________________________________________________________________________________________________ _ Correspondence: Mary E. White-Scharf, Tel: 617-241-5200 ext. 209, Fax 617-241-8780, E-mail: Mary.WhiteScharf@Biotransplant.com

Summary The success o f organ transplantation i s limited by the availability o f donor organs and the requirement for long-term immunosuppression. Use of animal organs would circumvent the organ shortage, and the swine is the most likely candidate to serve as donor for xenotransplantation. Establishing specific tolerance t o donor organs would eliminate the need for chronic immunosuppression. Specific tolerance to transplantation antigens across major histocompatibility complex (MHC) barriers has been demonstrated by establishing mixed bone marrow chimerism in the recipient. An alternative approach to establishing chimerism is the use of gene therapy to transduce autologous bone marrow cells with a vector containing one or more MHC antigens. Previous studies in miniature swine have shown that tolerance can be established across allogeneic barriers when a single MHC antigen is shared between donor and recipient. A gene therapy approach is particularly attractive in xenotransplantation since the availability of MHC-inbred miniature swine as donors allows the use of a single vector for transferring swine MHC genes into recipient progenitor cells. In this article, we review the basics for using a gene therapy approach to achieve specific immune tolerance and describe the models being used to evaluate its efficacy in allogeneic as well as in xenogeneic organ transplantation.

I. Introduction Gene transfer methodology for treating human disease was first used in 1990 under an FDA approved protocol for treating patients with adenosine deaminase deficiency (Reviewed in Anderson, 1992; Miller, 1992). Since then, over 100 protocols have been approved by the Recombinant Advisory Committee in the United States (Hodgson, 1995) for a variety of applications. Some of these applications have involved transplant of bone marrow cells which have been transduced with vectors carrying replacement genes for enzyme deficiencies or defects in hematopoietic cell function (Karlsson, 1991; Hoogerbrugge et al., 1996). Recent studies in large animal models suggest that gene therapy approaches can be used to establish tolerance to allogeneic antigens, enabling the transplant and long-term survival of solid organs between fully mismatched donor/recipient pairs (Emery et al., 1997). This application should be extremely useful in xenotransplantation where the donor species is well defined immunologically, such as with 333

MHC inbred miniature swine, and the supply of organs is not limited (Sachs, 1994, Greenstein and Sachs, 1997).

II. Current status of clinical transplantation In order to understand the potential application of gene therapy in solid organ transplantation, it may be helpful to review briefly some of the current issues in clinical transplantation. The development of new immunosuppressive therapies in recent years has resulted in a dramatic increase in patient survival rates for the first five years following organ transplantation. Unfortunately, this increase in success has enhanced the problem of meeting the demand for available human donor organs. Each year, the demand for donor organs increases at a rate disproportionate to the number of human organs available (United Network for Organ Sharing, Scientific Registry, March 1996). Furthermore, the increase in early survival rates has not been sustained long-term (>5 years). Those

White-Scharf et al: Applications of Gene Therapy in Transplantation surviving long-term frequently experience a decrease in the quality of life as a result of life-long immunosuppression and episodes of chronic rejection. Prolonged immunosuppression often leads to increased infections, an increased incidence of cancer, and complications associated with drug toxicity. Such complications lead to repeat hospitalizations and increased healthcare costs. Generally speaking, it could be said that there are two major limitations on the current success of transplantation: the shortage of human donor organs available and the requirement for long-term immunosuppression.

A. Overcoming the problem of organ shortage The first limitation, that of organ shortage, has not been diminished by increased education and presumed consent laws. It could be eliminated, however, by the use of animal organs. Several groups are currently pursuing the technology to enable xenotransplantation. The miniature swine appear to be the optimal donor for xenotransplantation for a variety of reasons (Sachs, 1994). Their size is comparable to that of a human. The physiology of swine organs and immune system is remarkably similar to that of humans (Cooper et al., 1991). Their reproductive patterns are such that organs are readily available. Miniature swine have a lower potential for zoonotic infections than do primates as witnessed by the fact that they have co-existed with humans for thousands of years. The large majority of pig diseases are known, have been characterized, and can be eliminated from source herds by barrier housing techniques (Michaels and Simmons, 1994; Fishman, 1994). Furthermore, the use of swine organs engenders less ethical concern than would the use of primate organs. Over ninety million pigs are used annually for food in the U.S. alone.

III. Avoiding long-term immunosuppression The second limitation, long-term immunosuppression, could be eliminated by developing clinical protocols for the induction of specific immune tolerance to donor organs. It has been demonstrated that specific tolerance to transplantation antigens across major histocompatibility complex (MHC) barriers can be achieved by establishing allogeneic or xenogeneic mixed bone marrow chimerism in the recipient (Sharabi et al., 1990; Sharabi et al., 1992; Sykes et al., 1994; Kawai et al., 1995). Hematopoiesis gives rise to lymphoid precursors which migrate to the thymus. Within the thymic environment, T cells develop and undergo both positive and negative selection to maintain the so-called “protective” T cells while eliminating those which react with “self” antigens. This 334

results in a tolerance to “self”. Mixed bone marrow chimerism can be established by engrafting pluripotent donor progenitor cells into an immunosuppressed recipient. In this case, both donor and recipient progenitors undergo selection in the thymus and become educated to recognize each other as "self". That implies that an organ derived from the same donor as the bone marrow and subsequently transplanted into the mixed marrow recipient will be treated as "self". Such tolerance induction has been observed in both rodents (Ilstadt, 1984, Sharabi and Sachs 1989) and large animals (Smith et al., 1992; Kawai et al., 1995) which have intact immune systems capable of reacting to foreign antigens. This is evidenced by the fact that organs from an unrelated or third party donor are rejected (reviewed in Sachs, 1995).

IV. Structural and functional similarities between porcine and human MHC The general use of large animal models to explore the importance of MHC gene matching in graft survival has been difficult because of the lack of genetically defined animal models. The MHC inbred miniature swine are uniquely well suited for such studies because of the similarity of the pig MHC locus (SLA) to its human HLA counterpart and because of the availability of MHC recombinant strains (Pennington et al., 1981). Three partially inbred strains of miniature swine have been derived which are fully inbred at the SLA locus and which have the defined haplotypesa, c, and d. Furthermore, four additional SLA haplotypes (f, g, h, and j), with recombinations between the originally defined class I and class II loci, have been obtained and bred to homozygocity (F i g u r e 1 ). Pulsed field analysis of the pig class II region allowed accurate mapping of the locus and showed similar class II gene distribution between pig and human (LeGuern, C., unpublished data). Porcine class I (Singer, 1982; Singer et al., 1987) and class II (Sachs et al., 1988; Pratt et al., 1990) genes have also been described. The class II region encodes polymorphic cell surface molecules, composed of heterodimers consisting of an alpha and a beta chain, which are involved in the molecular control of immune responses. Three major class II subregions (DR, DQ and DP) have been identified in man to encode functional cell surface class II molecules whereas only two of them (DR and DQ) appear to be functional in miniature swine (Pratt et al., 1990). Analysis of class II cDNA clones corresponding to the DRA/B and DQA/B genes from two different SLA haplotypes ( c and d) indicate that sequence variability is distributed similarly to that observed for the human class II sequences. As in human, the pig DRA sequence is

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V. MHC class II genes and tolerance induction to transplanted organs

Figure 1. Origin of inbred miniswine.

conserved among various MHC haplotypes (Hirsch et al., 1992) whereas the DRB (Gustafsson et al., 1990a) and both the DQA (Hirsch et al., 1990) and DQB sequences (Gustafsson et al., 1990b) are polymorphic.

Transplantation studies on genetically defined miniature swine have demonstrated that sharing of MHC class II antigens between donor and recipient pairs is critical for insuring long-term renal allograft survival (Pescovitz et al., 1984a; Kirkman, 1979; Rosengard et al., 1992). In approximately 35% of miniature swine, long-term specific tolerance spontaneously develops to one-haplotype class I plus minor antigen disparate, but class II matched, renal allografts in the absence of exogenous immunosuppression (Pescovitz et al., 1983; Pescovitz et al., 1984a). If, however, the kidney recipients were treated with a short course of cyclosporine (CsA : 10 mg/kg i.v. for 12 days) starting the day of transplantation, they accepted long-term (> 100 days) twohaplotype class I mismatched renal allografts (i.e., gg dd) in 100% of the cases (F i g u r e 2 ). Non-treated control animals rejected class II only matched grafts in 13.7 Âą 0.9 days and cyclosporine-treated recipients showed a delayed but consistent rejection of fully mismatched grafts (reviewed in Gianello and Sachs, 1996) (Figure 2).

Figure 2. Effects of CsA on renal allograft survival across selective MHC barriers (Gianello and Sachs, 1996).

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Gene Therapy and Molecular Biology Vol 1, page 336 Importance of class II matching for the outcome of kidney allografts has also been reported in clinical transplantation (Opelz et al., 1991; Ichikawa et al., 1993). Rodent studies designed to look at the effects of class II matching on graft survival have yielded mixed results (Aizawa, 1984; Paris and Gunther, 1980). It should be noted, however, that the tissue distribution of class II gene expression on rodent vascular endothelium (Benson et al., 1985) differs from that found in humans (Daar et al., 1984) and in large animals (Pescovitz et al., 1984b) so that mechanisms of graft rejection may differ (Hart and Fabre, 1981a; Hart et al., 1981b). The singular importance of class II sharing between donor and recipient points to the likely feasibility of using a gene transfer approach clinically to provide sharing of class II. In other words, if a recipient could be made to express the same class II as that of the donor organ, transplantation of that organ under the coverage of a short course of high dose CsA should result in prolonged graft survival. The applicability of such an approach in allogeneic organ transplantation will depend on the number of class II genes required to provide for the genetic diversity of the human population. Rough estimates of five to six prototypic class II genes, corresponding to the major phylogenetic families of class II sequences for DRB and DQ, could be envisioned, although this has not yet been tested. The practicality of such an approach for xenotransplantation is obvious, especially when the organ

donors are as genetically well defined as the MHC inbred miniature swine.

VI. Use of gene therapy in a miniature swine allogeneic transplantation model A. Protocol for tolerance induction An allogeneic miniature swine transplantation model has been developed to evaluate the efficacy of a gene therapy approach to provide sharing of class II on an MHC class I disparate background. A diagram of the experimental protocol is shown in F i g u r e 3 . Donor (SLAd) and recipient (SLAc ) animals are chosen to differ at both class I and class II loci. BM is collected from the SLAc recipient and transducedin vitro with a recombinant retrovirus vector for a class IId gene matching that of the future kidney donor. The transduced marrow is then infused back into the recipient pig which has been conditioned with lethal irradiation. Following full reconstitution with the class II-engineered bone marrow, the SLAc recipient receives a fully mismatched SLAd kidney transplant, which is matched only to the class IId transgene previously introduced through the retrovirus, under a 12 day course of CsA (Figure 3). Serum creatinine levels are then monitored as a read-out for kidney function (Emery et al., 1997).

Figure 3. Tolerance induction by class II gene transduction of bone marrow.

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Figure 4. Retrovirus vector for SLA class II DR allogeneic gene transfer.

B. Construction of recombinant vectors for pig Class II The utility of retroviral vectors in mediating gene transfer for the introduction of new genetic material into the hematopoietic compartment has now been demonstrated by a number of groups in rodents (Gilboa et al, 1986; Bodine et al, 1989; Sykes et al, 1993; Fraser et al, 1995; Mayfield et al., 1997) as well as in large animals (Emery et al., 1993; Banerjee et al., 1997a) and humans (Lu et al., 1993; von Kalle et al., 1994; Conneally et al., 1996). Since retroviral vectors have a high efficiency of chromosomal integration once inside of a cell, they insure transmission of the gene to future progeny of the targeted cell. It appears, however, that stable genome integration of recombinant proviruses requires cell division (Harel et al., 1981, Miller and Miller, 1990), a prerequisite not fulfilled by mostly quiescent hematopoietic early progenitors and stem cells. Although the requirements for long-term expression of allogeneic class II in this model have not been determined, stable expression of the trangene(s) would seem to be preferable, based on the results of allotransplant studies between recombinant haplotype pigs as described above (Rosengard et al., 1992). An initial construct, named GS4.5, was engineered to express both the neomycin phosphotransferase drugresistance gene (Neo) as well as the pig DRBd allelic cDNA (Figure 4) (Shafer et al., 1991). Since the DRA nucleotide sequence is non-polymorphic, it was assumed that the recipient endogenous DRAc chain would associate with the donor DRBd product for expression of allogeneic class II heterodimers. The SLA-DRBd cDNA transgene is under the control of the thymidine kinase (Tk) promoter. The expression cassette was inserted into the 3' long terminal repeat (LTR) of the N2A double-copy vector (Hantzopoulos et al, 1989) in the same transcriptional orientation as that of the retrovirus. Recombinant retrovirus particles were first produced in the ecotropic cell line GP+E86 (Markowitz et al., 1988) and then transferred to the amphotropic packaging cell line PA317 (Miller and Buttimore, 1986). Extensive selection of clones resulted 337

in a stable producer cell line which yielded viruscontaining supernatants at a titer of 3 x 106 G418resistant colony forming units (CFU) per ml. A highly sensitive marker-rescue assay (Miller and Rosman, 1989) was used to confirm the absence of replication-competent helper virus (Emery et al., 1993) in all preparations of amphotropic DRB-virus.

C. Transduction of swine cells To demonstrate that the GS4.5 vector could transduce and express recombinant SLA class II in or on cells of swine origin, primary swine fibroblast cultures were incubated with high-titered virus supernatant without selection for three to four days and assayed by Northern blotting using probes for either Neo or DRB. The DRBspecific transcript pattern observed was similar to that resulting from RNA derived from the producer cell line indicative of correct expression of the integrated DRBprovirus (not shown). Whole swine BM cells were then harvested and cultured in the presence of viral supernatants at an estimated multiplicity of infection of 3 to 5. To assess for bone marrow (BM) transduction, a colony assay for swine colony-forming-unit-granulocyte-macrophage (CFU-GM) was developed. Titration studies were first performed to test the plating efficiency of swine CFU-GM with increasing concentrations of the neomycin drug analog G418. In these studies (Emery et al., 1993), colony formation was inhibited at G418 concentrations of 1 mg/ml. Transduced BM cells were then plated in methylcellulose cultures in the presence or absence of 1.2 mg/ml active G418 to assess the efficiency of transduction of CFU-GM. In addition, they were also plated directly into long-term bone marrow cultures (up to 5 weeks) followed by CFU-GM assay to determine if early progenitors were also transduced. Transduction efficiencies estimated from the initial CFU-GM cultures ranged from 4% to 14% in 9 experiments. Only slightly lower numbers were obtained in the CFU-GM assays of the long-term cultures. These ranges of efficiency were later confirmed by PCR assay using DR-specific probes (Emery et al., 1993). These data indicate that early

White-Scharf et al: Applications of Gene Therapy in Transplantation progenitors were transduced and predict that engraftment of these cells in vivo should result in long-term presence of the gene.

D. Addition of growth factors to optimize transduction of bone marrow cells At the time these initial experiments were performed, pig-specific cytokines were not available. Cytokines from man and mouse were therefore screened for crossreactivity with pig BM cells. Mouse c-kit ligand (KL) and human GM-CSF were stimulatory for pig cells but, as expected, mouse or human IL-3 was not. Surprisingly, PIXY321, a fusion molecule consisting of human IL-3 and GM-CSF (gift from Dr. D.E. Williams, Immunex Corp.), exhibited more activity than did similar molecular concentrations of GM-CSF alone, suggesting that the fused IL-3 portion of the molecule was stimulating pig BM cells. When KL and PIXY321 were added to cultures during transduction, the transduction efficiency was increased almost 3-fold (Emery et al., 1997).

Table 1: MLR Analysis of Lymphocyte Responses from Animals 10736 and 10807 (Emery et al., 1997) Responder Cells

Blocking Ab

Table 2: Summary of Experimental Animals

% Relative

Experimental :

Response

Naive cc

Anti-DR Anti-DQ Anti-DR + DQ

41 50 10

10736

Anti-DR Anti-DQ Anti-DR + DQ

52 15 1

Naive cc

Anti-DR Anti-DQ Anti-DR + DQ

52 73 18

10807

Anti-DR Anti-DQ Anti-DR + DQ

75 79 12

possible to test directly the DR-dependent reactivity of T cells harvested from engineered animals. Therefore, mixed lymphocyte experiments were performed in the presence of either anti-DR or anti-DQ specific antibodies (Table 1). Cells from either control (#10807) or experimental (#10736) pigs were used as responder cells in a mixed lymphocyte response (MLR) to cells bearing the same DR allele as the donor DRB transgene. Antibodies specific for either DR or DQ were added to the cultures. When the anti-DR antibody was added either to cells from the control or experimental pig, no effect on proliferation was observed. When anti-DQ was added, no effect was observed on the response of the control pig; however, the response of the experimental pig to the allogeneic cells was inhibited. Control blocking MLR experiments performed with both antibodies confirmed complete inhibition. These data indicate that the immune cells from pig #10736, which received autologous BM cells engineered with the allogeneic DRB transgene, were unresponsive to the donor DR antigen (Emery et al., 1997), and support the use of gene therapy to induce specific immune tolerance and long-term graft survival by enabling the expression of donor class II determinants in a class I, class II-disparate recipient.

Animal Number

Host Haplotype

DR Vector Allele

Cytokines*

Ktx Donor H apl o type

10660 10680 10697 10736

c d d c

d c c d

Control: 10807

*BMC were treated (+) or not treated (-) with cytokines at the time of transduction (Emery et al., 1997).

E. DR matching â&#x20AC;&#x153;turns offâ&#x20AC;? alloreactive T cells

F. Kidney survival in miniature swine engrafted with Class II targeted bone marrow cells

The putative effect of in vivo DRB transgene expression on T cell reactivity was assessed in vitro on target cells expressing class II molecules related to the transgene products. Since there are no recombinant class II haplotypes which separate DR from DQ, it was not

Autologous BM cells from five miniature swine were transduced in the presence or absence of cytokines and then infused back into the original pigs which underwent a split dose of 10 Gy given on two consecutive days just

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Gene Therapy and Molecular Biology Vol 1, page 339

Figure 5. Construction of polycistronic retroviral vectors (Banerjee et al, 1997).

prior to BM transplant (Table 2). Three pigs (#10660, 10680, and 10697) received autologous BMC transduced with allogeneic class II in the absence of cytokines. One (#10736) was reconstituted with DRB-transduced BMC in the presence of cytokines, and, finally, a control animal (#10807) received BM cells transduced with syngeneic class II in the absence of cytokines. Each animal was infused with approximately 9 x 10 7 transduced cells per kg of body weight. Engraftment occurred within 12 Âą 4 days post BMT based on the criteria of a white blood cell count of 1000/mm 3. Progenitor colony formation was estimated on aliquots of BM taken prior to infusion and from serial rib biopsies taken post-infusion. The transduction efficiencies prior to infusion ranged from 6.5% to 25.9% with the highest being associated with the bone marrow transduced in the presence of cytokines. The initial levels of G418 resistant CFU-GMs were maintained for at least 12-22 weeks post BM transplant after which they declined substantially (Emery, D.W., 1997). Following full reconstitution of the recipient with the DRB engineered BM cells (4-6 months post BM transplant), each animal was challenged with a fully allogeneic kidney only DR matched to the introduced transgene. According to the protocol presented in Figure 3, a 12 day treatment with CsA was administered, beginning at the time of allogeneic kidney transplant. Function of renal allografts was measured by serum creatinine levels. The control pig # 10807, which expressed a syngeneic DRB transgene in BM derived cells, underwent an early rejection episode which was followed by progressive renal failure. It eventually died 120 days post-transplant. Of the three pigs reconstituted with noncytokine treated BM cells, one rejected its transplant on day 8 while still under CsA coverage; the other two survived 22 and 40 days, respectively. In contrast, the pig whose BM was transduced in the presence of growth factors, and who showed a high initial transduction rate (25.9% G418r CFU), accepted its kidney long-term and showed no sign of cellular rejection. It exhibited normal kidney functions until it was sacrificed on day 995 postBM transplant (Emery et al., 1997).

339

VII. Use of gene therapy in a pig to primate xenotransplant model Since matching of class II genes between graft donor and recipient, through gene therapy appeared to control T cell dependent responses in the miniswine allotransplantation model, similar studies have been initiated in a pig to primate xenotransplantion model to assess the extent to which class II sharing may also provide specific unresponsiveness to xenogeneic antigens. Transfer and expression of porcine class II DR antigens into monkey bone marrow cells was first accomplished. In contrast to the allotransplantation studies in the miniature swine in which the DRa chain sequence is conserved, expression of pig DR in other species requires that both the DRA and DRB genes be transferred.

A. Construction of Vector for Pig Class II In order to insure transfer of the DRA and DRB genes into the same cell, a recombinant polycistronic vector was generated (Banerjee et al., 1997a) (Figure 5). This vector enables the expression of multimeric proteins from a single transcript by using internal ribosomal entry sites (IRES) for independent initiation of translation from internal cistrons. The plasmid pL7gCAT (gift from Dr. B. Seed, Massachusetts General Hospital, Boston, MA) was derived from the pLN backbone (Miller and Rosman, 1989). Derivatives of this vector which initially contained three copies of the immunoglobulin heavy chain binding protein IRES (BiP) (Macejak, 1990) were found to be unstable in transduction experiments. Thus, two of the three BiP cassettes were eliminated and the remaining cellular-derived BiP was used in combination with the viral IRES derived from the encephalomyocarditis virus (EMCV) (Jang, 1988). In addition, the murine myeloproliferative sarcoma virus (MPSV) enhancer sequence (Johnson et al., 1989) was incorporated into the 5' end of the 3' LTR in order to increase the tropism of

White-Scharf et al: Applications of Gene Therapy in Transplantation

F i g u r e 6 . Ex vivo transduction of primate bone marrow cells with recombinant retrovirus for SLA gene transfer.

vector expression toward hematopoietic tissues as reported (Stocking, 1994). An enhancerless SV40 origin of replication sequence was also inserted upstream of the 3' LTR to allow testing of preliminary recombinant constructs using transient expression in COS cells (Aruffo and Seed, 1987). Porcine DRB and DRA sequences were generated by PCR amplification of cDNA clones (Gustafsson 1990a, Hirsch et al. 1992). The sequence for resistance to neomycin (Neo) was inserted downstream of the DRA cDNA to allow for selection with G418. High titer virus producer clones (> 1x 106 pfu/ml) were derived by transfection/transduction of appropriate ecotropic/amphotropic packaging lines according to standard protocols (Banerjee, 1997a). Analysis of selected virus producer clones demonstrated stability of particle expression over an eight to ten week period. These clones were cultured in roller bottles and production was monitored by G418 resistant CFU titers. The viral titer averaged approximately 4 x 107 CFU/ml during eight weeks of culture. No replication-competent retroviruses were detected by S+L- assay on PG4 cells (Banerjee et al. 1997a). Virus producer clones were

340

ultimately selected for their ability to express cell surface class II heterodimers (not shown).

B. Transduction of non-human primate cells Bone marrow was harvested from a Cynomolgus monkey and enriched for CD34+ cells by selective binding to a Ceprate column (CellPro Inc.) (F i g u r e 6 ). The enriched population was then plated onto autologous stromal cells which had been established 3 weeks prior to transduction. Cultures were exposed twice to retrovirus containing culture supernatants in the presence of polybrene and the human recombinant cytokines SCF, IL3 and IL-6 for 18 hours. Aliquots were tested for transduction by plating into CFU assays in the presence of G418. All showed G418r colonies (15.5% +/- 5.6%). Transduced cells were then infused back into the monkey from which they were originally taken. Both DNA- and RT-PCR assays were performed to confirm transduction and expression of the transgene according to the scheme detailed in Figure 7. Two sets of DRB and DRA specific primers were used in a nested

Gene Therapy and Molecular Biology Vol 1, page 341

Figure 7. Primers for PCR detection of construct.

PCR reaction to generate a fragment that initiated in the DR sequences and spanned the IRES. Primers specific for the Neo resistance gene have also been generated. In addition, PCR primers specific for the beta-actin gene were used as controls in parallel PCR reactions for monitoring the quality of the cDNA templates. All bone marrow cultures transduced with the pig DR vector showed a clear PCR positive signal when assayed in vitro (not shown).

C. Engraftment of transduced cells In contrast to miniature swine recipients which received lethal irradiation, a non-myeloablative conditioning regimen which made use of T cell depleting reagents was applied to monkey recipients of transduced bone marrow cells. The protocol included non-lethal total body irradiation (TBI) of 300 Rads, thymic irradiation of 700 Rads, and anti-thymocyte globulin (ATGAM) on days -3, -2, and -1. Administration of CsA was initiated on day 0 and continued for 28 days. Recombinant human GM-CSF was given subcutaneously from day 0 to day 14. Peripheral blood and BM aspirates were collected periodically and either assayed directly by PCR or plated into CFU assays from which colonies were assayed by PCR. RT-PCR and DNA-PCR products generated using the nested primers were detected in all of the peripheral 341

blood samples assayed through week 56 post-BM transplantion indicating that transcription of pig DR cDNA persisted long-term in peripheral blood cells of the monkey. Furthermore, the amplification of PCR products of predicted size indicated that no apparent genomic rearrangement had occurred within the region of the proviral genome containing the DR sequences. The frequency of expression was estimated by assaying colonies derived from BM aspirates taken at 4 weeks and 25 weeks post-BM transplantation. Frequencies for these time points were estimated to be approximately 2% and 1%, respectively (Banerjee, et al. 1997b). These data show that long-term stable engraftment was achieved in the Cynomolgus monkey.

VIII. Conclusions The potential for using gene therapy to establish specific immune tolerance and to enable long-term graft survival has been demonstrated in the miniature swine allotransplantion model. Studies have shown that sharing of a single class II DR gene between a kidney donor and the recipient results in specific immune unresponsiveness for organs expressing the shared gene. These promising results led us to believe that a gene therapy approach could be adapted to clinical transplantation. In the longterm, this approach could also be valuable in

White-Scharf et al: Applications of Gene Therapy in Transplantation xenotransplantation in order to control the T cell dependent anti-xenogeneic immune responses. Xenotransplantation, however, is complicated by additional issues such as managing the natural antibodies which exist in primates and which react with the tissues of discordant species (i.e., pig) to cause hyperacute rejection. The pilot primate experiment described here shows that long-term expression of class II genes can be achieved in non-human primates using a non-lethal, clinically acceptable protocol and points to the appropriateness of a gene therapy approach in achieving shared antigen expression across allogeneic and xenogeneic barriers. It has yet to be determined if tolerance to xenogeneic antigens can be achieved by sharing only class II. Thus, the possibility of transferring additional pig genes such as class I genes is currently under investigation. It is our conviction that the benefits of having a genetically well-defined unlimited supply of organs justify the additional research required to facilitate implementation of xenotransplantation.

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Pescovitz, M. D., Auchincloss Jr., H., Thistlethwaite Jr., J. R., Sachs, D. H. (1 9 8 3 ). Transplantation in miniature swine: acceptance of class I antigen mismatched renal allografts. Transplant Proc. 15, 1124-1126.

Singer, D. S., Camerini-Otero, R. D., Satz, M. L., Osborne, B., Sachs, D. H., and Rudikoff, S. (1 9 8 2 ). Characterization of a porcine genomic clone encoding for a major histocompatibility antigen: Expression in mouse L cells. P r o c . N a t l . Acad. S c i . USA 79, 1403-1407.

Pescovitz, M. D., Thistlethwaite Jr., J. R., Auchincloss Jr., H., Ildstad, S. T., Sharp, T. G., Terrill, R., and Sachs, D. H. (1 9 8 4 a ). Effect of class II antigen matching on renal allograft survival in miniature swine. J . E x p . Med. 160, 1495-1505. Pescovitz, M. D., Sachs, D. H., Lunney, J. K., and Hsu, S. M. (1 9 8 4 b ). Localization of class II MHC antigens on porcine renal vascular endothelium. T ra n s p la ntation 37, 627-630. Pratt, K., Sachs, D. H., Germana, S., El-Gamil, M., Hirsch, F., Gustafsson, K., and LeGuern., C. (1 9 9 0 ). Class II genes of miniature swine. II. Molecular identification and characterization of beta genes from the SLA c haplotype. I m m u n o g e n e t i c s 31, 1-6. Rosengard, B. R., Ojikutu, C. A., Guzzeta, P. C., Smith, C. V., Sundt III, T. M., Nakajima, K., Boorstein, S. M., Hill, G. S., Fishbein, J. M., and Sachs, D. H. (1 9 9 2 ). Induction of specific tolerance to class I-disparate renal allografts in miniature swine with cyclosporine. T r a nspl a n t atio n 54, 490-487. Sachs, D. H., Germana, S., El-Gamil, Gustafsson, K., Hirsch, F., and Pratt, K. (1 9 8 8 ). Class II genes of miniature swine. I. Class II gene characterization by RFLP and by isolation from a genomic library. I m m u n o g e n e t i c s 28, 22-29. Sachs, D. H. (1 9 9 4 ). The pig as potential xenograft donor. Veterinary Immunol. I m m u n o p a t h o l . 43, 185191. Sachs, D. H. (1 9 9 5 ). Mixed chimerism as an approach to transplantation. In T r a n s p l a n t a t i o n I m m u n o l o g y , F. H. Bach and H. J. Auchincloss, eds. (New-York: John Wiley & Sons, Inc.), pp. 219-225. Shafer, G. E., Emery, D. E., Gustafsson, K., Germana, S, Anderson, W. F., Sachs, D. H. and LeGuern, C. (1 9 9 1 ). Expression of a swine class II gene in murine bone marrow cells by retroviral-mediated gene transfer. P r o c . N a t l . A c a d . S c i . U S A 88, 9760-9764. Sharabi Y., Abraham, V. S., Sykes, M., and Sachs, D. H. (1 9 9 2 ). Mixed allogenic chimeras prepared by a nonmyeloablative regimen: requirement for chimerism to maintain tolerance. B o n e Marrow Transplant 9, 191-197.

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Singer, D. S., Ehrlich, R., Satz, L., Frels, W., Bluestone, J., Hodes, R., and Rudikoff, S. (1 9 8 7 ). Structure and expression of class I MHC genes in the miniature swine. Veterinary Immunol. Immunopathol. 17, 211221. Smith, C. V., Nakajima, K., Mixon, A., Guzetta, P. C., Rosengard, B. R., Fishbein, J. M., and Sachs, D. H. (1 9 9 2 ). Successful induction of long-term specific tolerance to fully allogeneic renal allografts in miniature swine. Transplantatio n 53, 438-444. Sykes, M., Sachs, D. H. Nienhuis, A. W., Pearson, D. A., Moulton, A. D. and Bodine, D. M. (1 9 9 3 ). Specific prolongation of skin graft survival following retroviral transduction of bone marrow with an allogeneic major histocompatibility complex gene. T r ans pla nt at i on 55, 197-202. Sykes, M., Lee, L. A., Sachs, D. H., (1 9 9 4 ). Xenograft tolerance. I m m u n o l o g i c a l R e v i e w s 141, 245-276. von Kalle, C., Kiem., H. P., Goehle, S., Darovsky, B., Heimfeld, S., Torok-Storb, B., Storb, R., and Schuening, F. G. (1 9 9 4 ). Increased gene transfer into human hematopoietic progenitor cells by extended in vitro exposure to a pseudotyped retroviral vector. B l o o d 84.9, 2890-2897.

Gene Therapy and Molecular Biology Vol 1, page 345 Gene Ther Mol Biol Vol 1, 345-363. March, 1998.

Myoblast transfer as a platform technology of gene therapy Peter Law, Tena Goodwin, Qiuwen Fang, George Vastagh, Terry Jordan, Tunja Jackson, Susan Kenny, Vijaya Duggirala, Charles Larkin, Nancy Chase, William Phillips, Glenn Williams, Michael Neel, Tim Krahn, and Randall Holcomb Cell Therapy Research Foundation, 1770 Moriah Woods Blvd., Suite 16-18, Memphis, TN, 38117, USA

__________________________________________________________________________________ Correspondence: Peter Law, Tel: (901) 681-9045, Fax: (901) 681-9048, E-mail: cell@attmail.com Keywords: Myoblast transfer; Clinical trials; Gene therapy; Duchenne muscular dystrophy; Viral vectors

Summary Myoblasts divide profusely, and fuse during muscle regeneration, interiorizing MHC-I antigens and inserting myonuclei with the normal genome into muscles o f genetically deficient recipients, where any replacement gene can be stably integrated and naturally expressed. Myoblasts are the natural source and vehicle for many gene therapies. Myoblast transfer therapy is completing US FDA Phase II clinical trials for Duchenne muscular dystrophy.

I. Introduction The National Institute of Standards and Technology has recently announced that tissue engineering will likely be the key to treating genetic diseases and degenerative disorders that accounted for 50% of the $1+ trillion U.S. health care cost in 1995 (Schwartz, 1997; Langer and Vacanti, 1993; Nerem and Sambanis, 1995). Among the many programs of tissue engineering, gene therapy has been hailed as the medicine of the 21st century. Despite the nearly universal belief that gene therapy will ultimately allow the treatment of currently incurable diseases and conditions, its potential remains largely unfulfilled (Hillman et al., 1996). Only when a safe and effective gene delivery technology has been proven in humans can the full potential of gene therapy be realized. To date, over 3000 subjects worldwide have received gene therapies among the 280+ protocols approved. Data indicate that no single vector will serve all systems. In examining gene transfer methods mediated by particle bombardment (Jiao et al., 1993; Sautter et al., 1991), liposomes (Stewart et al., 1992; Ray and Gage, 1992), calcium phosphate precipitation (Ray and Gage, 1992; Albert and Tremblay, 1992), and electroporation (Ray and Gage, 1992; Albert and Tremblay, 1992; Puchalski and Fahl, 1992), one can conclude that transduction efficiency 345

is extremely low and variable. The level of transgene expression depends on the promoter strength in a particular cell type. Only liposomes, together with retroviruses, adenoviruses, adeno-associated viruses and myoblasts have been used in clinical trials.

A. Vectors 1. Liposomes Cationic liposome/DNA complexes gain cellular entry via receptor-mediated endocytosis (Stewart et al., 1992; Trubetskoy et al., 1992). Assuming the transgene escapes digestion by the endosome, it has no built-in mechanism to get across the nuclear membrane and is therefore nonintegrative. The minimal and transient expression of the transgene is the result of random targeting, integration, and regulation. Liposomes have the advantage of being non toxic and can therefore be used in large quantities and repeatedly (Brenner, 1995).

2. Viruses The viral vectors were the first to gain widespread scientific applications. Notable was "the first federally approved gene therapy protocol, for correction of adenosine deaminase (ADA) deficiency, began on 14 September 1990"( Anderson, 1990, 1992, 1995).

Law et al: Myoblast transfer as a platform technology been used for in vivo transfer of factor IX cDNA to the liver. Although therapeutic levels of factor IX were obtained, the expression decayed in a few weeks, possibly due to immune response and gene inactivation (St. Louis and Verma, 1988). Gene therapy with viral vectors has been developing rapidly, but judging from the results of cystic fibrosis and brain tumor clinical trials, it is still a young discipline (Rosenfeld and Collins, 1996; Alton and Geddes, 1994 ). Since the main thrust of this chapter is on myoblast transfer therapy (MTT), additional details of nonmyoblastic single gene manipulations can be found in the books entitled "Gene Therapy - A Primer for Physicians" (Culver, 1996) "Somatic Gene Therapy" (Chang, 1994) and "Gene Therapy for Neoplastic Diseases" (Huber and Lazo, 1994).

Retroviral vectors can transduce dividing cells with integration into host DNA. They integrate randomly and may cause mutation and cell death. They exhibit no toxicity. Although they can house larger transgenes than adenoviruses and adeno-associated viruses, the capacity is less than 10 kb. They are unstable in primate complement and cannot be targeted to specific cell types in vivo (Brenner, 1995; Cornetta et al., 1991). Adeno-associated viruses and adenoviruses have shown considerable promise and are widely used. They can accommodate a broad range of genetically modified genes; are efficiently taken up by non-dividing cells in vivo; do not integrate into chromosomal DNA, thus reducing the risk of insertional mutagenesis; and are amenable to redirected tissue targeting (Morsey and Caskey, 1997). All viruses can cause harm when they revert to wild type and become replication-competent (Brenner, 1995; Coutelle et al., 1994; Curiel et al., 1996). Dose-dependent inflammation occurred after nasal (Knowles et al., 1995) or lung (Crystal et al., 1994) administration of the cystic fibrosis transmembrane conductance regulator (CFTR) cDNA conjugated with adenoviral vectors. The low efficacy, if any, is what one would have expected of pioneering studies. However, the risk to benefit ratio cannot be ignored. Also viruses produce antigens. When exposed to the host immune system, through leakage, secretion or cell damage, these antigens trigger immune reactions against the transduced cells. Certain viral elements are also toxic. These three inherent problems post almost insurmountable difficulties that prohibit the safe and efficacious clinical use of viral vectors at the present except for terminal cases. To raise caution, the FDA has mandated viral vector validation of every batch to be used on humans.

5. Myoblasts

3. Plasmids Single gene manipulation, often exercised ex vivo, has been used in vivo. Recombinant genes by themselves were shown to have been taken up and expressed in murine skeletal myofibers (Wolff et al., 1990; Ascadi et al., 1991; Davis et al., 1993) and cardiac myocytes (Leinwand and Leiden, 1991) following intramuscular injections. Gene expression is invariably low despite different delivery conditions and methods (Wolff et al., 1991). This approach lacks basis and evidence of gene integration and regulation.

4. Combinations A more logical approach is to include viral or cellular transcriptional regulatory sequences to effect expression. In the prophylactic treatment of hemophilia A, a retroviral factor-VIII cDNA conjugate was used to induce secretion of the blood-clotting factor in athymic mice from transduced human skin fibroblasts implanted (Hoeben, 1995). Both adenoviral (Smith et al., 1993) and Herpes Simplex virusderived (Miyanohara et al., 1992) vectors have similarly 346

Although genetic ailments constitute less than 2% of all human diseases, far more currently incurable diseases are the result of inadequate genetic predisposition and/or haphazard interactions between multiple genes. Symptoms precipitate when a regulatory or a structural protein is either missing or malfunctional. Without knowing these defect(s) or how they can be corrected, tissue engineering will favor genome replacement rather than single gene(s) replacement. The cell knows more than we do. Furthermore, for a gene therapy to be effective and efficient, transgene expression requires appropriate targeting into a specific cell type, integration onto a specific site on a specific chromosome, and regulation by factors that are the products of other genes. This chain of events involves numerous cofactors many of which are produced transiently during embryonic development but not in adulthood. This is where the approach of single gene manipulation is conceptually inadequate because it cannot provide these cofactors. In complex systems, one hardly knows what they are. Again only transfer of the whole normal genome can allow the orderly provision of these cofactors necessary for the transgene expression. Finally secondary degenerative changes often accompany the primary protein defect. Additional structural and/or regulatory protein(s) are lost (F i g . 1 ). Even if single gene manipulation replaces the primary protein deficit, transduced cells still degenerate because of the secondary changes. These latter proteins can only be replaced by re-transcribing the normal genome inserted. Myoblasts are muscle-building cells endogenous to the human body. Contained within the nucleus of each human myoblast is the normal genome with over 100,000 normal genes that determine cell normality and cell characteristics. Less than 10% of the gene actions is known. Myoblasts is the only somatic cell type in the body capable of natural cell fusion. Through this process, they insert their nuclei, and therefore all of the normal genes, into multinucleated myofibers of the host to effect genetic repair (F i g . 2 ).

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F i g . 1 . Diagram of some of the known genetic factors in DMD muscle cells that differ from normal muscle cells. These include genes for membrane structural proteins that are decreased or absent in DMD, dystrophin (DIN), dystrophin-related-proteins (DRP) and dystrophin-associated glycoproteins (DAG), genes for enzymes elevated in serum levels of DMD patients, creatine phosphokinase (CPK), aldolase (ALD) and aspartate transaminase (AST), and genes for mitochondrial (Mito) differences.

F i g . 2 . Immunocytochemical localization of donor (stained, white arrowheads) and host (unstained, dark arrowheads) nuclei in longitudinal muscle sections. A and B are normal and dystrophic controls, respectively. C is from a dystrophic muscle 18 months after normal myoblast injection. A mosaic fiber (M) is demonstrated by the presence of both stained and unstained nuclei.

1984; Appleyard et al., 1985). These combined properties render myoblasts superior for gene transfer. Being endogenous cells, myoblasts do not produce the adverse reactions of viral vectors.

The transfer of genetic material and information occurs in vivo, with the myoblasts serving as the source and the vehicle to effect gene transfer. Myoblasts are the only cells that divide extensively (Law et al., 1997a), migrate (Law et al., 1992), fuse naturally to form syncytia (Law et al., 1992), interiorizing major histocompatibility complex class I (MHC-1) antigens after fusion (Daar et al., 1984; Appleyard et al., 1985), and develop up to 50% of human body weight. Myoblast recipients need no more than two months of immunosuppression after MTT because mature myotubes and myofibers do not exhibit MHC-1 antigens (Daar et al.,

II. Myoblast Transfer Therapy (MTT) technology MTT is a platform technology of gene therapy and tissue engineering. The procedure consists of culturing large quantities of myoblasts from muscle biopsies of genetically normal human donors. Cultured myoblasts are 347

Law et al: Myoblast transfer as a platform technology injected into patient's muscles while the patient is under general anesthesia. An immunosuppressant is administered following the procedure to minimize donor cell rejection. The injection injury activates regeneration of host myofibers, allowing them to fuse with the injected myoblasts, thus forming genetically mosaic multinucleated myofibers (F i g . 2 ) (Law et al., 1988a,b, Chen et al., 1992). In addition, injected myoblasts fuse among themselves, forming genetically normal myofibers (Law et al., 1988a,b; Chen et al., 1992). Thus, MTT delivers the normal nuclei, the genetic software and hardware in total, into muscles of the genetically defective host, where the critical transgene is naturally and stably integrated, regulated and expressed. Since the fusion process is a natural occurrence, there should not be any problem with specificities of integration, complementation, regulation, and expression of the normal genome inserted. It is not necessary to know which gene(s) is responsible for the defect. Abnormal gene identification is time-consuming and expensive. Furthermore, the injection of normal myoblasts directly into the host muscle eliminates any uncertainty of tissue targeting. Natural transcription of the normal genome within the donor nuclei following MTT ensures orderly replacement of any protein deficiency resulted from single gene defects or from haphazard polygenic interactions, much of which is unknown.

Since MTT incorporates all of the normal genes into the dystrophic myofibers to repair them, it should exert similar effects regardless of which gene is abnormal or which protein is missing. Accordingly, MTT should be as beneficial to the murine dystrophies showing laminin !2 2J mutation in the dy and dy phenotypes (Sunada et al., 1995) as DMD showing dystrophin deletion (Hoffman et al., 1987), given adjustments from mouse to human.

IV. Animal experiments To develop a treatment we need to know the pathogenesis of the disease. By comparing the electric (Law and Atwood, 1972; Law et al., 1976) and ultrastructural properties (Mokri and Engel, 1975; Law et al., 1983) of normal vs. dystrophic myofibers, the genetic defects in muscular dystrophy were established to result from membrane deterioration and dysfunction. Using a normal/dystrophic parabiotic mice model with crossreinnervation of muscles, it was demonstrated that the dystrophic nervous system would support normal muscle development (Law et al., 1976; Saito et al., 1983). Without such knowledge, it would be imprudent to attempt strengthening dystrophic muscles with normal myogenic cell transfer. Earlier developmental work of MTT consisted of two approaches that were disparate but complementary. These are the demonstration of safety and efficacy of transferring normal myogenic cells into the dy2Jdy2J dystrophic mice

III. Muscular dystrophies: the testing ground Muscular dystrophies are genetic diseases of progressive muscle degeneration. Debilitating and fatal, these hereditary degenerative diseases deprive their sufferers of a normal quality of life and life span. Duchenne muscular dystrophy (DMD) confines boys to wheelchairs by age 12 and claims their lives by 20. Second in prevalence only to cystic fibrosis, DMD afflicts one in every 3300 male births worldwide (Emery, 1991). As with any hereditary degenerative disease, DMD treatment will require repairing degenerating cells and replenishing dead cells. MTT is unique in treating the muscular dystrophies in that it transfers the normal genome to repair degenerative myofibers and it provides normal cells to replenish degenerated myofibers. As such, MTT is a combined cell/gene therapy. Potentially, not only can MTT prevent further weakening, it can also increase muscle strength. Like murine dystrophy, DMD serves as a disease model to test MTT as a cell/gene therapy in treating hereditary degenerative diseases. MTT is being developed to repair degenerating cells and to replenish degenerated cells of the muscles in all of the neuromuscular diseases affecting over one million people worldwide. In a broad sense MTT is tested for its feasibility, safety, and efficacy to integrate the normal human genome into genetically abnormal patients. 348

(Law, 1978; Law and Yap, 1979; Law, 1982) and the examination of the developmental fate of donor cells in normal mice (Partridge et al., 1978; Watt, 1982; Watt et al., 1982). The dy2Jdy2J dystrophic mice share a common gene defect of laminin !2 mutation with congenital muscular dystrophy, the most severe form of human dystrophies (Sunada et al., 1995). It was not until 1989 that a study of MTT on mdx mice was first published (Partridge et al., 1989; Karpati et al., 1989). The majority of evidence in support of MTT safety and efficacy is derived from previous studies using the dy2Jdy2J mice (Law et al., 1988a,b; Chen et al., 1992; Law, 1978; Law and Yap, 1979; Law, 1982; Law et al., 1990b,d). This was at a time when neither the golden retriever muscular dystrophy (GRMD) nor the xmd canine dystrophy was known. Dystrophic dogs are available to a few laboratories that have not produced any significant results with MTT (Kornegay et al., 1992). Central to MTT is the correlation of genetic and phenotypic improvement at the cellular and at the whole muscle levels. These studies play an essential role in the elucidation of the mechanisms by which MTT exerts its beneficial effects on dystrophic muscles (Law et al., 1978; Law and Yap, 1979; Law, 1982; Law et al., 1988a,b; Chen et al., 1992; Partridge et al., 1989; Karpati et al., 1989; Law et al., 1990b,d).

Gene Therapy and Molecular Biology Vol 1, page 349 that donor cells survived and developed in the host muscles, using electrophoretic analyses of glucose phosphate isomerases (GPI), the genetic markers to identify hosts vs. donor cells. The use of cultured myoblasts with dystrophic mice eventually appeared. In the first study, primary myoblast cultures from limb-bud explants of normal mouse embryos were injected into the soleus muscles of histocompatible dystrophic hosts (Law et al., 1988,b). In the second study, clones of normal myoblasts were injected into the leg and intercostal muscles of histoincompatible hosts with cyclosporine-A (CsA) as a host immunosuppresant (Law et al., 1988a). Using GPI as genotype markers, donor myoblasts were shown to have fused among themselves, developing into normal myofibers. They also fused with dystrophic host myogenic cells to form mosaic myofibers of normal phenotype (Law et al., 1988a,b; Law et al., 1990a,c). These two mechanisms of genetic complementation were shown to be responsible for improvement in muscle genetics, structure, function and animal behavior of the test dystrophic mice (Law et al., 1988a,b; Law, 1978; Law and Yap, 1979; Law, 1982; Law et al., 1990b,d). Prolongation of the life-spans of the myoblast-injected dystrophic mice was demonstrated (Law et al., 1990b,d). The improvement persisted despite CsA withdrawal. Morgan et al. (1988) reported the synthesis of trace amounts of phosphorylase kinase (PhK) in about 5% of the myoblast-injected muscles of the PhK-deficient mice. Although there have been frequent claims of supplying normal muscle precursor cells to alleviate hereditary myopathies, no evidence of any structural or functional improvement after transplantation was presented. With the discovery that the absence of the gene product dystrophin is the cause of DMD (Hoffman et al., 1987) and mdx mouse dystrophy, a new biochemical marker became available to demonstrate MTT efficacy (Partridge et al., 1989; Karpati et al., 1989; Chen et al., 1992). With implantation of cultured normal myoblasts into muscles of immunosuppressed mdx mice, MTT was shown to convert mdx myofibers from dystrophin-negative to -positive (Partridge et al., 1989; Karpati et al., 1989). The study demonstrates biochemical improvements in the mdx mouse model, an additional evidence to confirm the efficacy of MTT. Given the use of inbred mice that afford histocompatible MTT, the reality is that fully matched human donors and dystrophic recipients are rarely available. MTT would thus necessitate the inclusion of host immunosuppression to facilitate myoblast survival after transfer. Cyclosporine (Cy) is the most widely documented immunosuppressant in transplantation studies (Kahan and Bach, 1988). Availability of FK506 in the late 80's was limited (Starzl et al., 1991). Typically, host mice were primed 1 week with CsA injected subcutaneously every day at 50 mg/kg body weight before receiving myoblasts. The same CsA treatment continued for 6 months after MTT (Law et al., 1988b).

The demonstration that cultured cells survived, developed and functionedin vivo after implantation into an organ of a genetically abnormal mammal bridges the gap between in vitro and in vivo cell biology. This was first achieved with myoblast transfer (Law et al., 1988a,b). The foremost study in adult dystrophic mice was aimed at producing mosaic muscles containing normal, dystrophic and mosaic myofibers from the normal and dystrophic minced muscle mixes (Law, 1978). It focused on incorporating the "missing" gene and its product(s) into genetically defective cells through cell transplantation and natural cell fusion, the result of which is strengthened dystrophic muscles (Law, 1978) having a gene defect similar to human congenital muscular dystrophy (Sunada et al., 1995). The result contradicts the study of Partridge and Sloper (1977) who concluded, in transplanting normal minced muscles into normal hosts, that little or none of the regenerates was of donor origin. Eventually, fusion between host and donor myogenic cells of normal genotypes using skeletal muscle grafts were demonstrated with genotype marker (Partridge et al., 1978). Although this latter study did not involve dystrophic animals, it was inferred that MTT was a distinct development with potential applicability to hereditary myopathies. In a later study, muscles of newborn normal mice were grafted into recipient soleus muscles of dystrophic mice. Results obtained 6 months after the grafting indicated that the grafts survived, developed, and functioned in the dystrophic environment. The regenerates had larger crosssectional areas and more muscle fibers than the contralateral dystrophic solei. MTT increased the mean twitch tension of adult dystrophic muscles to that of the normal (Law and Yap, 1979). The concept of replenishing lost cells and repairing degenerative cells through the production of genetic mosaicism using MTT was firmly established (Law and Yap, 1979). An important finding was that myoblasts cultured from muscle biopsies of adult normal rats could survive and develop in the original donor after implantation (Jones, 1979). MTT with cultured myoblasts became the logical development since myoblasts do not require neuronal and capillary connections to survive and develop, and since myoblasts can fuse to effect genetic repair. A convenient way to obtain normal myoblasts in mice is through dissection of limb-bud mesenchyme of day-12 embryos. Dissected mesenchyme was surgically implanted into the solei of dy2Jdy2J mice. Host and donors were histocompatible. Contralateral solei served as controls. Six to seven months postoperatively, the myoblastimplanted solei exhibited greater cross-sectional area, total fiber number, better cell structure, and twitch and tetanus tensions than their contralateral controls (Law, 1982). The incorporation and fusion of allogeneic muscle precursor cells in vivo were further explored using normal mice (Watt, 1982). The implants consisted of minced muscle mixes or newborn muscles (Watt et al., 1982; Watt et al., 1984; Morgan et al., 1988). It was confirmed 349

Law et al: Myoblast transfer as a platform technology Aside from donor cell survival in an immunologically hostile host, cell fusion is the key to strengthening dystrophic muscles with MTT. To improve the fusion rate between host and donor cells, various injection methods aimed at wide dissemination of donor myoblasts were tested and compared. The goal was to achieve maximum cell fusion with the least number of injections. The results indicate that delivery of myoblasts is best conducted by diagonal placement of needle into the host muscle with ejaculation of the myoblasts as the needle is withdrawn. This method of myoblast injection yields even and wide distribution of donor myoblasts with a high rate of cell fusion. Myoblasts injected perpendicular to myofiber orientation are partially distributed. Myoblasts injected longitudinally through the core of the muscles and parallel to the myofibers are poorly distributed (Law et al., 1994b). Thus myoblast injection method regulates cell distribution and fusion.

V. Clinical trials Gene therapy encompasses interventions that involve deliberate alteration of the genetic material of living cells to prevent or to treat diseases (Kessler et al., 1993). According to this FDA definition, the first MTT on a DMD boy on February 15, 1990 marked the first clinical trial on human gene therapy (Hooper, 1990). In addition to fulfilling their primary muscle-building mission, the myoblasts served as the source and the transfer vehicles of normal genes to correct the gene defects of DMD. The protocol was approved by four institutional review boards (Law, et al., 1990c). Subjects and parents gave informed consents. The safety and efficacy of MTT was assessed by injecting the left extensor digitorum brevis (EDB) muscle of a 9-yr-old DMD boy with about 8 x 10 6 myoblasts. Donor myoblasts were cloned from satellite cells derived from a 1 g rectus femoris biopsy of the normal, adoptive father. Cyclosporine was administered for three months at a dose of 5-7 mg/kg body weight divided into two daily oral doses. Donor myoblasts survived, developed, and produced dystrophin in myofibers biopsied from the myoblastinjected EDB 92 days later. Dystrophin was not found in the contralateral sham-injected muscle. This first case suggested that MTT offered a safe and effective means for alleviating biochemical deficit(s) inherent in muscles of DMD (Law et al., 1990a). A pioneering work (Anderson, 1990; see also Brenner, 1995; Karlsson, 1991) is often considered as the "first human gene therapy"; correction of the ADA deficiency study began on September 14, 1990 (Anderson, 1990), two months after the MTT correction of the DMD gene defect was published (Law et al., 1990a). In the ADA protocol, T cells from a patient with a severe combined immunodeficiency disorder (SCID) were transduced with functional ADA genes ex vivo and returned to the patient 350

after expansion through culture. In the MTT protocol, primary culture of myoblasts derived from a muscle biopsy of a normal donor was injected into a muscle of the DMD subject to produce in vivo nuclear complementation. Both gene therapies utilize cell transplantation to treat diseases. However, it is pointed out that the ADA protocol involved genetic modification and correction of the patients T cells with the adenosine deaminase gene whereas in the DMD protocol normal donor cells were used which were not genetically modified ex vivo. Six years after the foremost MTT, dystrophin was found in the myoblast-injected muscle but not in the sham-injected muscle (F i g u r e 3 , Law, 1997). Six years is the longest period through which any gene therapy has sustained positive results. Despite cyclosporine withdrawal at 3 months after MTT, myofibers expressing foreign dystrophin were not rejected. This is because dystrophin is located in the inner surface of the plasma membrane, and because mature myofibers do not exhibit MHC-1 surface antigens. Not only has the result demonstrated MTT overall safety and efficacy in this single case, it also shows stability in the integration, regulation and expression of the inserted dystrophin gene. The presence of dystrophin in the myoblast-injected but not in the sham-injected muscle provided unequivocal evidence of the survival and development of donor myoblasts in the myoblast-injected muscle. In a randomized double-blind study involving three subjects, myoblast-injected EDBs showed increases in tensions whereas sham-injected EDBs showed reductions (Law et al., 1991a,b). Both immunocytochemical staining and immunoblot revealed dystrophin in the myoblastinjected EDBs. Dystrophic characteristics such as fiber splitting, central nucleation, phagocytic necrosis, variation in fiber shape and size, and infiltration of fat and connective tissues were less frequently observed in these muscles. Sham-injected EDBs exhibited significant structural and functional degeneration and no dystrophin. Throughout the study, there was no sign of erythema, swelling or tenderness at the injection sites. Serial laboratory evaluation including electrolytes, creatinine, and urea did not reveal any significant changes before or after MTT. To reconcile these positive results with less convincing ones (Gussoni et al., 1992; Huard et al., 1992; Karpati et al., 1993; Mendell et al., 1995; Miller et al., 1992; Morandi et al., 1995; Tremblay et al., 1993), several issues need to be addressed. To begin with, the use of large quantities of pure live myoblasts is a pre-requisite of successful MTT. Except for one study (Law et al., 1992), there is no published pictorial evidence to substantiate the purity, myogenicity and viability of the injected myoblasts as claimed. Myoblast cultures are usually contaminated with fibroblast overgrowth. MTT with such impure culture could lead to deposition of connective tissues rather than myofiber production. Culturing 50 billion pure human myoblasts for MTT from two grams of muscle biopsy has

Gene Therapy and Molecular Biology Vol 1, page 351 only been reported by our team (Law et al., 1997a). Other teams work at ranges of hundreds of millions of myoblasts. In most studies (Gussoni et al., 1992; Karpati et al., 1993; Mendell et al., 1995; Miller et al., 1992; Morandi et al., 1995) myoblasts were transported frozen, chilled for over two hours from the site of harvest before being injected. Since myoblasts have a high metabolic rate, they could not have survived for two hours without significant nutrients, oxygen and proper pH, being closely packed in saline within a vial for transport. Determination of cell viability before MTT were not conducted in these studies. Our myoblasts were injected into the subject within minutes of harvest, at the same location without transport. MTT studies that reported failure (Gussoni et al., 1992; Huard et al., 1992; Karpati et al., 1993; Mendell et al., 1995; Miller et al., 1992; Morandi et al., 1995; Tremblay et al., 1993) subscribed to the fallacy of making 55 to 330 injections into a muscle the size of an egg, traumatizing indiscriminately the underlying nerves, muscle, and vasculature. These injection traumas boosted macrophage access and host immune responses (Guerette et al., 1995). They also induced fibrosis (Chen et al., 1988). Surviving myoblasts fused within three weeks in small mouse muscles (Chen et al., 1992). A nerve with multiple trauma could not regenerate soon enough through scar and connective tissues to innervate the newly-formed myotubes in a large human dystrophic muscle. Stabilization of muscle contractile properties in a similar situation is achieved by 60 days in the rat, and functional return is incomplete (Carlson, 1983). Non-innervated myotubes died within one week. Whatever few myotubes that developed in the unsuccessful MTT studies could not compensate for the traumatized myofibers. In the study yielding positive results, 5 to 8 x 108

has to be made to satisfy the unavoidable scavenger process. As reflected in the small numbers of myoblasts injected in unsuccessful studies, it appears that either such allowance was not considered or that the teams were not able to produce larger quantities of pure myoblasts. Although myoblast loss can be minimized by downregulating macrophage activity (Guerette et al., 1997), such additional compromisation of the host immune system may lead to higher risk of infection, since MTT subjects are already taking immunosuppressants. The less successful MTT teams focused on immunosuppression to prevent T-lymphocyte proliferation and antibody production without overcoming the primary hurdle of providing enough pure and live myoblasts. A basic study indicates that cyclophosphamide did not permit myoblast engraftment in the mouse (Vilquin et al., 1995), and a MTT clinical trial was conducted without success using cyclophosphamide immunosuppression (Karpati et al., 1993). Cyclosporine (Law et al., 1990a) and potentially FK506 (Kinoshita et al., 1994) remain the immunosuppressants of choice for MTT. Results could have been more positive if either was employed in the study of Tremblay et al. (Huard et al., 1992; Tremblay et al., 1993). All of these single muscle MTT studies had begun before the FDA established policies and regulations for cell/gene therapies. Our studies are the only ones that received permission for an investigational new drug application (IND) on MTT for treatment of multiple muscles. As a cell/gene therapy, all American MTT clinical trials must come under FDA purview. Beginning with 8 million myoblasts into a small foot muscle, our team proceeded to test 5 billion cells into 22 leg muscles, 25 billion cells into 64 body muscles, and now 50 billion cells into 82 muscles (T a b l e 1). With over 150 procedures having been conducted, the complete safety of the MTT procedure has been proven. There have been no adverse reactions or side effects.

pure myoblasts were delivered with eight injections into the biceps brachii without nerve injury (Law et al., 1994a, 1997a). Contrarily, in another study, 55 sites, each 5 mm apart, distributed in 11 rows and 5 columns, were injected throughout the depth of each biceps of 5- to 9- year old boys (Mendell et al., 1995). This was repeated monthly for six months. Axonal sprouts, myotubes and neuromuscular junctions that take six weeks to mature (Fex and Jirmanova, 1969) were repeatedly traumatized by a total of 330 injections until the biceps, with or without myoblast/cyclosporine, were irreversibly damaged or destroyed. The result: no functional difference between myoblast- and sham-injected muscles (Mendell et al., 1995). Once injected, the myoblasts are subjected to scavenger hunt by macrophages for up to three weeks. This is because myoblasts exhibit MHC-1 surface antigens (Friedlander and Fischman, 1979; Fang et al., 1994) that become absent after cell fusion. The latter occurs between one to three weeks after myoblast injection (Chen et al., 1992). An allowance in the number of injected myoblasts

Table 1 . Dose escalation protocols of MTT and the number of subjects receiving such procedures.

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F i g . 3 . Immunocytochemical demonstration of dystrophin in DMD muscles 6 yr after MTT. Dystrophin absent in sham-injected EDB muscle (A , C ), but present in the contralateral myoblast-injected muscle (B , D ). Dystrophin was immunocytochemically localized at the sarcolemma (arrows). Dystrophin demonstrated at low (E) and high (F) magnification in normal control muscle. Cross-section; bar = 100Âľm.

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F i g . 4 . Dystrophin immunocytochemistry showing the presence of dystrophin in (A) normal control and in (C,E,G) muscle biopsy specimens of three subjects. Dystrophin is absent in (B) Duchenne's muscular dystrophy control and in (D,F,H) contralateral biopsy specimens from the same subjects.

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F i g . 5 . (A , C , E ) Three dystrophin-positive muscle biopsy specimens exhibit less dystrophic characteristics than the contralateral dystrophin-negative biopsy specimens (B , D , F ). Dystrophic characteristics include increases in fat and connective tissue, fiber splitting, central nucleation, round and oval fibers.

took cyclosporine for six months after MTT. More than 88% of the injected ankle plantar flexor muscles showed either increase in strength or no further deterioration at 9 months after MTT (Law et al., 1992, 1993).

The five billion myoblast cell protocol. The 5-billion myoblast MTT protocol was tested in 32 DMD boys aged 6-14 yr. Through 48 injections, 5 billion myoblasts were transferred into 22 major muscles in both lower limbs under general anesthesia. Only four donors were histocompatible with their recipients. All subjects

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F i g . 6 . Dose-dependent responses to MTT of plantar flexion with greater increase in maximum isometric force using the 50-billion MTT protocol than with the 25billion MTT protocol. Both protocols show efficacy in strengthening the plantar flexion when compared to the natural history control.

Table 2. Percentage increases over a one-year natural history control in the maximum isometric force of the plantar flexor muscles at 3, 6, 9, 12, and 15 months after the administration of the 25-billion MTT protocol or the 50-

billion MTT protocol.

The 25 billion myoblast cell protocol. Under FDA purview, MTT is completing Phase II clinical trials on DMD. The whole body trial (WBT) consisted of injecting 25 billion myoblasts in two MTT procedures separated by 3 to 9 mo. Each procedure delivered up to 200 injections or 12.5 billion myoblasts to either 28 muscles in the upper body (UBT) or to 36 muscles in the lower body (LBT). A randomized doubleblind portion of the study was conducted on the biceps brachii or quadriceps. Subjects took oral cyclosporine for 3 months after each MTT. One infantile facioscapulohumeral dystrophy and 40 DMD boys aged 6 to 16 received WBT in the past 36 months with no adverse reaction. Nine months after MTT immunocytochemical evidence of dystrophin were demonstrated in 18 of the 20 DMD subjects biopsied (F i g . 4 ). Dystrophin positive sections showed less dystrophic characteristics than dystrophinnegative ones (F i g . 5 ). Forced vital capacity increased by

33.3% and maximum voluntary ventilation increased by 28% at 12 months after UBT (Law et al., 1997a). Plantar flexion showed an increase of 45% in maximum isometric contraction force in 12 months in the DMD subjects when compared to the natural deterioration (F i g . 6 , Table 2). Behavioral improvements in running, balancing, climbing stairs and playing ball were noted (Law et al., 1995; Law et al., 1996; Law et al., 1997a,c,d). Notable was a 16-yr-old DMD subject who continued to walk without assistance and capable of driving an automobile by himself. 50 Billion myoblast cell protocol. The current study involves a one time injection of 50 billion myoblasts into 82 muscles with 179 skin punctures, approved by the FDA for subjects with DMD, Becker MD and Limb-girdle MD (Law et al., 1997d). Twenty-nine subjects who underwent this protocol have experienced no adverse reaction.

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Law et al: Myoblast transfer as a platform technology repairs muscle cell membrane leakage of enzymes. This contention is further substantiated by similar findings with another muscle enzyme AST, aspartate aminotransferase (F i g . 8 ). The breakthrough came when a 29-yr-old Becker MD (BMD) subject began to walk, with his hands being held, beginning at 2.5 months after the 50-billion MTT. He had previously been diagnosed repeatedly with BMD. He had been non-ambulatory and required the use of a wheelchair for over four years as documented in his medical record. He began walking with assistance a total of eight steps at 3 months after MTT. This ability increased with time, now reaching 60 steps at eight months after MTT. He began to stand and walk with his crutches at four months after MTT (F i g . 9).

For the 22 DMD subjects aged 5 to 16, there was a significant increase in the maximum isometric force generated by the plantar flexor muscles at 3, 6, and 9 months after MTT (F i g . 6 , T a b l e 2 ). This functional improvement is more pronounced with the 50-billion MTT than with the 25-billion MTT, indicating that it is dose-dependent. Thus, in the 25billion MTT, 800 million myoblasts were injected into the plantar flexors, producing a mean 61% increase in force at 15-months after MTT. With the 50 billion MTT, 50% more myoblasts were injected, projecting a 10% greater increase in force at 15 months after MTT (F i g . 6 , T a b l e 2).

VI. Future perspectives As an universal gene transfer vehicle with which the entire human genome can be integrated into patient's muscles, myoblasts have shown promise in studies of the following diseases: Cardiomyopathy. Labeled cultured myoblasts engrafted and formed structures resembling desmosomes, intercalated discs, fascia adherents junctions, and gap junctions in myocardia of dogs (Chiu et al., 1995), rats (Murry et al., 1996) and mice (Robinson et al., 1996) when MTT was delivered intramuscularly (Chiu et al, 1995; Murry et al., 1996) or intraarterially (Robinson et al., 1996). Donor muscle regenerates exhibited cardiac-like properties such as central nucleation (Chiu et al., 1995), fatigue resistance, slow twitching, and were capable of twitch and tetanus contractions when stimulated (Murry et al., 1996). Similar results were obtained when cardiomyocytes were injected in dystrophic mice and dogs (Koh et al., 1995), rats (Li et al., 1996) and swine (Van Meter et al., 1995). These findings, together with established MTT safety, pave the way to MTT clinical trial in treating myocardial degeneration and dysfunction.

F i g . 7 . Serum creatine kinase (CK) level of DMD subjects increased before MTT and decreased after MTT.

Insulin-resistant diabetes mellitus. Commonly known as Type II diabetes, this disease is genetically predisposed and afflicts 90% of the diabetic population. Virtually all identical siblings of these patients develop the disease, and the genetic defect can be traced to the GLUT4 gene deletion. The major sequela of insulin resistance is decrease muscle uptake of glucose, due to the moderate decrease in insulin receptors on muscle cell surface. Conceptually MTT can add genetically normal myofibers with normal insulin receptors. It can also genetically repair the patients' myofibers and produce normal insulin receptors on the heterokaryons. Basic research is need to test this hypothesis on diabetic rats.

F i g . 8 . Serum aspartate aminotransferese (AST) level of DMD subjects increased before MTT and decreased after MTT.

Elevated serum creatine kinase (CK) has traditionally been used to diagnose muscle degeneration, notable in DMD (Heyck et al., 1966). The 22 DMD subjects, mean ages 10.7-yr-old and, median age 9.9 yr-old, showed a 19.3% increase in serum CK within 3 months before MTT (F i g . 7 ). This trend was reversed after MTT, and the serum CK declined at a steady rate of 48.7% over 12 months. This result provides strong evidence that MTT 356

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F i g . 9 . First muscular dystrophy subject ever to walk after wheelchair bound for years. (A). The 29-yr-old BMD subject had been wheelchair-bound for over 4 years. (B , C , D , E ) He began to walk with his hands held at 2.5 months after the 50billion MTT. (F) At 4 months after MTT, he was able to walk on crutches for about 20 steps.

Leiden, 1991) and allograft rejection (Lau et al., 1996). MTT has produced a new frontier in medicine.

Bone/cartilage degeneration. During embryonic development, mesenchymal progenitor cells differentiate into myoblasts, osteoblasts, chondrocytes and adipocytes under controls of various regulatory factors. Ectopic bone formation in muscle has been achieved through implantation of bone morphogenetic protein (BMP). BMP-2 was shown to convert the differentiation pathway of clonal myoblasts into the osteoblast lineage (Katagiri et al., 1994). This opens new ways to treat conditions of bone degeneration such as the degeneration of tooth pulp, hip, bone/joint, and long bone fractures. Given the ability to mass-produce myoblasts that can be transformed into osteoblasts, and potentially chondrocytes, the difficulty of proliferating osteoblasts and chondrocytes can be overcome. Cultured autologous chondrocytes can be used to repair deep cartilage defects in the femorotibial articular surface of the human knee joint (Brittberg et al., 1994). The use of normal or transduced myoblasts as the source and vehicles for gene delivery has found application in the potential treatments of restenosis (Morishita et al., 1995), soft tissue deformities (Teboul et al., 1995), hemophilias (Dai et al., 1992; Yao et al., 1994), anemia (Hamamori et al., 1994), muscle trauma (Almeddine et al., 1994), human growth hormone deficiency (Barr and

VII. Our vision MTT implementation can benefit from development of the following programs (Law, 1994):

the

Controlled cell fusion. It will be useful to be able to control, initiate or facilitate cell fusion once myoblasts are injected. This is to minimize loss of myoblasts from macrophages whose presence is unavoidable if the patient is to have some immune protection. As the myoblasts are injected intramuscularly into the extracellular matrix, injection trauma causes the release of basic fibroblast growth factor (bFGF) and large chondroitin-6-sulfate proteoglycan (LC6SP). These latter growth factors stimulate myoblast proliferation. Unfortunately, they also stimulate the proliferation of fibroblasts that are already present in increased amount in the dystrophic muscle. That is why it is necessary to inject as pure as possible fractions of myoblasts in MTT without contaminating fibroblasts.

357

Law et al: Myoblast transfer as a platform technology be delivered to remote points of utilization around the world. F i g 1 0 shows the effectiveness of such a medium developed in our foundation. Fifty billion myoblasts can be shipped at 4o C for four days with 90% viability.

Controlled cell fusion can be achieved by artificially increasing the concentration of LC6SP over the endogenous level. In addition, insulin or insulin-like growth factor I (IGF-1) may facilitate the developmental process, resulting in the formation of myotubes soon after myoblast injection. Enhanced fusion of myoblasts into myotubes had been achieved with the use of PDO98059 (Coalican et al., 1997) and ED2+ macrophages conditioned medium (Massimino et al., 1997). The use of these compounds in the cell culture medium and in the injection medium will likely lead to greater MTT success.

C e l l banks. The automated cell processors will constitute only a part of the cell banks. The current thought is to obtain donor muscle biopsies from young adults aged 8 to 22 to feed the inputs. Each donor has to undergo a battery of tests that are time-consuming and expensive. From the test results and from the donorâ&#x20AC;&#x2122;s physical conditions, one can determine if the donor cells are genetically defective or infected with viruses and/or bacteria. Human fetal tissues can potentially provide greater supplies of cells. However, aside from ethical issues surrounding abortion, it is difficult to determine the genetic normality of the cells. Muscle primordia of fetus derived from in vitro fertilization of genetically welldefined background may be an alternative. Sperm and ova can be recovered from healthy individuals that are known for their muscle strength and mass. In vitro fertilization will be followed by embryo culture to a specific developmental stage (day 26 to day 56 gestation) of the embryos. The muscle primordia that are rich in myoblasts can then be dissected out to feed the automated cell processors.

Superior c e l l lines. These cell lines should be highly myogenic, nontumorigenic, nonantigenic, and will develop very strong muscles. The superior cell lines will bypass the use of immunosuppressant, and will provide a ready access for patients who do not have a donor. A unique property of myoblasts is their loss of MHC-I antigens soon after they fuse. The immunuosuppression period depends on how soon the myoblasts lose their MHC-I antigens after MTT. Even more ideal is the establishment of a myoblast cell line in which MHC-I antigens are absent. In human myoblasts cultured from normal muscle biopsies, some 91.7% of the myoblasts reacted with MHC-I MAb (monoclonal antibodies). The remaining 8.3% of the myoblasts, that were negative for MHC-I antigen expression were successfully separated by cytofluorometry. The lack of MHC-I antigens on these latter myoblasts may enhance survival of these myoblasts in recipients after MTT (Fang et al., 1994).

VIII. Conclusion This chapter describes the landmark development of the first gene therapy study in humans. Through natural cell fusion, myoblasts transfer the human genome into dystrophic muscle cells to effect phenotype repair. The innovative cell transplantation procedure also revitalizes the degenerative organ by providing living cells of normal genotype to replenish cell loss. The result is potentially a new form of medicine. The conceptual approaches of single gene transfer and myoblast transfer toward treatment of hereditary degenerative diseases are compared. As more scientists continue to recognize myoblasts as a stable source of genes and a safe and efficient gene transfer vehicle, MTT application will extend far beyond the treatment of neuromuscular diseases. This chapter provides insights to guide future development of MTT in battling against genetic and acquired diseases that presently have only diagnoses but no treatment.

Automated cell processors. The great demand for normal and transduced myoblasts, the labor intensiveness and high cost of cell culturing, harvesting and packaging, and the fallibility of human imprecision will soon necessitate the invention and development of automated cell processors capable of producing huge quantities of viable, sterile, genetically well-defined and functionally demonstrated biologics. This invention will be one of the most important offspring of modern day computer science, mechanical engineering and cytogenetics. The intakes will be for biopsies of various human tissues. The computer will be programmed to process tissue(s), with precision controls in time, space, proportions of culture ingredients and apparatus maneuvers. Cell conditions can be monitored at any time during the process and flexibility is built-in to allow changes. Different protocols can be programmed into the software for culture, controlled cell fusion, harvest and package. The outputs supply injectable cells ready for cell therapy or shipment. The cell processor will be selfcontained in a sterile enclosure large enough to house the hardware in which cells are cultured and manipulated.

Acknowledgment Clinical trials are supported by public donations with FDA approval for direct cost recovery.

Transport medium. A transport medium that can sustain the survival and myogenicity of myoblasts in package for up to four days will allow the cell packages to 358

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F i g . 1 0 . Transport medium effectiveness as demonstrated by myoblast survival and myotube formation. (A). Myoblasts before a 50-billion MTT showing 99% viability using the vital stain erythrocin B,1% at pH 7.23. (B ). Myoblast left-over from a 50-billion MTT maintained in the transport medium for 4 days at 4o C and stained with erythrocin B. The sample showed 90% viability. (C). Cells in B were put back into culture for 2 days before feeding fusion medium. (D). Cells in C in fusion medium for 1 day, showing myoblast fusion (arrow). (E). Cells in C in fusion medium for 2 days, showing myotubes (arrow). (F). Cells in C in fusion medium for 5 days, showing extensive myogenic capability in myotube formation (arrows).

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Gene Therapy and Molecular Biology Vol 1, page 365 Gene Ther Mol Biol Vol 1, 365-379. March, 1998.

Constitutive activation of fibroblast growth factor receptors in human developmental syndromes Melanie K. Webster and Daniel J. Donoghue* Department of Chemistry and Biochemistry and Center for Molecular Genetics, University of California, San Diego, La Jolla, California 92093-0367 __________________________________________________________________________________________________ * Correspondence: Daniel J. Donoghue, Tel: (619) 534-2167, Fax: (619) 534-7481, E-mail: ddonoghue@ucsd.edu

Keywords: FGFR3; Thanatophoric Dysplasia; skeletal malformation; achondroplasia; receptor tyrosine kinase Abbreviations: FGFR, fibroblast growth factor receptor; FGF, fibroblast growth factor; TD, thanatophoric dysplasia

Summary Fibroblast growth factor receptors (FGFRs) represent specific receptors for the fibroblast growth factors (FGFs), a family of at least 13 polypeptides. Ligand/receptor interactions between FGFs and their receptors are involved in many fundamental biological processes, particularly cell growth and differentiation during chondrogenesis and myogenesis. The four different human FGFR genes encode related glycoproteins with a common structure consisting of an N-terminal signal peptide, three immunoglobulin (Ig)-like domains, a single transmembrane domain, and an intracellular split t y r o s i n e - k i n a s e d o m a i n . F G F s , a c t i n g i n concert with heparan sulfate proteoglycans, bind to FGFRs and result in their activation, involving homo- or hetero-dimerization of receptors, leading to trans-phosphorylation of the kinase domains. The activated receptors can then phosphorylate various intracellular proteins involved in signal transduction, although much remains to be learned concerning these signal transduction pathways downstream of activated FGFRs. Many mutations in different domains o f FGFR1, FGFR2 and FGFR3 have recently been identified as causing various human craniosynostosis and dwarfism syndromes, and the molecular consequences of these mutations are beginning to be unraveled. Craniosynostosis syndromes, characterized by premature ossification and fusion of the cranial sutures of the skull, arise primarily from mutations in the extracellular domain FGFR2, although specific mutations i n other FGFRs may also underlie related craniosynostosis syndromes. Skeletal dwarfism syndromes, characterized by disproportionate short stature and macrocephaly, arise predominantly from mutations in FGFR3 and include achondroplasia, the most common genetic form o f dwarfism, as w e l l as the thanatophoric dysplasias (type I and type II). Recent studies demonstrate that a common mechanism, constitutive activation of receptor signaling, underlies most of these disorders. The mutations responsible for the craniosynostosis and skeletal dwarfism syndromes map variously to either the extracellular domain, the transmembrane domain, or the tyrosine kinase domain of these receptors, suggesting multiple mechanisms of aberrant receptor activation. An overview of the developmental consequences arising from mutations in FGFR family members will be presented, including an examination of the molecular mechanisms underlying these defects.

the skull, and endochondral ossification, which occurs at the growth plates of the vertebrae, the pelvis, and the long bones of the extremities. In the process of intramembranous ossification, primitive mesenchymal cells differentiate into osteoblasts (bone-forming cells) that secrete a collagen-glycosaminoglycan matrix which

I. Skeletal Development The development of the human skeleton is a highly complex and regulated process. Osteogenesis (bone formation) includes both intramembranous ossification, responsible for development and fusion of the flat bones of

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Webster and Donoghue: FGFR Activation in Developmental Syndromes subsequently becomes calcified. In contrast, endochondral ossification is a two-step process in which mesenchymal cells first condense and differentiate into chondrocytes that secrete a cartilaginous template. As the chondrocytes proliferate, hypertrophy and die in an ordered sequence, osteoblasts carried by infiltrating blood vessels deposit bone matrix to replace the degrading cartilage template (Gilbert et al., 1994). Reflecting the complexity of skeletal development, more than 150 different disorders of osteochondrogenesis have been described (Spranger, 1992). For instance, defects may occur in the condensation or differentiation of the mesenchyme, in the structure or regulation of components of the extracellular matrix, and/or in the normal proliferation and maturation of the chondrocytes (Mundlos and Olsen, 1997a,b). Recently, the specific genes defective in some of these human skeletal disorders have been identified (Reardon, 1996), and many encode proteins falling into three categories: structural proteins, including several different collagens (Mundlos and Olsen, 1997a,b); transcription factors, including Sox9 (Wagner et al., 1994; Foster et al., 1994), MSX2 (Jabs et al., 1993) and Twist (El Ghouzzi et al., 1997; Howard et al., 1997); and growth factors and their receptors, including the fibroblast growth factor receptors (FGFRs) (Webster et al., 1996). In this review we will focus on skeletal and cranial malformation syndromes associated with mutations in FGFRs.

F i g u r e 1 . T he FGFR family . The overall structure of the four human FGFR family members is shown, together with their chromosomal locations.

Fibroblast growth factors (FGFs) comprise a family of structurally-related heparin-binding proteins with pleiotropic actions. For instance, they are able to stimulate proliferation in certain cell types, and induce, inhibit or maintain the differentiated phenotype of other cell types. FGFs also exhibit potent neurotrophic and angiogenic activities, and play key roles in embryogenesis (Johnson and Williams, 1993). FGFs, in association with heparan sulfate proteoglycans, bind with high affinity to the extracellular domain of a family of four transmembrane tyrosine kinases, FGFR1-FGFR4, shown schematically in Figure 1.

ing ligand stimulation, it is still unclear exactly how the signal from FGFR is transmitted to Ras (Mohammadi et al., 1996a). Recent studies suggest that tyrosine phosphorylation of both FRS2 and Shc, causing recruitment of the Ras activator complex, Grb2/Sos, to the plasma membrane, links FGFR1 activation to the Ras/MAPK pathway (Kouhara et al., 1997), although a physical association of FGFRs with these proteins has not been demonstrated. Ligand activation of FGFR1 also leads to phosphorylation of and association with PLC-! (Mohammadi et al., 1991), although PLC-! " dependent phosphatidylinositol hydrolysis does not appear to be required for either FGF-dependent mitogenesis (Mohammadi et al, 1992) or for differentiation (SpivakKroizman et al, 1994b; Muslin et al., 1994). There is significant homology in the catalytic domains of all four FGFRs, suggesting that they regulate many of the same signaling pathways, although differences have been observed in the strength of signals generated by activation of the different receptors (Ornitz and Leder, 1992; Wang et al., 1994).

This binding results in receptor homo- or heterodimerization, leading to trans-phosphorylation and activation of the intracellular kinase domains (SpivakKroizman et al., 1994a; Ullrich and Schlessinger, 1990). The activated receptors can then phosphorylate substrate molecules which transmit signals into the cell. Some of the effectors involved in FGFR signaling have been identified, and include components of the Ras/MAPK pathway. In contrast to many other receptor tyrosine kinases that directly interact with adaptor proteins follow-

The extracellular domains of the FGFRs are also highly homologous, and are comprised of three immunoglobulin-like (Ig-like) domains containing characteristic cysteine residues, with an acidic region between the first and second Ig domains. Alternative splicing is common within the extracellular domain of FGFRs, and forms of the receptors possessing only the second and third Ig domains are recognized (Johnson et al., 1991). FGFs bind to Ig-2 and Ig-3 and the linker region between these two Ig domains, although it is the choice of

II. Fibroblast growth factor receptors: signaling, ligand-binding, and expression

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Gene Therapy and Molecular Biology Vol 1, page 367 exon (IIIb or IIIc) in the 3' half of the third Ig domain of FGFR1-FGFR3 that is critical in determining the ligandbinding specificities of each receptor (Johnson and Williams, 1993). Of the nine well-characterized FGFs, only FGF-1 (aFGF) binds with high affinity to all receptor isoforms, and certain FGF/FGFR interactions, such as FGF-7 (KGF) with FGFR2b, are highly specific (Ornitz et al., 1996).

Local application of several different FGFs can also induce the formation of ectopic limb buds in the chick, which develop into complete limbs with a normal skeletal structure (Cohn et al., 1995). Consistent with a key role for FGFs and FGFRs in bone development, transgenic mice overexpressing FGF-2 exhibit a variety of skeletal malformations resembling human chondrodysplasia syndromes, including shortening and flattening of the long bones and macrocephaly (Coffin et al., 1995). On the other hand, targeted disruption of the murine FGFR3 gene results primarily in an expansion of the zones of proliferating and hypertrophic chondrocytes at the bone growth plates, resulting in enhanced growth of the long bones and vertebrae (Deng et al. 1996; Colvin et al. 1996).

The biological activities of the four FGFRs are determined not only by their differences in signaling and ligand-binding affinities, but also by their distinct spatial and temporal expression patterns. For instance, FGFR1 is expressed in the primitive ectoderm of the postimplantation embryo, and FGFR1-deficient embryos die prior to gastrulation (Deng et al, 1994). During organogenesis, FGFR1 is widely expressed throughout the mesenchymal tissues, including the limb buds, whereas FGFR2 is expressed in the surface ectoderm and epithelia (Orr-Urtreger et al., 1991; Peters et al., 1992). The two different isoforms of FGFR2, KGFR and bek, are expressed in different layers of the developing skin, with bek also highly expressed in bones of the vertebrae, limbs, skull and ribs (Orr-Urtreger et al., 1993). Unlike FGFR1 or FGFR2, FGFR3 expression is less widespread in early embryogenesis, and is restricted to the glial cells of the brain, the differentiating hair cells of the cochlear duct, and the cartilage of the vertebrae, skull and long bones (Peters et al., 1993). FGFR4 expression is most apparent in the endoderm of the developing gut, liver and lung, in skeletal muscle, and in the endochondral cartilage of the ribs and the olfactory and auditory regions (Stark et al., 1991).

IV. Mutations in FGFRs are associated with human skeletal dysplasias The initial clue that FGFRs are involved in human cranial and skeletal disorders came from genetic linkage studies. The gene for achondroplasia, the most prevelant form of short-limb dwarfism, was mapped by several groups to 4p16.3, a chromosomal region that includes the FGFR3 gene (Le Merrer et al., 1994; Velinov et al., 1994). A recurrent mutation in this gene was rapidly confirmed in virtually all patients with achondroplasia (Shiang et al., 1994; Rousseau et al, 1994). At about the same time, certain autosomal dominant craniosynostosis syndromes were mapped to the FGFR2 locus on 10q25 (Reardon et al., 1994; Jabs et al., 1994; Schell et al., 1995), and to chromosome 8p11.2-p12, a region that included the FGFR1 gene (Muenke et al., 1994). Various mutations were identified in these candidate genes (see below), suggesting that they were directly responsible for the craniofacial and digital anomalies observed in these syndromes.

III. Role of FGFs and FGFRs in bone development As noted above, all four receptors are expressed to some extent in developing bone. Specifically, FGFR1, FGFR2 and FGFR3 have overlapping expression patterns in prebone cartilage rudiments, whereas in later stages of bone formation, FGFR1 is expressed primarily in osteoblasts and hypertrophic cartilage, FGFR2 is expressed in the perichondrium/periostium and in the presumptive bone marrow, and FGFR3 expression is confined to resting cartilage (Peters et al., 1993). These observations suggest that individual FGFRs play distinct and important roles in skeletogenesis.

V. FGFR mutations in craniosynostosis syndromes The flat bones of the skull of the neonate are normally discrete, enabling molding and overlap to occur during compression in the birth canal as well as allowing the skull to grow in parallel with the growth of the brain. The fibrous sutures between these bones gradually interdigitate and close with bony bridging, although complete fusion of the cranial and facial sutures takes place over the lifetime of the individual (Cohen, 1997). Craniosynostosis, a relatively common birth defect, occurs as a result of premature ossification and fusion of one or more of the cranial sutures. Several related syndromic forms of craniosynostosis have been identified, which all share characteristic craniofacial features including abnormal head

FGFs are clearly intimately involved in bone and limb development. They act as potent mitogens for chondrocytes, yet they also inhibit chondrocyte terminal differentiation (Kato and Iwamoto, 1990). Additionally, FGFs have been shown to enhance extracellular matrix formation by chondrocytes and to accelerate vascular invasion and ossification of growth plate cartilage (Baron et al., 1994). 367

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Figure 2. The locations of point mutations in FGFR1, FGFR2 and FGFR3 giving rise to craniosynostosis syndromes. Abbreviations: AB, acid box; Ig, immunoglobulinlike domain; Kinase, split tyrosine kinase domain; SP, signal peptide; TM, transmembrane domain. The numbers indicate the amino acid residue number at the approximate boundaries of each domain.

causing Pfeiffer syndrome when expressed in FGFR1 (Muenke et al. 1994), Apert syndrome when expressed in FGFR2 (Wilkie et al. 1995b), and a non-syndromic craniosynostosis when expressed in FGFR3 (Bellus et al. 1996; Muenke et al., 1997). Often, the identical mutation results in clinically distinct disorders in different individuals; for instance, substitutions at Cys342 in the Ig3 domain of FGFR2 lead to Pfeiffer, Crouzon or Jackson-Weiss syndromes (Wilkie et al., 1995a; Meyers et al., 1996; Park et al., 1995; Reardon et al., 1994; Rutland et al., 1995; Steinberger et al., 1995), although these phenotypes usually breed true within families. It is also of interest that substitutions leading to craniosynostosis are found both in FGFR2 exon IIIa, in which case they are expressed in both bek and KGFR forms of the receptor, and also in exon IIIc, in which case they are exclusive to the bek isoform, without apparent phenotypic differences.

shape, protruding eyes, and midface underdevelopment. These syndromes can be distinguished by the pattern of associated limb involvement. For instance, in Crouzon syndrome there is no apparent malformation of the hands or feet, whereas in Pfeiffer syndrome the thumbs and great toes are broad and medially deviated, and in Apert syndrome there is severe and symmetric fusion of the bones of the hands and feet. Jackson-Weiss syndrome has a high degree of phenotypic variability, but generally is associated with anomalies of the feet (Cohen, 1986). Craniosynostosis syndromes are occasionally associated with skin disorders. For instance, Crouzon syndrome with acanthosis nigricans is a distinct syndrome involving hypertrophy of the skin and hyperpigmentation (Meyers et al., 1995). Beare-Stevenson cutis gyrata is an often-lethal craniosynostosis disorder characterized by furrowed skin, hyperpigmentation, and abnormalities of the digits, the umbilical cord and the anogenital region (Hall et al., 1992).

VI. FGFR mutations in dwarfism syndromes

Surprisingly, given the distinct expression patterns of the different FGFRs discussed previously, related craniosynostosis syndromes have been mapped to point mutations in FGFR1, FGFR2 and FGFR3. The mutations found in FGFR-related craniosynostosis syndromes are shown diagrammatically in F i g u r e 2 . For instance, a Pro-Arg substitution in the linker region between Ig-like domains 2 and 3 occurs in each of the three receptors,

Several related forms of short-limb dwarfism, including achondroplasia, hypochondroplasia, and thanatophoric dysplasia, have recently been linked to mutations in different structural and functional domains of FGFR3 (Rousseau et al., 1994; Shiang et al., 1994; Superti-Furga

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Figure 3. The locations of point mutations in FGFR3 giving rise to dwarfism syndromes. Numbers and symbols are as described in the legend to Fig. 2.

et al., 1995; Ikegawa et al., 1995; Tavormina et al., 1995a,b; Rousseau et al., 1996; Rousseau et al., 1995). The mutations found in FGFR3 in these dwarfism syndromes are shown in Figure 3.

The underlying defect in each of these dwarfisms appears to be varying degrees of disruption of the normal proliferation and differentiation of chondrocytes at the epiphyseal plates of the long bones.

Achondroplasia is characterized by disproportionate shortening of the long bones, macrocephaly, spinal curvature, and midface underdevelopment. Although individuals with achondroplasia are of normal intelligence, they often have delayed motor development and an increased incidence of respiratory problems (Wynne-Davies et al., 1985). This disorder, affecting about 1 in 26,000 individuals (Oberklaid et al., 1979), is usually sporadic, but when familial is transmitted as an autosomal dominant trait with complete penetrance. Homozygous achondroplasia is usually lethal shortly after birth, due to impaired development of the ribs resulting in early respiratory failure. Thanatophoric dysplasia (TD) is a severe disorder resembling homozygous achondroplasia, and can generally be divided into two subclasses. TDI is distinguished by the presence of femoral bowing, whereas the femurs are straight in TDII. Furthermore, TDII is invariably associated with cloverleaf skull (a form of multiple suture craniosynostosis), whereas cloverleaf skull occurs only occasionally in TDI individuals (Langer et al., 1987). On the milder end of the spectrum of short-limb dwarfisms, hypochondroplasia is quite similar to achondroplasia except the skeletal abnormalities observed are considerably less severe (Wynne-Davies et al., 1985).

The mutations responsible for all cases of achondroplasia identified to date reside in the transmembrane domain of FGFR3, and result in either a Gly380Arg substitution (Rousseau et al., 1994; Shiang et al., 1994), or, much more rarely, a Gly375Cys substitution (Superti-Furga et al., 1995; Ikegawa et al., 1995). In contrast, three distinct types of point mutations give rise to TDI: mutations to Cys at the extracellular/transmembrane domain junction (Tavormina et al., 1995b; Rousseau et al., 1996); mutations to Cys in the linker region between Ig-like domains 2 and 3 (Tavormina et al., 1995a,b); and mutations that allow readthrough of the stop codon (Rousseau et al. 1995). A Gln540Lys substitution in the proximal portion of the split tyrosine kinase domain is found in many patients with hypochondroplasia (Bellus et al., 1995), and a Lys650Glu mutation in the kinase activation loop is responsible for all cases of thanatophoric dysplasia type II identified to date (Tavormina et al., 1995b).

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Webster and Donoghue: FGFR Activation in Developmental Syndromes Several of the mutations that arise in the extracellular domain of FGFR2 and FGFR3 in bone development disorders are caused by mutations that either destroy or create Cys residues. For instance, in Crouzon and Pfeiffer syndromes, mutations at each of the conserved Cys residues at the base of the third Ig domain of FGFR2 have been observed (Wilkie et al., 1995a; Meyers et al., 1996; Oldridge et al., 1995; Reardon et al., 1994; Rutland et al., 1995; Steinberger et al., 1995). Normally, these residues are presumed to be involved in a disulfide bond, which stabilizes the Ig domain structure, and disruption of this bond appears to underlie constitutive activation of FGFR2 observed in Crouzon mutants (Galvin et al., 1996). In Xenopus FGFR2, mutation of either of these Cys residues results in FGF-independent signaling, as measured by induction of mesoderm in animal pole explants (Neilson and Friesel 1995; Neilson and Friesel 1996). Furthermore, the mutant receptors form covalent homodimers, exhibit increased tyrosine autophosphorylation, and are unable to bind ligand. These data suggest that destruction of one of the paired Cys residues not only disrupts normal folding of the third Ig domain, but also mimics ligand binding by constitutively dimerizing the receptor.

Figure 4. Focus formation assay of chimeric FGFR3/Neu receptors containing TDI mutations. Chimeric FGFR3/Neu constructs containing interlinker region mutations were transiently transfected into NIH3T3 cells, which were subsequently scored for focus formation. (A) mock-transfected cells (nontransformed); (B) wild type FGFR3; (C) R248A; (D) R248C; (E) S249C; (F) activated Neu containing the mutation V664E as a positive control.

There are numerous examples of mutations within the third Ig domain of FGFR2 that cause craniosynostosis syndromes yet do not directly alter Cys residues. Some of these mutations, including Trp290Gly and Thr341Pro, involve residues that are predicted to play an important structural role in the correct folding of the Ig domain . These mutations were also demonstrated to result in ligand-independent FGFR2 activation and receptor dimerization (S. C. Robertson and D. J. Donoghue, unpublished data), presumably by destabilizing the disulfide-bond that normally forms within the third Ig domain, instead allowing disulfide bond formation to occur between receptor monomers.

VII. Constitutive FGFR activation underlies both craniosynostosis and dwarfism syndromes Many different mutations in FGFR1, FGFR2 and FGFR3 have been identified in craniosynostosis and dwarfism syndromes, and it was initially unclear whether these mutations resulted in loss of receptor function (either through dominant negative effects or through loss of ligand binding capacity), alteration of receptor localization (perhaps due to misfolding), or enhanced signaling capacity. Evidence has recently been accumulating that suggests that each of these mutations leads to constitutive activation of receptor signaling, although by different mechanisms, and that this may explain, at least in part, the similar biological consequences of such different FGFR mutations. The effects of mutations in the extracellular, transmembrane and kinase domains of FGFRs will be discussed separately in this review.

Mutations in the conserved Arg-Ser-Pro tripeptide in the Ig2-Ig3 linker region of FGFR1, FGFR2 and FGFR3 have also been observed in craniosynostosis and dwarfism syndromes (see Figures 2 and 3). Some of these mutations have been examined in the context of chimeric receptors, where the extracellular domain is derived from FGFR and the intracellular domain is derived from the proto-oncogene Neu. In these constructs, signaling through the Neu tyrosine kinase is used as reporter for receptor activation, and results in the formation of transformed foci in NIH 3T3 fibroblasts, which can be easily scored. TDI mutations that create unpaired Cys residues in the Ig2-Ig3 linker region of FGFR3, including Arg248Cys and Ser249Cys, have been engineered in FGFR3-Neu chimeric receptors and shown to cause ligandindependent signaling through the Neu tyrosine kinase

VIII. Extracellular domain mutations

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Webster and Donoghue: FGFR Activation in Developmental Syndromes F i g u r e 5 . Ligand independent dimerization of TDI mutants of FGFR3. Lysates from [35S]labeled cells were immunoprecipitated and electrophoresed under nonreducing and reducing conditions through SDSPAGE gradient gels. Proteins were transferred to nitrocellulose and immunoblotted using FGFR3 antiserum. (Top) Non-reducing gel. The positions of dimeric and monomeric forms of FGFR3 are indicated. (Bottom) Reducing gel. Monomeric FGFR3 is shown, indicating equivalent expression of all constructs.

(d'Avis et al., 1997). Figure 4 presents a typical transformation assay of chimeric FGFR3/Neu receptors carrying TDI mutations. When examined in the context of full-length FGFR3, mutations such as the TDI mutations Arg248Cys and Ser249Cys also lead to constitutive receptor dimerization, which can be readily observed by SDS-PAGE under non-reducing gel conditions, as shown in Figure 5. A control mutation, Arg248Ala, does not cause receptor activation or dimerization, implying that it is the introduction of a novel disulfide bond, as opposed to an alteration of the structure in this region, that is responsible for TDI. The consequences of other interlinker mutations to non-Cys residues in craniosynostosis disorders are still unknown. However, in contrast to Ig3 domain mutations, Ig2-Ig3 linker domain mutations apparently do not abolish FGF binding (Neilson and Friesel, 1996; Naski et al., 1996). Perhaps these mutations affect the sensitivity or specificity of the receptor to small amounts of ligand, which could also play a role in their abnormal regulation in vivo.

The introduction of an Arg residue into the normally hydrophobic transmembrane domain of FGFR3 has been shown to be responsible for the vast majority of cases of achondroplasia (Rousseau et al., 1994; Shiang et al., 1994). Interestingly, this Gly380Arg mutation is in an analogous position to the Val664Glu mutation that activates the proto-oncogene Neu (Bargmann et al., 1986). F i g u r e 6 presents an alignment of the transmembrane domain sequences of FGFR3 and Neu, showing the location of activating mutations. Activation of Neu has been proposed to involve stabilization of the receptor in a dimeric conformation due to hydrogen bond formation, leading to elevated receptor tyrosine kinase activity and cellular transformation (Sternberg and Gullick 1989). It was demonstrated that the achondroplasia mutation has similar functional consequences, as evidenced by the fact that substitution of the transmembrane domain of Neu with the transmembrane domain of mutant human FGFR3 causes ligand-independent signaling through Neu (Webster and Donoghue, 1996). Consistent with this model, resi-

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F i g u r e 6 . A c t i v a t i n g m u t a t i o n s i n t h e F G F R 3 t r a n s m e m b r a n e d o m a i n . An alignment of the transmembrane domains (gray) of rat Neu and human FGFR3 is presented, showing the location of activating mutations. The mutation in the Neu oncogene (associated with rat neuroglioblastoma) is shown in black. The mutations in FGFR3 giving rise to thanatophoric dysplasia type I are shown in blue, achondroplasia in red, and Crouzon syndrome with acanthosis nigricans in green.

Figure 7. Oncoproteins and receptors activated by mutations in the transmembrane domain. Amino acids are shown that allow activation of p185c-neu when substituted at residue 664, activation of BPV-E5 when substituted at residue 17, and activation of FGFR3 when substituted at residue 380. Those substitutions that allow activation in these three different systems share the property that they are strongly polar, in an otherwise hydrophobic membrane environment, and thus share the ability to participate in hydrogen bond formation that may stabilize dimer formation.

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Webster and Donoghue: FGFR Activation in Developmental Syndromes dues with side chains capable of participating in hydrogen bond formation, but not hydrophobic residues, also activated the chimeric receptor at this position. Additionally, the Gly380Arg mutation activated ligandindependent kinase activity of full-length FGFR3 (Naski et al., 1996; Webster and Donoghue, 1996). Figure 7 presents the mutations that occur within the transmembrane domain of three different oncoproteins or receptors which can result in activation (Chen et al., 1997). The activating mutations in these cases are all polar residues capable of participating in hydrogen bond formation to stabilize receptor dimers. A dramatically different phenotype, Crouzon syndrome with the associated skin disorder, acanthosis nigricans, results from a Ala391Glu substitution just 11 residues away from the site of the principal achondroplasia mutation in the transmembrane domain of FGFR3 (Meyers et al., 1995) We have observed that this mutation is also activating in the context of the chimeric system described above (Chen et al., 1997), presumably by stabilization of dimers due to hydrogen bonding. It will be of interest, however, to determine whether this mutant FGFR3 in some way affects signaling through FGFR2, as the phenotype of this disorder is much more characteristic of craniosynostosis syndromes resulting from FGFR2 mutations than of dwarfism syndromes typically arising from FGFR3 mutations.

F i g u r e 8 . I n v i t r o k i n a s e a c t i v i t y o f FGFR3 mutants causing achondroplasia and TDII. The TDII mutant FGFR3 is constitutively active as a tyrosine kinase. NIH3T3 cells transiently expressing either a vector control (Mock), wild-type FGFR3, the Gly380Arg mutant causing achondroplasia, or the Lys650Glu mutant causing were lysed and immunoprecipitated with FGFR3 antiserum. (Left) Immunoblot of immunoprecipitates with FGFR3 antiserum followed by horseradish peroxidaseconjugated secondary antiserum and ECL development, showing comparable levels of receptor expression. (Right) Autophosphorylation assay. Immunoprecipitates were subjected to in vitro kinase reactions in the presence of ![32 P]ATP, and analyzed by SDS-PAGE and autoradiography. Cells expressing the TDII mutant receptor construct exhibited significantly increased autophosphorylation relative to the achondroplasia mutant.

A number of mutations resulting in the creation of Cys residues at the junction of the extracellular and transmembrane domains of FGFR3 warrant mention. Substitution by Cys at residues 370, 371 and 373 of FGFR3 has been observed in the lethal dysplasia, TDI, whereas a Gly375Cys mutation, just two residues away, is found in rare instances of achondroplasia. It has recently been demonstrated that these juxtamembrane TDI mutations result in the formation of stable, disulfide-linked receptor dimers and induce high levels of expression of a cfos-luciferase reporter construct (d'Avis et al., 1997). In contrast, it is possible that the milder achondroplasia mutation, occurring more deeply within the lipid bilayer of the cell, might result in the formation of weaker dimers and thus less pronounced signaling through FGFR3. Indeed, in a chimeric system involving the extracellular domain of the platelet-derived growth factor receptor, and the transmembrane and intracellular domains from the Gly375Cys mutant FGFR3, these receptors did not signal in a ligand-independent fashion, although they were responded more rapidly and robustly to ligand (Thompson et al., 1997).

involve the point mutation, Lys650Glu, in the activation loop of the FGFR3 kinase domain. Several groups have demonstrated that expression of this mutant receptor in mammalian cells leads to strong, constitutive activation of the tyrosine kinase activity of the receptor (Naski et al., 1996; Webster et al., 1996), to a much greater extent than seen for the achondroplasia mutation, suggesting that there may be a correlation between the degree of receptor activation in vitro and the clinical severity of the phenotype. Figure 8 presents an in vitro kinase assay of immunoprecipitated FGFR3 receptors, showing modest activation for the achondroplasia mutant compared with profound activation for the TDII mutant receptors.

X. Kinase domain mutations As opposed to TDI, where a variety of mutations occur in the extracellular domain of FGFR3, all cases of TDII 373

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F i g ur e 9 . M o d el fo r th e effect of point mutations o n F G F R f unct io n . (a) Normal ligand-dependent activation leads to regulated signals for proliferation and differentiation of bones. (b , c , d) Certain extracellular domain mutations activate the receptors through the formation of aberrant disulfide bonds, indicated by S-S, leading to constitutive dimerization. Transmembrane domain mutations result in hydrogen (H)-bonded FGFR dimers. Mutations in the activation loop of the kinase domain result in conformational changes that activate receptor tyrosine kinase activity. Constitutive signaling through inappropriately activated FGFRs results in premature maturation of the bones of the skeleton and cranium.

XI. Relevance of FGFR mutations in skeletal disorders to human cancers

Substitutions at position 650 and at neighboring positions indicate that the Lys650Glu mutation mimics the activating conformational changes that normally accompany autophosphorylation at conserved Tyr residues within the activation loop (Webster et al., 1996). Recently, a second mutation at this residue, Lys650Met, has been identified in Skeletal-Skin-Brain Dysplasia (SSBD) (Tavormina et al., 1997), a severe disturbance in endochondral bone growth and neural and skin development. Despite the involvement of the same residue as that mutated in TDII, and similarly high levels of in vitro receptor activation (Tavormina et al., 1997), there is no observed cloverleaf skull in SSBD, and usually this disorder is not lethal.

Although patients with skeletal dysplasias caused by activating FGFR germ-line mutations do not have an apparent increase in tumor frequency, enhanced signaling through FGFRs has been implicated in tumor progression. For instance, amplification or ectopic expression of genes encoding FGFs and FGFRs has been found in neoplastic cells (Adnane et al., 1991; Hattori et al., 1990; Kobrin et al., 1993; Yamanaka et al., 1993; MacArthur et al., 1995; Delli Bovi et al., 1987; Goldfarb et al., 1991; Marics et al., 1989), and overexpression of FGFs results in morphological transformation of cells co-expressing FGFRs in vitro. (MacArthur et al., 1995; Delli Bovi et al., 1987; Goldfarb et al., 1991; Marics et al., 1989). Interestingly, some of the identical activating mutations as those found in the severe skeletal dysplasias TDI, TDII and SSBD have recently been identified in human multiple myeloma (Chesi et al., 1997). In these tumors and cell lines, a translocation leading to the juxtaposition of FGFR3 near the IgH switch region was observed, resulting in the selective overexpression of the mutant FGFR3 allele. We have recently confirmed that a highly activated kinase-domain derivative of FGFR3 (Lys650Glu) can transform NIH3T3 fibroblasts, suggesting a causative role for activated FGFR3 in the development of certain cancers (Webster and Donoghue, 1997).

A mutation that results in the substitution Asn540Lys in the kinase domain of FGFR3 causes a mild form of dwarfism, hypochondroplasia (Bellus et al., 1995). The prediction would be that this mutation is also able to activate the receptor in a ligand-independent fashion, but probably to a lesser degree than other activating mutations, based on the relative severities of the associated skeletal disorders. The crystal structure of the tyrosine kinase domain of FGFR1 in the inactive conformation was recently solved, which suggested that Asn540 is normally hydrogen bonded to His535 (Mohammadi et al., 1996b). Disruption of this bond would thus be predicted to stabilize the active conformation of the receptor.

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Gene Therapy and Molecular Biology Vol 1, page 375 Why, then, do activating germ-line mutations in FGFR3 cause defects in skeletal, cranial and skin development, rather than cancers? Perhaps in these affected tissues, FGFR3 activation is coupled to signaling pathways leading to differentiation or growth arrest, rather than proliferation. For instance, constitutive activation of STAT1 and elevated expression of the cell cycle inhibitor p21 WAF1/CIP1 has been observed in cartilage cells from a TDII fetus but not a normal fetus (Su et al., 1997). In other cells from TDII patients, such as fibroblast and lymphoid cells where FGFR3 signaling may be coupled to mitogenesis, the level of expression of the receptor may not be sufficient to stimulate the unregulated proliferation necessary for tumor development, consistent with our observation that only greatly enhanced levels of signaling result in morphological transformation of cells (Webster and Donoghue, 1997).

heterodimerization partners, and downstream effectors might ultimately determine the severity of the phenotype and the precise tissues affected. Finally, studies described here imply a normal role for FGFRs in restraining premature maturation at the growth plates of long bones and at the sutures of the skull. It is as yet unknown at which of the highly regulated steps of proliferation and differentiation constitutive FGFR activation acts to disrupt normal bone maturation. It will be very useful to develop transgenic mice expressing mutant FGFR proteins to help define the normal roles of FGFRs in skeletal and cranial development. Such in vivo systems will allow the examination of developmental changes specifically due to mutant receptors, in the context of the normal complement of heterologous FGFRs, ligands, and effectors, which is a limitation in the interpretation of data from current in vitro assay systems. As opposed to certain genetic skeletal disorders which appear to be due to loss-of-function mutations, such as Saethre-Chotzen syndrome (El Ghouzzi et al., 1997; Howard et al., 1997) and campomelic dysplasia (Wagner et al., 1994; Foster et al., 1994), the craniosynostosis and dwarfism syndromes discussed in this review are due, at least in part, to gain-of-function mutations in FGFRs. As such, gene replacement therapy is not expected to be of consequence in the treatment of these disorders in the foreseeable future. Nonetheless, an ability to correlate specific FGFR mutations with particular phenotypic consequences, arising from research described in this review, is already proving useful for diagnostic and genetic counseling purposes.

XII. Perspectives and future directions The recognition that constitutive FGFR activation appears to underlie many human dwarfism and craniosynostosis disorders is an important first step in understanding the role of FGFRs in normal human development. As described above, this activation may occur by a number of distinct mechanisms, depending on which structural domain of the receptor is involved. These different mechanisms are shown schematically in Figure 9. A number of important questions remain, however, and addressing these questions will provide exciting challenges to molecular and developmental biologists for many years to come. For instance, it is unclear how phenotypically similar craniosynostosis syndromes can arise from mutations encoding three different FGFRs (and splice variants thereof), with different spatial and temporal patterns of expression. This observation implies a certain overlap in function of FGFR1, FGFR2 and FGFR3 during development, and might additionally suggest an ability of one receptor to affect signaling through a heterologous receptor. In fact, one study suggests that mutant FGFR alleles may also function in a dosage-dependent dominantnegative fashion to inactivate ligand-dependent signaling from wild-type FGFR alleles (Nguyen et al., 1997).

Acknowledgements We thank Laura Castrejon for excellent editorial assistance and all lab members for their many valuable comments and suggestions concerning experimental design and preparation of this manuscript. This work was supported by grant DE 12581 from the National Institutes of Health.

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Gene Therapy and Molecular Biology Vol 1, page 381 Gene Ther Mol Biol Vol 1, 381-398. March, 1998.

Genes involved in the control of tumor progression and their possible use for gene therapy Georgii P. Georgiev 1, Sergei L. Kiselev 1, and Evgenii M. Lukanidin 1,2 Institute of Gene Biology, Russian Academy of Sciences, 34/5 Vavilov Street, 117334 Moscow, Russia; and

1 2

Danish Cancer Society, Department of Cancer Molecular Biology, Copenhagen, Denmark.

__________________________________________________________________________________________________ Correspondence: Georgii P. Georgiev, Fax: +7-095-135 41 05, E-mail: georg@biogen.msk.su

Summary Three major groups o f genes may be used for cancer gene therapy: (i ) oncogenes and tumor suppressor genes; (i i ) genes involved in the control of tumor progression and metastasis; and ( i i i ) genes encoding proteins protecting the organism from tumor cells. Each group contains numerous genes, and the discovery of new important genes is an exciting prospect in cancer research. We are working on the search and characterization of the genes over- or under-expressed i n metastatic comparing to non-metastatic tumors of the same origin. Two mouse systems are being u sed: ( i ) VMR-0 (non-metastatic mammary adenocarcinoma cells) - VMR-100-Liv and VMR-100-Ov cells (metastatic preferentially to the liver or ovaries, respectively); and (i i ) CSML-0 - CSML-100 (mammary adenocarcinoma cells non-metastatic and metastatic to the lungs, respectively). Several different genes were found to be over-expressed in metastatic cells, but only few of them were shown t o be necessary and sufficient for maintaining the metastatic phenotype using stably transfected cells and/or transgenic animals. Among them are the m t s 1 and c-met g e n e s . T h e m t s 1 gene, encoding a calcium-binding protein of 101 amino acids of the S-100 family, was extensively characterized. Its expression induced a number o f changes i n c e l l functions connected with cytoskeleton features, attachment properties of the cell, mesenchyme formation and possibly tumor vascularization. As a multifunctional regulator, the m t s 1 gene i s a promising target for gene therapy of cancer. Other genes identified are over-expressed only in few metastatic tumors and do not seem to be connected directly with the acquisition o f the metastatic phenotype. However, during the transfection experiments some interesting features emerged for these genes, raising the possibility of their exploitation in cancer gene therapy. The most interesting is the tag7 gene encoding a new cytokine, 182 amino acids long, with a far distant relation to cytokines of the TNF-Lymphotoxin family. The tag7 gene is expressed in lymphoid cells, in a limited set of other normal cells, and in f e w c a n c e r c e l l s i n c l u d i n g m y e l o m a s . T h e T a g 7 p r o t e i n i s s e c r e t e d t o the culture medium and possesses a strong cytotoxic activity inducing apoptosis. VMR-0 cells were stably transfected with a construct containing the tag7 g e n e u n d e r c o n t r o l o f t h e C M V p r o m o t e r . T h e o r i g i n a l V M R - 0 tumors killed mice i n one month after subcutaneous transplantation; animals displayed large n e c r o t i c f o c i a t t h i s s t a g e . H o w e v e r , t h e V M R - 0 / tag7 c e l l s , s y n t h e s i z i n g v e r y l o w a m o u n t s of Tag7 protein, exhibited dramatically different growth properties: they grew much slower; even after 4 months, no mice were killed by tumors arising from the transplanted cells and no necrotic foci were formed. Histological analysis of VMR-0/ tag7 t u m o r s s h o w e d a s t r o n g i n h i b i t i o n i n m i t o t i c rates and an enhanced rate o f apoptosis compared t o VMR-0 tumors. The tumors induced by transplantation of a mixture of VMR-0 and VMR-0/tag7 cells also grew much slower than VMR-0 c e l l s a l o n e , s u g g e s t i n g a n a c t i v a t i o n o f t h e immune system against tumor (tumor vaccination effect), which may be mediated through induction of CTL cells. Experiments with nude mice gave s i m i l a r r e s u l t s . I n f a c t a t l a t e r s t a g e s o f d e v e l o p m e n t i n n u d e m i c e , VMR-0/tag7 tumors were completely eradicated. It seems that the effect o f tag7 expression i s complex and includes 381

Georgiev et al: Genes involved in the control of tumor progression in gene therapy activation of an immune response as well as a direct cytotoxicity. The higher tag7 expression in culture cells is incompatible with cell survival. Experiments are in progress for further elucidating the role of Tag7 and its exploitation for the development of tumor vaccines.

for rapid growth or other properties useful for the cell itself, it has some good chances for survival and multiplication. Such changed cells may replace the original population of tumor cells over time. This phenomenon is known as tumor progression, that usually leads to appearance of a more malignant phenotype. Usually the same tumor contains cells with different genotypes and several clones obtained from the same tumor may differ in the level of malignancy.

I. Introduction Three major groups of genes may be used for cancer gene therapy: (i) oncogenes and tumor suppressor genes; (ii) genes involved in the control of tumor progression and metastasis; and (iii) genes encoding proteins protecting the organism from tumor cells. Each group contains numerous genes, and the discovery of new important genes is an exciting prospect in cancer research. We are working on the search and characterization of the genes over- or under-expressed in metastatic comparing to non-metastatic tumors of the same origin. Below, we briefly summarize the general data on genes and proteins involved in the control of tumor progression (for more information see review articles in the Reference list). Thereafter, we present the data obtained in our laboratories on two genes from the second and third groups mentioned above. At least one of these genes and its protein product may be used for the gene therapy of cancer.

It should be pointed out that in some cases, the malignant phenotype can appear just at the first stage of tumor development, as for example, in mouse mammary tumors induced by activation of the neu oncogene. However, in many cases, the malignancy develops in the course of tumor progression. One of the major features of malignant tumor cells is the ability to give metastases, i.e. new foci of tumor growth in distantly located regions of the organism. The process of tumor progression and tumor metastasis is very complex and includes several independent steps: 1) vascularization of a primary tumor node; 2) detachment of tumor cell from the primary focus; 3) invasion into surrounding tissues including the blood vessels; 4) transfer to the new site and arrest at this site; 5) adhesion to the endothelial cells; 6) extravasation; 7) vascularization of a novel focus and its invasive growth at the new place. Each step depends on new special properties of tumor cells, such as ability to induce angiogenesis, detachment from or attachment to cell aggregates, cell invasiveness and cell motility (F i g . 1 ).

II. Tumor progression and tumor metastasis The tumor is not a static formation. It is developing and changing constantly. This depends on the genetic instability of the tumor. As a result of transformation, tumor cells acquire a partial independence from regulatory signals arising from neighboring cells and grow more or less independently of these signals. Another important feature is the elimination of the cells with damaged DNA. Normally such cells cannot overcome the cell cycle checkpoints and progress through the apoptotic process leading to their death. The p53 protein plays an important role in the direction of damaged cells along the apoptotic way. Many mutations in the p53 gene, frequently occurring in tumors, lead to the loss in the ability to induce apoptosis and to down regulate cell proliferation. As a result, cells become able to survive after DNA damage, and this results in the increase in mutation rate in such cell populations. Interestingly, another protein involved in the control of apoptosis, the product of the bcl-2 gene which down regulates apoptosis, is a potential oncogene.

Each step is a complex one and is controlled by a number of genes and proteins. Therefore, one can expect a number of genes/proteins to be involved in the control of tumor metastasis. Several activated oncogenes themselves can generate a metastatic phenotype. However, in many other cases, the processes of oncogenesis and metastasis are uncoupled, and special genes are responsible for the appearance of the metastatic phenotype. The genes for tumor progression and metastasis may be separated into two groups: effector genes and upstream regulatory genes. The protein products of effector genes directly determine the invasiveness and other features of the metastatic tumor, while proteins encoded by the genes in the second group act in an indirect way. They either control the expression of different effector genes or control some general cell function indirectly determining the features characteristic of malignant tumor cell.

This, and possibly some other processes, result in accumulation of different types of mutations in tumor cells: translocations, loss of heterozygosity, point mutations and transpositions. Consequently, this leads to an accumulation of heterogeneity in tumor cell populations. If the mutated cell acquires some advantage 382

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F i g . 1 . The schematic presentation of tumor progression and metastasis. Some proteins activating (+) or suppressing (-) tumor progression are indicated.

metastases. In such cases, surgical removal of the primary focus may lead to induction of metastasis. The control of angiogenesis by antiangiogenic polypeptides, like angiostatin, endostatin and some other agents is an important approach for cancer therapy (see this volume).

III. Some main steps of tumor progression and some genes involved in their control A. Angiogenesis. Before vascularization, or angiogenesis, takes place the tumor can only grow up to 2 mm in diameter due to the shortage of nutrition. After vascularization has occurred, tumor cells can grow to much larger dimensions. In addition, tumor cells from vascularized tumors can penetrate into the blood vessels and this is the first step for metastasis development. The invasion of endothelial cells into the tumor node and formation of capillary sprout are influenced by many different cytokines produced either by tumor cells or by normal inflammatory cells whenever inflammation accompanies tumor development. There are positive and negative cytokines. Among positive cytokines inducing tumor angiogenesis are fibroblast growth factors, especially bFGF, vascular endothelial growth factor (VEGF), interleukin-8 (IL-8) and many others. On the contrary, cytokines such as interferons (IFN-! and IFN-"), angiostatic cartilage-derived inhibitors and several others inhibit angiogenesis in tumor foci. The production of corresponding cytokines by tumor cells depends on the complex interactions between tumor and surrounding host cells. Sometimes, angiostatic cytokines are produced by primary tumor itself and this prevents overgrowth of

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B. Detachment of tumor cells from the original cell cluster. The next important step in metastasis is the detachment of tumor cells from aggregates to enter the blood vessel. Cell aggregation depends on several factors, the most important being represented by cadherins, immunoglobulins, integrins and selectins. Aggregation of the cells plays an especially important role in the progression of the cancers, or epithelial tumors, as epithelial cells originally form firm aggregates and have very little motility. They bind to each other and to the basal membrane. Adherence junctions between normal and tumor epithelial cells depend on the cadherin family of proteins. The best characterized member of the family, Ecadherin, is a ca. 120 kDa protein with a large N-terminal extracellular part containing four calcium-binding domains, a transmembrane domain and a C-terminal intracellular domain. The extracellular domains of E-cadherins located on different cells interact in a calcium-dependent way. The C-terminal domain interacts with the C-terminal region of "-catenin (plakoglobin), whose N-terminal part binds to !-

Georgiev et al: Genes involved in the control of tumor progression in gene therapy catenin which interacts with the cytoskeleton. This chain of protein-protein interactions firmly holds epithelial cells at their position.

activator and its inhibitor, as well as gualuronidase and several other enzymes destroying extracellular matrix. The genes encoding these degradative enzymes are frequently activated in metastatic and, in general, in invasive cancer cells, although the correlation is not absolute. This group of enzymes may obviously play a role in degradation of extracellular matrix synergizing to the overgrowth and invasion of cancer cells, and in particular their invasion into blood vessels.

There is a strong correlation between cell behavior and E-cadherin content (Vlaminckset al. 1991). In transfection experiments, stable transfection and expression of the Ecadherin gene strongly suppresses the metastatic phenotype of different tumor cell lines. On the other hand, antibodies to E-cadherin make the tumor cells more aggressive in some cases. Mutations in the E-cadherin and "-catenin genes leading to their inactivation have been observed in some tumors and found to be associated with enhancement of metastatic phenotype. Down regulation of E-cadherin gene expression leads to a similar effect. Detachment from the cell aggregate leads to the aquisition by epithelial cells of several properties typical of mesenchymal cells: mesenchymal transformation of epithelial cells takes place. This phenomenon correlates neatly with the increase of invasiveness and appearance of metastatic phenotype in tumor cells (see also below).

D. Attachment to endothelial cells (arrest in capillary bed) and extravasation. The process of attachment of tumor cells to endothelial cells is induced by cytokines produced in tumor or inflammatory cells. These are IL-1, TNF, lipopolysaccharides (LPS) etc. Weak attachment is mediated by selectins. Synthesis of E-selectin is induced in endothelial cells. Its extracellular part interacts with tumor cell or leukocyte carbohydrates through an N terminal lectin-like domain. Strong attachment is mediated by interaction of integrins of tumor cells with the members of immunoglobulin superfamily located on the surface of endothelial cells. For example, !4"1 integrin usually present in melanomas and sarcomas binds to VCAM-1 (vascular cell attachment molecule), while !6"1 (present in colon carcinomas) and !6"4 (present in lung carcinomas) bind to ICAM-1 (intercellular cell attachment molecule). These interactions are responsible for a firm attachment.

C. Invasive growth. Different groups of genes/proteins are involved in determination of invasive tumor growth. Examples include the tyrosine protein kinase-type receptors, the autocrine motility factor (AMF) and its receptor and different types of degradative enzymes. Among the genes encoding tyrosine kinase receptors, c-met, c-neu, c-ret and c-ros were shown to be associated with the invasiveness of tumor cells. In more detail, the pair consisting of the ligand, scattering factor (SF)/hepatocyte growth factor (HGF), and of the receptor, c-Met, was studied. Their interaction leads to the induction of liver morphogenesis and at the same time to the increase of motility and invasiveness of tumor cells into the collagen matrix. The synthesis of both c-Met and SF/HGF is increased in different malignant tumors, in particular, in many cases of metastatic human breast cancer. In contrast to effector proteins, the system SF/HGF-cMet is part of the control proteins determining signal transduction resulting in the change of expression of several different genes.

Several reports have appeared indicating the special role in tumor metastasis of the CD-44 transmembrane hyaluronate receptor. Both metastatic and non-metastatic tumors contained the major variant of this protein, but only metastatic cells contained some minor variants of the protein characterized by the presence of additional domains, that were found to be responsible for intercellular interactions. The appearance of such variants was a result of alternative splicing that led to inclusion into mRNA of additional small exon(s). Stable transfection of nonmetastatic cells with the construct expressing the CD-44 variant in some cases led to the enhancement of metastatic potential, although some opposite results were also obtained. Since both the attachment and detachment of cells play a role in metastasis just at different stages of the process, these controversial results may not be too surprising. Further experiments are needed before final conclusions can be reached.

AMF is a 64 kD protein that interacts with the receptor, 78 kD glycoprotein, gp78, and activates cell motility. The synthesis of the components of this ligandreceptor system is activated in parallel with progression of some tumors, for example, bladder carcinoma. A wealth of data have been obtained on the role of different degradative enzymes and factors in tumor progression. Examples of degradative enzymes include different metalloproteinases and serine proteases, such as cathepsins, collagenases, stromelysins; plasminogen

Many other genes/proteins important for tumor progression have been described; the examples mentioned here are those of extensively studied. Some additional genes will be mentioned in further discussion. 384

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T a b l e 1 . The genes with changed expression detected in the VMR-0 - VMR-100 and CSML-0 CSML-100 pairs of tumor cells and/or tumors Cell-line system / The gene or protein VMR-100 / VMR-0 tag7 * c-met novel serine threonine protein kinase* LAR (tyrosine protein phosphatase) MHC class I H2-L-a antigen Na,K-ATPase, catalytic sub-unit Prothymosin Cathepsin D MMTV LTR CSML-100 / CSML-0 mts1* ly6 new Semaphorin*

Metastatic tumors comparing to non-metastatic selectively expressed over-expressed selectively expressed over-expressed over-expressed over-expressed under-expressed over-expressed over-expressed selectively expressed selectively expressed selectively expressed

*Novel, previously undetected genes.

IV. Search for new genes involved in the control of tumor metastasis and the systems used. Each of above mentioned genes plays a certain role in the acquirement by tumor cells of an invasive and metastatic phenotype. Probably, in different cases, different genes may be involved; one could expect that a number of genes controlling metastasis are still unknown. In particular, this may include upstream genes that are not directly involved in cell functioning but control other genes or the activity of proteins. An important direction for further studies is to discover such genes and to understand the role of their protein products in tumor progression and metastasis. In this respect, the most interesting are genes that either play a key role in producing a metastatic phenotype in various tumors or which can potentially be exploited for metastasis diagnostics and/or treatment. Different approaches can be used in the search of new genes involved in the control of tumor progression. One approach is to search for genes, whose protein products are directly connected with tumorigenesis and metastasis. In this case, one can usually expect to find the “effector genes” encoding proteins directly participating in corresponding cell functions. Another approach is a less direct, but still may give interesting results. It is the search for genes over-expressed or under-expressed in metastatic tumor cells compared to non-metastatic tumor cells of the same origin. In an ideal case, one can discover a gene belonging to a class of upstream genes with wide functions in the generation and maintenance of the metastatic phenotype. In many other cases, some “occasional” genes may be fished out, that are differentially expressed only in few types of tumor cells. Yet, in many cases, these new genes may become valuable 385

for understanding several aspects of tumor progression and, more important, in development of the methods for gene therapy. For example, one such gene identified from our experiments, tag7, seems to belong to the group of genes encoding proteins protecting the organism from tumor cells, and its transfer to cancer patient could constitute an approach for gene therapy. Another gene discovered in our laboratory, mts1, may play an important role in metastasis as an upstream regulator gene. Two mouse systems elaborated in the Russian Oncology Center (E. Revasova and V. Senin) have been used in our experiments. Both use the cell lines obtained from spontaneous mammary adenocarcinomas. The first is represented by a pair of VMR-0 non-metastatic cells and VMR-100 metastatic cells. Originally, VMR-0 cells were obtained and maintained as a cell line by subcutaneous transplantation. Occasionally, metastatic foci appeared, and the cells from these foci were taken for preparing cell culture. As a result of such selection, highly metastatic cell lines were obtained with preferential metastasis to the liver (VMR-100-Liv cells) or to the ovaries (VMR-100-Ov cells). Another pair are the CSML-0 and CSML-100 cells, non-metastatic and highly metastatic to the lungs, respectively, obtained in about the same way, also from spontaneous mammary adenocarcinoma. These two pairs of cell lines were further used for screening of the genes differentially expressed in metastatic cells. Two technologies were used: subtraction of cDNA libraries at the earlier stage and the mRNA display method at the later stage of the screening methodology. The mRNA display method, although giving a lot of false clones, is still much easier to apply and, ultimately, more genes of interest may be obtained with this technique. Several different genes were found to be over-expressed or under-expressed in metastatic cells in the two pairs

Gene Therapy and Molecular Biology Vol 1, page 386

F i g . 2 . Nucleotide sequence of the tag7 gene (exons and upstream sequences) and amino acid sequence of Tag7 protein. The transcription factorbinding sites, promoter, initiation and termination codons and AATAAA signal are underlined. The symbol / means borders between exons. In the amino acid sequence, the homologies to the TNF-Lymphotoxin family are underlined.

A. Structure of the tag7 gene.

described above (Table 1). One of them, the mts1 gene was found to be over-expressed in many different metastatic tumors and also shown to be necessary and sufficient for maintaining the metastatic phenotype at least in some tumor cells in experiments on stably transfected cells and on transgenic animals. Several other new genes may arise; however, additional experiments on stable transfection of cells with the corresponding gene constructs followed by phenotypic analysis, are required.

The tag7 gene was obtained by the mRNA display method, as a gene over-expressed in VMR-100-Liv and VMR-100-Ov tumors growing in mice after subcutaneous transplantation, as compared to tumors induced by VMR-0 cells (Kustikova et al. 1996). The full-length cDNA clone as well as a genomic clone were obtained and sequenced. The putative Tag7 protein consists of 182 aminoacids with no homology in the data-base (F i g . 2 ). Three exons were determined. The upstream region of the tag7 gene contains several binding sites for well known transcription factors, Ets1, NF#B, Sp-1 and MyoD. It was noticed that the arrangement of these sites was similar to that in the gene encoding Lymphotoxin-". A careful manual comparison of the Tag7 and TNF-Lymphotoxin amino acid sequences revealed a very low level of homology in different regions (F i g . 2 ), suggesting Tag7 to be a far distant relative of the members of TNF-Lymphotoxin family of cytokines.

Some of the discovered genes correspond to those described before, and are mostly effector genes (see Table 1). Among the upstream genes, the c-met gene seems to be an important regulator of tumor cell invasiveness and, as a result, of metastatic behaviour. It is the receptor for the SF/HGF (see above). Interestingly, the difference in expression of c-met between VMR-100 and VMR-0 cells appeared only in vivo. The effect can be mimicked in cell culture after contact with stromal cells. The c-met gene expression is used as a test for the level of malignancy.

The tag7 gene is located on the seventh mouse chromosome in the A3 region, i.e. in a different chromosome than the TNF gene cluster. The 7A3 chromosomal region is interesting, as it has a genetic relation to the development of such autoimmune diseases, as lupus erythematosus.

Finally, some of the discovered genes were overexpressed only in few types of metastatic cells, and therefore, they could not play a general role in determining the metastatic phenotype. This was confirmed in transfection experiments. Among them are the gene encoding a novel serine-threonine protein kinase and the tag7 gene, both over-expressed in VMR-100 cells. Although, transfection with the tag7 construct did not induce a metastatic phenotype, it strongly influenced the properties of tumor cells. Therefore, this gene and its protein product were studied in more detail.

B . Expression of the tag7 gene in normal tissues and tumors. High expression of the tag7 gene is not a general characteristic feature of metastatic tumors (F i g . 3 ). For example, CSML-100 cells do not express the tag7 gene, whereas CSML-0 cells do. Most metastatic and nonmetastatic tumor cells tested do not express the gene at all. Thus, it is clearly not a marker for the metastatic pheno-

V. A novel cytokine, Tag7, and its properties 386

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Exceptionally, the human multiple myeloma cells all express the tag7 gene at a rather high level. Strong tag7 expression in VMR-100 cells takes place only in tumors in vivo. In cells in culture, the level of tag7 mRNA is very low, suggesting that active tag7 expression depends on interactions between tumor and host (possibly stromal) cells.

F i g . 3 . Northern blot hybridization of RNAs from cell culture and tumors with the tag7 probe type.

Expression of the tag7 gene takes place in several tissues of normal organism (F i g . 4 ). The highest signal after Northern blot hybridization was obtained in the spleen and lungs. It is actively expressed in isolated lymphoid cells, circulating monocytes, thymocytes, splenocytes and resident peritoneal macrophages. The level of tag7 mRNA synthesis and Tag7 protein accumulation in cultured splenocytes is moderately enhanced by LPS induction (but not by IL-2 or PHA). The LPS effect on TNF and Lymphotoxin-a expression is much stronger and faster. Interestingly, in situ hybridization reveals the active tag7 expression in some specific cell sets in different organs, for example in Purkinje cells of cerebellum and in some neurons of hippocampus (F i g . 4 ). Its expression was found in the duodenal cells of 7-8 day embryos.

F i g . 4 . Expression of the tag7 gene in normal tissues. A. Northern blot analysis of RNAs prepared from different mouse tissues. B-E. In situ hybridization of adult mouse tissues with tag7 cRNA probe. B, hippocampus; C, cerebellum Purkinje cells, PC, are intensively labeled; C, intestinal section; E, the same after RNase digestion.

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amount of Tag7 protein can be obtained from the conditioned medium from the cultured cells producing small amount of Tag7. The problem of obtaining of native Tag7 from inclusive bodies has not yet been solved.

VI. Possible exploitation of the tag7 gene for antitumor gene therapy A. Influence of tag7 expression on the growth of VMR-0 tumors. The possibility to use the tag7 gene for cancer gene therapy was revealed from transfection experiments aimed to analyse the role of Tag7 in tumor metastasis. VMR-0

F i g . 5 . Tag7 p r o t e i n e x i s t s i n s o l u b l e and c e l l - a s s o c i a t e d f o r m s . Western blot analysis with affinity-purified antibodies to Tag7 protein. 1, Freshly isolated splenocytes; 2, 3, Splenocytes in culture after 0.5 h. LPS induction; 4, 5, The same after 24h induction; 6, 7, VMR100-Liv cells after LPS stimulation. 1, 2, 4, 6, Tag7 protein from the cells; 3, 5, 7, Tag7 protein from the culture medium. 8, Recombinant Tag7 protein.

C. Properties of the Tag7 protein. The recombinant Tag7 protein was obtained in inclusive bodies of E. coli and used for preparing polyclonal and monoclonal antibodies. Western blot analysis showed the presence of Tag7 protein both in the cells and in the culture medium (the major part) of Tag7-producing cells (F i g . 5 ). Thus, Tag7 is a secreted protein. Tag7 protein possesses a rather strong cytotoxic activity in respect to several cell lines, in particular, mouse L929 and human Jurkatt and MCF-7 cells (F i g . 6). Affinity-purified polyclonal or monoclonal antibodies destroy the cytolytic activity of Tag7 protein, while antibodies to TNF and Lymphotoxin-" do not. Vice versa, antibodies to Tag7 are not efficient in preventing the cytotoxic effect of TNF. The cytotoxicity of Tag7 is higher than that of the best commercial TNF preparation at the same concentration. The cytotoxicity of Tag7 protein is mediated through apoptosis as deduced from cytological analysis and the appearance of oligonucleosomal DNA repeats in the nuclei of target cells (F i g . 6 ).

F i g . 6 . C y t o t o x i c i t y o f T a g 7 p r o t e i n . The L929 cells were incubated with Tag7 from VMR-100-Liv supernatant or with commercial TNF. In some experiments the indicated antibodies were added.

High level of cytotoxicity of Tag7 protein creates a number of experimental problems. Synthesis of recombinant Tag7 in the periplasm of bacterial cells kills them. In transfection experiments, the cells producing high amount of Tag7 rapidly die. Therefore, only a limited

A. Cell death assayed by trypan blue staining; B . Appearance of nucleosome repeats after incubation of cells with TNF and Tag7.

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Gene Therapy and Molecular Biology Vol 1, page 389 cells were stably transfected with a construct containing the tag7 gene under control of the CMV promoter. Two stable cell lines, VMR-0/tag7, were obtained, both expressing tag7 at a low level. Yet, the level of expression in one cell line was two- three-fold higher than in another. It should be pointed out that the growth rate of VMR0/tag7 cells in culture was the same as of control VMR-0 cells. However, the attempts to obtain the higher level of tag7 expression in VMR-0 cells led to the inhibition of growth of the cells in culture followed by their death at later stages. The VMR-0/tag7 cells (106) were subcutaneously transplanted to isogeneic mice. The untreated VMR-0 cells or cells transfected with neomycin gene alone were used as a control. The original VMR-0 tumors grow fast at the site of injection and kill mice in one month after subcutaneous transplantation. The tumors contain large necrotic foci at this stage (F i g . 7 ).

F i g . 7 . Influence of tag7 expression on the tumor cell growth. A. Northern blot hybridization of RNAs from different VMR/tag7 cell lines (SX4 and SX12) with tag7 probe. The level of tag7 expression in transfected cells is much lower than in VMR-100-Liv. B . 10 6 VMR-0 cells ( ), mock-transfected VMR-0/Neo (!) and tag7 transfected SX4 (") and SX12 ( $ ) cells were subcutaneously injected to 10, 5, 10 and 10 A/Sn mice, respectively. The mean values of tumor size were determined at different periods after injection. SX4 cells expressed tag7 at higher level, than SX12 cells. (#). Inhibition of SX4 effect by purified polyclonal antibodies to Tag7 (3 mice). ($), 10 6 VMR-0 cells were coinjected with 10 6 SX4 cells (3 mice). *Animals of these groups died 4-5 weeks after injection.

The VMR-0/tag7 cells have a dramatically changed growth properties. They grow much slower. Even after 4 months, no mice were killed by the tumor. No necrotic foci were formed, even at later stages, when the tumors reached a large size. Histological analysis of VMR-0/tag7 tumors recovered strong inhibition of mitotic rate and activation of apoptosis frequency comparing to VMR-0 tumors (Table 2). Growth inhibition was much stronger in the case of VMR-0/tag7 cell line producing higher amount of Tag7. The injections of antibodies to Tag7 accelerated the tumor growth at the period of their application (F i g . 7 ). The tumors induced by transplantation of the mixture of VMR-0 (10 6) and VMR-0/tag7 (10 6) cells also grow much slower, than VMR-0 cells alone (F i g . 7), suggesting the activation of immune system against tumor cells (tumor vaccination effect), which may be realized through formation of CTL cells. This interpretation is supported by observation, that the growth of tumor cells of another line (CSML-100) is not inhibited by cotransplantation with VMR-0/tag7 cells.

T a b l e 2 . Influence of the tag7 expression on the growth properties of VMR-0 cells in vivo in isogenic mice. Tumor cells

Mitotic cells 3-5%

Apoptotic cells <0.5%

(ii ) VMR-0/tag7

<1%

5-8%

Ratio, (ii ):(i)

ca. 1 / 5

ca. 2 0 / 1

(i) VMR-0

389

Ratio M:A ca. 10/1 ca. 1/10 ca. 1/100

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F i g . 8 . The scheme with a possible explanation of the inhibition of tumor growth by activation of cytolytic T lymphocytes. APC-antigen-presenting cells. Filled circles, Tag7; empty circles, tumor antigens.

B. On the mechanism of tumor growth inhibition by tag7 expression. It is usually accepted that tumor cells expressing some cytokines, in particular GM-CSF or IL-2, attract the antigen presenting cells (APC). Then, APC convert naive T-lymphocytes into cytolytic T-lymphocytes (CTL) recognizing the antigens present in tumor cells and attacking both primary foci and metastases. One can suggest, that VMR-0/tag7 cells possess the same properties (F i g . 8 ). To check this, the experiments with nude mice were performed as they lack or have a very weak system for T lymphocyte response. The results were very similar. VMR-0/tag7 cells grow much slower, than VMR-0 cells. VMR-0 cells killed mice in 2 months, i.e. later than in the case of isogenic mice. However, the general properties of growth and tumor morphology remain unchanged (F i g . 9). The VMR-0/tag7 cells grow in nude mice even slower. The tumors increased in size at the beginning but later on they frequently decreased in size. Some waves of growth followed by degeneration could be observed. Finally either

F i g . 9 . Nude mice injected with 106 VMR-0 (A) or SX4 (B ) cells after 2 months of tumor development.

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Gene Therapy and Molecular Biology Vol 1, page 391 small tumors remained or complete dissolving of the tumor took place (F i g . 9 ). Histological analysis again shows strong inhibition of mitotic rate and at least tenfold increase in frequency of apoptosis. Typical pictures of apoptosis are observed under electron microscopy (EM) (F i g . 1 0 ). It seems that the effect of tag7 expression is rather complex and at least includes activation of an immune response mediated by CTL cells and direct cytotoxicity of the Tag7 protein. Experiments are in progress for more precise understanding of Tag7 action and for its application to cancer treatment.

VII. The mts1 gene A. General properties of the mts1 gene.

F i g . 1 0 . EM pictures of mitotic (M) and normal (N) cells in VMR-0 tumor and apoptotic cells (A) in SX4 tumor transplanted to nude mice.

Another extensively studied gene is the mts1 gene. It may play an important role in the control of tumorprogression and appearance of metastases at least in some tumors. The mts1 gene has been discovered in experiments on cDNA libraries subtraction using CSML-100 and CSML-0 cell lines (see above). The mts1 gene is transcribed to a 0.55 kb mRNA, which was abundant in CSML-100 cells and absent from CSML-0 cells. This mRNA was detected in many metastatic tumor cell lines of different origin, but not in non-metastatic tumors, although several exceptions could be observed. Therefore, the gene was called as mts1, a gene encoding Metastasin 1 protein, Mts1 (Ebralidze et al. 1989). The cDNA was sequenced and the protein structure was deduced. Mts1 was found to be a protein 101 amino acid long with two typical calcium-binding domains (F i g . 11). It belongs to the S-100 sub-family of calciumbinding proteins. The mts1 gene was described at about the same time in several other groups under different names, but without any relation to tumor metastasis. The mts1 has also been cloned from the human genome. Human Mts1 protein differs from its mouse counterpart just by 7 amino acid substitutions. The mts1 gene is expressed in several normal tissues: embryonic fibroblasts, trophoblasts, and lymphoid cells, in particular, in T-lymphocytes and activated macrophages. At least some of these cells possess invasive properties. The level of mts1 expression can be readily modulated by different lymphokines or calcium ionophores (Grigorian et al. 1993).

F i g . 1 1 . Nucleotide sequence of the mts1 gene coding sequence and amino acid sequence of Mts1 protein. In the amino acid sequence, the calcium-binding domains are underlined.

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No sequence rearrangement usually takes place during the change of tumor phenotype to a metastatic one, as deduced from Southern blot hybridization experiments. The only exception was observed in the VEHI-3 cell line (myelomonocytic leukaemia), where a deleted copy of IAP retrovirus-like mobile element was found to be inserted into the first intron of the mts1 gene. As a result, the

Georgiev et al: Genes involved in the control of tumor progression in gene therapy transcription was started within the IAP LTR, and chimeric mRNA was synthesized, while the protein remained unchanged (Tarabykina et al. 1996). Therefore, activation of mts1 mRNA synthesis should usually result from changes in concentration of certain trans-regulatory factors. Finding of such factors responsible for mts1 activation may lead to discovery of new genes involved in the creation of metastatic phenotype, acting upstream in respect to the mts1 gene.

with binding of activating proteins to the enhancer. The effect should be indirect as the enhancer core sequence does not contain CpG dinucleotides. The second cis-regulatory element is represented by the sequence TGACTCG differing from the consensus AP-1 binding sequence (TGACTCA) by one base substitution. As a result of substitution, a CpG dinucleotide appears that is the subject for deoxycytidine methylation. Both methylated and non-methylated sequences interact with nuclear proteins, as followed from band shift experiments. The protein binding to methylated sequence is different from that binding to non-methylated one and is much more abundant in nuclear extracts. The consensus AP-1 binding sequence competes only with the methylated element. This suggests that only the methylated sequence binds AP-1 factor. The conclusion was proved in supershift experiments with antibodies to Jun and Fos proteins. Thus, methylation of CpG creates a novel site for AP-1 binding. In the mts1 gene, methylated AP-1 binding element plays the role of a transcription silencer of a moderate strength. This inhibition seems to play a role in vivo, as a particular CpG sequence in CSML-0 cells is not methylated, while in CSML-0 cells, it is completely methylated (Tulchinsky et al. 1996).

The mts1 gene consists of three exons and two introns. The first exon is small and does not contain translated sequences. The gene is located in the gene cluster containing several other members encoding proteins belonging to S-100 family. The distances between genes in the cluster are rather small. Examination of the mts1 upstream region up to the 3â&#x20AC;&#x2122;-end of the neighbor gene of the cluster has led to the conclusion that cis-regulatory elements are not present but instead a TATA-box containing promoter. All cis-regulatory elements have been found within the first intron. Several well known positive and negative cis-regulatory element binding was determined as well as some new transcription factors have been found (F i g . 1 2 ) (Grigorian et al. 1993, Tulchinsky et al. 1996, 1997). The first element (from the 5â&#x20AC;&#x2122;-end of the intron) is the enhancer of a moderate strength possessing no homology

The third cis-regulatory element is located further downstream and is represented by the GGGGTTTTTCCAC sequence, related to NF#B-binding sites. The sequence does actually bind NF#B (p50/p50 and p50/p65), but this binding does not change mts1 transcription, at least in experiments with transient expression. On the other hand, the same sequence binds another factor, p200, of a higher molecular weight. As was shown in experiments with different constructs, the latter was responsible for activation of transcription in transient transfection assays. The p200 concentration in CSML-100 extract is about tenfold higher, than that in CSML-0 cells. In vivo footprinting showed the occupancy of the DNA sequence only in CSML-100 cells. These data suggest the role of the factor in the in vivo activation of mts1 transcription (Tulchinski et al., 1997). Closely to the previous element, a fourth element is located, which contains a microsatellite motif and is protected from nucleases in both in vivo and in vitro footprinting assays. It binds a protein interacting with microsatellite. It seems to play in vivo a role of a moderate transcription activator, as follows from stable transfection experiment (Prokhourtchuk et al., in preparation).

F i g . 1 2 . Regulatory region of the mts1 region located in the first intron.

with known enhancers. It binds a novel transcription factor, which is present in both CSML-100 and CSML-0 cells. However, in vivo the protein binding was detected only in CSML-100 cells. Thus, the enhancer selectively works in metastatic cells in spite of the presence of activating protein in both. One can suggest the involvement of structural changes in chromatin. Actually, the test for DNA methylation showed the absence of mts1 methylation in CSML-100, while in CSML-0 cells the mts1 gene was heavily methylated. The latter may interfere

The fifth positive cis-regulatory element binds a novel protein which is present only in CSML-100 cells. Therefore, it may represent a metastasis-specific transcription factor. Its cloning is in progress. Finally, the sixth regulatory element is represented by the enhancer binding AP-1 protein. In CSML-100 it is a FRA-JunD 392

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T a b l e 3 . Results of transfection experiments with the mts1 gene constructions Cells Phen mts1 Mts1 in otype construction transfected cells CSML-100 M Antisense Decrease OHS (human)* M Ribozyme Decrease CSML-0 NM Sense Absence Line 1 + DMSO NM Sense Appearance Rama 37 (rat)** NM Sense Appearance MCF-7 (human) NM Sense Appearance Transgenic mice; NM Sense Appearance spontaneous adenocarcinoma

complex. CSML-0 cells are FRA-deficient, and this may play an important role in the transcription control.

Change of metastatic phenotype Strong decrease Strong decrease No change Strong increase Strong increase Increase Strong increase

Another technology to switch off the gene expression is to use a construct encoding ribozymes, i.e. RNAs that specifically cleave a particular RNA. The ribozyme specifically cleaving human mts1 RNA at the second exon was transfected into human osteosarcoma (OHS) cells. The control OHS cells gave metastases to bone marrow of nude rats after intracardiac injection. The stable transfectants strongly suppressed the metastatic phenotype. The Mts1 protein content in such cells was decreased (Maelandsmo et al. 1996).

Thus, the first intron of the mts1 gene contains a complex regulatory system, which is sensitive to methylation. Some factors present in this system may play an important role in the control of metastatic behaviour of tumors.

B. The role of mts1 expression in tumor metastasis.

The reverse experiment with CSML-0 cells stably transfected with a construct expressing sense mts1 mRNA gave negative results. However, in spite of active mts1 transcription, these cells did not contain Mts1 protein. Thus, in addition to the control of mts1 expression at the transcription level, the control at translational level is also important and some cells can not translate mts1 mRNA. Therefore, these experiments are non interpretable until the translation suppression is overcome.

The central question is whether the over-expression of the mts1 gene in tumor cell can or can not change the phenotype from non-metastatic to metastatic and vice versa, i.e. whether the presence of Mts protein is casual for metastatic behavior of the tumor cell or an occasional coincidence. Certainly, the over-expression of any cellular gene could not be expected to constitue the only factor responsible for metastasis (see above), but some genes on certain background of expression of other genes may become indispensable for that. These â&#x20AC;&#x153;key genesâ&#x20AC;? can be detected in transfection experiments that allow to switch on or off the gene functioning.

Yet sense constructs were successfully tested in three other cell lines. One of them is line 1 cells, that are mouse small cell lung carcinoma cells highly metastatic to lungs upon intravenous injection. However, after dimethylsulfoxide (DMSO) treatment, they lose the ability to metastasize. DMSO treatment was also shown to strongly inhibit mts1 expression. Sense constructs were transfected to these cells and the transfectants, actively expressing exogenous mts1 gene, acquired the ability to give metastases even after DMSO treatment. The latter did not interfere with mts1 expression governed by MSV-LTR control elements (Grigorian et al. 1993).

Several cell systems were used in such experiments (Table 3). First, CSML-100 cells were transfected with the construct containing mts1 cDNA in antisense orientation under the control of Moloney sarcoma virus promoter/enhancer element present in its LTR. The cell lines actively expressing antisense RNA were selected and used for subcutaneous transplantation to isogeneic mice. They had a dramatically decreased metastatic potential compared to highly metastatic CSML-100 cells. Instead of hundreds of metastatic foci expected in the lungs of mice subcutaneously injected with the original CSML-100 cells, either no foci or single metastases appeared after transplantation of the same cells but expressing mts1 antisense RNA. Thus, mts1 expression was necessary for maintaining a metastatic phenotype in CSML-100 cells (Grigorian et al. 1993).

A strong increase in metastatic potential was found in rat mammary epithelial Rama37 cells after stable transfection with the mts1-expressing constructions. The original cells are benign and do not metastasize, while transfectants gave metastases to lungs and lymph nodes (Davies et al. 1993). Finally, experiments on the well-characterized human mammary adenocarcinoma MCF-7 cells were performed.

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Georgiev et al: Genes involved in the control of tumor progression in gene therapy MCF-7 cells are rather benign. They can grow after transplantation to nude mice only when supported with estrogen and only when transplantation into the mammary fat pad is performed. The growth is non-invasive and no metastases can be observed. MCF-7 cells do not contain any significant amount of Mts1 protein. Only a low level of mts1 expression could be observed in stromal cells. The expression of the exogenous mts1 gene induced by stable transfection with the construct containing the mts1 gene strongly changed the properties of the MCF-7 cell growth. First, their growth in nude mice became hormoneindependent. Second, they could grow after just subcutaneous transplantation. Third, an invasive growth at the primary focus could be detected. Fourth, metastases to regional lymph nodes and small-size metastases to the lungs were observed. The tumor cells contained varying amounts of Mts1 protein (Grigorian et al. 1996). Thus, in all mentioned cases, the appearance or disappearance of Mts1 protein led to a significant modulation of metastatic phenotype in the expected direction.

obtained in experiments with transgenic animals (Ambartsumian et al. 1996) (F i g . 1 3 ). Transgenic mice were obtained with the construct containing the mts1 gene under control of the MMTV-LTR promoter/enhancer

Yet, a weak point in transfection experiments is the heterogeneity of the cell population used for transfection. For example, CSML-0 cells consist of three morphologically distinct cell types that are reproduced after cloning from individual cells. It can not easily be excluded that only cells with pre-existing differences in metastatic potential have been selected during transfection experiments. Some other approaches should also be used. The most clear evidence for the casual role of the mts1 gene expression for creation of metastatic phenotype was

F i g . 1 3 . The scheme of the experiment with transgenic animals.

F i g . 1 4 . Staining of the non-transgenic (A) and transgenic tumor (B ) with antibodies to Mts1 and the metastasis of transgenic tumor to the lungs (C).

(#), mammary adenocarcinomas; ( ), metastases; (!) presence of Mts1 protein.

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Gene Therapy and Molecular Biology Vol 1, page 395 T a b l e 4 . Appearance of metastases in spontaneous and transplanted tumors depending on the presence of actively expressed mts1 transgene Type of the tumor

Mice line and generation

Spontaneous mammary adenocarcinoma

Tg463 (F1-F4) Tg507 (F1) Transgenic total Non-transgenic Transgenic metast. Transgenic non-met. Transgenic total Non-transgenic

Transplantation to nude mice

Number of tumors 23 5 28 21 5 2 7 2

Number of tumors with metastases 9 (39%) 2 (40%) 11 (40%) 1 (5%) 3 2 5 (70%) 0 (0%)

transgenic tumor cells at the primary focus as well as in the metastatic foci. The concentration varied in a wide range among different cells even in the same tumor. Nontransgenic tumor cells in neither case contained Mts1 protein. The stromal cells in the both types of tumors contained the same small amounts of Mts1 expressed from endogenous gene.

element. Transgenic mice expressed exogenous mts1 in several tissues. The highest level of expression was found in lactating mammary glands, where the MMTV promoter is very active. The endogenous mts1 gene is not expressed in lactating mammary gland. Interestingly, the phenotype of transgenic mice was not changed compared to normal mice. Even the presence of a high amount of Mts1 protein in lactating mammary glands did not interfere with their functioning and no mammary gland tumors could be observed. Obviously, the mts1 gene is not an oncogene.

All mentioned experiments clearly demonstrate that in some tumors mts1 expression is necessary and sufficient for the acquisition of the metastatic phenotype. It is quite clear that mts1 can not be responsible for all metastatic phenotypes, as several metastatic tumors, in particular one which appeared among the non-transgenic tumors, do not express mts1. However, the described results show that the mts1 gene is one of the key metastatic genes. The question arises, what is a possible mechanism of the action of Mts1 protein.

Thereafter, the transgenic mice were crossed with mice from the GRS/A strain characterized by a high incidence of mammary gland tumors appearing after several cycles of pregnancy and lactation. These tumors are non-metastatic. As transgenic mice were heterozygous, only half of the offspring carried the transgene, while another half represented the control group. The tumors appeared with the same high frequency in both groups and they were morphologically indistinguishable. The tumor growth rate also did not depend on the presence of the transgene.

C. A possible biological role of the Mts1 protein.

However, a dramatic difference in the metastatic potential of tumors from the two groups was found. As was mentioned above, non-transgenic tumors never metastasize. Just in only one case (out of 21 tested), nontransgenic tumor gave metastases to lungs, but this probably depended on certain additional genetic changes. On the other hand, 40% of transgenic tumors were metastatic (Table 4). Then, the tumors were subcutaneously transplanted to athymic mice to determine their metastatic phenotype. Both transgenic tumors that had metastasized before and transgenic non-metastasizing tumors gave rise to lung metastases. Thus, 40-50% incidence of metastasis is an intrinsic feature of spontaneous mammary carcinomas expressing the mts1 gene. Non-transgenic tumors never metastasized after transplantation, with the above mentioned exception.

To answer the question, intensive studies of the protein had to be performed. For this, one needs high amounts of protein. It was obtained in a bacterial system with oligohistidine tail that allowed an easy purification of the protein on nickel columns. The purified protein was used for preparing polyclonal and monoclonal antibodies. Western blot analysis and immunostaining of cells with these antibodies showed the Mts1 protein to be localized in the cytoplasmic fraction, like other calciumbinding proteins. Experiments on fractionation of cell extracts suggested the presence of a significant fraction of Mts1 protein in the cytoskeleton. To understand Mts1 function, attempts to determine the targets of Mts1 protein were performed. The in vivo labeled proteins which bind Mts-1 were immunoprecipitated with antibodies to Mts1 and the proteins specifically precipitated by these antibodies were

The distribution of Mts1 protein was detected with the aid of immuno-staining (F i g . 1 4 ). Mts1 was found in

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F i g . 1 5 . Interaction of Mts1 with carboxy-terminus of heavy chain of non-muscle myosin.

heavy chain of non-muscle myosin was tested. Mts1 specifically inhibited phoshorylation of serine residue no. 1917 by protein kinase C without any effect on other phosphorylation sites and without interference with casein protein kinase action. The target serine residue is located just inside the binding region for Mts1 (F i g . 1 5 ). One can suggest that at least one of Mts1-induced effects is an inhibition of this phosphorylation reaction. The latter was claimed to play a role in non-muscle myosin functioning putatively leading to changes in cell motility. This may be a possible way for changing the metastatic phenotype of tumor cells. However, for the time being, this is just a hypothesis.

analyzed. This approach resulted in the isolation of different components of myosin complex. On the other hand, antibodies to myosin co-precipitated Mts1. Ultracentrifugation of cell extracts on sucrose gradients also demonstrated cosedimentation of a rather significant fraction of Mts1 with a much heavier myosin complex suggesting their reversible association. After double immuno-staining with antibodies to Mts1 and myosin, one can see the exact coincidence in fluorescence distribution for both antibodies. Mts1 interacts only with the heavy chain of non-muscle myosin as followed from different overlay experiments and using antibodies specific for different types and different chains of myosin. Mts1 does not interact with the light chain and with heavy chain of smooth muscle myosin (Kriajevska et al. 1994).

Another consequence of interaction between Mts1 and carboxy-terminal part of non-muscle myosin is the solubilization of the non-muscle myosin. The simple addition of a native Mts1 to the myosin polypeptide precipitate at physiological salt concentration completely solubilized the precipitate, confirming the role of Mts1 in myosin disaggregation.

To further analyze Mts1-myosin interaction, different fragments of the heavy chain of non-muscle myosin (HCNMM) were prepared using recombinant DNA and tested for interaction with Mts1 in protein overlay experiments. Only the carboxy-terminal part of HCNMM did react. Analysis of the deletions obtained in this part of HCNMM showed that the only peptide responsible for interaction with Mts1 was located between the 1908 and 1938 aminoacid residues (F i g . 15). Then the effect of such binding on protein kinase mediated phoshorylation of

It should be pointed out that myosin is not the only target for Mts1. In particular, the method of binding to affinity column revealed another protein, p37, interacting with the Mts1 column. The p37 protein binds to Mts1 in a calcium-dependent manner. Interestingly, this binding 396

Gene Therapy and Molecular Biology Vol 1, page 397 strongly changes the interaction of Mts1 with calcium. Two calcium-binding domains in Mts1 act cooperatively and in general the affinity to calcium is increased. Possibly, p37 may play some role in the control of Mts1 functions connected with binding of calcium ions (Dukhanina et al. in press). Another important function of Mts1 protein may be excerted via mesenchymal transformation of epithelial cells. Strutz et al (1995) found, that expression of the mts1 gene in epithelial cells might induce their mesenchymal transformation. We have found that appearance of Mts1 proteins in the cells of transgenic mice was accompanied by the loss of E-cadherin. The reverse correlation between Mts1 and E-cadherin content may be especially important as E-cadherin is one of most clear-cut “antimetastatic proteins” with well understood function (Vlamincks et al. 1991). The mechanism of Mts1 influence on E-cadherin remains unclear. Finally, Onishchenko et al. (1997) recently observed the destabilization of blood vessels in tumors expressing mts1; this means that endothelial cells of blood vessels may constitute another target of Mts1 and this additional phenotypic effect of Mts1 may be effected through its influence on tumor vascularization and on the state of endothelial cells. Thus, Mts1 may be a multifunctional regulator of cell functions connected with the acquisition of an invasive metastatic phenotype. The mts1 gene may be one of the critical genes for metastasis development and, therefore, a promising target for cancer gene therapy.

VIII. Conclusions In general, this study summarizes our results on the search for new genes and proteins controlling tumor progression. Here we described explicitly two examples of such genes. One gene, (mts1), tentatively assigned to the second class (tumor progression genes), and another gene, (tag7), putatively belonging to the third class (genes for biological defense against tumors) were discovered in this research program together with many other, yet less characterized genes. The tag7 gene seems to be a promising factor to be used for gene therapy or even for the direct treatment of tumors by its product, Tag7 protein. We propose that the mts1 gene may also find a place in the constellation of target genes for cancer gene therapy.

Acknowledgements. This work was supported by Moscow Program for Cancer Treatment, Russian Fund for Basic Researches, INTAS and PECO grants

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References. 1. Review articles: Anderson MW, Reynolds SH, You M, Maronpot RM (1 9 9 2 ) Role of proto-oncogene activation in carcinogenesis. Environ Health Perspect 98,13-24 Folkman J (1 9 9 2 ) Inhibition of angiogenesis. S e m i n . C a n c e r B i o l. 3, 65-71. Barbacid M (1 9 8 7 ) ras genes. A n n . R e v . B i o c h e m . 56, 779-827. Georgiev GP, Kiselev SL, Lukanidin EM Tumor progression and metastasis. (1 9 9 7 ) In “Genome structure and f u n c t i o n ” (Nicolini C, ed.) Kluwer Acad. Publ., Netherlands, pp217-237. Gunthert U, Birchmeier W, Eds. (1 9 6 6 ) Attempts to understand metastasis foemation. II. Regulatory factors (Current t o p i c s i n M i c r o b i o l . Immunol.) Springer-Verlag, Berlin- Heidelberg, v. 213/II. Vogelstein B, Kinzler KW (1 9 9 3 ) The multistep nature of cancer. Trends Genet. 9, 138-141. White E (1 9 9 6 ) Life, death, and the pursuit of apoptosis. G e n e s D e v . 10, 1-15. 2. Experimental papers: Ambartsumian NS, Grigorian MS, Larsen IF, Karlstrom O, Sidenius N, Rygaard J, Georgiev G, Lukanidin E (1 9 9 6 ) Metastasis of mammary carcinomas in GRS/A hybrid mice transgenic for the mts1 gene. O n c o g e n e 13, 16211630. Davies BR, Davies MPA, Gibbs FEM, Barrachlough R, Philip S, Rudland PS (1 9 9 3 ) Induction of the metastatic phenotype by transfection of a benign rat mammary epithelial cell line with the gene for p9Ka, a rat calciumbinding protein, but not with the oncogene EJ-ras-1. O n c o g e n e 8, 999-1008. Grigorian MS, Tulchinsky EM, Zain S, Ebralidze AK, Kramerov DA, Kriajevska MV, Georgiev GP, Lukanidin EM (1 9 9 3 ) The mts1 gene and control of tumor metastasis. Gene 135, 229-238. Grigorian M, Ambartsumian N, Lykkesfeldt AE, Bastholm L, Elling F, Georgiev G, Lukanidin E (1 9 9 6 ) Effect of mts1 (S100A4) expression on the progression of human breast cancer cells. Int. J. Cancer 67, 831-841 Ebralidze A, Tulchinsky E, Grigorian M, Afanasyeva A, Senin V, Revasova E, Lukanidin E (1 9 8 9 ) Isolation and characterization of a gene specifically expressed in different metastatic cells and whose deduced gene product has a high degree of homology to a Ca2+-binding protein family. G e n e s D e v . 3, 1086-1093. Kriajevska MV, Cardenas MN, Grigorian MS, Ambartsumian NS, Georgiev GP, Lukanidin EM (1 9 9 4 ) Non-muscle myosin heavy chain as a possible target for protein encoded by metastasis-related mts-1 gene. J . B i o l . Chem. 269, 19679-19682.

Georgiev et al: Genes involved in the control of tumor progression in gene therapy Kustikova OS, Kiselev SL, Borodulina OR, Senin VM, Afanasyeva AV, Kabishev AA (1 9 9 6 ) Cloning of the tag7 gene expressed in metastatic mouse tumors. Genetika (in Russian) 32, 621-628. Maelandsmo GM, Hovig E, Skrede M, Kashani-Sabet M, Engebraaten O, Florenes VA, Myklebost O, Grigorian M, Lukanidin E, Skanlon KJ, Fodstad O (1 9 9 6 ) Reversal of the in vivo metastatic phenotype of human tumor cells by an anti-CAPL (mts1) ribozyme. Cancer Res . 56, 54905498. Onischenko A, Chenard MP, Lefebvre O, Bruyneel E, Rio MC (1 9 9 6 ) Defective tumor vascularization induced by metastasin 1 expression. I n v a s i o n M e t a s t a s i s 16, 160-168 Strutz F, Okada H, Lo CW, Danoff T, Carone RL, Tomaszewsky JE, Neilson G (1 9 9 5 ) Identification and characterization of a fibroblast marker: FSP1. J . C e l l B i o l . 130, 393-405. Tarabykina S, Ambartsumian N, Grigorian M, Georgiev G, Lukanidin E (1 9 9 6 ) Activation of mts1 transcription by insertion of a retrovirus-like IAP element. Gene 168, 151-155. Tulchinsky E, Georgiev G, Lukanidin E (1 9 9 6 ) EM Novel AP1 binding site created by DNA-methylation. O n c o g e n e 12, 1737-1745. Tulchinsky E, Prokhortchouk E, Georgiev G, Lukanidin E (1 9 9 7 ) A kappaB-related binding site is an integral part of the mts1 gene composite enhancer element located in the first intron of the gene. J . B i o l . C h e m . 272, 48284835. Vlemincks K, Vakaek L, Mareel M, Fiers W, VanRoy F (1 9 9 1 ) Genetic manipulation of E-cadherin expression by epithelial tumor cells reveals an invasion suppressor role. C e l l 66, 107-119.

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Gene Therapy and Molecular Biology Vol 1, page 399 Gene Ther Mol Biol Vol 1, 399-406. March, 1998.

The Apoptin gene of chicken anemia virus in the induction of apoptosis in human tumorigenic cells and in gene therapy of cancer*. Mathieu H.M. Noteborn 1,2, Astrid A.A.M. Danen-van Oorschot2 and Alex J. van der Eb2 1

Leadd BV, and 2 Laboratory for Molecular Carcinogenesis, Department of Medical Biochemistry, Leiden University, P.O. Box 9503, 2300 RA, Leiden, The Netherlands. __________________________________________________________________________________________________ Correspondence: Mathieu Noteborn, Tel: 31-71-527-87 36, Fax: 31-71-527-17 36, E-mail:: leadd@leadd.nl *Apoptin ! is a registered trademark by Leadd BV, Leiden, The Netherlands

Summary Apoptosis is an active physiological process for the elimination of superfluous or altered cells from a developing or adult organism. Aberrations in the apoptotic process cause various diseases, e.g. tumor formation. Chemotherapy for the treatment of cancer often exerts its cytotoxic effect via the induction of apoptosis and its success depends mainly on the presence of functional p53 and the absence of an overexpressed Bcl-2 proto-oncogene. Unfortunately, in more than 50% of the tumors functional p53 is lacking, and/or Bcl-2 is overexpressed, which often results in resistance to anti-cancer therapy. Apoptin !,1 , a protein derived from chicken anemia virus (CAV), can induce apoptosis in cultured (human) cells derived from various tumors, e.g., osteosarcoma, lymphoma, leukemia, hepatoma, melanoma, and from tumors of breast, colon, and lung. Tumor cells lacking p53 can undergo Apoptin-induced apoptosis, and over-expression of Bcl-2 even stimulates Apoptininduced apoptosis. Downstream inhibitors of the p53-pathway, like Bcl-2, BAG-1 and CrmA, do not inhibit Apoptin-induced apoptosis, which indicates that Apoptin induces c e l l death via a different pathway or that Apoptin acts at a step far downstream the apoptotic cascade. Interestingly, Apoptin fails to induce apoptosis in human primary lymphoid, dermal, epidermal, endothelial and smooth muscle c e l l s . Co-expression o f a transforming agent with Apoptin in normal diploid cells results in apoptosis. The fact that Apoptin induces a p53-independent, and Bcl-2-stimulated type of apoptosis in human tumor cells, but not in normal diploid cells, renders Apoptin a potential anti-tumor agent. Gene-therapy strategies based on viral vectors expressing Apoptin are currently under development.

phagocytose these dying cells (F i g u r e 1 ; Wyllie et al., 1980; White, 1996). The apoptotic process can be initiated by a variety of regulatory stimuli (Wyllie, 1995; White, 1996; Levine, 1997). Changes in the rate of cell survival play an important role in, e.g., oncogenesis, which is caused by enhanced proliferation but also by decreased cell death (Kerr et al., 1994; Paulovich, 1997).

I. Introduction Apoptosis is an active and programmed physiological process for eliminating superfluous, altered or even malignant cells (Earnshaw, 1995; Duke et al., 1996). Apoptosis is characterized by shrinkage of cells, segmentation of the nucleus, condensation and cleavage of DNA into domain-sized fragments, in most cells followed by internucleosomal degradation. The apoptotic cells fragment into membrane-enclosed apoptotic bodies. Finally, neighboring cells and/or macrophages will rapidly

Several oncogenes and tumor-suppressor genes have been found to play a role in either induction or inhibition of apoptosis. The tumor suppressor p53, for example, can

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Gene Therapy and Molecular Biology Vol 1, page 400

Figure 1 . Schematic illustration of the subsequent morphological events, which occur during the apoptotic process. 1. Scheme of a normal cell before undergoing apoptosis. 2. The cytoplasmic membrane starts to form blebbing structures and the DNA begins to condensate. 3. The DNA condensates adjacent to the still intact nuclear membrane. 4. The nucleus becomes segregated, which is (5) subsequently followed by separation of the apoptotic cell. The complete cell content is surrounded by membrane structures, which are called apoptotic bodies. 6. These apoptotic bodies are phagocytosed by neighbouring cells and/or macrophages.

induce either cell-cycle arrest or apoptosis upon DNA damage. In many tumors, however, the p53 function is lost as a result of mutation or deletion. The protooncogene Bcl-2, which inhibits apoptosis, has been found to be activated in several lymphomaâ&#x20AC;&#x2122;s due to a translocation.

tumor agent, its tumor-specific activity and the first genetherapy experiments.

Apoptosis plays a role not only in the development of tumors but also in the treatment of cancer. Conventional therapies using cytotoxic agents often act via induction of apoptosis. In many instances, the induction of apoptosis requires functional p53 and, consequently, loss of p53 function, correlates with resistance to these therapies. For example, melanomas, lung, prostate and colon cancers often have mutated p53, resulting in a poor response to chemotherapy (Lowe et al., 1994; Levine, 1997). Overexpression of Bcl-2 in lymphomas due to a t(14;18) translocation or in chronic myelogenous leukemia due to formation of the Bcr-abl fusion product by t(9;22) translocation leads to enhanced protection against apoptosis, and sometimes to resistance against (chemo)therapy (McDonell et al., 1995).

Chicken anemia virus (CAV) is an avian pathogen of worldwide importance. The clinical signs of CAV in young chickens are increased mortality, loss of body weight and hemorrhages. Severe depletion of cortical thymocytes and erythroblastoid cells results in transient immunodeficiency and anemia (Coombes and Crawford, 1996).

II. Apoptin!, a CAV-derived protein induces apoptosis

The depletion of thymocytes observed after CAV infection is based on apoptosis (Jeurissen et al., 1992; Noteborn, 1993). DNA isolated from the thymus of infected chickens shows the apoptosis-specific laddering pattern, which is not observed in DNA isolated from the thymus of non-infected chickens. Electron-microscopic analysis of the cortex ten days after infection shows cells containing condensed chromatin adherent to the nuclear membrane, and apoptotic bodies in the cytoplasm of epithelial cells.

This has led to a search for new therapies, leading for example to restoration of the function of p53. Adenovirusor retrovirus-mediated transfer of p53 inhibits the growth of certain tumor cells in tissue culture and in mouse models (Sandig et al., 1997; Gomez-Manzano et al., 1996; Yang et al., 1995). However, restoring p53 function may not be effective in tumors which have a block in the apoptosis pathway, e.g. those which over-express Bcl-2.

CAV is a small non-enveloped virus containing a single-stranded DNA genome of 2.3 kb (Noteborn and Koch, 1995). From the CAV genome, a singlepolycistronic mRNA is transcribed, with three open reading frames which partially or completely overlap. These three reading frames are indeed used, and the CAV mRNA encodes three completely different proteins VP1 (51.6 kDa), VP2 (24.0 kDa) and VP3 (13.6). The products show no homology to each other or to any other known proteins. VP1 is the only capsid protein, whereas VP2 seems to be required for capsid formation (Noteborn and Koch, 1995).

The apoptosis-inducing protein Apoptin is the basis for an alternative candidate strategy, since it induces apoptosis in various human tumor cell lines, including those lacking p53 and/or overexpressing Bcl-2. In this review we discuss the potential of Apoptin as an anti-

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Gene Therapy and Molecular Biology Vol 1, page 401 To establish which CAV protein is responsible for the induction of apoptosis, plasmids encoding either VP1, VP2 or VP3 were transfected into cultured chicken mononuclear cells. The cells were analysed by indirect immunofluorescence with specific antibodies, and its DNA was stained with propidium iodide (PI), which stains DNA strongly when it is intact, but weakly and/or irregularly when it is apoptotic. Expression of VP3 alone was sufficient for the induction of apoptosis as observed in CAV infection. Therefore we renamed VP3 as Apoptin. Soon after transfection, Apoptin is dispersed throughout the nucleus. Somewhat later, when the cells become apoptotic, Apoptin becomes aggregated in the nucleus or its remnants. At this point in time, nucleosomal laddering can be seen in the DNA of Apoptin-expressing cells, whereas DNA from cells transfected with a control plasmid remains intact (Noteborn, 1994). Preliminary results indicate that VP2 also has some apoptotic activity, although much weaker than Apoptin. Surprisingly, VP2 enhances Apoptin-induced apoptosis in transformed cells. Expression of VP1 did not result in apoptosis (Noteborn, unpublished data).

undergo apoptosis, and the process can still be reversed. Many independent signal-transduction pathways can be used, but they all converge in the third phase: the execution of apoptosis. When the cell-death effectors are activated, the cell crosses the point of no return, and undergoes apoptosis rapidly. An important regulator of apoptosis is the tumorsuppressor protein p53, which, among others, is involved in the response to excessive DNA damage caused by cytotoxic agents. On the other hand, it does not seem to be important in the regulation of apoptosis during embryonal development, since p53-knockout mice develop normally (Levine, 1997). To determine whether p53 is involved in Apoptininduced cell death, Apoptin was transiently expressed in human tumor cells either lacking p53, expressing a mutant form, or synthesizing wild-type p53. In all three cell lines, Apoptin was able to induce apoptosis to the same extent, indicating that it does not need functional p53 (Zhuang et al., 1995a). In consistence with this, the adenovirus E1B55K protein, an inhibitor of p53, did not decrease the apoptotic activity of Apoptin (Zhuang et al., unpublished data).

Apoptin consists of 121 amino acids and is rich in proline, serine and threonine residues. It contains a hydrophobic region, resembling a nuclear export signal (Wen et al., 1995; Fischer et al., 1995) and two positively-charged regions representing nuclear localization signals (Noteborn et al., 1987). A mutant Apoptin protein in which one of the two nuclear-localization signals was deleted, showed significantly reduced apoptotic activity, and was localized partially in the cytoplasm (Zhuang et al., 1995, 1995a).

IV. Bcl-2 inhibits p53-induced, but not Apoptin-induced apoptosis. The proto-oncogene Bcl-2 acts downstream of p53 and is shown to inhibit p53-mediated, but also p53independent apoptosis (White et al., 1996). Zhuang and colleagues studied the effect of several apoptosis inhibitors on Apoptin-induced apoptosis (Zhuang et al., 1995, 1995a).

It seems to be essential for induction of apoptosis by Apoptin that it co-localizes with the chromatin. The basic regions of Apoptin may allow interaction with nucleic acids, explaining the nuclear localization. The presence of Apoptin in the chromatin structure, and its high proline content, may cause disturbance of the supercoil organization, which could then result in apoptosis. Another possibility is that Apoptin acts as a transcriptional activator of genes, which directly or indirectly mediate apoptosis.

Apoptin could still induce apoptosis in human hematologic malignant cells expressing high levels of Bcl2 (DoHH-2) or BCR-ABL (K562), both inhibitors of p53mediated cell death. In DoHH-2 cells, Apoptin induced cell death even faster than in K562 cells. Another inhibitor of apoptosis is BAG-1, a Bcl-2binding protein that is functionally but not structurally homologous to Bcl-2. In some cell types, Bcl-2 and BAG1 cooperate to inhibit apoptosis, whereas Bcl-2 alone has only a minor effect. We therefore tested the effect of BAG1, alone or in combination with Bcl-2, on Apoptin-induced apoptosis (Danen-van Oorschot, 1997a). Like Bcl-2, BAG1 did not inhibit cell death induced by Apoptin, and neither did the combination of Bcl-2 and BAG-1. Surprisingly, Apoptin-induced apoptosis is enhanced by Bcl-2 and, to a lesser extent, by BAG-1. In parallel experiments, BAG-1 and/or Bcl-2 did inhibit p53-induced apoptosis.

III. Apoptin-induced apoptosis is p53independent Apoptosis is a physiological process which is as tightly and extensively regulated as cell proliferation. Apoptosis can be divided into three phases (Vaux and Strasser, 1996). It starts with some stimulus, for example cytotoxic drugs, growth-factor deprivation, or binding of a ligand to a cell-surface receptor (e.g. Fas). In the second phase, the stimulus is detected and a transduction signal is produced. In this phase, the cell decides whether or not to

In conclusion, Bcl-2 overexpression stimulates Apoptin-induced cell death. From a therapeutic point of view this feature is important, because lymphomas carry 401

Noteborn et al: The Apoptin gene in apoptosis and in gene therapy of cancer an overexpressed Bcl-2 gene which results in their derailment and resistance to chemotherapeutic agents.

V. Caspases are differently involved in Apoptin- versus p53-induced apoptosis In general, a step further down in the apoptotic decision cascade are the caspases, which are specific cysteine-proteases (also called ICE-like proteases). Caspases can cleave themselves, other caspases or target proteins, such as PARP, lamins and the 70-kDa U1 small ribonucleoprotein (Rao and White, 1997; Nicholson, 1996). Thus far, one assumes that all apoptotic pathways converge in the activation of the cascade of caspases, which seems to be part of the execution phase. To determine whether caspases are also involved in Apoptin-induced cell death, the cowpox-virus protein CrmA was co-expressed with Apoptin. CrmA mainly inhibits the activity of caspase 1, but also of other caspases, and thereby prevents apoptosis. Co-expression of CrmA with Apoptin, however, could not prevent apoptosis, whereas in-parallel experiments it was revealed that p53-induced cell death was suppressed by CrmA (Danen-van Oorschot, unpublished results). Similar results were obtained with modified peptides, known to specifically inhibit caspases, also those more downstream in the cascade. These data indicate that Apoptin-induced apoptosis does not involve caspases, or at least not those interfering in the p53-apoptotic pathway. Alternatively, Apoptin may act downstream of the caspases. As Apoptin was found to co-localize with chromatin, it may bind to DNA and disturb its structure, or even act as an endonuclease. However, no evidence of binding to DNA has been found so far (Noteborn, unpublished results). Another possible explanation is that Apoptin associates with DNA-binding proteins, and indirectly influences the chromatin structure. We are currently trying to identify proteins that associate with Apoptin via the yeast-two-hybrid system. A schematic overview of the pathways via which Apoptin and p53 induce apoptosis, is shown in Figure 2.

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Figure 2 . Schematic representation of the Apoptinpathway versus the p53 pathway of apoptosis induction. The adenovirus E1B 55K protein inhibits p53 activity but not apoptin-induced apoptosis. The Apoptin-pathway is strongly stimulated by Bcl-2 and to some extent by BAG-1, whereas in contrast, the p53-pathway is inhibited by both Bcl-2 and BAG-1. The caspase-inhibitors CrmA and peptide zVAD inhibit p53, but not Apoptin-induced apoptosis.

VI. Apoptin induces apoptosis in human tumor cells, but not in normal cells The fact that Apoptin does not need the tumor suppressor p53 to induce apoptosis, and is even stimulated by the proto-oncogene Bcl-2 makes Apoptin an interesting candidate anti-tumor agent. To explore this potential, the apoptotic effect of Apoptin has been examined in many human and other mammalian tumor cell lines of different origins. In all examined tumorigenic/transformed cell lines Apoptin was proven to induce apoptosis. Some of these cell lines are ones derived from breast and lung tumors, from neuroblastoma, hepatoma, melanoma, lymphoma, leukemia, or colon carcinoma (see Table 1 and T a b l e 2). In some tumor cell lines, programmed cell death was induced faster than in others, but apoptosis had always reached 90-100% at 5-6 days after transfection. Interestingly, Apoptin did not induce apoptosis in normal, diploid (human) cells. We have examined human keratinocytes, vascular endothelial cells, smooth muscle cells, primary T cells and two types of human diploid fibroblasts under tissue-culture conditions, and murine and rat embryonal fibroblasts: in none of these cells Apoptin was able to induce apoptosis (Table 3). Among the above cell lines, several ones do not express (functional)

Gene Therapy and Molecular Biology Vol 1, page 403 T a b l e 1 . Human tumorigenic and transformed cell lines in which Apoptin induces apoptosis.

T a b l e 3 . Normal diploid cells in which Apoptin does NOT induce apoptosis

Cell line 1. MCF-7 2. Saos-2 3. U2OS 4. SLCC-1 5. SLCC-2 6. Hep3B 7. HepG2 8. G401 9. KG-1 10. K562 11. DoHH-2 12. Jobo-O 13. Pre

Human cells 1. T cells

14. Post 15. NW-18 16. SCC-15 17. SVK14 18. HaCaT 19. 911 20. 293 21. HT29

Type Breast tumor Osteosarcoma, p53Osteosarcoma, p53+ Small lung cell carcinoma Small lung cell carcinoma Hepatoma, p53Hepatoma, p53+ Kidney rhabdoid tumor Acute myeloid leukemia Acute myeloid leukemia, Bcr-abl Immunoblastic B-lymphoma Epstein-Barr virus transformed B cells SV40-transformed pre-crisis fibroblasts SV40-transformed post-crisis fibroblasts SV40-transformed tumorigenic fibroblasts Squamous cell carcinoma SV40-transformed keratinocytes Spontaneously transformed keratinocytes Adenovirus-5-transformed embryonal retinoblasts Adenovirus-5-transformed embryonal retinoblasts Colon carcinoma

2. HUVEC 3. HSMC 4. FSK-1 5. VH10 6. VH25 Non-human cells 7. REF 8. MEF-1 9. MEF-2

4. CC-531 5. Cos-7 6. MDDC- MSB1 7. HD-11

Type Rat embryo fibroblasts Mouse embryo fibroblasts, p53+ Mouse embryo fibroblasts, p53-

p53, indicating once again that Apoptin induces apoptosis independently of p53. It might be expected that tumorigenic and/or transformed derivatives from the above primary cell types also are resistant to Apoptin-induced apoptosis. We, therefore, examined the effect of Apoptin in transformed cells derived from normal diploid fibroblasts and keratinocytes. Apoptin was found to induce apoptosis in VH-10 fibroblasts and keratinocytes that were transformed with SV40, and also in spontaneously immortalized keratinocytes. Apparently, these cells had become susceptible to Apoptin-induced cell death upon transformation (Danen-van Oorschot et al., 1997). This result indicates that Apoptin specifically induces apoptosis in transformed and tumorigenic cells, but not in normal diploid cells. Even more fascinating is the observation by Zhang and Noteborn (unpublished data) that co-expression of the transforming agent large T antigen of DNA tumor virus SV40 and Apoptin in normal diploid human cells results in Apoptin-induced cell death. An early event during tumorigenesis seems to be the switch for Apoptininduced apoptosis. All these experiments have been carried out under cell-culture conditions, and it is now of great interest to determine whether Apoptin shows the same specificity in vivo.

Table 2. Non-human tumorigenic and transformed cell lines in which Apoptin can induce apoptosis. Cell line 1. N1E-115 2. P19 3. BRK-Xho

Type Phytohemagglutinin-stimulated primary T cells Umbilical-cord vascular endothelial cells Smooth muscle cells Epidermal keratinocytes Diploid fibroblasts Diploid fibroblasts

Type Murine neuroblastoma Murine embryonal teratocarcinoma Adenovirus-5-transformed baby rat kidney cells Rat colon carcinoma Monkey SV40-transformed kidney cells Avian lymphoma Avian myeloblastosis virus (AMV)transformed cells.

VII. Apoptin is differential located in normal diploid cells versus transformed and/or tumorigenic cells. An explanation why Apoptin specifically induces apoptosis in transformed and tumorigenic cells came from the analysis of its localization by indirect immunofluorescence. In tumorigenic/transformed cells 403

Noteborn et al: The Apoptin gene in apoptosis and in gene therapy of cancer Apoptin is localized in the nucleus, initially in finely dispersed form, and when the cell becomes apoptotic it starts to aggregate. In normal diploid cells, however, Apoptin is found predominantly as fine granules in the cytoplasm. As mentioned above, a truncated version of Apoptin, lacking one of the putative nuclear localization signals, is also localized mainly in the cytoplasm, but in a more diffused form. This truncated Apoptin has a strongly reduced apoptotic activity. These observations suggest that the nuclear localization of Apoptin is an important factor in its ability to induce apoptosis. It is possible, however, that nuclear localization as such is not sufficient and that, in addition, Apoptin also needs to be modified in order to be able to trigger the apoptotic event in the nucleus. The fact, that the transforming SV40 large T antigen translocates, besides Apoptin, also the tumor-related Hepatitis B virus HBx protein into the nucleus (Doria et al., 1995), suggests that Apoptin-induced apoptosis is linked to cell transformation. Paradoxically, the nuclear location of HBx will result in carcinoma formation, whereas nuclear Apoptin induces apoptosis. The answer to the interesting question why Apoptin is differential located in normal versus transformed cells might help to unravel the mechanism of Apoptin-induced apoptosis and also one of the (early) steps in tumorigenesis.

VIII. Apoptin is a potential anti-tumor agent Treatment of tumors with cytotoxic agents has been shown to kill tumor cells, often via the induction of apoptosis. Many tumors, however, are resistant to induction of apoptosis by such chemotherapy due to the loss of functional p53 or to overexpression of Bcl-2. For these categories of tumors, the development of new, effective therapies would be very welcome. The results obtained so far with Apoptin suggest that this viral protein is a promising candidate for the treatment of tumors, including those that lack functional p53 or overexpress Bcl-2. The apoptosis inhibitor Bcl-2 not only fails to block, but even seems to enhance Apoptin-induced apoptosis. Furthermore, Apoptin induces apoptosis in transformed/tumorigenic cells, but does not in normal diploid cells grown under tissue-culture conditions. In conclusion, these observations imply that Apoptin is a serious candidate as an anti-tumor agent. In order to use Apoptin as an anti-tumor agent, genetherapy strategies can be used. A major problem that needs to be overcome is the efficient delivery of Apoptin throughout the entire tumor and to all metastases.

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Presently, the most commonly used gene delivery systems are viral vectors because of their high efficiency. Retroviral vectors are used in the majority of approved gene-transfer protocols (reviewed by Roth and Cristiano, 1997). The advantage of this system is that the viral DNA can stably integrate into the host genome, which ensures long-term expression of the introduced gene. However, this may also be disadvantageous because of the risks associated with random integration. Nevertheless, retroviral vectors can infect only dividing cells and they are, therefore, potentially useful for the treatment of cancer (Vile et al., 1996). Other commonly applied vectors are based on human adenovirus 5 (Ad5), which can infect many different cell types and does not require cell division. They are specifically targeted to the respiratory and gastrointestinal epithelia, as well as to the liver. This system is less suitable for long-term expression as adenoviral DNA does not integrate into the host genome. This feature has the advantage that no risk exists for insertional mutagenesis. These properties, and the fact that adenoviral vectors have a high transduction efficiency and can be produced at high titers, renders these vectors particularly suitable for gene therapy against cancer (Ginsberg, 1996; Kozarsky and Wilson, 1993; Roth and Cristiano, 1997). Rodent parvoviruses like H-1 and MVM viruses (that can also grow in human cells) have been shown to exert an oncosuppressive activity, i.e. they inhibit the formation of spontaneous, as well as chemically or virally induced tumors in laboratory animals. Cancer cells provide these viruses with an environment beneficial to their multiplication, which means that they appear to be oncotropic. However, these rodent parvoviruses are not always able to prevent the cancer cells from multiplying. Therefore, Dinsart et al. (1996) have developed a system for recombinant parvoviral particles expressing cytotoxic agents. In collaboration with Dinsart and Cornelis, DKFZ, Heidelberg, Germany, we are developing a recombinant parvovirus expressing Apoptin. The first aim will be to examine whether parvovirus-expressed Apoptin will kill those tumor cell lines, that are resistant to lysis by H1 and/or MVM parvovirus. It was unclear, initially, whether viral vectors expressing the Apoptin gene could be produced at all, since the required helper cells for growing the replicationdefective viral vectors are transformed cell lines and might be susceptible to apoptin-induced apoptosis. Indeed, the murine packaging cell lines PA317 and Psi-crip expressing Apoptin were killed within four days after the onset of recombinant retrovirus production (Noteborn et al., unpublished results). Hence, retroviral vectors carrying the gene encoding Apoptin need to be produced in a batchwise fashion.

Gene Therapy and Molecular Biology Vol 1, page 405 Fallaux, F.J., Kranenburg, O., Cramer, S.J., Houweling, A., Van Ormondt, H., Hoeben, R.C., and Van der Eb, A.J. (1 9 9 6 ). Characterization of 911: a new helper cell line for the titration and propagation of early region 1-deleted adenoviral vectors. Hum. Gene Ther. 7, 215-222.

At the moment, we are investigating a recombinant replication-deficient adenovirus expressing Apoptin. The transformed helper cells (Fallaux et al., 1996) used for growing adenoviral vectors, as expected, also underwent apoptin-induced cell death. Fortunately, however, the lytic cycle of the adenovirus vector seemed to proceed faster than the induction of apoptosis by Apoptin, resulting in recombinant-Apoptin viral stocks with high titers (Pietersen et al., unpublished data).

Fischer, U., Huber, J., Boelens, W.C., Mattaj, I.W., and Luhrmann, R. (1 9 9 5 ). The HIV-1 Rev activation domain is a nuclear export signal that acesses an export pathway used by specific cellular RNAs. C e l l 82, 475-483. Ginsberg, H.S. (1 9 9 6 ). The ups and downs of adenovirus vectors. B u l l . N . Y . A c a d . M e d . 73, 53-58.

Animal studies with the recombinant-apoptin adenovirus will reveal whether in-vivo expression of Apoptin will indeed have anti-tumor activity and no toxic side effects. Preliminary results of preclinical trials assaying the in-vivo toxicity of Apoptin, show that adenovirus-expressed Apoptin causes only very minor toxic side effects, if any. Currently, anti-tumor studies using perfusion of isolated rat livers containing colonmetastases are under way.

Gomez-Manzano, C., Fueyo, J., Kyritsis, A.P., Steck, P.A., Roth, J.A., McDonell, T.J., Steck, K.D., Levin, V.A., and Yung, W.K.A. (1 9 9 6 ). Adenovirus-mediated transfer of the p53 gene produces rapid and generalized death of human glioma cells via apoptosis. Cancer R e s . 56, 694-699. Jeurissen, S.H.M., Wagenaar, F., Pol, J.M.A., Van der Eb, A.J., and Noteborn, M.H.M. (1 9 9 2 ). Chicken anemia virus causes apoptosis of thymocytes after in vivo infection and of cell lines after in vitro infection. J . V i r o l . 66, 7383-7388.

Ackowledgements We would like to thank Hans van Ormondt for critically reading the manuscript, Josephine Dorsman, Marjolijn van der Eb, Alexandra Pietersen, Paul Smits and Yinghui Zhang for kindly providing their data on Apoptin expression in transformed and/or normal diploid cells. This research was partially made possible by grants from the Dutch Cancer Foundation (Grant no. 95.1094), the Netherlands Ministry of Economic Affairs, and Aesculaap BV, Boxtel, The Netherlands.

Kerr, J.F.R., Winterford, C.M., and Harmon, B.V. (1 9 9 4 ). Apoptosis: Its significance in cancer and cancer therapy. Cancer 73, 2013-2026. Kozarsky, K.F. and Wilson, J.M. (1 9 9 3 ). Gene therapy: adenovirus vectors. C u r r . O p i n . G e n e t . D e v . 3, 499503. Levine, A.J. (1 9 9 7 ). p53, the cellular gatekeeper for growth and division. C e l l 88, 323-331. Lowe, S., W., Bodis, S., McClathey, A., Remington, L., Ruley, H.E., Fisher, D.E., Housman, D.E., and Jacks, T. (1 9 9 4 ). p53 status and the efficacy of cancer therapy in vivo. S c i e n c e 266, 807-810.

References Coombes, A.L., and Crawford, G.R. (1 9 9 6 ). Chicken anaemia virus: a short review. W o r l d â&#x20AC;&#x2122; s P o u l t r y S c i . J . 52, 267-277.

McDonell, T.J., Meyn, R.E., and Robertson, L.E. (1 9 9 5 ). Implications of apoptotic cell death regulation in cancer therapy. S e m i n . C a n c e r B i o l . 6, 53-60.

Danen-van Oorschot, A.A.A.M., Den Hollander, A., Takayama, S., Reed, J., Van der Eb, A.J., and Noteborn, M.H.M. (1 9 9 7 a ). BAG-1 inhibits p53-induced but not apoptin-induced apoptosis. A p o p t o s i s , In press.

Nicholson, D.W. (1 9 9 6 ). ICE/CED3-like proteases as therapeutic targets for the control of inappropriate apoptosis. N a t u r e B i o t e c h n . 14, 297-301.

Danen-van Oorschot, A.A.A.M., Fischer, D.F., Grimbergen, J., Klein, B., Zhuang, S.-M., Falkenburg, J.H.F., Backendorf, C., Quax, P.H.A., Van der Eb, A.J., and Noteborn, M.H.M. (1 9 9 7 ). Apoptin induces apoptosis in human transformed and malignant cells but not in normal cells. P r o c . N a t l . A c a d . S c i . U S A 94, 5843-5847.

Noteborn, M.H.M. and Koch, G. (1 9 9 5 ). Chicken anemia virus infection: molecular basis of pathogenicity. A v i a n P a t h o l . 24, 11-31. Noteborn, M.H.M., Arnheiter, H., Richter-Mann, L., Browning, H., and Weissmann, Ch. (1 9 8 7 ). Transport of the murine Mx protein into the nucleus is dependent on a basic carboxy-terminal sequence. J . I n t e r f e r o n R e s . 7, 657-669.

Dinsart, C., Cornelis, J., and Rommelaere, J. (1 9 9 6 ). Recombinant autonomous parvoviruses: New tools for the gene therapy of cancer. Chemica OGGI C h e m i s t r y Today 14, 31-38.

Noteborn, M.H.M., Todd, D., Verschueren, C.A.J., De Gauw, H.W.F.M., Curran, W.L., Veldkamp, S., Douglas, A.J., McNulty, M.S., Van der Eb, A.J., and Koch, G. (1 9 9 4 ). A single chicken anemia virus protein induces apoptosis. J . V i r o l . 68, 346-351.

Doria, M., Klein, N., Lucito, R., and Schneider, R.J. (1 9 9 5 ). The hepatitis B virus HBx protein is a dual specificity cytoplasmic activator of Ras and nuclear activator of transcription factors. EMBO J. 14. 4747-4757.

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Noteborn et al: The Apoptin gene in apoptosis and in gene therapy of cancer Noteborn, M.H.M., Van der Eb, A.J., Koch, G., and Jeurissen, S.H.M. (1 9 9 3 ). VP3 of the chicken anemia virus (CAV) causes apoptosis. In: Vaccines 93: Modern approaches to new vaccines including prevention of AIDS (Ginsberg, H.S., Brown, F., Chanock, R.M., and Lerner, R.A., Eds.) CSHL Press, Cold Spring Harbor, USA,. pp 299-304. Paulovich, A.G., Toczyski, D., Hartwell, H. (1 9 9 7 ). When checkpoints fail. C e l l 88, 315-321. Rao, L. and White, E. (1 9 9 7 ). Bcl-2 and the ICE family of apoptotic regulators: making a connection. Curr. Opin. G e n . D e v . 7, 52-58. Roth, J.A. and Cristiano, R. (1 9 9 7 ). Gene therapy for cancer: What have we done and where are we going? J . N a t l . Cancer Inst. 89, 21-39. Sandig, V., Brand, K., Herwig, S., Lukas, J., Bartek, J., and Strauss, M., (1 9 9 7 ). Adenovirally transferred p16/ink4/cdkn2 and p53 genes cooperate to induce apoptotic tumor cell death. Nat. Med. 3, 313-319. Vaux, D.L. and Strasser, A. (1 9 9 6 ). The molecular biology of apoptosis. P r o c . N a t l . A c a d . S c i . U S A 93, 22392244. Vile, R.G., Tuszynski, A., and Castleden, S., (1 9 9 6 ). Retroviral vectors. From laboratory tools to molecular medicine. M o l . B i o t e c h n o l . 5, 139-158.

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Wen, W., Meinkoth, J.L., Tsien, R.Y., and Taylor, S.S. (1 9 9 5 ). Identification of a signal for rapid export of proteins from the nucleus. C e l l 82, 463-473. White, E. (1 9 9 6 ). Life, death, and the pursuit of apoptosis. G e n e s & D e v . 10, 1-15. Wyllie, A.H. (1 9 9 5 ). The genetic regulation of apoptosis. Curr. Opin Gen. Dev. 5, 97-104. Yang, C., Cirielli, C., Capogrossi, M.C., and Passaniti, A. (1 9 9 5 ). Adenovirus-mediated wild-type p53 expression induces apoptosis and suppresses tumorigenesis of prostatic tumor cells. Cancer Res. 55, 4210-4213. Zhuang, S.-M., Shvarts, A., Van Ormondt, H., Jochemsen, A.G., Van der Eb, A.J., and Noteborn, M.H.M. (1 9 9 5 a ). Apoptin, a protein derived from chicken anemia virus, induces p53-independent apoptosis in human osteosarcoma cells. Cancer Res. 55, 486-489. Zhuang, S.-M., Landegent, J.E., Verschueren, C.A.J., Falkenburg, J.H.F., Van Ormondt, H., Van der Eb, A.J., and Noteborn, M.H.M. (1 9 9 5 ). Apoptin, a protein encoded by chicken anemia virus, induces cell death in various human hematologic malignant cells in vitro. Leukemia 9S, 118-120.

Gene Therapy and Molecular Biology Vol 1, page 407 Gene Ther Mol Biol Vol 1, 407-418. March 1998.

Tissue-specific triple ribozyme vectors for prostate cancer gene therapy Dale J. Voeks1, Gary A. Clawson2, and James S. Norris 1 1Medical University of South Carolina, Charleston, SC; 2Penn State University, Hershey, PA __________________________________________________________________________________________________ Correspondence: Dr. James S. Norris, Department of Microbiology and Immunology, Medical University of South Carolina, 171 Ashley Avenue, Charleston, South Carolina 29425-2230, Tel: (803) 792-4421, Fax: (803) 792-2464, E-mail: jim_norris@smtpgw.musc.edu

Summary Ribozymes are naturally occurring catalytic RNAs which can be targeted to bind and cleave other RNA molecules with high specificity. Ribozyme-mediated inhibition of gene expression at the message level represents a powerful therapeutic tool. Implement of ribozymes in gene therapy has focused mainly on HIV and cancer. Use in HIV protocols has been widespread due to the wealth of targets offered by a well-characterized RNA retrovirus. Ribozyme utility in cancer has centered on oncogene inhibition in tumors with well defined genetics. As molecular mechanisms of prostate cancer are poorly understood, ribozyme targets must be universal with either delivery or expression firmly restricted to the area of interest. Overall, continued research on ribozyme design, delivery vehicles, tissue specificity, and the genetic makeup of disease will allow ribozymes to fulfill their considerable potential as therapeutic agents.

satellite RNA of tobacco ringspot virus (sTobRV) (Buzayan et al., 1986) and lucerne transient streak virus (Forster and Symons, 1987) are better suited for manipulation of cleavage activity and specificity. The catalytic domains were termed "hammerhead" or "hairpin" owing to their secondary structure. Uhlenbeck (1987) showed that the hammerhead ribozyme could cleave in trans. Haseloff and Gerlach (1988) defined the consensus sequences of the hammerhead ribozyme required for sitespecific cleavage of target RNA through in vitro mutagenesis studies of the plus strand of sTobRV. These early studies paved the way for research on the design of hammerhead ribozymes against any gene containing a cleavage target.

I. Introduction The regulation of gene expression through inhibition at the protein, message, or DNA level offers not only insight into biologic function but also a powerful treatment modality in disease. Antisense oligonucleotides have previously been demonstrated to have utility at this level of regulation (Reviewed in Helene and Toulne, 1990). More recently, research in ribozyme design and implement in therapy has expanded exponentially. Ribozymes are RNA enzymes capable of specifically binding to and cleaving complementary RNA molecules allowing for inhibition of gene expression at the message level.

B. Hammerhead ribozyme design

A. Ribozyme background

The basic hammerhead ribozyme (see Figure 1) is composed of a catalytic core of 24 conserved bases and three self associating helices. Helix I and III serve as substrate recognition and binding sites by acting in antisense to the target sequence. The site of cleavage for the ribozyme was initially shown to require a NUX sequence where N is any nucleotide and X is A, C, or U (Ruffner et al., 1990) with GUC predominantly favored as the cleavage site in naturally occurring self-cleavage (Haseloff and Gerlach, 1988). The ribozyme cleaves 3' of this ribonucleotide triplet with the presence of divalent

The discovery that certain naturally occurring RNAs had catalytic self-cleaving activity (Cech and Bass, 1986; Altman, 1987; Forster and Symons, 1989) changed our conception about the origin of life (Joyce, 1989). RNA catalysis proved that enzymatic activity was not restricted to proteins. The original ribozyme was a self-splicing intervening sequence in the pre-rRNA of Tetrahymena thermophilia whose only requirement for activity was the presence of a divalent metal cation (Cech and Bass, 1986). However, ribozymes originating from the plant viroids 407

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F i g u r e 1 . Hammerhead ribozyme. The basic design is composed of three hybridizing helices and a conserved catalytic core. Helices I and III recognize and bind the target RNA sequence by antisense base pairing. Cleavage occurs 3' of the NUX sequence.

efficiency in cleaving long RNA transcripts (presumably due to secondary/tertiary structure considerations) (Bertrand et al., 1994). But, enhanced cleavage of longer RNA transcripts was achieved using oligonucleotide facilitators which interact with the substrate at the termini of the ribozyme (Jankowsky and Schwenzer, 1996). Other substrate concerns can be addressed by accurate predictions of target secondary structure to identify susceptible NUXcontaining cleavage sites for ribozyme design. The next stage in improved ribozyme efficiency (without taking into account the rate-limiting step of delivery) is increasing intracellular activity of the ribozyme. There are two general methods for introducing ribozymes into the cell: (1) incorporation of DNA that codes for the ribozyme into viral or non-viral vectors, or (2) delivery by lipid or other means of chemically synthesized ribozymes. Under the latter technique, the ribozymes are very susceptible to ribonuclease attack once inside the cell. A number of structural modifications can improve stability. Substitution of deoxynucleotides in helices I and III increased stability and catalytic activity (Taylor et al., 1992). The introduction of phosphorothioate linkages in stems I, II, and III further protected from degradation (Shimayama et al., 1993). With the first method, a gene encoding the ribozyme is cloned into a vector to take advantage of the cellular transcriptional machinery for ribozyme production following delivery. Cloning along with erratic initiation or termination of transcription introduces anomalous flanking sequences both upstream and downstream of the ribozyme recognition sequences capable of interfering with catalytic activity by non-specific hybridization or increased secondary structure (Fedor and Uhlenbeck, 1990). Several strategies utilizing cis-acting ribozymes have been proposed to generate a consistent RNA transcript with defined 5' and 3' ends (Ruiz et al., 1997; Ventura et al., 1993; Chowrira et al., 1994; Taira et al., 1991). In a

cations fulfilled by Mg+2 or Mn+2 as the only requirement (Pyle, 1993). The overall cleavage reaction consists of three phases. (1) The ribozyme flanking (recognition) sequences bind to the RNA substrate by Watson-Crick base pairing to properly align the catalytic core. (2) Phosphodiester cleavage results in the formation of a 2',3' cyclic phosphate on the 5' fragment and a free 5'-hydroxyl on the 3' fragment. (3) Substrate dissociation and product release from the flanking sequences allow for multiple turnover of the ribozyme (Reviewed in Scanlon et al., 1995). Identification of variables affecting these steps to improve the efficiency of the ribozyme is an active area of research. Ribozymes differing in number and sequence of RNA in their hybridizing helices were shown to vary greatly in cleavage kinetics (Fedor and Uhlenbeck, 1990). The optimal length of ribozyme flanking sequences is still controversial. The goal is minimized mismatch pairing with maximized product dissociation (the cleavage step is unaffected by length of recognition sequence). Increasing the number of paired bases resulted in stronger binding and slow product release which limited turnover (Herschlag and Cech, 1990). Strong binding also reduced specificity by slowing the dissociation of mismatched RNA substrates compromising ribozyme discrimination (Herschlag, 1991). Although a target sequence length of 15 bases may be considered unique in the human genome, multiple studies have demonstrated the optimal length to be 12-14 bases (Bertrand et al., 1994; Goodchild and Kohli, 1991). In addition, the RNA binding protein hnRNPA1 and the nucleocapsid protein of HIV-1 enhanced the rate of binding and product release if the length of the flanking sequences was not more than 14 bases (Bertrand and Rossi, 1994). However, some findings support longer flanking sequences for activity in vivo (Crisell et al., 1993). Another variable affecting hybridization is substrate structure (Scarabino and Tocchini-Valentini, 1996). Hammerhead ribozymes have demonstrated reduced 408

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Figure 2. Autocatalytic double ribozyme vector. The 5' and 3' cis-acting ribozymes were designed to bind and undergo self-cleavage at GUC sequences by homology of 13 bases thereby releasing the internal trans-acting ribozyme with defined 5' and 3' ends. The DNA template for the internal ribozyme can be inserted into the BglII site.

prominent design, the 5' and 3' cis-acting ribozymes undergo autocatalytic self-cleavage thereby releasing the internal trans-acting ribozyme as a short uniform RNA (see Figure 2) (Clawson et al., 1997). Under this approach, trans-acting ribozyme sequences targeting either single or multiple cleavage sites can be connected in tandem with flanking cis-acting ribozymes to counteract two inherent difficulties. (1) A large excess of ribozyme over substrate is necessary for a significant reduction in target RNA (Cotten and Biernstiel, 1989; Cameron and Jennings, 1989). (2) It may be beneficial to target more than one site of a given substrate due to the presence of genetic variablity in target sites and the inaccessibility of certain sites (Reviewed in Sarver et al., 1990; Rossi et al., 1990). These multimeric ribozymes are liberated as individual units in a cell by autocatalysis to either increase ribozyme concentration or target multiple sites (Ohkawa et al., 1993).

In early studies, a ribozyme directed against gag and expressed in HeLa CD4+ cells challenged with HIV-1 reduced gag transcript and p24 levels (Sarver et al., 1990). Other studies have shown ribozyme-mediated protection from HIV-1 infection by targeting the 5' leader sequence (regulatory region conserved in most viral strains) (Weerisinghe et al., 1991; Dropulic, 1992). A hairpin ribozyme also targeted to this region under the control of a strong promoter decreased p24 levels and drastically reduced proviral DNA formation (Yu et al., 1993; Yamada et al., 1994). Because a base change in the recognition or cleavage sequence of the target could abrogate ribozyme effect, the high mutation rate of HIV-1 resulting from the error-prone activity of its reverse transcriptase (Roberts et al., 1988) poses a problem for ribozyme therapy. However, the use of multimeric ribozymes to simultaneously target different sites can offset this potential concern. In this regard, multiple hammerhead ribozymes with different flanking sequences targeted against the HIV-1 genome increased cleavage in proportion to the number of connector units (Ohkawa et al., 1993). In conclusion, ribozymes exhibit significant promise in gene therapy of AIDS; but, as with all gene therapy, improvements in a number of aspects such as delivery are required for successful clinical translation.

II. Ribozymes in gene therapy Research in ribozyme technology coupled with that in delivery vehicles implicates ribozymes as potential therapeutic agents. Ribozyme-mediated inhibition of gene expression at the message level allows the mRNA of nearly any gene to be targeted for destruction. Possible applications are only limited by molecular knowledge of the disease. The majority of ribozymes implemented for therapy thus far have focused on HIV and cancer.

B. Cancer Cancer is a multi-step process involving sequential genetic activations and inactivations of certain key elements involved in cell proliferation, differentiation, and apoptosis. The altered genes fall into one of two groups: oncogenes or tumor suppressor genes. Oncogenes are often overexpressed or mutated in the signal transduction pathway leading to uncontrolled cell growth. Because the process works through an RNA intermediate, targets are available for ribozyme activity. The majority of ribozyme gene therapy of cancer has focused on inhibition of specific oncogene expression in tumors with a relatively defined genetic basis. The ras family of G proteins plays a vital role in the signal transduction pathway which converts extracellular

A. HIV-1 HIV-1 is a retrovirus with an RNA genome and, consequently, has numerous potential ribozyme cleavage sites. Ribozyme activity against HIV-1 infection would theoretically be effective at two stages in the life cycle of the virus: (1) directly following infection when the viral genome is still in RNA form, and (2) following integration when viral transcript production begins (Reviewed in Akhtar and Rossi, 1996; Yu et al., 1994). As an added benefit, ribozymes are designed to cleave viral RNA leaving cellular transcripts unaffected. 409

Voeks et al: Triple ribozyme vectors for prostate cancer gene therapy page410 signals into an intracellular response mediated by nuclear transcription factors. The ras gene is mutated in approximately 90% of pancreatic adenocarcinomas (Almoguera et al., 1988), approximately 50% of adenocarcinomas of the colon (Bos et al., 1987), and approximately 45% of melanomas (Ball et al., 1994). Because of this high prevalence, the ras oncogene is a popular target for knockout in gene therapy. A hammerhead ribozyme targeted to a predominant mutation in the 12th codon of H-ras (GGC--GUC) can discriminate the mutated from the normal gene and exert profound antineoplastic effects (Kashani-Sabet et al., 1992). EJ bladder carcinoma cells transfected with the ras ribozyme under transcriptional control of the human !-actin promoter showed a decreased level of H-ras gene expression, reduced cell growth, and diminished tumorigenicity in nude mice (Kashani-Sabet et al., 1992; Tone et al., 1993). In human melanoma cells, transfection of the ras ribozyme resulted in decreased H-ras gene expression, reduced cell proliferation, and also exhibited a more differentiated phenotype (Ohta et al., 1994/1996). Tranfection into NIH3T3 cells transformed by the mutant H-ras gene demonstrated decreased focus formation and protection from future transformation (while not affecting normal 3T3 growth) (Koizumi et al, 1992; Funato et al., 1994). The ras ribozyme displays great therapeutic potential due to its prevalence in a number of tumors and the ability to target a tumor-specific mutation while leaving normal cells unaffected. The c-fos protein complexes with c-jun to form the AP-1 transcription factor at the distal end of many signal transduction pathways and regulates a number of genes involved in proliferation, apoptosis, and drug resistance. The c-fos oncogene plays a leading role in the development of resistance to chemotherapeutic drugs which currently represents a serious limitation in the treatment of a number of cancers. Resistance to cisplatin (one of the most widely used anti-neoplastic agents) appears to be caused by overexpression of the c-fos oncogene (Scanlon et al., 1989/1990; Kashani-Sabet et al., 1990a/1990b). A ribozyme designed to cleave c-fos was transfected into an ovarian carcinoma cell line resistant to cisplatin resulting in regained sensitivity to cisplatin treatment due to downregulation of c-fos as well as c-fos regulated genes which direct DNA synthesis and repair (DNA polymerase !, topoisomerase I, and metallothioneein IIB) (Scanlon et al., 1991/1994; Funato et al., 1992). The MDR drug resistant phenotype is caused by overexpression or amplification of the mdr-1 gene which also has an AP-1 site signifying regulation by c-fos (Teeter et al, 1991; Gottesman and Pasten, 1993). mdr-1 encodes a membrane p-glycoprotein involved in drug efflux. The same drug resistant cell line found to overexpress c-fos also overexpresses mdr-1; and transfection of the c-fos riboyzme decreased mdr-1 levels along with levels of c-fos which restored sensitivity to MDR agents (Scanlon et al., 1994). Multiple groups have also produced mdr-1 ribozymes and showed their efficacy in restoring drug sensitivity in a number of cell lines 410

(Kobayashi et al, 1994; Holm et al., 1994; Scanlon et al., 1994; Kientopf et al., 1994). Another prime ribozyme target is the bcr-abl gene present in more than 95% of chronic myelogenous leukemias (CML) (Kurzrock et al., 1988). The Philadelphia chromosome results from the reciprocal translocation between chromosmes 9 and 22 fusing the bcr and abl genes to form an augmented tyrosine kinase with transforming ability. Several groups targeted the breakpoint of the fusion gene for ribozyme cleavage resulting in varying levels of reduced cell growth in CML cell lines transfected with the ribozyme (Snyder et al., 1993; Shore et al., 1993; Lange et al., 1994). However, ribozyme-mediated cleavage of bcr-abl may not be entirely specific to the fusion gene (Wright et al., 1993). Several groups have overcome wild type mRNA cleavage and therefore regained tumor-specificity by altering the recognition sequences of the ribozyme (Kearney et al., 1995; James et al., 1996). The studies reviewed here of oncogene inhibition by ribozymes clearly demonstrate their future role as antineoplastic agents in gene therapy protocols. They have proven their superiority to antisense approaches (currently used in several clinical trials) due to multiple catalytic turnover (Reviewed in Kijima et al, 1995). And potential targets in cancer therapeutics are limited only by our current understanding of the disease. a. Prostate Cancer Prostate cancer is the most frequently diagnosed cancer in U.S. males and the second leading cause of cancer death (Parker et al., 1997). Although already a major health problem with predicted dramatic increases in incidence and mortality over the next 15 years, the molecular mechanisms behind the disease's initiation and progression remain largely a mystery. Therapeutic modalities thus far are limited and largely ineffective. Current treament of prostate cancer includes watchful waiting, radical prostatectomy, radiation therapy, hormonal therapy, and limited chemotherapy. Recurrence rates and mortality, especially with non-localized late stage disease are very high (Reviewed in Cersosimo and Carr, 1996). Further, approximately one-third of patients are in advanced stages of the disease at the time of diagnosis (Reviewed in Konety and Getzenberg, 1997). Additional therapeutic options are clearly necessary. Though a number of genes such as Rb and p53 have been found mutated in late stage disease (Kubota et al., 1995; Dahiya et al., 1996), a consensus sequential series of activations and inactivations of oncogenes and tumor suppressor genes has not been elucidated in prostate cancer as in other cancers such as in the colon (Cho and Vogelstein, 1992). Because specific genetic alterations are relatively unknown, target selection for ribozyme gene therapy must move beyond inhibition of oncogene expression. The target must be more universal to affect all prostate cancers and not just subsets harboring a specific oncogene or tumor suppressor gene dysfunction.

Gene Therapy and Molecular Biology Vol 1, page 411 RNA pol1-mediated transcription (Cavanaugh et al., 1995). Similarly, a ribozyme targeted to bind and cleave the message of RNA pol1 would compromise cellular viability by preventing proper formation of the protein synthesis machinery through a shutdown or reduction in ribosomal RNA production (see Figure 3). Increased effect should be observed in neoplastic cells due to their augmented growth rate.

In this regard, the transcription of ribosomal DNA is completely dependent on activated RNA polymerase I (RNA pol1) and its associated factors. Ribosomal genes comprise only 1% of the genome but account for 40% of total cellular transcription (Reviewed in Moss and Stefanowsky, 1995). This immense udertaking is a prerequisite for growth and development (Reviewed in Moss and Stefanowsky, 1995). Indeed, the tumor suppressor gene Rb may repress proliferation by disrupting

F i g u r e 3 . R N A p o l y m e r a s e I t a r g e t e d r i b o z y m e . A ribozyme targeted to bind and cleave the message of RNA polymerase I leads to a shutdown or significant reduction in ribosomal RNA transcription. The disruption in the protein synthesis machinery results in diminished cellular growth and compromised viability.

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F i g u r e 4 . RNA polymerase I secondary structure prediction. To identify potential cleavage sites for the transacting ribozyme, the sequence of the RNA pol1 transcript was searched for the presence of the nucleotide triplet GUC. Secondary RNA structure was then predicted using the program MFOLD and susceptible regions of the transcript sequence containing GUC were identified. The region at nucleotide 457-459 indicated by the arrow was chosen as the target cleavage site.

ability. However, application of the RNA pol1 triple ribozyme to cancer gene therapy, in general, and prostate cancer gene therapy, in particular, requires highly restricted targeting of the construct to the tissue of interest to prevent collateral damage in other tissues.

A ribozyme was targeted to subunit AC40 of RNA pol1 (see Figure 4) and cloned into the BglII site between the 5' and 3' autocatalytic ribozymes to form a RNA pol1 targeted triple ribozyme (TR) (Clawson et al., 1997). In vitro, the construct displayed predicted autocatalytic cleavage in cis to release the internal ribozyme targeted to cleave RNA pol1 in trans (Clawson et al., 1997). Transfection of TR into mouse fibroblast cells resulted in a 50% decrease in RNA pol1 message levels (50% transfection efficiency) (Clawson et al., 1997). Further, the diminution of RNA pol1 mRNA caused by the activity of the triple ribozyme was sufficient to markedly reduce cell population (below that of a mutant non-catalytic version of the ribozyme acting in antisense fashion) thus implicating ribosomal transcription as an important mediator of cell proliferation and viability (Clawson et al., 1997). Thus, ribozyme-mediated knockout of this housekeeping gene has cell killing

b. Tissue Specificity Targeting can occur at two levels: (1) targeted delivery by restricted entry of DNA into cells, or (2) targeted expression by restricted transcription of the introduced DNA. Through implemention of these strategies both a local and systemic response to primary and distant tumors can be achieved. In the former approach, research is focused on engineering surface components of viral and non-viral vectors to recognize specific target cell types or tissues (Reviewed in Miller and Vile, 1995; Harris and Lemoine, 1996). The latter approach of directed 412

Gene Therapy and Molecular Biology Vol 1, page 413 expression of the therapeutic gene to specific cell types can be accomplished by coupling to a tumor- or tissue-specific promoter. Tumors often sustain the ability to produce proteins specific for the tissue from which the neoplasm arose (Hart, 1996). To exploit this transcriptional specificity, essential promoter regions are defined and used to restrict expression of the exogenous gene of interest. Although transcriptional targeting is considered to be tissue-specific, a number of promoters can also be thought of as tumorspecific if the normal tissues are not essential for viability or accessible to the introduced DNA (Deonarain et al., 1995). Promoters of potential use for gene therapy of cancer include "-fetoprotein for liver tumors and c-erbB2 for breast or pancreatic tumors (Deonarain et al., 1995), tyrosinase or tyrosinase related protein for melanoma and polymorphic epithelial mucin for ovarian carcinoma (Hart, 1996), and surfactant protein promoter for lung cancer (Smith et al., 1994). The tyrosinase promoter was used to express a ribozyme targeted to the ras mutation in human melanoma cells and found to be superior in suppressing the melanoma phenotypes over a conventional promoter (Ohta et al., 1996). The expression of certain proteins is strongly restricted to prostate tissue. Prostate-specific antigen (PSA) is a serine protease with homology to kallikreins (Watt et al., 1986). It is considered the most sensitive marker available for diagnosis and management of prostate cancer (Cersosimo and Carr, 1996). The biologic role of PSA is to lyse seminal fluid, and it was recently found to cleave the insulin-like growth factor binding protein-3 which then releases insulin-like growth factor serving as a mitogen (Cohen et al., 1994). Of therapeutic interest is the nearly exclusive production of PSA by both normal and, in most cases, neoplastic luminal prostatic epithelial cells (Reviewed in Peehl, 1995). The ability of the androgen regulated promoter region of the PSA gene to target heterologous gene expression to protate tissue is of critical import. Transgenic mice bearing a 6-kb PSA promoter region driving expression of a reporter construct demonstrated prostate-specific expression mirroring the expression patterns seen in humans (Cleutjens et al., 1997). A shorter segment of the PSA promoter was able to drive expression of a reporter in LNCaP cells without activity in lines of non-prostatic origin (Lee et al., 1996; Pang et al., 1995). Overall, the PSA promoter has significant potential in prostate cancer gene therapy; and research continues in the identification of promoter regulatory elements necessary for tissue specificity and maximum expression (Zhang et al, 1997; Pang et al., 1997). Similarly, rat probasin is a hormonally regulated protein predominantly found in the dorsolateral region of the prostate (Spence et al., 1989). Transcriptional activity of probasin (mediated by androgens) begins in the prostate between 2 and 7 weeks of age and increases with sexual maturity (Matusik et al., 1986). Transgenic studies with a minimal rat probasin promoter region driving a reporter

construct revealed highly specific expression restricted to the lateral, dorsal, and ventral lobes of the prostate with very limited expression also observed in the anterior prostate and seminal vesicles but nowhere else in the body (Greenberg et al., 1994). Maximal expression occurred in the mouse dorsolateral prostate, significant due to the region's correlation to the peripheral zone of the human prostate where the majority and most invasive forms of human cancer arise (Reviewed in Price, 1963). Subsequent research demonstrated that a larger fragment of the probasin promoter containing androgen and zinc regulatory regions was able to direct higher levels of expression specifically to the prostate in transgenic mice (Yan et al., 1997). Probasin promoter expression patterns in human tissue have yet to be resolved; but, at minimum, the promoter sequence has utility in the transcriptional targeting of prostate cancer gene therapy constructs for evaluation of effect in animal model systems. Under the aforementioned classification system for transcriptional targeting, prostate tissue is considered nonessential; thereby defining prostate-specific promoters as tissue-specific and tumor-specific (Deonarain et al., 1995). The coupling of cytotoxic genes to prostate-specific promoters is a feasible approach to prostate cancer therapy. The cell killing effects of the previously designed RNA

Figure 5 . Seap a s s a y - - p r o b a s i n p r o m o t e r . TRAMP cells were transfected with PBSeap or Seap Basic (control) in triplicate. Samples of culture media were taken daily and the seap assay performed. RLU/sec values of the PBSeap and Seap Basic transfected cells were averaged and control transfected cell values subtracted. Initiation of maximal expression was observed on day two posttransfection.

413

Voeks et al: Triple ribozyme vectors for prostate cancer gene therapy page414 using a cationic liposomal carrier as vehicle. Both single administration and repeated administration on three consecutive days resulted in a consistent reduction of tumor growth between day two and day six postadministration followed by a resumption of normal tumor growth (Voeks et al., 1997). Delivery of reporter constructs under the same conditions indicated that gene transfer by liposomal vehicle was low (unpublished). However, the reduced tumor burden following injection of the probasin-driven triple ribozyme offers the promise of an enhanced, longer-lasting effect by employing a more efficient delivery vehicle. Prostate epithelial cell-specific knockout of a key gene in cellular growth and viability has the capability of treating cancer confined to the prostatic capsule and, more importantly, the potential to track metastasized tumor cells without realizing collateral damage in other tissues. The initial reduction in tumor burden by liposomal delivery of the probasin-driven triple ribozyme indicates the possibility for translation of prostate-specific RNA pol1 ribozyme therapy to human cancer if increased levels of cell killing can be attained with improved delivery. Also, additional promoters can be utilized should PB not display similar properties in human cancer cells or lack the ability to drive expression in advanced stages of the disease. F i g u r e 6 . T ra n s fec tio n a s s a y - - p r o b a s i n - R N A p o l 1 t r i p l e r i b o z y m e ( t u r n o v e r r a t e ) . TRAMP cells were plated at 8x10 5 for either mock transfection with liposome only (Lipo Cntrl), transfection with the probasindriven triple ribozyme vector (PBTR), or transfection with the identical construct without the internal RNA pol1 ribozyme (PBDR). The population and turnover rate of the PBTR transfected cells were lower at all time points. (Note: transient transfection with efficiency of 25-30%)

III. Conclusions The rapid emergence of ribozyme technology for the specific control of gene expression harbors great potential in gene therapy. Antisense approaches have been widely utilized in therapeutic research and a number of clinical trials initiated. Ribozymes hold clear advantages over antisense methodology evidenced by their increasing application in the treament of disease, most notably HIV and cancer. At present, there are two ongoing HIV ribozyme clinical trials, and widespread ribozyme entry into a broad range of clinical trials should soon occur. However, additional research is necessary to successfully advance ribozyme therapy to the clinical setting. For instance, extensive in vivo animal-based evaluation is required. Ribozyme activity and specificity need to be further optimized. Even with stronger research emphasis, delivery remains the major rate-limiting step in gene therapy protocols. Also, improved molecular knowledge of malignancies will identify additional ribozyme targets. And advances in targeting through vector design coupled with elucidation of key transcriptional elements will greatly improve ribozyme therapy. The future of ribozymes in the growing field of gene therapy appears promising.

pol1 targeted triple ribozyme can be restricted to prostatic epithelium and neoplastic derivatives if placed under transcriptional control of the minimal probasin promoter. A recently developed mouse prostate tumor cell line (TRAMP) provides an opportunity to evaluate the probasin-driven triple ribozyme construct (PBTR). The probasin promoter was able to drive expression of heterologous genes in this cell line as judged by transient transfection of a probasin-driven Seap reporter construct. Initiation of maximal expression occurred between day one and two with slightly increasing intensity therafter (see F i g u r e 5 ) (Voeks et al., 1997). Transfection of the prostate tumor cells with the PBTR construct reduced cellular proliferation beginning between day one and two as compared to control transfected cells (see F i g u r e 6 ) (Voeks et al., 1997). Because transfection was transient and at low efficiency, profound deviation in cell number only occurred for a brief time before returning to normalcy (presumably when the transduced cells were removed from the population). To evaluate the therapeutic potential of PBTR on tumor tissue in vivo (TRAMP xenografts), the PBTR construct was administered by direct intra-tumoral injection

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Voeks, D.J., Staley, M.C., Greenberg, N.M., Matusik, R.J., Clawson, G.A., and Norris, J.S. (1 9 9 7 ). Prostate cancer gene therapy using targeted expression of ribozymes against the AC40 subunit of RNA po11. In Cancer Gene Therapy, L. Savage, ed. (Southborough, MA, IBC), in press.

Scanlon, K.J., Wang, W.Z., and Han, H. ( 1 9 9 0 ) . Cyclosporin A suppresses cisplatin-induced oncogene expression in human cancer cells. Cancer Treat. Rev. S u p p l . 17, 27-35.

Watt, K.W., Lee, P.J., Mâ&#x20AC;&#x2122;Timkulu, T., Chan, W.P. and Loor, R. ( 1 9 8 6 ) . Human prostate-specific antigen: structural and functional similarity with sereine proteases. P r o c . N a t l . A c a d . S c i . U S A 83, 3166-3170.

Scarabino, D., and Tocchini-Valentini, G.P. ( 1 9 9 6 ) . Influence of substrate structure on cleavage by hammerhead ribozyme. FEBS Lett. 383, 185-190. Shimayama, T., Nishikawa, F., Nishikawa, S., and Taira, K. (1993). Nuclease-resistant chimeric ribozymes containing deoxyribonucleotides and phosphorothioate linkages. N u c l e i c A c i d s R e s . 21, 2605-2611.

Weerasinghe, M., Liem, S.E., Asad, S., Read, S.E., and Joshi, S. ( 1 9 9 1 ) . Resistance to human immunodeficiency virus type 1 (HIV-1) infection in human CD4+ lymphocytederived cell lines conferred by using retroviral vectors expressing an HIV-1 RNA-specific ribozyme. J . V i r o l . 65, 5531-5534.

Shore, S.K., Nabissa, P.M., and Reddy, E.P. ( 1 9 9 3 ) . Ribozyme-mediated cleavage of the BCRABL oncogene transcript: in vitro cleavage of RNA and in vivo loss of P210 protein-kinase activity. O n c o g e n e 8, 3183-3188.

Wright, L., Wilson, S.B., Milliken, S., Biggs, J., and Kearney, P. ( 1 9 9 3 ) . Ribozyme-mediated cleavage of the bcr/abl transcript expressed in chronic myeloid leukemia. E x p . H e m a t o l . 21, 1714-1719.

Smith, M.J., Rousculp, M.D., Goldsmith, K.T., Curiel, D.T., and Garver, R.I. Jr. ( 1 9 9 4 ) . Surfactant protein A-directed toxin gene kills lung cancer cells in vitro. Hum. Gene Ther. 5, 29-35.

Yamada, O., Yu, M., Yee, J.K., Kraus, G., Looney, D., and Wong-Staal, F. ( 1 9 9 4 ) . Intracellular immunization of human T cells with a hairpin ribozyme against human immunodeficiency virus type 1. Gene Ther. 1, 38-45.

Snyder, D.S., Wu, Y., Wang, J.L., Rossi, J.J., Swiderski, P., Kaplan, B.F., and Forman, S.J. ( 1 9 9 3 ) . Ribozymemediated inhibition of bcr-abl gene expression in a Philadelphia chromosome-positive cell line. B l o o d 82, 600-605.

Yan, Y., Sheppard, P.C., Kasper, S., Lin, L., Hoare, S., Kapoor, A., Dodd, J.G., Duckworth, M.L., and Matusik, R.J. ( 1 9 9 7 ) . A large fragment of the probasin promoter targets high levels of transgene expression to the prostate of transgenic mice. Prostate , in press.

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Voeks et al: Triple ribozyme vectors for prostate cancer gene therapy page418 Yu, M., Ojwang, J., Yamada, O., Hampel, A., Rappaport, J., Looney, D., and Wong-Staal, F. ( 1 9 9 3 ) . A hairpin ribozyme inhibits expression of diverse strains of human immunodeficiency virus type 1. P r o c . N a t l . Acad. S c i . U S A 90, 6340-6344. Yu, M., Poeschla, E., and Wong-Staal, F. ( 1 9 9 4 ) . Progress towards gene therapy for HIV-1 infection. Gene Ther. 1, 13-26. Zhang, S., Murtha, P.E., and Young, C.Y. ( 1 9 9 7 ) . Defining a functional androgen responsive element in the 5â&#x20AC;&#x2122; far upstream flanking region of the prostate-specific antigen gene. B i o c h e m . B i o p h y s . R e s . Commun. 231, 784-788.

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Gene Therapy and Molecular Biology Vol 1, page 419 Gene Ther Mol Biol Vol 1, 419-434. March, 1998.

A novel system for selection of intracellularly active ribozymes using the gene for dihydrofolate reductase (DHFR) as a selective marker in Escherichia coli Satoshi Fujita1 , 2 , 3 , ¶ , Makiko Hamada1 , 2 , 4 , ¶ , Yoshifumi Jigami2 , 4 , Hideo Kise3 , Kazunari Taira1 , 2 , 5 1

2 National Institute for Advanced Interdisciplinary Research and National Institute of Bioscience & Human Technology, 3 4 5 Tsukuba Science City 305, Japan; and Institute of Materials Science, Institute of Biological Sciences and Institute of Applied Biochemistry, University of Tsukuba, Tennoudai 1-1-1, Tsukuba Science City 305, Japan. ¶

The first two authors contributed equally to this work.

________________________________________________________________________________________________________________________ 5

Correspondence: Kazunari Taira at the Institute of Applied Biochemistry , Phone/Fax: +81-298-53-4623, E-mail: taira@nibh.go.ip

Summary If ribozymes are to be exploited i n v i v o , it is necessary to select ribozymes that are functional in the intracellular environment. Ribozymes selected in the intracellular environment should retain their function i n v i v o as well as i n v i t r o . We have devised a novel system for selection of active ribozymes from pools of active and inactive ribozymes using the gene for dihydrofolate reductase (DHFR) as a selective marker. In the DHFR expression vector, a sequence encoding either an a c t i v e o r a n inactive ribozyme was connected either upstream (5'-connected active or inactive ribozyme) or downstream (3'-connected active or inactive ribozyme) of the gene for DHFR. Each plasmid was designed such that, when the ribozyme was active, the ribozyme would cleave the t a r g e t s i t e a n d , a s a r e s u l t , t h e r a t e o f p r o d u c t i o n o f DHFR would be high enough t o endow resistance to trimethoprim (TMP). In the case of both 5'-connected and 3'-connected ribozymes, the active ribozyme did indeed cleave the primary transcript i n v i v o , whereas inactive ribozymes had no cleavage activity. We confirmed that c e l l s that harbored the active ribozyme-coding plasmid grew faster in the presence of a fixed concentration of TMP than the corresponding cells that harbored an inactive ribozyme-coding plasmid. Consequently, when cells were transformed with a mixture that consisted of active ribozyme-coding and inactive ribozyme-coding plasmids at a ratio of 1:1, it was mainly the cells that harbored the active ribozyme that survived in the presence o f T M P . T h e s e r e s u l t s i n d i c a t e d t h a t o u r p o s i t i v e s e l e c t i o n s y s t e m i n v i v o was functional and that, moreover, if the background "noise" could be removed completely in the future, it might usefully complement existing selection systems i n v i t r o .

high substrate specificity. Therefore, ribozymes (as well as antisense technologies) appear to have potential as tools for suppressing the expression of specific genes (Cameron and Jennings, 1989; Sarver et al., 1990; Uhlmann and Peyman, 1990; Erickson and Izant, 1992; Heidenreich and Eckstein, 1992; Murray, 1992; Ojwang et al., 1992; Rossi, 1992; Ohkawa et al., 1993). They are expected to be useful in gene therapy for some diseases that are caused by the expression of abnormal mRNA, which include diseases caused by infectious agents such as HIV (Sarver et

I. Introduction Catalytic RNAs, known collectively as ribozymes, were discovered in the early 1980s in the group I intron of Tetrahymena by Cech and as the RNA subunit of RNase P by Altman (Cech et al., 1981; Guerrier-Takada et al., 1983). Various types of ribozyme have been identified, including group II introns; hammerhead, hairpin and hepatitis delta virus ribozymes; and ribosomal RNA. Natural ribozymes have RNA-cleavage activity and exhibit 419

Fujita et al. Selection of Intracellularly Active Ribozymes using DHFR al., 1990; Heidenreich and Eckstein, 1992; Ojwang et al., 1992; Ohkawa et al., 1993; Leavitt et al., 1994). There are several strategies for inhibiting the expression of specific genes during transcription and translation, as follows. (i) At the DNA level, before transcription from the template DNA, a triple helix or a repressor peptide can be used (Blume et al., 1992; Roy, 1993; Gee et al., 1994; Mayfield et al., 1994; Choo et al., 1994). (ii) After transcription, antisense RNA/DNA or a ribozyme can be used to inhibit translation at the RNA level. (iii) After translation, antibodies or inhibitors can be used at the protein level. The hammerhead ribozyme belongs to the class of molecules known as antisense RNAs (hereafter, the term ribozyme refers exclusively to hammerhead ribozymes unless otherwise noted). However, because of the short extra sequences that form the so-called catalytic loop, it can act as an enzyme. Since the substrate specificity of antisense and ribozyme molecules is high, antisense and ribozyme strategies seem likely to have some value as therapeutic agents (Erickson and Izant, 1992; Murray, 1992; Eckstein and Lilley, 1996).

ex ponential enrichment, and the selected nucleic acids are referred to as aptamers. The method takes advantage of a process that mimics evolution, namely, mutation, amplification and selection. A pool of completely random RNAs is subjected to selection. Selected functional RNAs are amplified as double-stranded DNAs and the next generation of RNAs is transcribed from these template DNAs. Then the transcribed RNAs are subjected to selection in the next cycle. In efforts to engineer specific ribozymes, this method was successfully used (Beaudry and Joyce, 1992; Pan and Uhlenbeck, 1992; Lehman and Joyce, 1993; Nakamaye and Eckstein, 1994; Cuenoud and Szostak, 1995; Ishizaka et al., 1995). New functional ribozymes with ligase, kinase, amino-acid cleavage or selfalkylating activities have already been selected by this method (Bartel and Szostak 1993; Lorsch and Szostak 1994; Dai et al., 1995; Wilson and Szostak 1995). One might be able to select very active ribozymes using this method. However, the functional ribozyme selected in vitro might not always be the same as the best ribozyme in the cellular environment, in which there are potential inhibitory factors, a limited concentration of mandatory Mg2+ ions, and so on (Denman et al., 1994; Kawasaki et al., 1996). Moreover, while the activity of a ribozyme is associated with its specific structure, reverse transcriptase is known to be inhibited by certain secondary structures (Tuerk et al., 1992). Therefore, there is always a risk of missing the most effective ribozymes during selection in vitro.

When the hammerhead ribozyme was engineered such that it could cleave specific RNA sequences â&#x20AC;&#x153;in transâ&#x20AC;? (Uhlenbeck, 1987; Haseloff and Gerlach, 1988), it was postulated that this ribozyme might be much more effective than simple antisense molecules in several respects (Uhlenbeck, 1987; Haseloff and Gerlach, 1988; Walbot and Bruening, 1988; Maddox, 1989; Inokuchi et al., 1994). However, because the activity and stability of ribozyme are very dependent on the cellular environment (Chen et al, 1997), ribozymes have not yet proven their superiority to antisense molecules and the practical use of ribozymes in therapy has not yet been achieved. There seem to be several reasons for the low activity of ribozymes in vivo. (i) Various cellular proteins might exist in vivo that inhibit their catalytic activity (Parker et al., 1992; Taira and Nishikawa et al., 1992). (ii) The intracellular concentration of Mg2+ ions is much lower than that used in vitro in assays of ribozyme activity (Silver and Clark, 1971; Flatman, 1984; Romani and Scarpa, 1992). (iii) Several cellular RNases contribute to the instability of ribozymes (Olsen et al., 1991; Pieken et al., 1991; Heidenreich and Eckstein, 1992; Paolella et al., 1992; Taylor et al., 1992; Shimayama et al., 1993). (iv) Unlike the case of some proteinaceous enzymes, it seems unlikely that ribozymes reach their target sites by a sliding mechanism. Many attempts have been made to overcome some of these problems, for example, the chemical modification and substitution of nucleotides to improve the stability and activity of ribozymes (Thomoson et al., 1996).

In this report, we describe a novel method for screening in vivo that was designed to identify new hammerhead ribozymes with high activity. This method avoids the above mentioned disadvantages of selection in vitro. We chose the gene for dihydrofolate reductase (DHFR) as the selective marker. DHFR is an essential proteinaceous enzyme on the pathway to thymidylic acid (Blakley and Benkovic, 1985). Because the synthesis of DNA is required by all proliferating cells, inhibition of this process is one of the most effective ways of controlling cell division. Several drugs, such as methotrexate (MTX) and trimethoprim (TMP), are potent inhibitors of DHFR and, consequently, they inhibit DNA synthesis and the multiplication of cells (Iwakura et al., 1982; Blakley and Benkovic, 1985; Taira et al., 1987; Taira and Benkovic, 1988). We designed our vector in such a way that the level of expression of DHFR would be high when a ribozyme successfully cleaved its target site. Thus, our method involves positive selection and operates as follows. When an inhibitor of DHFR, such as TMP, is present in the culture medium at a certain concentration, DHFR-producing clones, which have already been transfected by a DHFR expression vector, would be expected to survive and grow more rapidly than nonexpressing clones (Iwakura et al., 1983). Moreover, there is a direct correlation between the level of expression of DHFR and the strength of resistance to TMP. When the level of expression of DHFR exceeds the inhibitory capacity of TMP, E. coli cells can proliferate on TMPcontaining plates. Furthermore, we can regulate the

For the rapid selection of functional sequences in vitro from a population of random sequences, Gold, Szostak, and Joyce and their respective groups developed a novel method (Ellington and Szostak, 1990; Robertson and Joyce, 1990; Tuerk and Gold, 1990). This genetic selection system is sometimes called SELEX, an abbreviation for s ystematic e volution of l igands by

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Gene Therapy and Molecular Biology Vol 1, page 421 toxicity of TMP by changing its concentration. Therefore, if we can control the level of expression of the gene for DHFR, which depends on the activity of a ribozyme, we should be able to select ribozymes, that are active in the cellular environment by monitoring resistance to TMP (Fujita et al., 1997).

strong SD sequence. If the ribozyme failed to cleave the target site, a ribosome would be expected to associate with the strong SD sequence for the frame-shifted AUG and the subsequent translation would not produce DHFR. However, when the ribozyme cleaves the target site, the strong SD sequence and frame-shifted initiation codon are disconnected and a weak SD sequence associated with the correct initiation codon for DHFR within the DHFR mRNA becomes operational, with resultant production of DHFR.

We placed a ribozyme-encoding sequence either upstream or downstream of the gene for DHFR. We report here that clones that survived at a chosen concentration of TMP harbored mostly active ribozymes.

To avoid any readthrough from upstream regions, an “all stop codon” sequence (TAACTAACTAA) was introduced between the ribozyme and the strong SD sequence. In this region, three stop codons should terminate translation in all possible frames. Furthermore, we introduced a terminator sequence (Yanofsky, 1981; Iwakura and Tanaka, 1992) downstream of the DHFR gene to facilitate the analysis of transcripts. If the active ribozyme were to attack the target site and cleave the primary transcript, we should be able to detect cleaved transcripts by Northern blotting analysis.

II. Results A. Design and construction of the screening vector with a 5'-connected ribozyme As a first step towards our goal of designing a screening system in E. coli, we attempted to distinguish between two vectors, one of which contained an active ribozyme and one of which contained an inactive ribozyme as a result of a single base substitution (F i g . 1 A ). The active ribozyme sequence was the same as that of the wildtype hammerhead ribozyme and the inactive ribozyme sequence differed from the active ribozyme by a single G5 to A 5 or A 14 to G 14 mutation within the catalytic core of the ribozyme. These mutations completely abolish the activity of the ribozyme (Ruffner et al,. 1990; Inokuchi et al., 1994). Because transcription and translation are coupled in E. coli, the ribozyme must cleave its target site before completing translation of DHFR. Therefore, in two types of vector, the active ribozyme sequence and the inactive ribozyme sequence were connected, separately, upstream of the gene for DHFR (F i g . 2). If the ribozyme were targeted to the gene for DHFR itself, the growth of cells that had been transformed by the vector with the active ribozyme should be slower in the presence of inhibitors of DHFR, such as trimethoprim (TMP) and methotrexate (MTX), because of a lower rate of production of the essential enzyme DHFR. Then clones surviving in the presence of TMP or MTX would turn out to have a vector with an inactive ribozyme sequence, in other words, this method corresponds to negative selection. We need a positive selection method to find active ribozyme sequences. Therefore, we took advantage of a frame shift in the AUG codon. We introduced, from the upstream to the downstream direction, between the ribozyme-coding and DHFR-coding sequences, an efficient Shine-Dalgarno (SD) sequence, a frame shift initiation codon that was out of frame relative to the gene for DHFR, a target site for the ribozyme and the correct initiation codon for the gene for DHFR. In our vectors, the ribozyme was not targeted to the gene for DHFR itself but to the region between the two AUG codons, one of which was the original initiation codon of the DHFR gene itself and the second of which (the pseudo-initiation codon) was located upstream of the original initiation codon to introduce a frame shift. The second frame-shifted AUG triplet was associated with a

B. Discrimination of active ribozymes from inactive ribozymes, connected on the 5' side of the DHFR gene, in the presence of TMP Taking advantage of the direct relationship between the level of expression of DHFR and the strength of resistance to TMP (Iwakura et al., 1983), we constructed a system for screening active ribozymes. Among several concentrations of TMP tested, we found that, at 70 µg of TMP per ml of culture medium, E. coli cells that had been transformed with the active ribozyme expression vector grew more rapidly and made larger colonies than cells transformed with the inactive ribozyme expression vector. Figure 3 shows the difference in growth rates between the active ribozyme- and inactive-ribozyme expressing colonies at 27˚C and 37˚C. Since E. coli strain HB101, which we used in this study, produces a low level of endogenous DHFR, formation of background colonies (noise) could not be avoided. Since the difference in growth rates between the active ribozyme- and inactive ribozyme-expressing colonies was greater at 27˚C than at 37˚C (F i g . 3B), the selection of active ribozymes described below was made at 27˚C in the presence of 70 µg of TMP and 100 µg of ampicillin per milliliter. Since active ribozyme-expressing colonies grew more rapidly, as expected, than inactive ribozyme-expressing colonies, we performed a random screening assay according to the procedure outlined in F i g u r e 4 . In this assay, equimolar amounts of active ribozyme- and inactive ribozyme-coding plasmids were mixed and competent HB101 cells were transformed with the mixture. The transformed cells were divided into two portions and each portion was plated either on an ampicillin-containing plate (100 µg/ml) or on a plate that contained both ampicillin (100 µg/ml) and TMP (70 µg/ml). After incubation for 421

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Figure 1. Secondary structures of the 5'-connected ribozyme (A) and the 3'-connected ribozyme (B ). A single point mutation (G5 to A5 or A14 to G14 ; circled) eliminates the ribozyme activity (Ruffner et al., 1990; Inokuchi et al., 1994). Note that the catalytic loop that contains G 5 and A14 captures Mg 2+ ions since a hammerhead ribozyme is a metalloenzyme (Dahm et al., 1993; Pyle, 1993; Uebayasi et al., 1994; Bassi et al., 1995; Sawata et al., 1995; Amontov and Taira, 1996; Kumar et al., 1996; Orita et al., 1996; Zhou et al., 1996a,b; Zhou et al., 1997).

Figure 2. The 5â&#x20AC;&#x2122;-connected ribozyme expression vector. The plasmid vector has two ATG codons, one of which is a pseudoinitiation codon, located upstream of the authentic AUG codon, which is the initiation codon for the DHFR gene. If an active ribozyme is introduced upstream of the DHFR-coding region and if, upon transcription, the primary transcript is cleaved by this cis-acting ribozyme at the predetermined site between the two AUG codons, the excised mRNA can produce DHFR. Otherwise, the translation of the primary transcript starts at the pseudo-initiation codon, which is associated with a strong Shine-Dargano sequence and is out of frame with respect to the DHFR gene.

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Figure 3. (A), Colonies of E. coli HB101 cells that had been transformed with the 5'-connected active (left) or inactive (right) ribozyme expression plasmid. In the presence of 70 µg/ml TMP, colonies expressing an active ribozyme (left) grew faster than colonies (right) that expressed an inactive ribozyme. The difference in growth rates between the active ribozyme- and inactive ribozyme-expressing colonies was greater at 27 °C (top) than at 37 °C (bottom). (B ), Distribution of colonies according to their diameters. About 4,000 colonies of the types shown in Figure 3A were divided into 11 classes based on the diameter of colonies. The difference in growth rates between the active ribozyme- and inactive ribozyme-expressing colonies was greater at 27 °C (left) than at 37 °C (right). Since E. coli strain HB101 that we used in this study produces a low level of endogenous DHFR, formation of background colonies could not be avoided.

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Gene Therapy and Molecular Biology Vol 1, page 424 Figure 4. Schematic diagram of the in vivo selection system. Competent cells are transformed with a mixture of equimolar amounts of active ribozyme- and inactive ribozyme-expressing plasmids. In the absence of selective pressure (Amp plate), both active ribozyme- and inactive ribozyme-expressing colonies are expected to grow at the same rate. By contrast, active ribozymeexpressing colonies are expected to grow faster on the Amp plus TMP plate.

T a b l e 1 . Numbers of selected colonies with 5’-connected and 3’-connected active and inactive ribozymes on trimethoprimcontaining and/or ampicillin-containing plates 5’-connected ribozyme

3’-connected ribozyme

Ampicillin plate Trimethoprim plate

Trimethoprim plate

(70 µg/ml)

(130 µg/ml)

(133 µg/ml)

(140 µg/ml)

"G5 and A5 " mixture Active ribozyme

Inactive ribozyme

Active ribozyme

Inactive ribozyme

"A14 and G14 " mixture

Plates were incubated at 27°C for 2 to 3 days, and then larger colonies were picked up at random. Trimethoprim plates contained 70 µg of TMP and 100 µg of Amp per ml for 5 ’-connected ribozyme screening and they contained 130 µg, 133 µg or 140 µg TMP and 100 µg of Amp per ml for 3’-connected ribozyme screening. Ampicillin plates contained 100 µg Amp per ml without TMP.

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one or more days, rapidly growing colonies were picked up at random from both plates. In order to check the reproducibility of our results, we picked up only ten colonies from each plate every day. Then, after minipreparation of plasmid DNA, we determined the sequences of the ribozyme-coding regions of the selected clones. Table 1 summarizes the sequencing results for the selected clones from more than seven independent experiments. Clones selected in the presence of TMP harbored mainly the active ribozyme: in the case of the "G5 and A5" mixture, only one out of 76 sequences turned out to encode an inactive ribozyme. By contrast, colonies selected in the absence of TMP (in the presence of Amp only) yielded active ribozyme and inactive ribozyme sequences at a ratio of 1:1. Similar results were obtained in the case of the "A 14 and G14" mixture.

In order to confirm that the phenotypic differences shown in T a b l e 1 really originated from a single base change and not from any other mutations within the DHFR gene, we sequenced several clones in their entirety, including the DHFR-coding region, and we also exchanged the HindIII-AccIII fragment (see F i g . 2 ) that contained each ribozyme sequence between the selected active and inactive clones. Since (i) no mutation was detected in the DHFR gene and (ii) the exchanged constructs had the opposite respective phenotypes, we were able to conclude that the phenotypic differences shown in Table 1 originated from the single base mutations. Thus, we confirmed that the selection pressure exerted by TMP was useful for identification of a single base change within the catalytic core of the ribozyme, which was correlated with the presence or absence of ribozyme activity, which, in turn, was correlated with the level of expression of DHFR.

C. Detection by Northern blotting analysis of a fragment cleaved by the 5'connected ribozyme In order to confirm that the above-described phenotypic differences were associated with the cleavage activity of the ribozyme, we performed Northern blotting analysis with total RNA from E. coli HB101 cells that had been transfected with the ribozyme expression vectors. Northern blotting analysis is the most direct method for the identification of cleavage activities of ribozymes in vivo. However, since cleaved fragments tend to undergo rapid degradation in vivo, Northern blotting analysis has failed in the past to detect some cleaved fragments (Sioud and Drlica, 1991; Ferbeyre et al., 1995). The results of our Northern analysis are shown in Figure 5. As can be seen in lane 1, both the intact primary transcript and the cleaved fragment were detected in the analysis of total RNA extracted from cells that contained the active ribozyme vector. However, no cleavage activity was detected when we analyzed the total RNA extracted from cells that contained the inactive ribozyme vector (lane 2). Although the lane corresponding to the inactive ribozyme (lane 2) appears to include a weak signal with the same mobility as the cleaved fragment, the signal did not represent a cleavage product, as demonstrated below by primer extension analysis (F i g . 6 ). The identification of the bands was based on the mobilities of RNA sizemarkers.

F i g u r e 5 . Northern blotting analysis of the 5’-connected ribozyme expression vector. Ten micrograms of total RNA from E. coli cells, transformed with the ribozyme expression vector shown in Figure 2, were subjected to electrophoresis in a 1.8% MetaphorTM agarose gel. After transfer to a membrane filter, the RNA was allowed to hybridize with the synthetic oligonucleotide probe (40-mer), which was complementary to part of the DHFR gene. Lane 1 : 5’-connected active ribozyme, with G5 at the catalytic core. Lane 2 : 5’connected inactive ribozyme, with A 5 at the catalytic core. The active ribozyme expression vector produced an excised short fragment (lane 1) but there is no truncated fragment in lane 2, which corresponds to the inactive ribozyme expression vector. Lane 1 also shows the intact primary transcript. Lengths of fragments were consistent with the expected values, as estimated from a standard curve of mobilities of RNA size-markers. The numbers indicate the lengths of fragments (in nucleotides) as determined by reference to size-markers (not shown).

Why did we detect the cleaved fragments when others have failed? In our case, the target site of the ribozyme was located upstream of the DHFR gene (F i g . 2 ), and the DHFR mRNA itself remained intact before and after the ribozyme-mediated cleavage. Protection (by the binding of ribosomes, etc.) from digestion by RNases, which must be an intrinsic property of the sequence of DHFR mRNA, allowed the mRNA to remain unchanged after the ribozyme-catalyzed cleavage.

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Fujita et al. Selection of Intracellularly Active Ribozymes using DHFR ribozyme. In the second case, we used plasmids without the "all stop codon" region but with either an active (lane 3) or the G 5 to A 5-mutated inactive (lane 4) ribozyme. As judged from the sequencing ladders that were analyzed simultaneously, exactly the expected target sites were cleaved by the active ribozymes (lanes 1 and 3). By contrast, no cleavage products were detected with the inactive ribozyme constructs (lanes 2 and 4), strongly supporting the conclusion obtained from the results in F i g u r e 5 . A fragment with one extra base was also observed for each transcript. These fragments can, most probably, be explained by the characteristics of reverse transcriptase, which has a "snap back" feature and which can incorporate one extra nucleotide independently of the template (Frohman et al., 1990). It should be noted that, since the reaction mixture for the reverse transcriptase reaction contained Mg2+ ions, some of the initial transcripts (intact mRNA) might have undergone ribozyme-mediated cleavage during reverse transcription. However, since there were no products other than the expected ones, we can safely conclude that cleavage occurred specifically at the predetermined target site in vivo.

E. Construction of a screening system using a 3'-connected ribozyme In our original construct, as mentioned above, the ribozyme sequences were inserted on the 5' side of the gene for DHFR so that the ribozyme would be transcribed upstream of the target site of the ribozyme. We examined the 5'-side ribozyme first for the following reasons. In the case of prokaryotes such as E. coli, transcription is coupled with translation so that, if the target RNA had been transcribed prior to transcription of the ribozyme, there was less of a chance that the ribozyme would cleave the target site. Moreover, polysomes could protect a target site, located downstream of a strong SD sequence, from attacks by ribozymes. Therefore, we placed the ribozyme upstream of its target site simply to allow transcription of the ribozyme prior to the transcription of the target site and before its protection by polysomes. In this system, regulation of the level of expression of DHFR by a ribozyme seemed easily controllable.

F i g u r e 6 . Primer extension analysis of the 5â&#x20AC;&#x2122;-connected ribozyme expression vector. Five micrograms of total RNA were used as template for reverse transcription, with a 5'-endlabeled synthetic oligonucleotide as primer. After transcription, the labeled transcribed product was subjected to electrophoresis on an 8% polyacrylamide gel. Lane 1: active ribozyme with the "all stop codon" was used as template. Lane 2: inactive ribozyme with the "all stop codon" was used as template. Lane 3: active ribozyme without the "all stop codon". Lane 4: inactive ribozyme without the "all stop codon". Both lane 1 and lane 3 include cleaved fragments. On the other hand, no cleaved fragments are visible in lanes 2 and 4. The exact site of cleavage was determined by reference to the sequencing ladders.

D. Identification of the cleavage site by primer extension analysis

However, there might be a critical defect associated with introduction of a ribozyme upstream of the DHFR gene. During actual screening for active ribozymes on the 5' side from a pool of random sequences, there might be the danger of selecting sequences that are not related to the activity of ribozymes. Such sequences might include sequences that regulate transcription, for example, promoter sequences and anti-terminators, or sequences that yield tertiary structures that promote re-initiation among others. If such sequences were selected from the random pool, they might affect the level of expression of the DHFR protein and, as a result, the resistance of E. coli to TMP. To avoid these possibilities, we must place the ribozyme downstream of the DHFR gene. If the activity

Although the intact and cleaved mRNAs (F i g . 5) were determined to be of the anticipated sizes by reference to RNA size-markers, the exact site of cleavage could not be determined by Northern blotting analysis. In order to confirm that the cleaved fragment shown in Figure 5 was really produced by the action of the ribozyme, we performed primer extension analysis (F i g . 6 ). In these experiments, we used two different sets of constructs. In one case, we used the plasmids shown in Figure 2 that contained the "all stop codon" region and either the active (lane 1) or the G5 to A5-mutated inactive (lane 2)

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Figure 7. Construction of the 3'-connected ribozyme expression vector. The ribozyme region and the "all stop codon" between the promoter and SD sequence in the 5'-connected ribozyme expression vector were cut out and then inserted between the gene for DHFR and the terminator sequence. The inactive ribozyme sequence differed from the active sequence by a single G5 to A 5 mutation within the catalytic core of the ribozyme, as in the 5'-side ribozyme construct. The target site of the 3'-connected ribozyme was the same as that of the 5'-connected ribozyme.

(5'-connected and 3'-connected ribozyme vectors), we replaced the HindIII-AccIII region by a linker that had the same length in nucleotides as the corresponding region that contained the ribozyme and the "all stop codon". As a result, the final 3'-connected ribozyme expression vector contained, from the upstream to the downstream region, the promoter, the linker, the SD sequence, the pseudoinitiation codon, the ribozyme target site, the original initiation codon for the DHFR gene, the DHFR gene, the ribozyme-coding region and the terminator (F i g . 7 ). The inactive ribozyme sequence differed from the active one by a single G 5 to A 5 mutation within the catalytic core of the ribozyme as in the 5'-side ribozyme construct. The target site of the 3'-connected ribozyme was exactly the same as that of the 5'-connected ribozyme.

of the 3'-side ribozyme were as high as that of the 5'-side ribozyme, there would clearly be an advantage to using the 3'-side ribozyme because accidental selection of the abovementioned regulatory sequences would be avoided. Our preliminary data indicate that some 3' ribozymes are more effective than the corresponding 5' ribozymes in some eukaryotic cells (Ohkawa and Taira, unpublished results). In eukaryotic cells, after mRNA has been transcribed in the nucleoplasm, the mRNA moves to cytoplasm and is translated into protein there. Thus, in eukaryotic cells unlike in prokaryotic cells, ribozymes might have a better chance of encountering their target site since transcription and translation are not coupled. At any rate, we felt that it was worth examining 3'-side ribozymes in prokaryotic cells also to determine whether we could achieve the same or greater selective power than that obtained with 5'-side ribozymes.

The newly constructed 3'-connected ribozyme was then examined by optimizing the level of discrimination between active and inactive constructs as a function of the concentration of TMP.

We constructed 3'-connected ribozyme expression vectors that contained either an active ribozyme or an inactive ribozyme sequence. These vectors were based on the 5'-connected ribozyme expression vectors (F i g . 7). The ribozyme region and the "all stop codon" between the promoter and the SD sequences of the 5'-connected ribozyme expression vector were cut out and then inserted between the DHFR gene and the terminator sequence. In order to maintain the same distance between the promoter and the strong SD sequence in the two kinds of construct

Figure 8 shows the difference in growth rates between active ribozyme- and inactive ribozyme-expressing colonies at 27Ë&#x161;C at 130 Âľg of TMP per ml of culture

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Fujita et al. Selection of Intracellularly Active Ribozymes using DHFR contained both ampicillin (100 µg/ml) and TMP (125-140 µl/ml). Faster growing colonies were picked up at random from both plates and ribozyme sequences were confirmed. The results are shown in Table 1. For some unknown reason, the level of background colonies was very sensitive to the concentration of TMP and, therefore, reproducibility was lower than with the 5' ribozyme construct. In general, selection was better when freshly prepared TMP was used. Nevertheless, we did achieve limited success even though we could not eliminate the background colonies (Table 1). To confirm that the 3'-side active ribozyme cleaved the target site in vivo and that the phenotype reflected the ribozyme's cleavage activity, as well as to compare the efficiency of cleavage between the 5'-side and 3'-side ribozymes, we performed Northern blotting analysis (F i g . 9). As mentioned above, the level of the transcript was higher when the 3'-side ribozyme was used than with the 5'-side ribozyme. As indicated in lanes 2 and 4, both 5'and 3'-connected ribozymes recognized and cleaved the target site. The cleavage efficiencies were nearly identical for the two types of ribozyme, 24% by the 5'-side ribozyme and 23% by the 3'-side ribozyme. It is noteworthy that polysomes did not seem to inhibit the action of the 3'-connected ribozyme, in our specific construct.

F i g u r e 8 . Colonies of E. coli HB101 cells that had been transformed with the 3'-connected ribozyme expression plasmid. G5 (Active ribozyme): Active ribozyme. A5 (Inactive ribozyme): Inactive ribozyme with G5 replaced by A. C5 (Inactive ribozyme): Inactive ribozyme with G 5 replaced by C. T 5 (Inactive ribozyme): Inactive ribozyme with G5 replaced by U. In the presence of 130 µg/ml TMP, colonies expressing the active ribozyme grew more rapidly than colonies that expressed inactive ribozymes.

III. Discussion Successful selection in vitro of tailored RNA has been reported by others and is of considerable current interest (Beaudry and Joyce, 1992; Pan and Uhlenbeck, 1992; Gray and Cedergren, 1993; Lehman and Joyce, 1993; Nakamaye and Eckstein, 1994; Cuenoud and Szostak, 1995; Ishizaka et al., 1995). However, efforts aimed at construction of selection systems in vivo have met with only limited success (Ferbeyre et al., 1996; Fujita et al., 1997). For use of ribozymes in vivo, we need RNAs that function optimally in the intracellular environment. Tsuchihashi and Herschlag reported that a protein derived from the p7 nucleocapsid (NC) protein of HIV-1 can facilitate cleavage by a ribozyme (Tsuchihashi et al., 1993; Herschlag et al., 1994). Other proteins also probably facilitate ribozymecatalyzed cleavage (Bertrand and Rossi, 1994). There have been a few reports of the successful ribozyme-mediated inactivation of genes in Saccharomyces cerevisiae (Parker et al., 1992; Taira and Nishikawa, 1992; Egli and Braus, 1994; Ferbeyre et al., 1995; Ferbeyre et al., 1996). The difficulties encountered in attempts to characterize ribozyme action in Saccharomyces cerevisiae suggest the existence of inhibitory factors in yeast. Under such circumstances, it is obviously desirable to be able to select ribozymes that function in the presence of such putative

medium. E. coli cells that had been transformed with the active ribozyme-expressing vector grew more rapidly and made larger colonies than the cells that had been transformed with the inactive ribozyme-expressing vector, as we had observed previously with the 5'-connected ribozyme construct (F i g . 3 ). For some unknown reason, cells harboring the C 5-inactive ribozyme vector grew more rapidly than cells with the other inactive ribozyme vector. Among the several concentrations of TMP tested, we found that the difference in colony size between active ribozyme- and inactive ribozyme-expressing clones was greatest at a concentration of TMP of 125-140 µg per ml of culture medium. This range of concentrations is higher than the 70 µg of TMP per ml of culture medium used in the assay with the 5' construct. The increased resistance to TMP might have originated from an increased level of the transcript (see F i g . 9 ) and a higher rate of production of DHFR. The level of mRNA might have changed since the sequence of the Hind III/Acc III region strongly influenced the rate of transcription, as confirmed in experiments with different kinds of linker (data not shown). We found that the shorter was the linker, the higher was the level of the transcription. We then carried out a random screening assay for the 3' ribozyme construct, following the procedure used for the 5' ribozyme construct, as outlined in F i g u r e 4 . E. coli cells transformed with the active ribozyme- or inactive ribozyme-coding plasmid were plated on a plate that contained ampicillin (100 µg/ml) or on a plate that

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Gene Therapy and Molecular Biology Vol 1, page 429 rescued. In this first attempt, we were unable to control the extent of the toxicity of RNase T1. We next chose a potentially more controllable gene as a selective marker, namely, the gene for DHFR (Iwakura et al., 1983; Fujita et al., 1997). As stated in the Introduction, DHFR is essential for DNA synthesis (Blakley and Benkovic, 1985). Moreover, there exists a direct relationship between the level of expression of DHFR and the strength of resistance to TMP (Iwakura et al., 1983). We tested the feasibility of use of the gene for DHFR with the constructs shown in F i g u r e s 2 and 7. We examined two types of ribozyme, an active and an inactive ribozyme, each located on the 5' or 3' side relative to the target site.

Figure 9. Northern blotting analysis for comparison of the cleavage efficiency between the 5’-connected ribozyme and the 3’-connected ribozyme. Ten micrograms of total RNA from E. coli cells, transformed with the 5'-connected or 3'connected ribozyme expression vector, were subjected to electrophoresis in a 1.8% Metaphor TM agarose gel. After transfer to a membrane filter, the RNA was allowed to hybridize with a synthetic oligonucleotide probe (40-mer) that was complementary to part of the gene for DHFR. Lane 1: 5’-connected inactive ribozyme, with A5 at the catalytic core. Lane 2: 5’-connected active ribozyme with G5 at the catalytic core. Lane 3: 3’-connected inactive ribozyme, with A 5 at the catalytic core. Lane 4: 3’-connected active ribozyme with G 5 at the catalytic core. Both active ribozyme expression vectors produced the excised short fragment (lanes 2 and 4), but no such fragment was produced by inactive ribozymes (lanes 1 and 3). Cleavage efficiencies were the same in lanes 2 and 4.

In the case of the 5'-side ribozyme construct, at a fixed concentration of TMP of 70 µg/ml, E. coli cells harboring the active ribozyme expression vector grew faster than those harboring the inactive ribozyme expression vector (F i g . 3 ). Then we prepared a mixture of active ribozyme and inactive ribozyme expression vectors in equimolar amounts and plated E. coli cells that had been transformed with the mixture on LBM plates that contained TMP at 70 µg/ml. After incubation at 27 °C for 2 to 3 days, colonies were harvested and DNA sequences were examined to determine whether each clone contained the sequence of an active or an inactive ribozyme. In this way, we were able to judge whether our method for selecting active ribozymes had any statistical significance. Since, for the most part, active ribozymes were selected in the presence of TMP (Table 1), DHFR appeared more suitable as a selective marker than RNase T1. We also demonstrated, by Northern blotting and primer extension analyses (F i g s . 5 and 6 ), that the active ribozymes were fully functional in vivo; they cleaved the primary transcript of the DHFR gene specifically and at the predetermined site only. In both of these analyses (F i g s . 5 and 6), the mutant ribozyme (G5 to A 5) had no cleavage activity. Another change, that eliminated ribozyme activity was the single base change at A14. With the A 14/G14 system, we were also able to select active ribozymes in the presence of TMP (Table 1). Taking all our results into account, we can conclude that the difference in phenotypes of the clones originated from only the single base mutation at the catalytic core of the hammerhead ribozyme F ( i g . 1 ).

F i g u r e 1 0 . Schematic representation of the design of a plasmid for the in vivo selection system. When the ribozyme is active, it can prevent expression of the toxin.

inhibitory factors in vivo. To this end, we attempted to construct a positive selection system in vivo based on the general scheme shown in Figure 10. When a toxin is expressed, cells harboring the gene for the toxin should be killed. If mRNA for the toxin can be successfully cleaved by the ribozyme that is co-expressed with the mRNA for the toxin, then cells harboring active ribozymes should survive and should form colonies. Consequently, all surviving colonies should harbor information about the sequences of active ribozymes. In our first attempt, we selected the gene for RNase T1 as the gene for the toxin. However, despite considerable effort, we failed to generate any plasmids that corresponded to the one shown in F i g u r e 1 0 when RNase T 1 was used as the selective marker. No constructs with a gene for RNase T1 were rescued from transformedE. coli cells. Only frame-shifted constructs, with aborted production of RNase T1, were

Our analysis of the construct shown in Figure 2 confirmed the possibility of selecting active ribozymes in vivo with DHFR as a selective marker. However, in its present form, the method for selection of an active mutant ribozyme from a completely randomized large pool is inadequate: the background noise might easily obscure identification of an active mutant from a large pool of inactive molecules. Furthermore, randomized sequences on the 5' side might influence the levels of transcription and translation (data not shown). In order to avoid problems associated with changes in levels of translation that are not related to the function of the ribozyme, we constructed the 3'-connected ribozyme vectors (F i g . 7 ). In the case of the

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Fujita et al. Selection of Intracellularly Active Ribozymes using DHFR ribozyme expression vectors were constructed by modifying the DHFR expression vector pTZDHFR20 (Iwakura et al., 1995).

3' ribozyme constructs, we had to use a higher concentration of TMP because of the higher level of transcription. At concentrations of TMP of 125-140 µg/ml, E. coli cells harboring the active ribozyme expression vector grew faster than those harboring the inactive ribozyme expression vector (F i g . 8). We prepared a mixture of active ribozyme and inactive ribozyme expression vectors and applied the same random screening procedure as in the case of the 5'-connected ribozyme. The E. coli cells transformed with the mixture were plated on LBM plates that contained TMP at 125-140 µg/ml. We picked up rapidly growing colonies at random and examined the ribozyme sequences of selected colonies (Table 1). As compared with the noise with the 5' construct, the background noise in the case of 3'-connected ribozyme could not be reduced, even though, for the most part, active ribozymes could also be selected in the presence of TMP.

B. Synthesis of oligonucleotides and construction of plasmids We prepared ten kinds of oligodeoxynucleotide for construction of 5'-connected ribozyme expression vectors [active-ribozyme linkers (forward, 5'-AGC TTA ACT AAT TGA ATT CCT GAT GAG TCC CTA GGG ACG AAA CCA TGG ACT AAC TAA CTA AT-3'; and reverse, 5'-CCG GAT TAG TTA GTT AGT CCA TGG TTT CGT CCC TAG GGA CTC ATC AGG AAT TCA ATT AGT TA-3'), inactive-ribozyme (G5 to A 5 ) linkers (forward, 5'-AGC TTA ACT AAT TGA ATT CCT AAT GAG TCC CTA GGG ACG AAA CCA TGG ACT AAC TAA CTA AT-3'; and reverse, 5'-CCG GAT TAG TTA GTT AGT CCA TGG TTT CGT CCC TAG GGA CTC ATT AGG AAT TCA ATT AGT TA-3'), inactive-ribozyme (A14 to G14 ) linkers (forward, 5'-AGC TTA ACT AAT TGA ATT CCT GAT GAG TCC CTA GGG ACG AGA CCA TGG ACT AAC TAA CTA AT-3'; and reverse, 5'-CCG GAT TAG TTA GTT AGT CCA TGG TCT CGT CCC TAG GGA CTC CTT AGG AAT TCA ATT AGT TA-3'), pseudo-ATG linkers (forward, 5'-CCG GAA AAG GAG GAA CTT CCA TGG TCG AAT TCA ACC TAT ATG ATC AGT CTG ATT GCG GCG-3'; and reverse, 5'-CTA GCG CCG CAA TCA GAC TGA TCA TAT AGG TTG AAT TCG ACC ATG GAA GTT CCT CCT TTT-3'), and 3'terminator linkers (forward, 5'-TCG AGC GTC GTT AAA GCC CGC CTA ATG AGC GGG CTT TTT TTT TTA G-3'; and reverse, 5'-GAT CCT AAA AAA AAA AGC CCG CTC ATT AGG CGG GCT TTA ACG ACG C-3' )]. The oligodeoxynucleotides were synthesized with a DNA synthesizer (model 392; Applied Biosystems, Foster City, CA) and purified by chromatography on OPC columns (oligonucleotide purification columns; Applied Biosystems). We also similarly synthesized and purified six kinds of oligodeoxynucleotide for construction of 3'-connected ribozyme expression vectors [primers for PCR for copying the 5'-connected active ribozyme (forward, 5'AGA CGT ATC TCG AGC GTC GTT AAA ACT AAT TGA ATT CCT GAT GAG TCC -3'; and reverse, 5'- GCG TAC GTG GAT CCT AAA AAA AAA AGC CCG CTC ATT AGG CGG GCT TTA GTT AGT TAG TCC ATG GTT TCG TCC CTA -3'), primers for PCR for copying the 5'-connected inactive ribozyme (forward, 5'-AGA CGT ATC TCG AGC GTC GTT AAA ACT AAT TGA ATT CCT AAT GAG TCC-3'; and reverse, 5'-GCG TAC GTG GAT CCT AAA AAA AAA AGC CCG CTC ATT AGG CGG GCT TTA GTT AGT TAG TCC ATG GTT TCG TCC CTA-3'), and linkers for the replacement of the 5’-connected ribozyme (forward, 5'-CCG GAG TCA TGG TAG CAA GGT TTC CGC AAA ATT GTT CGT GAC CAT CAC ATA ACC TAG CGG ACA3'; and reverse, 5'-AGC TTG TCC GCT AGG TTA TGT GAT AAT CAC GAA CAA TTT TGC GGA AAC CTT GCT ACC ATG ACT-3'). A single base change (G5 to A5 , or A14 to G 14 ) was introduced within the catalytic core of the active ribozyme (F i g . 1 ). These changes had been shown previously to destroy cleavage activity (Ruffner et al., 1990; Inokuchi et al., 1994). Ribozyme linkers were "tailed" with a recognition sequence for restriction endonuclease HindIII at the 5’ end and with one for AccIII at the 3' end. Pseudo-ATG linkers were tailed with a recognition sequence for restriction endonuclease AccIII at the 5' end and with one for NheI at the 3' end. 3'Terminator linkers were tailed with a recognition sequence for restriction endonuclease XhoI at the 5' end and with one for BamHI at the 3' end. Each oligonucleotide linker was

Comparison of the efficiency of ribozyme-mediated cleavage in vivo between 5'- and 3'-connected ribozymes by Northern blotting analysis revealed that the efficiencies of cleavage were identical (F i g . 9 ) despite the fact that, in the case of the 3' construct, the target site had been transcribed prior to the ribozyme and the possibility existed of polysome-mediated protection against ribozymes (Zhang et al, 1997). Although the background noise could not be reduced by placing the ribozyme on the 3' side, it might be advantageous to improve the 3' ribozyme construct rather than the 5' construct if selections are to be made with a large pool of completely randomized RNA. In the case of the 3'-connected ribozymes, we can at least minimize effects on levels of transcription and translation. We have not yet optimized our positive selection system in vivo. We know that the cleavage activity of the ribozyme depends strongly on the target site. Among several possible target sites, we chose arbitrarily, in this study, one target site close to the initiation codon. Genes other than the gene for DHFR might also be more suitable as selective markers (the general positive selection system shown in Figures 2 and 7 might be applicable to genes other than the gene for DHFR). We are now trying to improve this system by removing the "noise", using several strategies that include the use of a DHFR-null strain. Nevertheless, as a first step toward the construction of a positive selection system in vivo, the present system allowed us successfully to identify a single base change that was associated with a change in ribozyme activity. While a bacterial cis-acting system is described in this report, it is clear that our approach might be adaptable to a trans-acting eukaryotic system, which would be of value for the development of ribozyme-mediated gene therapies for human diseases.

IV. Experimental procedures A. Bacterial strains and plasmids E. coli HB101 (recA13, supE44; Takara Shuzo Co., Kyoto) was used as the recipient for transformation. Several

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Gene Therapy and Molecular Biology Vol 1, page 431 denatured at 95˚C in a water bath and then gradually cooled to room temperature in TE buffer. After annealing, each linker set was then ligated to the digested vector pTZDHFR20 via its restriction sites and the tailed cohesive ends of the synthetic oligonucleotide linkers, for construction of 5'-connected ribozyme expression vectors (F i g . 2 ).

contained 30 mM Tris-HCl (pH 8.3), 15 mM MgCl2 , 8 mM DTT, 0.8 mM each dNTP, 6 units of human placental ribonuclease inhibitor, and 80 units of SuperScript RNaseH reverse transcriptase (Gibco BRL, Gaithersburg, MD) were added. The reverse transcriptase (RT) reactions were allowed to proceed at 42 °C for 60 min to avoid any influence of the secondary structure of the mRNA. After the RT reaction, 2 µl of stop solution, containing 95% formamide, 20 mM EDTA, 0.05% bromophenol blue and 0.05% xylene cyanol, were mixed with 3 µl of the reaction mixture and the resulting sample was fractionated on a 7 M urea-8% polyacrylamide gel. Four ddNTP sequencing reactions with the same [32 P]-labeled primer were fractionated together, creating sequencing ladders as markers.

The primers for PCR were complementary to the upstream region and downstream region of the ribozyme and were tailed with a recognition sequence for restriction endonuclease XhoI in the case of the forward primer and with one for BamHI in the case of the reverse primer. Linkers instead of a ribozyme were tailed with a recognition sequence for restriction endonuclease HindIII at the 5' end and with one for AccIII at the 3' end. For the construction of 3'-connected ribozyme expression vectors, the region that contained 5'-connected ribozymes were cut out from 5'-connected ribozyme vectors by restriction enzymes HindIII and AccIII and a linker was ligated to the digested vector, replacing the ribozyme portion. DNA fragments containing 5'-connected ribozyme sequences and restriction sites (XhoI and BamHI) were amplified by PCR and were cleaved at the restriction sites by XhoI and BamHI. Then, these fragments were ligated to the digested ribozymefree vector via the XhoI and BamHI restriction sites.

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C. Composition of culture media

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D. Northern blotting analysis Plasmid vector pTZDHFR, harboring both a ribozyme and a gene for DHFR, was used to transform E. coli HB101. After overnight incubation at 37 °C, total RNA was isolated with ISOGEN™ (Nippon gene Co., Toyama) from 2 ml of a culture of cells in 2x YT medium. Ten micrograms of total RNA per sample were denatured in glyoxal and dimethyl sulfoxide, subjected to electrophoresis in 1.8% Metaphor™ agarose gel (FMC Inc., Rockland), and transferred to a Hybond-N™ nylon membrane (Amersham Co., Buckinghamshire). The membrane was probed with a synthetic oligonucleotide (5'ATT CGC TGA ATA CCG ATT CCC AGT CAT CCG GCT CGT AAT C-3'; complementary to DHFR mRNA) that had been labeled with 32 P by T4 polynucleotide kinase (Takara Shuzo Co., Kyoto). Prehybridization and hybridization were performed in the same solution (5x SSPE, 50% formamide, 5x Denhardt's solution, 0.5% SDS, 150 mg/ml calf thymus DNA). Final washing was performed in 0.1x SSPE, 0.1% SDS at 70 °C for 30 min.

E. Primer extension analysis

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Comparison of the specificities and catalytic activities of conventional hammerhead ribozymes, Joyce's DNA enzymes, and novel dimeric minizymes with respect to the cleavage of BCR-ABL chimeric L6 (b2a2) mRNA Tomoko Kuwabara1,2,4, Masaki Warashina1,2,4, Aya Nakayama1,2,4, Makiko Hamada1,2,4, Sergei Amontov2, Yasuhiro Takasuka 3, Yuto Komeiji 3, and Kazunari Taira1,2,4, * 1National Institute for Advanced Interdisciplinary Research, 2National Institute of Bioscience & Human Technology, and 3Electrotechnical Laboratory, Agency of Industrial Science & Technology, Tsukuba Science City 305, Japan; 4Institute of Applied Biochemistry, University of Tsukuba, Tennoudai 1-1-1, Tsukuba Science City 305, Japan. Correspondence should be addressed to Prof. Kazunari Taira at the Institute of Applied Biochemistry, Phone/Fax: +81-298-53-4623, E-mail: taira@nibh.go.ip

Summary With the eventual goal of developing a treatment for chronic myelogenous leukemia (CML), attempts have been made to design hammerhead ribozymes that can specifically cleave BCR-ABL fusion mRNA. In the case of L6 BCR-ABL fusion mRNA (b2a2 type; BCR exon 2 is fused to ABL exon 2), which has no effective cleavage sites for conventional hammerhead ribozymes near the BCR-ABL junction, it has proved very difficult to cleave the chimeric mRNA specifically. Several hammerhead ribozymes with relatively long junction-recognition sequences have poor substrate-specificity. Therefore, we explored the possibility of using newly selected DNA enzymes that can cleave RNA molecules with high activity [Santoro & Joyce, (1997) Proc. Natl. Acad. Sci. USA, 94, 4262-4266] to cleave of L6 BCR-ABL fusion (b2a2) mRNA. By contrast to the results with the conventional ribozymes, the newly designed DNA enzymes, having higher flexibility for selection of cleavage sites, were able to cleave this chimeric RNA molecule specifically at sites close to the junction. We also designed a novel heterodimeric minizyme [Amontov & Taira, (1996) J. Am. Chem. Soc., 118, 1624-1628], in such a way that only in the presence of the L6 BCR-ABL (b2a2) mRNA junction the heterodimeric minizyme would form an active catalytic core. In the case of the novel heterodimeric minizyme, cleavage occurred only within the abnormal BCR-ABL mRNA, without any cleavage of the normal ABL or BCR mRNA. Thus, these chemically synthesized DNA enzymes as well as the novel heterodimeric minizyme seem to be potentially useful for application in vivo, especially for the treatment of CML. al., 1995; Zoumadakis and Tabler, 1995). Because of its small size and potential utility as an anti-virus agent, this ribozyme has been extensively investigated in terms of the mechanism of its action and possible applications in vivo (Server et al., 1990; Homann et al., 1993; Mulligan, 1993; Altman, 1993; Marschall et al., 1994; Sullivan, 1994; Cameron and Jennings, 1994; Ohkawa et al., 1993; Sun et al., 1995; Christoffersen and Marr, 1995; Ferbeyre et al., 1996; Kiehntopf et al., 1994; Kiehntopf et al., 1995; Thompson et al., 1995a; Thompson et al., 1995b; Tuschl et al., 1995; Kawasaki et al., 1996; Eckstein and Lilly, eds. 1996). For such applications, it is clearly necessary to direct the ribozyme specifically to the cellular RNA target of interest.

I. Introduction Hammerhead ribozymes are among the smallest catalytic RNAs. The sequence motif, with three duplex stems and a conserved "core" of two non-helical segments that are responsible for self-cleavage (cis action), was first recognized in the satellite RNAs of certain viruses (Symons, 1989). The trans-acting hammerhead ribozyme, which was designed by Uhlenbeck (1987) and Haseloff and Gerlach (1988), consists of an antisense section (stems I and stem III) and a catalytic domain with a flanking stem-loop II section (Haseloff and Gerlach, 1988). Such RNA motifs can cleave oligoribonucleotides at specific sites (most effectively at GUC) (Koizumi et al., 1988; Ruffner et al., 1990; Perriman et al., 1992; Shimayama et 435

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Figure 1. BCR-ABL translocations and fusion mRNAs. The two types of chromosomal translocation [K28type (upper panel) and L6-type (lower panel)] that are associated with CML and the corresponding fusion mRNAs are depicted. Boxes in violet represent BCR exons and boxes in green represent ABL exon 2. Dotted lines connecting BCR and ABL exons indicate alternative splicing pathways.

ribozyme-cleavage site, a GUU triplet, is located three nucleotides upstream from the chimeric junction (Fig. 1). A conventionally designed hammerhead ribozyme might be expected to cleave specifically the abnormal mRNA generated from K28 translocations. Indeed, several such examples have been reported (Shore et al., 1993; Snyder et al., 1993; Lange et al., 1993; Wright et al., 1993; Lange et al., 1994; Kearney et al., 1995; Leopold et al., 1995; Kronenwett et al., 1996; Leopold et al., 1996). By contrast, in the case of the b2a2 sequence, which results from L6 translocations, as well as some K28 translocations (Fig. 1), there are no triplet sequences that are potentially cleavable by hammerhead ribozymes within two or three nucleotides from the BCR-ABL junction (Kuwabara et al., 1997). In the sequence of b2a2 the closest ribozyme-cleavage sites in the vicinity of the BCR-ABL junction are located 7, 8, 9, and 19 nucleotides away from the junction (Fig. 2). A GUC triplet, which is generally most susceptible to cleavage by hammerhead ribozymes, is also located 45 nucleotides from the junction. In designing ribozymes that might cleave b2a2 mRNA, we must be sure to avoid cleavage of the normal

Chronic myelogenous leukemia (CML) is a clonal myeloproliferative disorder of hematopoietic stem cells associated with the Philadelphia chromosome (Nowell and Hungerford, 1960). The reciprocal chromosomal translocation t(9; 22) (q34; q11) can be subdivided into two types: K28 translocations and L6 translocations. They result in the formation of the BCR-ABL fusion gene which encodes two types of mRNA: b3a2 (consisting of BCR exon 3 and ABL exon 2) and b2a2 (consisting of the BCR exon 2 and ABL exon 2) (Rowley, 1973; Bartram et al., 1983; Heisterkamp et al., 1983; Groffen et al., 1984; Schtivelman et al., 1985; Schtivelman et al., 1986) (Fig. 1). Both of these mRNAs are translated into a protein of 210 kDa (p210 BCR-ABL) which is unique to the malignant cell phenotype (Konopka et al., 1984). For the design of ribozymes that will disrupt chimeric RNAs, it is necessary to target the junction sequence. Otherwise, normal mRNAs that share part of the chimeric RNA sequence would also be cleaved by the ribozyme, with resultant damage to the host cells. In the case of the BCR-ABL chimeric RNA sequence b3a2, a potential 436

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Figure 2 Nucleotide sequences of the conventional antisense-type ribozymes, DNA enzymes, and Rz37 targeted to the L6 BCR-ABL (b2a2) substrate. The sequence of L6 BCR-ABL near the junction is expanded. The BCR exon 2 sequence near the junction is depicted by capital letters and that of the ABL exon 2 sequence is shown in lower-case letters. The sites of cleavage by antisense-type ribozymes (81-mer Rz, 41-mer Rz and 52-mer Rz) and the control ribozyme, Rz37, are indicated by red lines and those of DNA enzymes by blue lines. The sites of cleavage by the dimeirc minizyme is identical to that of Rz37 and the recognition site for the dimeric minizyme is indicated in yellow line.

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Gene Therapy and Molecular Biology Vol 1, page 438 substrate-recognition sequences as the recognition arm of an active heterodimeric minizyme.

ABL mRNA itself. Previous attempts have involved a combination of a long antisense arm and the ribozyme sequence (Pachuk et al., 1994; James et al., 1996).

Since we were interested in cleaving b2a2 mRNA, we compared the specificity and catalytic activity of conventional hammerhead ribozymes, Joyce's DNA enzymes, and our novel heterodimeric minizyme with respect to the cleavage of BCR-ABL chimeric L6 (b2a2) mRNA.

In a recent study, Santoro and Joyce (1997) successfully selected DNA enzymes that can cleave RNA molecules with any sequence by using an in vitro selection procedure. Joyce's DNA enzymes are rather similar to the conventional hammerhead ribozyme. They consist of a catalytic domain of 15 deoxynucleotides and Mg2+ ions are necessary for catalytic activity, as in the case of hammerhead ribozymes, which are recognized as metalloenzymes (Dahm et al., 1993; Piccirilli et al., 1993; Yarus, 1993; Uchimaru et al., 1993; Steitz and Steitz, 1993; Pyle, 1993; Uebayasi et al., 1994; Sawata et al., 1995; Kumar et al., 1996; Taira et al., 1990; Zhou et al., 1996a; Zhou et al., 1996b; Pley et al., 1994; Scott et al., 1995: Scott and Klug, 1996; Scott et al., 1996; Pontius et al., 1997). The catalytic domain is flanked by two substrate-recognition domains of 7-8 deoxyribonucleotides each, and the RNA substrate is bound through Watson-Crick base pairing (Fig. 2). Joyce's DNA enzymes can be divided into two types. Type I DNA enzymes can cleave an RNA sequence at a phosphodiester bond located between an A residue and a G residue (DNA enzymes 1 and 2 in Fig. 2). The catalytic domain consists of a 4-nucleotides loop by the cleavage site and a stem-loop region that resembles the stem-loop II region of the hammerhead ribozyme. However, the former stem-loop region is essential for catalysis (Santoro and Joyce, 1997). The type II DNA enzymes can cleave an RNA sequence at a phosphodiester bond located between a purine and pyrimidine residue. In this case, the catalytic domain consists of 15 nucleotides (DNA enzyme 3 in Fig. 2). Such DNA enzymes can be expected to cleave almost any target RNA substrate. Indeed, Santoro and Joyce (1997) demonstrated that their DNA enzymes could cleave HIV-1 mRNAs.

II. Results and Discussion A. Design of catalytic molecules and selection of their target sites The design of conventional hammerhead ribozymes and their target sites for the specific cleavage of b2a2 mRNA were based on previously published results (Pachuk et al., 1994; James et al., 1996). Long antisense sequences of about 10 to 30 nucleotides in length, which could bind to and cover the junction region for some distance from the cleavage sites, were connected to binding sites of hammerhead ribozymes (81-mer, 41-mer and 52-mer Rz's in Fig. 2). The lengths of annealing arms are important for the activity of ribozymes because they influence the efficiency, as well as the specificity, of the cleavage reaction. In the case of a ribozyme that is directed against two non-contiguous sequences, the specificity is particular important if we are to avoid nonspecific cleavage of normal mRNAs. Among the conventional ribozymes depicted in Figure 2 , which were designed to cleave L6 BCR-ABL chimeric mRNA specifically, the 52-mer Rz and the 41-mer Rz had long binding arms, in the stem III region, of 20 and 12 nucleotides, respectively. In the case of the 81-mer Rz, the binding arms were 50 nucleotides in length in the stem III region and were connected to the ribozyme sequence by a 13-nucleotide spacer sequence that was noncomplementary to the substrate, to achieve greater flexibility of binding (Pachuk et al., 1994). The 52-mer Rz was designed to cleave the L6 BCR-ABL mRNA at the UUC triplet located 9 nts 3' of the junction (James et al., 1996). The 41-mer Rz was designed to cleave the substrate at the CUU triplet located 8 nts 3' of the junction. The 81-mer Rz was designed to cleave the substrate at the GUA triplet located 19 nts 3' of the junction. According to a published report (Pachuk et al., 1994), the 81-mer and 41-mer Rz's should have enhanced specificity for the chimeric b2a2 mRNA substrate. However, according to other studies (Hertel et al., 1996; Birikh et al., 1997), it seems that hammerhead ribozymes have cleavage ability even if the binding arm is as little as three nucleotides in

Recently, we discovered a novel motif of a minizyme (Amontov and Taira, 1996), a hammerhead ribozyme with short oligonucleotide linkers instead of stem/loop II (Kuwabara et al., 1996; Amontov et al., 1996; Sugiyama et al., 1996). Our previous study demonstrated that a minizyme with high-level activity forms a dimeric structure with a common stem II. Because of their dimeric structure, heterodimeric minizymes are capable of recognizing two independent sequences. We wondered whether it would be possible to design a novel heterodimeric minizyme that would form a catalytically competent structure, only in the presence of the L6 BCRABL (b2a2) mRNA junction, by the use of one of the

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Figure 3. Cleavage of normal and/or abnormal RNAs by ribozymes or dimeric minizymes. The sequence of a heterodimeric minizyme used in our previous study (Amontov and Taira, 1996) is depicted in the top panel. Minizyme left (MzL) and minizyme right (MzR) form a dimeric structure with common stem II. Formation of active heterodimeric minizymes in the presence of L6 b2a2 mRNA is depicted in the middle panal. One unit of the substrate-recognition sequences is used to recognize the abnormal BCR-ABL junction. One of the catalytic cores of the heterodimeric minizyme can be deleted completely to yield a "super dimeric minizyme" (right figure). The control ribozyme (Rz37), which targets the same site as the dimeric minizyme on L6 b2a2 mRNA, is expected to cleave not only the abnormal chimeric BCR-ABL mRNA but also the normal ABL mRNA since the cleavage site is located far from the BCR-ABL junction (bottom panel).

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Kuwabara et al: Specificities and catalytic activities of novel dimeric minizymes

Figure 4. Formation of active or inactive heterodimeric minizyme. In order to achieve high substrate-specificity, the heterodimeric minizyme should retain their active conformation only in the presence of the abnormal BCR-ABL junction (middle panel), while their conformation should ramain inactive in the presence or absence of the normal ABL mRNA (bottom panel). The novel minizyme, MzL (minizyme left) and MzR (minizyme right) should enable such conformational changes depending on the presence or absence of the abnormal b2a2 mRNA.

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Gene Therapy and Molecular Biology Vol 1, page 441 Figure 4 should enable such conformational changes depending on the presence or absence of the abnormal b2a2 mRNA.

length. As can be seen from Figure 2, the binding region of these antisense-type ribozymes to the normal ABL mRNA sequence consisted of at least six base pairs. Therefore, we could not exclude the possibility that such ribozymes might bind non-specifically to and cleave the normal ABL mRNA just as they specifically cleave the BCR-ABL (b2a2) mRNA. Moreover, longer substratebinding arms might lower the rate of dissociation from the substrate, with a resultant reduction in the ribozymeturnover rate. Therefore, we synthesized ribozymes (81mer, 41-mer, and 52-mer Rz's in Fig. 2) that were identical to those in the literature (Pachuk et al., 1994; James et al., 1996) and re-examined their specificities. As will be described in Materials and Methods, the 41-mer Rz was synthesized chemically and the 52-mer and 81-mer Rz's were generated by transcription from the corresponding DNA templates.

B. Specificity of cleavage of the chimeric BCR-ABL L6 (b2a2) substrate In order to examine the specificity of cleavage reactions catalyzed by conventional ribozymes, by Joyce's DNA enzymes (Pachuk et al., 1994; James et al., 1996; Santoro and Joyce, 1997), or by our novel heterodimeric minizyme (Amontov and Taira, 1996; Kuwabara et al., 1996; Amontov et al., 1996; Sugiyama et al., 1996; Kuwabara et al., 1997), we examined four types of RNA substrate (Figs. 5-7), namely, the normal ABL substrate, the chimeric BCR-ABL substrate, the normal BCR substrate, and a short ABL substrate with lengths, respectively, of 92, 121, 130, and 16 nucleotides. Enzymes with high specificity should cleave the chimeric BCR-ABL substrate (Figs. 5-6) or the 16-mer short ABL substrate in the presence of a pseudo-substrate that has the b2a2 junction sequence (Fig. 7), without cleaving the other RNAs. All kinetic measurements were made in 25 mM MgCl 2 and 50 mM Tris-HCl (pH 8.0), under enzymesaturating (single-turnover) conditions at 37째C (measurements of kcat or kobs), namely, our standard conditions for kinetic measurements (Kumar et al., 1996).

The chimeric (b2a2) mRNA substrate of 121 nucleotides in length, the normal ABL substrate of 92 nucleotides and the BCR substrate of 130 nucleotides were also generated by transcription. As a control, for the comparison of kinetic parameters, we also synthesized chemically Rz37 (Fig. 2), which can cleave the GUC triplet located 45 nucleotides downstream from the BCRABL junction. We note here that, since the cleavage site of Rz37 was located far from the BCR-ABL junction, Rz37 could cleave both normal ABL mRNA and chimeric BCRABL mRNA.

The results for the conventional antisense-type ribozymes are shown in Figure 5. As expected, all the conventional ribozymes cleaved the BCR-ABL substrate at the anticipated sites. However, in contrast to expectations based on previous reports (Pachuk et al., 1994; James et al., 1996), not only the control Rz37 but also the antisense-type ribozymes cleaved the normal ABL substrate within 1 hour. Moreover, the amounts of cleavage products obtained from the normal ABL mRNA with each ribozyme were almost the same as those obtained from the chimeric BCR-ABL substrate, indicating that these conventional ribozymes, with their relatively long antisense arms, recognized not only the abnormal BCR-ABL mRNA but also the normal ABL mRNA as substrates (Kuwabara et al., 1997). Therefore, nonspecific cleavage of normal ABL mRNA could not be avoided when we used conventionally designed ribozymes (Pachuk et al., 1994; James et al., 1996). In previous studies on these long antisense-type ribozymes, one part of the target site was designed to be accessible for annealing and served to direct ribozyme nucleation, while the other part recognized the cleavage triplet in the vicinity of the BCR-ABL junction, where specific cleavage of the hybrid mRNA occurred. We note that, in all cases, these conventional Rz's have regions of complementary binding to the normal ABL mRNA sequences of at least 6-8 nts. Previous studies of hammerhead ribozymes (Hertel et al., 1996; Birikh et al., 1997) demonstrated that cleavage of the substrate could occur when one of the substrate-

Examination of the BCR-ABL junction (b2a2 substrate) revealed the presence of several potential target sites, within three nucleotides of the junction, for Joyce's DNA enzymes (Santoro and Joyce, 1997). For example, the first AG sequence is located one nucleotide 5' of the junction. A second AG sequence is located two nucleotides 3' of the junction, and a GC sequence is located three nucleotides 3' of the junction. Therefore, we designed three kinds of DNA enzyme, each of which targeted one of these cleavage sites ( Fig. 2). For the AG cleavage sites, we used type I DNA enzymes (DNA enzyme 1 and DNA enzyme 2). A type II DNA enzyme was used for the GC cleavage site (DNA enzyme 3). DNA enzyme 1 should cleave the BCR region of the chimeric BCR-ABL mRNA, whereas DNA enzymes 2 and 3 should cleave the ABL region of the chimeric mRNA. Since a minizyme with high-level activity forms a dimeric structure with a common stem II (Amontov and Taira, 1996; Kuwabara et al., 1996; Amontov et al., 1996; Sugiyama et al., 1996), we decided to use one of the substrate-recognition sequences, within an active heterodimeric minizyme (Fig. 3), as the recognition arms for the abnormal BCR-ABL junction (b2a2 substrate). In order to achieve high substrate-specificity, the heterodimeric minizyme should retain their active conformation only in the presence of the abnormal BCRABL junction (b2a2 mRNA), while their conformation should ramain inactive in the presence or absence of the normal ABL mRNA (Fig. 4 ). The novel minizyme, MzL (minizyme left) and MzR (minizyme right), shown in 441

Kuwabara et al: Specificities and catalytic activities of novel dimeric minizymes

Figure 6. Gel electrophoresis showing cleavage by DNA enzymes. Specificity in cleavage by DNA enzymes was examined by using the normal ABL substrate (92 mer), the normal BCR substrate (130 mer) and the chimeric BCR-ABL substrate (121 mer). Each DNA enzyme (1 µM) and 2 nM 5'32P-labeled substrate were incubated at 37 °C for 60 minutes in a solution that contained 50 mM Tris-HCl (pH 8.0) and 25 mM MgCl2 and then subjected to electrophoresis on an 8% polyacrylamide/7M urea gel. Cleavage of the BCR-ABL substrate, at the AG sequence located 1 nt 5' of the junction, by DNA enzyme 1 generated a visible 5' fragment of 62 nts in length. Similarly, cleavage at the second AG sequence, located 2 nts 3' of the junction, by DNA enzyme 2 generated a fragment of 65 nts. Cleavage at the GC sequence located 3 nts 3' of the junction by DNA enzyme 3 generated a fragment of 66 nts. No cleavage of the normal ABL and BCR substrates occurred.

Figure 5. Autoradiogram after gel electrophoresis showing the non-specific cleavage of chimeric BCR-ABL mRNA, as well as of normal ABL mRNA, by conventional ribozymes. Specificity was examined with the normal ABL substrate (92 mer) and the chimeric BCR-ABL substrate (121 mer). Each ribozyme (1 µM) and 2 nM 5'-32P-labeled substrate were incubated at 37 °C for 60 minutes in a solution that contained 50 mM Tris-HCl (pH 8.0) and 25 mM MgCl2 and then the mixture was subjected to electrophoresis on an 8 % polyacrylamide/7M urea gel. Cleavage products from the normal ABL substrate (92 mer) were as follows. Non-specific cleavage, at the UUC triplet located 9 nts 3' of the junction, by the 52-mer Rz generated a 32P-labeled 5'-fragment of 43 nts in length. Similarly, non-specific cleavage, at the CUU triplet located 8 nts 3' of the junction, by the 41-mer Rz generated a visible fragment of 42 nts. Non-specific cleavage, at GUA located 19 nts 3' of the junction, by the 81-mer Rz generated a fragment of 54 nts. Non-specific cleavage, at GUC located 45 nts 3' of the junction, by Rz37 generated a fragment of 79 nts. Cleavage products from the BCR-ABL substrate (121 mer) were as follows. Cleavage by the 52-mer Rz generated a visible 5'-fragment of 72 nts in length. Similarly, cleavage by the 41-mer Rz generated a fragment of 71 nts. Cleavage by the 81-mer Rz generated a fragment of 83 nts. Cleavage by Rz37 generated a fragment of 108 nts in length.

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Gene Therapy and Molecular Biology Vol 1, page 443 reactions, demonstrating the expected high substratespecificity of these DNA enzymes. Our novel heterodimeric minizyme was expected to show high substrate specificity for the L6 BCR-ABL substrate, if and only if they form an active conformation in the presence of the abnormal b2a2 mRNA, as depicted in Figure 4. The specificity of dimeric minizyme was tested by incubating the minizymes with the 5'-32Plabeled short 16-mer substrate (S16) in the presence or absence of either a short 20-mer normal ABL pseudosubstrate (ABL pseudo-sub) or a short 28-mer BCR-ABL pseudo-substrate (BCR-ABL pseudo-sub) (Fig. 7). In order to obtain high resolution in the gel, we used synthetic short RNA substrates in this study. In this case, one part of the target site (b2a2 mRNA junction within the BCR-ABL pseudo-sub) was designed to be accessible by MzL and MzR for annealing and served to direct formation of the active dimeric minizyme (Fig. 4), while the other part recognized the cleavage triplet in the short 16-mer ABL substrate RNA, where specific cleavage of the latter RNA occurred (Fig. 7). It is to be noted that the cleavage activity of the novel heterodimeric minizyme was nearly identical with that of the control hammerhead ribozyme, Rz37. In terms of substrate specificity, no products of cleavage of the substrate were detected, in the absence of the BCR-ABL junction, demonstrating the expected high substrate-specificity of the heterodimeric minizyme. Since MzL or MzR alone, in the presence or in the absence of the pseudo-substrate, did not show any cleavage activity, the active species is clearly the heterodimeric form of the minizyme. Similar study demonstrated high substrate-specificity for the longer L6 BCR-ABL substrate (121-mer RNA) used in Figures 5 and 6 (data not shown).

Figure 7. Gel electrophoresis showing cleavage by dimeric minizymes. The specificity of dimeric minizyme-mediated cleavage was tested by incubating the minizymes with the 5'32P-labeled short 16-mer substrate (S16) in the presence or absence of either a short 20-mer normal ABL pseudo-substrate (ABL pseudo-sub) or a short 28-mer BCR-ABL pseudo-substrate (BCR-ABL pseudo-sub). Minizymes (MzL and MzR) were incubated at 0.1 µM with 2 nM 5'- 32P-labeled substrate (S16). When applicable, the concentration of pseudo-substrate, such as ABL or BCR-ABL, was at 1 µM. Reactions were usually initiated by the addition of 25 mM MgCl2 to a buffered solution that contained 50 mM Tris-HCl (pH 8.0) and enzyme together with the substrate, and each resultant mixture was then incubated at 37 °C for 60 minutes. The reaction mixture was subjected to electrophoresis on an 8 % polyacrylamide/7M urea gel.

In order to characterize in further detail the properties of the conventional hammerhead ribozyme, DNA enzymes and the novel heterodimeric minizyme, we obtained timecourses of the reactions that are shown in Figure 8. In terms of kinetic parameters (kcat), at least for this particular substrate, RNA enzymes including minizymes were more active than the DNA enzymes, although target sites differed among enzymes and no attempts were made to determine K M. It is to be mentioned, however, that, in terms of kcat/K M, DNA enzymes were shown previously to be more powerful than hammerhead ribozymes at least in the cleavage of HIV-1 mRNA (Santoro and Joyce, 1997).

binding arms was three nucleotides long. Thus, we would not expect substrate specificity from the conventionally designed ribozymes shown in Figure 2. In terms of substrate specificity, Joyce's DNA enzymes, as depicted in Figure 2, were expected to show high specificity for the L6 BCR-ABL substrate because all the target sites were located near the chimeric BCR-ABL junction (within three nucleotides of the junction). DNA enzyme 1 was designed to cleave the BCR region of the chimeric BCR-ABL mRNA, whereas DNA enzymes 2 and 3 were designed to cleave the ABL region of the chimeric mRNA. Therefore, as control substrates, both normal ABL mRNA and BCR mRNA were used in addition to the chimeric BCR-ABL mRNA (Fig. 6 ). The specificity of the newly designed DNA enzymes for the chimeric BCR-ABL substrate was tested by incubating the DNA enzymes with either the chimeric BCR-ABL substrate or the normal ABL or BCR substrate. As demonstrated in Figure 6, all the DNA enzymes cleaved the L6 BCR-ABL substrate at the anticipated cleavage site, producing products of the expected sizes. No products of cleavage of the normal ABL or the BCR substrate were detected in any of these

C. Potential gene therapy for treatment of chronic myelogenous leukemia (CML) The specific association of nucleic acid-based drugs, such as conventional ribozymes, Joyce's DNA enzymes, and our novel heterodimeric minizymes, with their targets via base pairing and subsequent cleavage of the RNA substrate suggest that these catalytic molecules might be useful for gene therapy. There are basically two ways to 443

Kuwabara et al: Specificities and catalytic activities of novel dimeric minizymes introduce ribozymes into cells. One such technique is a drug-delivery system (DDS) in which a chemically synthesized ribozymes (or DNA enzymes) are encapsulated in liposomes or other related compounds and delivered to target cells. Another way to introduce ribozymes into cells is by transcription from the corresponding DNA template (gene therapy). Current gene-therapy technology is limited primarily by the necessity for ex vivo manipulations of target tissues, namely, target cells must be removed from the body, engineered, and returned. Therefore, the limitations that determine the genetic diseases that can potentially be treated are directly linked to the limitations of current cell biology (Morgan and anderson, 1993). For the treatment of chronic myelogenous leukemia (CML) by nucleic acid drugs, in particular in the case of

the L6 translocations on which we focussed in this study, conventional antisense-type ribozymes are not the best choice because of the lack of substrate specificity. However, Joyce's DNA enzymes, despite their apparently lower cleavage activities, are suitable molecules for DDS, because DNA molecules are more stable than RNA molecules in vivo. The higher stability in vivo of DNA enzymes should counteract the apparent lower activity of the DNA enzymes because longer incubation resulted in a similar extent of cleavage of the chimeric BCR-ABL mRNA (Fig. 8). Since DNA enzymes are easier to synthesize, easier to handle, and more stable in vivo than ribozymes, we anticipate that all synthetic, catalytic nucleic acid drugs for DDS will turn out to be the DNA enzymes, with additional modifications for higher stability

Figure 8. Time courses for ribozyme, DNA enzyme, and dimeric minizyme-mediated cleavage reactions. For the cleavage reactions by the control ribozyme (Rz37) and the dimeric minizyme, a short substrate, S16, was used. For the cleavage reactions by DNA enzymes, a short substrate, S21, was used. Relative amounts of cleavage products (%) are plotted versus time. Calculated values of kcat are shown in Table 1.

Table 1. Kinetic parameters for the cleavage of short BCR-ABL substrate (S16 and S21)* Enzyme

kobs (min-1)

Rz37

> 0.5

Dimeric minizyme

0.1

DNA enzyme 1

0.012

DNA enzyme 2

0.003

DNA enzyme 3

0.012

*All reaction rates were measured, in 25 mM MgCl and 50 mM Tris-HCl (pH 8.0) under enzyme-saturating (single-turnover) conditions at 37 ˚C. In all 2 cases, kinetic measurements were made under conditions where all the substrate was expected to form a Michaelis-Menten complex, with high concentrations of enzymes (from 10 µM to 20 µM). Rate constants are averages from two sets of experiments.

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Gene Therapy and Molecular Biology Vol 1, page 445 CUU C-3'. The sequence of the 41-mer Rz was identical to that used in previous studies (Pachuk et al., 1994) Rz37, which targets the GUC triplet located 45 nucleotides (nts) 3' of the BCR-ABL junction had the following sequence: 5'-CAC UCA CUG AUG AGG CCG AAA GGC CGA AAC CCU GAG G-3'. Reagents for RNA synthesis were purchased from Perkin Elmer, Applied Biosystems. Oligonucleotides were purified as described in the user bulletin from ABI (no. 53; 1989) with minor modifications. Further purification was based on polyacrylamide gel electrophoresis as described previously (Shimayama et al., 1995).

and for higher affinity for their target molecules (Breaker, 1997). Nevertheless, current gene therapy technology is primarily limited by the necessity for ex vivo manipulations of target tissues and, practically, the technology is suited for RNA enzymes but not for DNA enzymes, because the former but not the latter can be transcribed in vivo. In this context, our novel heterodimeric minizymes are superior over the other nucleic acid-based drugs, because of their extreamly high substrate-specificity and high cleavage activity, for the treatment of chronic myelogenous leukemia (CML), especially in the case of L6 translocations.

B. Preparation of ribozymes by transcription DNA templates for ribozymes (52-mer Rz and 81-mer Rz) were synthesized chemically. Primers were also synthesized for each template, and the sense strand contained the T7 promoter. Downstream of the promoter sequence, we inserted no residues, such as two or three G residues for higher efficiency of transcription, so that the sequences of the products were identical to those reported in the literature (Pachuk et al., 1994; James et al., 1996). Products of PCR were gel-purified. T7 transcription in vitro and gel-electrophoretic purification of the ABL, BCRABL, BCR mRNA substrates were performed as described elsewhere (Shimayama et al., 1995).

D. Structure of dimeric minizymes We attempted to construct a molecular model of a dimeric minizyme, using the recently reported X-ray coordinates of the hammerhead ribozyme (Pley et al., 1994; Scott et al., 1995). Since stems II and III form a colinear pseudo-A-form helix, stem I of each component overlapped the other, so that, the model structure could be excluded as a result of extreme steric hindrance (Fig. 9a). Since kinetic studies and the above-mentioned substratespecificity unambiguously demonstrated that the active species is clearly the dimeric form and not the monomeric form of the minizyme, it should be possible for the hammerhead ribozyme to undergo conformational changes (Scott et al., 1996), leading to an alternative conformer that has fully active catalytic power. Indeed, our molecular modeling suggests such a candidate (Fig. 9b), in that the structure of the active portion is the same as that determined by X-ray analysis (Pley et al., 1994; Scott et al., 1995), however, the stem I has a slightly different orientation relative to that shown in Figure 9a. This demonstrates the flexibility of ribozymes in terms of their catalytically competent structures.

C. Preparation of target substrates, namely, ABL, BCR-ABL and BCR mRNAs, by transcription DNA templates for L6 BCR-ABL substrate RNA and for both the normal ABL and BCR substrate RNAs were synthesized chemically. The DNA oligodeoxynucleotide template for L6 BCR-ABL substrate RNA consisted of the sequence from 63 nts 5' of the BCR-ABL junction to 58 nts 3' of the BCR-ABL junction. The region of the DNA oligodeoxynucleotide template for the normal ABL substrate RNA extended from position 192 to position 283 of normal ABL cDNA. The DNA template for normal BCR substrate RNA extended from position 3234 to position 3363 of normal BCR cDNA. Primers were also synthesized for each template, and each sense strand contained the T7 promoter. The products of PCR were gel-purified. T7 transcription in vitro and gel-electrophoretic purification of the ABL, BCR-ABL, BCR mRNA substrates was performed as described elsewhere (Shimayama et al., 1995).

III. Experimental Procedures A. Synthesis of ribozymes, dimeric minizymes, and DNA enzymes

D. Activity assays of ribozymes, DNA enzymes, and dimeric minizymes

Ribozymes (41-mer Rz and Rz37), dimeric minizymes (MzL and MzR), DNA enzymes, and their corresponding short substrates (S16, S21, ABL Pseudo-sub and BCR-ABL Pseudosub) were chemically synthesized on a DNA/RNA synthesizer (model 394; Perkin Elmer, Applied Biosystems, Foster City, CA). S16, which contained GUC triplet located 45 nucleotides 3' of the BCR-ABL junction had the following sequence: 5'-CCU CAG GGU CUG AGU G-3'. S21, which contained 3 cleavage sites, each can be targeted by a respective DNA enzyme used in this stidy, had the following sequence: 5'-AAU AAG GAA GAA GCC CUU CAG-3'. ABL pseudo-sub which contained normal ABL mRNA sequence around the exon 1-exon 2 junction had the following sequence: 5'-UUA UCU GGA AGA AGC CCU UC3'. BCR-ABL pseudo-sub which contained BCR-ABL mRNA sequence around the BCR-ABL junction had the following sequence: 5'-CUG ACC AUC AAU AAG GAA GAA GCC

Assays of enzymatic activities were performed, in 25 mM MgCl2 and 50 mM Tris-HCl (pH 8.0), under enzyme-saturating (single-turnover) conditions at 37 °C, with incubation for 60 minutes. The substrates were labeled with [!- 32P]-ATP by T4 polynucleotide kinase (Takara Shuzo). Ribozyme or DNA enzyme was incubated at 1 µM with 2 nM 5'- 32P-labeled substrate (Fig. 5 and Fig. 6). The specificity of dimeric minizyme was tested by incubating the minizymes with the 5'32P-labeled short 16-mer substrate (S16) in the presence or absence of either a short 20-mer normal ABL pseudo-substrate or a short 28-mer BCR-ABL pseudo-substrate (Fig. 7 ). Minizymes (MzL and MzR) were incubated at 0.1 µM with 2 nM 5'- 32P-

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Gene Therapy and Molecular Biology Vol 1, page 446

Figure 9. CPK models and schematic representation of constructed dimeric minizymes. The constructed structures were base on X-ray coordinates, either from (a) URX059 or UHX026 (b) (see Experimental Procedures for details). The overview of the CPK dimeric minizyme: enzyme strands are colored magenta and purple, substrate strands are colored yellow and green. Views generated by rotating the molecule by 90° are also shown to emphasize the interaction between two stems I (center). Schematic models of dimeric minizymes are shown by cylinders, each representing a stem structure. Two active units are colored differently. The structure based on URX059 (a) causes severe steric hindrace between two stems I, whereas the other structure based on UHX026 (b) can avoid such steric hindrace. labeled substrate (S16). When applicable, the concentration of pseudo-substrate, such as ABL or BCR-ABL, was at 1 µM. Reactions were usually initiated by the addition of MgCl2 to a buffered solution that contained enzyme together with the substrate, and each resultant mixture was then incubated at 37 °C.

E. Kinetic analysis Reaction rates were measured, in 25 mM MgCl2 and 50 mM Tris-HCl (pH 8.0), under enzyme-saturating (single-turnover) conditions at 37 °C (measurements of kcat or kobs ). Reactions

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Gene Therapy and Molecular Biology Vol 1, page 447 were usually initiated by the addition of MgCl2 to a buffered solution that contained the enzyme together with the substrate, and each resultant mixture was then incubated at 37 °C. In all cases, kinetic measurements were made under conditions where all the available substrate was expected to form a MichaelisMenten complex, with high concentrations of enzymes (from 10 µM to 20 µM).

Cameron, F. H., and Jennings, P. A. (1994). Multiple domains in a ribozyme construct confer increased suppressive activity in monkey cells. Antisense Res. Dev. 4, 87-94. Christoffersen, R. E., and Marr, J. J. (1995). Ribozymes as human therapeutic agents. J. Med. Chem. 38, 2023-2037. Dahm, S. C., Derrick, W. B., and Uhlenbeck, O. C. (1993). Evidence for the role of solvated metal hydroxide in hammerhead cleavage mechanism. Biochemistry 32, 1304013045. F. Eckstein, and D. M. J. Lilly, eds. (1996). In Catalytic RNA, Nucleic Acids and Molecular Biology. Vol. 10, (Berlin, Germany: Springer-Verlag). Ferbeyre, G., Bratty, J., Chen, H., and Cedergren, R. (1996). Cell cycle arrest promotes trans-hammerhead ribozyme action in yeast. J. Biol. Chem. 271, 19318-19323. Groffen, J., Stephenson, J. R., Heisterkamp, N., de Klein, A., Bartram, C. R., and Grosveld, G. (1984). Philadelphia chromosomal breakpoints are clustered within a limited region, bcr, on chromosome 22. Cell 36, 93-99. Haseloff, J., and Gerlach, W. L. (1988). Simple RNA enzymes with new and highly specific endonuclease activities. Nature 334, 585-591. Heisterkamp, N., Stephenson, J. R., Groffen, J., Hansen, P. F., de Klein, A., Bartram, C. R., and Grosveld, G. (1983). Localization of the c-ab1 oncogene adjacent to a translocation break point in chronic myelocytic leukaemia. Nature 306, 239242 Hertel, K. J., Herschlag, D., and Uhlenbeck. (1996). Specificity of hammerhead ribozyme cleavage. EMBO J. 15, 3751-3757. Homann, M., Tzortzakaki, S., Rittner, K., Sczakiel, G., and Tabler, M. (1993). Incorporation of the catalytic domain of a hammerhead ribozyme into antisense RNA enhances its inhibitory effect on the replication of human immunodeficiency virus type 1. Nucleic Acids Res. 21, 2809-2814. James, H., Mills, K., and Gibson, I. (1996). Investigating and improving the specificity of ribozymes directed against the bcrabl translocation. Leukemia 10, 1054-1064. Kawasaki, H., Ohkawa, J., Tanishige, N., Yoshinari, K., Murata, T., Yokoyama, K. K., and Taira, K. (1996). Selection the best target site for ribozyme-mediated cleavage within a fusion gene for adenovirus E1A-associated 300kDa protein (p300) and luciferase. Nucleic Acids Res. 24, 3010-3016. Kearney, P., Wright, L. A., Milliken, S., and Biggs, J. C. (1995). Improved specificity of ribozyme-mediated cleavage of bcr-abl mRMA. Exp. Hematol. 23, 986-989. Kiehntopf, M., Brach, M. A., Licht, T., Petschauer, S., Karawajew, L., Kirschning, C., and Herrmann, F. (1994). Ribozyme-mediated cleavage of the MDR-1 transcript restores chemosensitivity in previously resistant cancer cells. EMBO J. 13, 4645-4652. Kiehntopf, M., Esquivel, E. L., Brach, M. A., and Herrmann, F. (1995). Ribozymes: biology, biochemistry, and implications for clinical medicine. J. Mol. Med. 73, 65-71. Koizumi, M., Iwai, S., and Ohtsuka, E. (1988). Cleavage of specific sites of RNA by designed ribozymes. FEBS Lett . 239, 285-288. Konopka, J. B., Watanabe, S. M., and Witte, O. N. (1984). An alteration of the human c-abl protein in K562 leukemia cells unmasks associated tyrosine kinase activity. Cell 37, 10351042.

Reactions were stopped at intervals by removal of aliquots from the reaction mixture and mixing them with an equal volume of a solution that contained 100 mM EDTA, 9 M urea, 0.1% xylene cyanol and 0.1% bromophenol blue. The substrate and the products of the reaction were separated by electrophoresis on an 8% polyacrylamide/7 M urea denaturing gel and were detected by autoradiography. The extent of cleavage was determined by quantitation of radioactivity in the bands of substrate and products with a Bio-Image Analyzer (BAS2000; Fuji Film, Tokyo).

F. Model building of dimeric minizymes Two hypothetical models of the dimeric minizyme were constructed from the coordinates of hammerhead ribozyme crystal structure [NDB entry URX059 (Scott et al., 1995) and UHX026 (Pley et al., 1994)]. In UHX026, the molecule 2 in the asymmetric unit was employed. Model building was performed as follows: the coordinates were transferred into PC-based software HyperChem (Hypercube, Inc.), and truncated at G10.1:C 11.1 pair in stem II, then, two copies of the truncated ribozyme were connected at the G10.1:C 11.1 pair to form a geometry of an A-type helix. For the construction of an altenative dimeric minizyme, based on coordinates of URX059, the GUAA loop at stem III was removed, and two residues were added to stem I for matching the length of stem I with that based on UHX026. Two types of dimeric form, derived either from URX059 or UHX026 are shown in Figure 9, together with schematic representation of the overall fold of each form of the dimeric minizymes.

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Pontius, B. W., Lott, W. B., and von Hippel, P. H. (1997). Observations on catalysis by hammerhead ribozymes are consistent with a two-divalent-metal-ion mechanism. Proc. Natl. Acad. Sci. USA 94, 2290-2294. Pyle, A. M. (1993). Ribozymes: a distinct class of metalloenzymes. Science 261, 709-714. Rowley, J. D. (1973). Letter: A new consistent chromosomal abnormality in chronic myelogenous leukaemia identified by quinacrine fluorescence and Giemsa staining. Nature 243, 290293. Ruffner, D. E., Stormo, G. D., and Uhlenbeck, O. C. (1990). Sequence requirements of the hammerhead RNA self-cleavage reaction. Biochemistry 29, 10695-10702. Santoro, S. W., and Joyce, G. F. (1997). A general purpose RNAcleaving DNA enzyme. Proc. Natl. Acad. Sci. USA 94, 42624266. Sarver, N., Cantin, E., Chang, P., Ladne, P., Stephens, D., Zaia, J., and Rossi, J. (1990). Ribozymes as potential anti-HIV-1 therapeutic agents. Science 247, 1222-1225. Sawata, S., Komiyama, M., and Taira, K. (1995). Kinetic evidence based on solvent isotope effects for the nonexistence of a proton-transfer process in reactions catalyzed by a hammerhead ribozyme: implication to the double-metal-ion mechanism of catalysis. J. Am. Chem. Soc. 117, 2357-2358. Schtivelman, E., Lifshitz, B., Gale, R. P., and Canaani, E. (1985). Fused transcript of abl and bcr genes in chronic myelogenous leukaemia. Nature 315, 550-553. Scott, W. G., Finch, J. T., and Klug, A. (1995). The crystal structure of an all RNA hammerhead ribozyme: a proposed mechanism for RNA catalytic cleavage. Cell 81, 991-1002. Scott, W. G., and Klug, A. (1996). Ribozymes: structure and mechanism in RNA catalysis. Trends Biochem. Sci. 21, 220224. Scott, W. G., Murray, J. G., Arnold, J. R. P., Stoddard, B. L., and Klug, A. (1996). Capturing the structure of a catalytic RNA intermediate: the hammerhead ribozyme. Science 274, 20652069. Shimayama, T., Nishikawa, S., and Taira, K. (1995). Generality of the NUX rule: kinetic analysis of the results of systematic mutations in the trinucleotide at the cleavage site of hammerhead ribozymes. Biochemistry 34, 3649-3654. Shore, S. K., Nabissa, P. M., and Reddy, E. P. (1993). Ribozymemediated cleavage of the BCRABL oncogene transcript: in vitro cleavage of RNA and in vivo loss of P210 protein-kinase activity. Oncogene 8, 3183-3188. Shtivelman, E., Lifschitz, B., Gale, R. P., Roe, B. A., and Canaani, J. (1986). Ribozyme-mediated inhibition of bcr-abl gene expression in a Philadelphia chromosome-positive cell line. Cell 47, 277-284. Snyder, D. S., Wu, Y., Wang, J. L., Rossi, J. J., Swiderski, P., Kaplan, B. E., and Forman, S. J. (1993). Ribozyme-mediated inhibition of bcr-abl gene expression in a Philadelphia chromosome-positive cell line. Blood 82, 600-605. Steitz, T. A., and Steitz, J. A. (1993). A general two-metal-ion mechanism for catalytic RNA. Proc. Natl. Acad. Sci. USA 90, 6498-6502. Sugiyama, H., Hatano, K., Saito, I., Amontov, S., and Taira, K. (1996). Catalytic activities of hammerhead ribozymes with a triterpenoid linker instead of stem/loop II. FEBS Lett . 392, 215-219.

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Gene Therapy and Molecular Biology Vol 1, page 451 Gene Ther Mol Biol Vol 1, 451-466. March, 1998.

Isolation and characterization of an RNA that binds with high affinity to Tat protein of HIV-1 from a completely random pool of RNA. Rika Yamamoto1 , 2 , 3 , Kazuo Murakami3 , Kazunari Taira1 , 2 , 3 , and Penmetcha K. R. Kumar 1 , 2 1National Institute of Bioscience and Human Technology, and 2National Institute for Advanced Interdisciplinary Research, AIST, MITI, and 3Institute of Applied Biochemistry, University of Tsukuba, Tsukuba Science City 305, Japan _______________________________________________________________________________________________ Correspondence: P. K. R. Kumar. Phone: +81-298-54-6085, Fax: +81-298-54-6095, E-mail: PKRKumar@nibh.go.jp

Summary The trans-activation (Tat) protein of human immunodeficiency virus type-1 (HIV-1) is vital for the replication of the virus. In a transcription assay i n v i t r o i n t h e p r e s e n c e o f a u t h e n t i c T A R RNA, we found that authentic TAR RNA inhibits transcription from a template based on the CMV early promoter in a manner that is not related to the Tat/TAR interaction. Using variants of TAR RNA, we identified potential sequences of RNA that seems to be responsible for the inhibition of transcription. In addition, we isolated an RNA aptamer that can bind with high affinity to Tat protein specifically. The isolated aptamer appears to include two TAR-like RNA motifs for higheraffinity binding to Tat peptides. The aptamer's high affinity for Tat peptides, as w e l l as the absence of any inhibitory effects on the transcription of unrelated genes, as opposed to those of TAR RNA, suggests that the novel RNA might be very useful as a Tat-specific decoy.

specific binding to Tat and for trans-activation, whereas loop sequences are necessary for trans-activation but are not essential for the binding of Tat in vivo (Feng and Holland, 1988; Berkhout and Jeang, 1989; Dingwell et al., 1989; Cordingly et al., 1990; Roy et al., 1990; Weeks et al., 1990).

I. Introduction The expression of genes encoded by human immunodeficiency virus type-1 (HIV-1) is regulated by the interaction of cellular factors and a viral trans-activator protein, Tat, with specific regulatory elements in the long terminal repeat (LTR) of HIV-1 (Gaynor, 1992). The HIV1 regulatory protein Tat binds to one of the regulatory elements in the LTR region, which is called the transactivating response region, TAR, (Rosen et al., 1985; Dayton et al., 1986; Fisher et al., 1986). This region is located immediately downstream from the site of initiation of transcription at the 5â&#x20AC;&#x2122;-end of all the viral transcripts (Berkhout et al., 1989). It is an RNA element consisting of 59 nucleotides (nt), which is the minimal motif that is sufficient for formation of a stable hairpin structure that allows binding of Tat in vivo (Rosen et al., 1985; Feng and Holland, 1988; Jakobovits et al., 1988). Tat effectively stimulates transcription after its binding to TAR RNA (Cullen, 1986; Peterline et al., 1986; Rice and Mathews, 1988). Deletion studies of TAR RNA revealed that so-called bulge residues are obligatory both for the

Tat is a small cysteine-rich nuclear protein consisting of 86 amino acids. It has two major domains, a cysteinerich region and highly basic region (Arya et al., 1985; Sodroski et al., 1985). The cysteine-rich region is essential for the function of this protein (Garcia et al., 1988; Kubota et al., 1988) and it has a metal-binding domain that probably mediates the metal-linked dimerization of Tat (Frankel et al., 1988). The basic region is responsible for the specific binding to TAR RNA (Weeks et al., 1990), as well as for nuclear localization (Dang and Lee, 1989; Endo et al., 1989). Tat belongs to a family of RNA-binding proteins that contain arginine-rich motifs for recognition of the respective cognate RNAs (Lazinski et al., 1989). A short peptide containing an arginine-rich region binds to TAR RNA with specificity similar to that of the intact

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Yamamoto et al. Tat Aptamer protein (Weeks et al., 1990; Calnan et al., 1991a). The tat gene product not only plays a key role in the transactivation of HIV-1 genes but also has a variety of effects on the growth and metabolism of the host cells (Ensoli et al., 1990, 1993). Moreover, Tat is now known to be important for the efficient reverse transcription of HIV-1 (Harrish et al., 1997).

experiments also support the model in which a Hoogsteen interaction is critical for the structure of TAR (Tao et al., 1997). Despite the discrepancy between results of NMR studies of TAR RNA from two different laboratories with respect to the formation of the base triple (as distinct from base pair), the general consensus on the structure of TAR RNA is in agreement about the approximate location of the bulge U in the major groove and the orientation of the functional groups in the TAR RNA.

Despite several studies on the stimulation by Tat of the trans-activation of expression of the HIV genome, the precise molecular mechanism by which it operates remains obscure. The rates of viral mRNA and protein synthesis induced by Tat in mammalian cells were estimated to be 100-fold higher than control rates (Hauber and Cullen, 1988). It has been reported that Tat functions as an antiterminator, an elongation factor in transcription and an enhancer of the initiation of transcription (for reviews, see Vaishav and Wong-Staal, 1991; Cullen, 1992; Jeang et al., 1993). Even though the exact function of Tat remains controversial, the emerging consensus appears to be that Tat functions as a promoter-specific elongation factor that modifies the transcription complex upon binding to TAR RNA. Several early studies showed that, for the stimulation of transcription by Tat, both cellular transcription factors and the integrity of the TAR RNA sequences are essential (for reviews, see Gaynor, 1992; Jones and Peterlin, 1994). However, the order of assembly of components of the transcription complex was not known until recently, it was unknown initially whether Tat binds to the transcription factors first and then binds to the TAR RNA or whether Tat binds initially to the TAR RNA with subsequent binding of transcription factors. Garcia-Martinez et al. (1997) suggested that Tat protein might associate with RNA polymerase II in the preinitiation complex and then the complexed Tat binds to TAR RNA, while stalled RNA polymerase II is passing through the TAR RNA.

Since the Tat protein has various functions in the life cycle of HIV-1, as well as in viral proliferation, it is an important and attractive target in efforts to develop weapons against HIV. Several genetic strategies have been tested, in the past, in attempts to repress the proliferation of HIV. Trans-dominant proteins, single-chain antibodies, antisense molecules, ribozymes, decoys (for review, see Yu et al., 1994) and use of the LTR of HIV to produce inducible and toxic gene products have all been tested in cells that were infected by HIV (Harrison et al., 1992). Combinations of these strategies (for example, a ribozyme and a decoy) have also been examined (Yuyama et al., 1994; Yamada et al., 1996). Although the expression and regulation of such therapeutic molecules might be possible in vivo, their constitutive expression could lead to cellular toxicity or to an immune response by the host against the engineered cells. This problem is especially significant in the case of toxins and suicide genes. Among various RNAbased strategies against HIV infection, the decoy strategy has a potential advantage over the use of other RNA inhibitors, such as short antisense RNAs and ribozymes, because the generation of escape mutants might be less frequent: alterations in Tat or Rev (HIV-1 protein) that prevent binding to a decoy would also prevent binding to native elements (such as RRE, the Rev-responsive element, and TAR sequences). Both RRE and TAR RNAs have been exploited as decoys and, in cell cultures, these decoys inhibited the replication of HIV by 80% to 97% (Graham and Maio, 1990; Sullenger et al., 1990; Lisziewicz et al., 1993).

A conformational change in TAR RNA upon binding of Tat is among the most intriguing of RNA-protein interactions. The conformational switch was clearly observed by circular dichroism (CD) and NMR studies (Tan and Frankel, 1992; Puglisi et al., 1992; Aboul-ela et al., 1995, 1996). A similar change in conformation of TAR RNA was achieved not only with Tat peptides but also by arginine alone. However, no conformational change was observed either with lysine or with variants of TAR that can no longer bind to Tat. Tat peptides and homopolymers of arginine that bind specifically to TAR RNA, as well as a peptide containing a central single arginine residue, were sufficient to reproduce wild-type trans-activation in studies designed to delineate the role of arginine (Calnan et al., 1991a,b). Further NMR studies on the TAR-argininamide complex suggested that TAR has a specific binding site for arginine (Puglisi et al., 1992). Arginine appeares to induce a structure where in a critical residue, U23, in a triple-base bulge makes a Hoogsteen interaction with an A•U base pair in the adjacent stem. This model was supported by replacing the U•A•U bonding in TAR by the isomorphous C•G•C bonding (Puglisi et al., 1993). Recent modification and mutagenesis

Although decoys might act as much more efficient inhibitors (with possible Ki values in the sub-nanomolar range) than other molecules, such as antisense RNAs and ribozymes, decoys might potentially be toxic to cells if they were to sequester cellular factors, in particular when the decoy RNA happens to include regions that can interact with cellular proteins. Several previous studies have shown that cellular factors, such as TRP-185 (Wu-Bear et al., 1995), Tat-SF1 (Zhou and Sharp, 1996), polymerase II (Wu-Bear et al., 1995, 1996) and others (Sheline et al., 1991; Rounseville and Kumar, 1992; Gatignol et al., 1991) bind efficiently to TAR RNA. Despite these studies, the effects of TAR RNA on the cellular machinery of the host cells have not been analyzed in detail either in vitro or in vivo. In the present study, we performed cell-free transcription assays in vitro in the presence of TAR RNA and demonstrated that authentic TAR RNA can inhibit transcription in a manner that is independent of the

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Gene Therapy and Molecular Biology Vol 1, page 453 Tat/TAR interaction. Furthermore, we identified important regions of TAR RNA that are responsible for the inhibition of transcription, namely, the loop, residues that surround the loop, the triple-base bulge, and the lower stem region of the TAR RNA. Since authentic TAR RNA of HIV-1 interacts with several cellular factors within the cell, in addition to inhibiting the transcription of unrelated genes, an authentic TAR decoy might not be the most suitable antagonist and specific inhibitor of Tat. In order to isolate more specific high-affinity RNA decoys that would bind to Tat, we used a recently developed technique of genetic selection in vitro (F i g . 1 , for reviews, see Gold et al., 1995; Osborne and Ellington, 1997) and isolated a high-affinity RNA motif that bound to Tat. The isolated motif, designated 11G-31 RNA, included conserved core elements of TAR RNA (F i g . 2 ) that have been identified as being necessary for binding to Tat. In addition, it has two TAR-like motifs opposite one another. The binding ability of a truncated mini 11G-31 RNA motif, a 37-mer, and of authentic TAR RNA were analyzed with Tat peptides in competitive binding assays, and the isolated RNA was found to bind with higher affinity. In addition, we performed a transcription assay in vitro in the presence of mini 11G-31 RNA to evaluate the effects of this RNA on the transcription of unrelated promoters.

A. The effects of TAR RNA in a cell-free transcription assay Studies both in vitro and in vivo with LTR-based templates and Tat have suggested that addition of exogenous TAR RNA and/or overexpression of TAR RNA can significantly inhibit trans-activation. Such significant inhibition might originate from a combination of two effects: a) the expressed or added TAR RNA might act as a decoy by sequestering Tat and interfering directly with the binding of Tat to the TAR in the LTR region after its transcription from LTR templates; and b) the TAR decoy might sequester transcription factors together with other important proteins, such as RNA polymerase II, that are unrelated to the Tat/TAR interaction. Since earlier decoy studies relied on LTR-based vectors, it was not possible to distinguish between the two possible scenarios.

F i g u r e 2 . TAR RNA of the core element of HIV-1. TAR motif is indicated by box and the core element that is required for binding to Tat protein is indicated by red.

In order to distinguish between the two possible effects of TAR RNA, we performed transcription assays in vitro with an extract of HeLa cell nuclei. This assay has been used routinely to study the Tat-mediated trans-activation of HIV-1 genes (Marciniak et al., 1990). We hoped that this method would provide some insight into Tat-mediated trans-activation and would also allow us to screen various inhibitors that might interfere with Tat-TAR interactions. To examine the interaction between TAR RNA and

Figure 1. Scheme for genetic selection in vitro.

II. Results and Discussion

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Gene Therapy and Molecular Biology Vol 1, page 454 Figure 3. Sequences and secondary structures of authentic TAR RNA and mutant TAR RNAs. Bases dissimilar to those in TAR RNA or deleted are indicated by boxes.

F i g u r e 4 . Inhibition of transcription from a CMV early promoter-driven template by authentic TAR RNA in an extract of HeLa cell nuclei. A, The template containing the early promoter of CMV was transcribed in the absence (lane 1) and in the presence (lanes 3 and 4) of 100 pmole of TAR RNA, or in the presence of 100 pmole of tRNA (total tRNA from yeast; lane 2). Singlestranded DNA markers were loaded in lane M. The newly synthesized transcript is indicated by the arrow. B, The relative levels of the transcript (364 nt) that was synthesized in vitro, as quantitated in four independent experiments (experimental variations are indicated by error bars).

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Gene Therapy and Molecular Biology Vol 1, page 455 inhibition (about 30%), while at 10 pmole the extent of inhibition by TAR RNA was similar to that (60-70%) that observed in the presence of 100 pmole of TAR RNA (data not shown). Although the levels of TAR RNA that we used appear to be rather high, similar concentrations of TAR RNA were used in the past to examine the decoy effects of TAR in cell-free transcription assays (Bohjanen et al., 1996). As can be seen in Figure 4, addition of 100 pmole of tRNA has either no effect or only a marginal effect. These results clearly demonstrated that inhibition of transcription by TAR RNA depended on the concentration of the RNA.

cellular factors, we performed transcription assays using a cytomegalovirus (CMV) early promoter-based template in the presence and in the absence of authentic TAR RNA (F i g . 3 ). As seen in Figure 4A, which shows a representative autoradiogram, the basal level of transcription (lane 1) from the CMV early promoter was greatly reduced by the addition of 100 pmole of authentic TAR RNA (lanes 3 and 4). Quantification of the results of four independent transcription experiments in the presence of TAR RNA revealed that transcription was inhibited by 60-70% (F i g . 4B). By contrast, after addition of a similar amount of tRNA (total tRNA from yeast) the basal level of transcription remained either unaffected or was only marginally reduced (about 10-20%; see F i g . 4). These results demonstrate that factors that are important in the transcription process can bind to TAR RNA and, therefore, that the TAR decoy can inhibit transcription in vitro. In analogous experiments based on the LTR promoter, we observed similar inhibition of transcription (data not shown). The effects of circular TAR RNA were analyzed recently by Bohjanen et al. (1996), who used an LTR promoter-based template and studied Tat-mediated transactivation under similar conditions to those that we employed here. They found that circular TAR RNA inhibited Tat-mediated trans-activation by 77% (Bohjanen et al., 1996). However, the inhibitory effect of circular TAR RNA could not be relieved by the addition of excess Tat (30-fold excess). The observed inhibition of transcription by TAR RNA in the present study, with the CMV promoter-driven template, was 60-70%, a value close to the 77% inhibition of trans-activation observed in earlier experiments with the LTR promoter. Therefore, we propose here that the effects of the TAR decoy observed earlier might have been due to inhibition of transcription, at least to some extent, rather than to sequestration of the Tat protein exclusively. In addition, in view of the above results, we cannot exclude the possibility that, upon expression of a TAR decoy in the host cell, transcription might be inhibited not only on templates that are dependent on Tat-mediated trans-activation but also on other templates, including those that encode housekeeping genes.

Figure 5. Dependence on the concentration of TAR RNA of the inhibition of transcription. Template containing the early promoter of CMV was transcribed in an extract of HeLa cell nuclei in the absence (lane 1) and in the presence of increasing concentrations of TAR RNA (lane 2, 0.01 pmole; lane 3, 1 pmole; and lane 4, 100 pmole). Newly synthesized transcripts is indicated by arrow.

B. Dependence of the inhibition of transcription on the concentration of TAR RNA Although the concentration of TAR RNA (100 pmole) tested in the above studies in vitro might be achievable in vivo with various expression vectors, we were interested in determining the minimum concentration of TAR RNA for inhibition. Therefore, we performed transcription reactions in vitro in the presence of concentrations of TAR RNA from 0.01-100 pmole. As seen in Figure 5, the inhibition of transcription was directly dependent on the concentration of TAR RNA. As expected, lower levels (0.01-1.0 pmole) of TAR RNA resulted in moderate

C. Inhibition of transcription by variants of TAR RNA In order to identify the regions of TAR RNA that are responsible for interactions with cellular transcription factors in vitro, we synthesized and tested four variants. Figure 3 shows mutant TAR-1 RNA with altered bases in the loop, mutant TAR-2 RNA with a substituted basepair (mutated bases are boxed), mutant TAR-3 RNA with a 455

Yamamoto et al. Tat Aptamer deletion of two bulge-bases, and mutant TAR-4 RNA with deletion of the lower stem. These variants were initially tested for their ability to bind to a Tat peptide (CQ peptide; see Experimental Procedures). Only TAR RNA variants with deletion or substitution of conserved bases (such as TAR-2 and TAR-3 RNAs) had significantly reduced affinity for the CQ peptide (F i g . 6 ). Both TAR-1 and TAR-4 RNAs bound efficiently to the CQ peptide (F i g . 6). However, the affinity for the CQ peptide of TAR-4 RNA was lower than the affinity of authentic TAR RNA. From these results it appeared, in harmony with previous results (Weeks and Crothers, 1991; Churcher et al., 1993), that the ability to bind to the CQ peptide was abolished only when the conserved residues in TAR RNA were replaced or missing.

particular, the TAR-3 RNA variant was a good control since this RNA was synthesized and processed under identical conditions to TAR RNA. Several transcription factors and cellular factors have been isolated from extracts of HeLa cell nuclei that bind to authentic TAR RNA and facilitate trans-activation in vitro. The importance of specific residues of TAR RNA for specific binding to such cellular factors and to proteins of the host cell has been evaluated in earlier studies and the reported results can be summarized as follow. a) Two distinct nuclear transcription factors, TRP-1 and TRP-2, were isolated (Sheline et al., 1991) that specifically recognize the loop and triple-base bulge residues of TAR RNA, respectively. b) A p140 protein and TRBP (TAR RNA-binding protein) were isolated from HeLa cell nuclei and were shown to interact specifically with the lower stem of authentic TAR RNA (Rounseville et al., 1992; Gatignol et al., 1991). c) For binding to polymerase II, both the loop and the triple-base bulge of TAR RNA were shown to be important (Wu-Baer et al., 1995, 1996). And, finally, d) TRP-185, a cellular factor appeared to bind specifically to the loop of TAR RNA (Wu-Baer et al., 1995, 1996).

Figure 6 . Binding of the Tat peptide (CQ) to TAR RNA and its variants. Relative amounts of complex formed (%) were analyzed after titration of increasing concentrations of CQ peptide with labeled RNAs and electrophoresis on nondenaturing gels, as described under Experimental Procedures. TAR RNA-CQ complex ( !), TAR-1 RNA-CQ complex (!), TAR-2 RNA-CQ complex (!), TAR-3-CQ complex (!), TAR4-CQ complex (!).

We next performed cell-free transcription reactions in the presence of authentic TAR RNA or of its variants. Among the parent and variant RNAs tested, authentic TAR RNA had the highest inhibitory effect on transcription from the CMV promoter. Substitution of bases in the loop sequence of TAR RNA (TAR-1), substitution of the conserved base-pair near the triple-base bulge (TAR-2), deletion of the triple-base bulge (TAR-3), and deletion of the lower stem (TAR-4) all halved the inhibitory effect of TAR on transcription. In order to test the validity of the observed inhibition by the TAR RNA variants in nuclear extracts, we performed four independent transcription experiments in the presence of TAR RNA variants and quantitated the amounts of transcript generated (F i g . 7 ). Each variant (TAR-1, TAR-2, TAR-3 and TAR-4) had a clearly reduced inhibitory effect on transcription. These variants of TAR RNA not only were helpful in attempts to identify regions that are important for interactions with cellular factors but also served as good internal controls. In

Figure 7 . Effects of mutant TAR RNAs on the transcription of a CMV early promoter-driven template in extracts of HeLa cell nuclei. The relative level (percentage) of transcript (364 nt) synthesized in four independent experiments was quantitated by reference to the control. Transcription was allowed to proceed in the presence of 100 pmole of TAR RNA or its variants (experimental variations are indicated by error bars).

Substitution or deletion of bases in the loop, of bases near the loop, of the triple-base bulge, and of the lower stem region of TAR RNA abolished the ability of TAR RNA to inhibit transcription. The loss of the ability of TAR RNA to inhibit transcription probably resulted from loss of the ability of cellular factors to bind to TAR RNA. Thus, the variants of TAR RNA used in our study had lost their ability to sequester cellular factors. In several earlier studies, mutations in the loop, bulge, and lower stem of TAR RNA were associated with marked defects in the 456

Gene Therapy and Molecular Biology Vol 1, page 457 replication of HIV-1 in human cell lines, as compared with that of wild-type HIV-1 produced under identical conditions, even though some of the variants could have interacted efficiently with Tat in vivo (Rounseville et al., 1996; Berkhout and Jeang, 1991; Harrich et al., 1994; Verhoef et al., 1997). Taken together, the results of earlier studies and our present results with the four TAR variants suggest that cellular factors interact efficiently with TAR RNA only when it retains its full integrity. Furthermore, we extended our inhibition studies with TAR RNA to an other human cell line, namely, Jurkat cells, and we observed similar inhibition of transcription to that described above in extracts of HeLa cell nuclei (data not shown).

folded by the Mulfold program (Zuker, 1989), 15 clones, representing about 40% of the population in the pool had a TAR-like motif (containing all core elements) in their randomized region. However, some of the clones has two TAR-like motifs, for example, 11G-31 (F i g . 8). Combinatorial analysis of TAR core elements predicts that at least one sequence should be found in every 2.16 x 106 nucleotides (Ferbeyre et al., 1997). Despite such a low probability of distribution of TAR core elements, we were able to isolate TAR-like elements from the random pool, and even a double-TAR element, probably because selective pressure was maintained during the entire selection procedure. Although the predominant selected aptamers that belonged to the two major classes contained two bulge residues (UC or UU), as opposed to three in the TAR of HIV-1, mutational analysis has revealed that at least two bulge residues are necessary for recognition of Tat (Weeks and Crothers, 1991). Moreover, TAR RNA of HIV-2 also contains two (UU or UA in two TAR motifs) bulge residues that allow efficient binding to the HIV-1 Tat peptide (Chang and Jeang, 1992). A similar selection procedure, using an RNA pool with a 30 nt random core, resulted in isolation of other structural forms (Tuerk and MacDougal-Waugh, 1993). In this case, selection might have been hampered by the short random-core region over the fixed sequences (for amplifications).

D. Isolation of an RNA aptamer with high affinity for Tat protein TAR RNA of HIV-1 binds to several cellular factors in the cell. In view of its inhibitory effects on the transcription of unrelated genes, as observed above, authentic TAR RNA might not be the most suitable antagonist and specific inhibitor of Tat. In order to isolate an RNA motif that binds to Tat specifically and with high affinity, we exploited a strategy for genetic selection in vitro using a pool of RNAs with a large random core sequence of 120 nt (120 N). In the first selection cycle, about 1013 RNA sequences were allowed to bind to the HIV-1 Tat protein at a molar ratio of protein to RNA of 1:10 in the binding buffer. In subsequent cycles, molar ratios of Tat and RNAs [the 120 N pool, specific competitors (either TAR RNA or selected pool RNA with a random core region of 12-18 nt having about 5% binding ability to Tat) and a non-specific competitor, tRNA] were manipulated in order to increase the stringency of selection (T a b l e 1). After each set of two cycles of selection, the RNA pool was analyzed in a filter-binding assay for binding to Tat. As the cycles progressed, levels of specific RNA aptamers that bound to Tat increased in the pool from 1% to 9%. Since the 10 14 variants in the 120 N pool could not encompass the entire range of possibilities (10 72) variants, mutagenic PCR was introduced after the ninth cycle to increase the diversity of functional molecules. However, with the introduction of mutagenic PCR, the number of binding species was reduced in the pool, probably as a number of the mutation of critical residues and, therefore, we cloned products from both the ninth and the eleventh cycle for the analysis of sequences.

E. Mini 11G-31 RNA binds efficiently to Tat-derived peptides Since a short peptide that contains an arginine-rich region binds to TAR RNA with specificity similar to that of the intact protein (Weeks et al., 1990; Calnan et al., 1991a), we used both the CQ (amino acids 37-72) and RE (amino acids 49-86) peptides in further studies. These peptides were synthesized chemically and purified to homogeneity (>95% purity) by reverse-phase HPLC.

In all, we sequenced 64 clones from the ninth and the eleventh cycles and divided the sequences from the eleventh cycle into four classes. Two major classes of sequences (two representative sequences, 11G-22 and 11G-31, are shown in F i g . 8 ) were derived from the eleventh cycle RNA pool, as compared to the ninth cycle, in which many sequences were unrelated. When these RNA sequences were

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Initially, a representative clone from each class was subjected to a competitive binding assay in the presence of a Tat peptide (CQ or RE; see Experimental Procedures) and authentic TAR RNA. RNA motifs with two TAR-like motifs, such as 11G-31 RNA, appeared to compete with TAR for bind to the Tat peptides (data not shown). In order to locate the binding region in the 11G-31 RNA (one of the RNAs with high affinity for the Tat peptides), we chemically synthesized a minimal RNA (mini 11G-31; 37 mer) that had two TAR-like motifs and analyzed its binding to Tat peptides on non-denaturing gels. Both CQ and RE peptides efficiently bound to mini 11G-31 RNA (F i g . 9 A and B).

Gene Therapy and Molecular Biology Vol 1, page 458 Table 1. Concentrations of RNA and protein used and the ability of the RNA pool to bind to Tat after each selection cycle. Cycle Concentration of Binding ability a number

Tat

pool RNA

tRNA

RNA#

1 2 3 4 5 6 7 8 9 10 11

ÂľM 0.50 0.50 0.50 0.50 0.50 0.50 0.50 0.50 0.50 0.33 0.17

5.0 1.5 1.5 3.0 1.5 5.0 3.0 5.0 5.0 5.0 2.5

3 5 10 40 50 50 50 50 50 25

1 5 10 7 10 7 14 14 14 7

% 0 0 0 0 0 0 0 0 0 0 0

2 4 5 4 6 5 6 7 9 7 9

Figure 8. Representative sequences and secondary structures from each class of RNAs. Regions resembling TAR core elements (for Tat binding) are shaded and residues are highlighted in either blue or red. Blue and red letters in mini 11G-31 RNA indicate the presence of two TAR core elements.

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residues in mini 11G-31 RNA and the RE peptide. F i g ur e . 9 A . Formation of complex between Tat peptides (CQ and RE) and TAR RNA or mini 11G-31 RNA. Binding reactions contained 5'-end labeled RNA (15,000 cpm) and 5, 10, 20, 40, 60, 80, or 100 nM Tat peptide. Complexes were separated from unbound RNAs by electrophoresis on 20% non-denaturing polyacrylamide gels. (A-1) TAR RNA and CQ peptide; (A2) TAR RNA and RE peptide; (A-3) mini 11G-31 RNA and CQ peptide; (A-4) mini 11G-31 RNA and RE peptide. B . Formation of complex between a bulge deletion variant, an RNA lacking the bulge residues in mini 11G-31 RNA, and Tat peptides (CQ and RE). (B-1) The RNA lacking the bulge residues in mini 11G-31 RNA and the CQ peptide; (B-2) the RNA lacking the bulge

the RE peptide (F i g . 9 B - 2 ). A similar experiment was performed with authentic TAR RNA, as well as with the bulge mutant RNA, to examine binding to the RE and CQ peptides and we observed that the CQ peptide efficiently distinguished the bulge variant, as compared to the RE peptide, a result consistent with previous observations (Churcher et al., 1993). From these studies it appears that the bulge residues in mini 11G-31 are important for the recognition of Tat peptides.

To compare the binding affinities of the aptamer, mini 11G-31, and TAR RNA to the CQ and RE peptides, we performed binding assay in which labeled RNAs were incubated with various concentrations of CQ or RE peptide, with subsequent separation of complexed and free RNA on a 20% non-denaturing polyacrylamide gel (F i g . 9A). The amount of each complex formed was calculated directly from the intensities of bands on the gel. Authentic TAR RNA formed a complex at a level of about 50% in the presence of 56 nM CQ peptide (F i g . 9 A - 1 ), whereas mini 11G-31 RNA efficiently formed the same amount of complex even at 14 nM CQ peptide (F i g . 9 A - 3 ). When we performed a similar analysis using RE peptide, authentic TAR RNA formed a complex at a level of about 50% in the presence of 23 nM RE peptide (F i g . 9 A - 2 ), whereas mini 11G-31 RNA formed the same amount of complex even at 3 nM RE peptide (F i g . 9 A - 4 ). These results suggest that the selected aptamer had higher affinity for the Tat peptides.

Our various studies indicated that core elements of TAR RNA were well conserved in the isolated aptamers that belonged to the two predominant classes, suggesting the importance of the conserved residues. The sequences of the selected RNAs, sequences containing either a single or a double TAR motif, confirm the details of all the core elements that were previously identified as being required for binding of authentic TAR RNA to Tat. Deletion of bulge residues from mini 11G-31 RNA completely abolished the binding of Tat peptides. In addition, the bulge U residue was found in the single and the double TAR motif. This motif probably forms a Hoogsteen base pair with A-U (Watson-Crick paired) residues to form a base-triple Uâ&#x20AC;˘Aâ&#x20AC;˘U as proposed for complexes of arginine or the Tat peptide and TAR RNA. Taken together, the recent mutational results of Tao et al. (1997) and our present results suggest that the arginine-binding motif of TAR can

In order to evaluate the importance of bulge residues, we synthesized an RNA that lacked the bulge residues in mini 11G-31 RNA and analyzed its binding in gel-shift assays with the CQ and RE peptides. No complex was formed even at a high concentration (200 nM) of CQ peptide (F i g . 9B-1). However, a small amount of complex was formed at high concentrations (>80 nM) of 459

Yamamoto et al. Tat Aptamer be summarized as 5' UXnGA, where the U residue is predicted to make a Hoogsteen interaction with the A residue, Xn indicates at least one unpaired nucleotide and the G residue forms a pocket for binding of arginine.

results of titration of Tat peptide and of the competitive binding assays suggest that the isolated aptamer not only interfered with Tat/TAR interactions but also efficiently trapped Tat peptides at sub-nanomolar concentrations. This property is clearly desirable for an efficient decoy of viral proteins.

F. Comparison of the relative affinities of TAR and mini 11G-31 for the Tat peptide

G. The double TAR-like RNA motif has enhanced affinity for the Tat peptide

When authentic TAR RNA and mini 11G-31 RNA were titrated with various amounts of Tat peptides, the aptamer was observed to bind efficiently to the Tat peptides. We next performed competitive binding assays to compare the affinities of authentic TAR RNA and mini 11G-31 RNA directly. The labeled aptamer and TAR RNA were incubated at a ratio of 1 : 1 with unlabeled aptamer and TAR RNA at various molar ratios (ranging from 408,000 nM) in the presence of 80 nM RE peptide. The reaction mixture was allowed to equilibrate at 30 °C for 12 hr and resolved on a 20% non-denaturing polyacrylamide gel. The amount of complex formed by the aptamer with the RE peptide was calculated for various ratios and we found that the amount of the aptamer-peptide complex fell by 50% when the molar ratio was one to eighty (mini 11G-31 : TAR = 40 nM : 3,200 nM). These results suggested that the affinity for the RE peptide of the aptamer might be about 80 times higher affinity (D1/2 " 3.2 µM) than that of authentic TAR RNA (F i g . 1 0 ). By contrast, no TAR-peptide complex was detected when the molar ratio of non-labeled aptamer to non-labeled TAR RNA was 1 : 1.

In order to define clearly the importance of the double TAR-like motif in the efficient binding to Tat peptides, we separated the two strands and deleted the loop sequences (F i g . 1 1 ).

F i g u r e 1 1 . Synthetic mini 11G-31 RNA duplexes. Bases deleted from duplex mini 11G-31 RNAs are indicated by boxes. Core elements of TAR RNA that are found in mini 11G31 RNA are indicated by blue and red letters. Blue and red nucleotides form the two TAR core elements.

Duplex RNA I, that could mimic mini 11G-31 RNA, was prepared by annealing two chemically synthesized 5' and 3' RNA oligomers, (20 mers). Duplex RNA II (with a deletion of the 3'-bulge residues U and C) was prepared by annealing 3' !UC and the 5' RNA oligomers. Duplex RNA III (with a deletion of the 5'-bulge residues U and U) was prepared by annealing the 3' and 5' !UU RNA oligomers. To prepare duplex RNA IV (with deletion of both pairs of bulge residues) both 3' !UC and 5' !UU RNA oligomers were annealed. After labeling of the 5'-end of the oligomer in each duplex, we equilibrated the CQ peptide (40 nM) with each duplex in binding buffer at 30 °C for 1 h. The products were resolved on a non-denaturing polyacrylamide gel (F i g . 1 2 ) and the amount of each complex was calculated as mentioned above. The duplex structure that contained both bulges (duplex RNA I) formed a complex at a level of about 80% at 40 nM (F i g . 12, lane 12) and mini 11G-31 RNA formed a similar amount of complex. Deletion of either the 3'-end bulge residues UU (in duplex RNA II); (F i g . 1 2 , lane 8) or the 5'-end bulge residues UC (in duplex RNA III); (F i g . 1 2 ,

F i g u r e 1 0 . Competitive binding assay. Formation of a complex when a mixture of 5'-end labeled mini 11G-31 RNA (20,000 cpm), unlabeled mini 11G-31 RNA (40 nM), and unlabeled authentic TAR RNA at various concentrations (40 to 8,000 nM) was allowed to bind with the RE peptide (80 nM) at 30 °C for 12 h. The mixture was fractionated on a nondenaturing gel as mentioned the legend to Figure 9.

In earlier studies, peptides derived from the argininerich region of HIV-1 appeared to bind to authentic TAR RNA at concentrations between 16 pM to 40 nM (Churcher et al., 1993; Long and Crothers, 1995). Both

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Gene Therapy and Molecular Biology Vol 1, page 461 lane 10) reduced by about 50% the amount of complex formed with the CQ peptide. After deletion of both pairs of bulge residues in the duplex (duplex RNA IV) no complex was formed in the presence of the CQ peptide (F i g . 1 2 , lane 4). The results obtained suggest that the ratio of aptamer to peptide in the complex was one to one and that both pairs of bulge residues did indeed play important roles in the efficient binding to the CQ peptide.

Figure 1 2 . Formation of complex between the Tat peptide CQ and duplex RNA I (mini 11G-31), duplex RNA II, duplex RNA III, or duplex RNA IV. Reaction mixtures contained either a duplex strand or RNA (one labeled RNA, usually the 5'-end-labeled oligomer and unlabeled second strand) alone or in the presence of 40 nM CQ peptide. Complexes were separated from unbound RNAs by electrophoresis on a 20% non-denaturing polyacrylamide gel. 3'!UC RNA oligo alone (lane 1) or in the presence of CQ peptide (40 nM) (lane 2); duplex RNA IV either alone (lane 3) or in the presence of CQ (lane 4); 3' RNA oligo either alone (lane 5) or in the presence of CQ (lane 6); duplex RNA III either alone (lane 7) or in the presence of CQ (lane 8); duplex RNA II either alone (lane 9) or in the presence of CQ (lane 10); duplex RNA I either alone (lane 11) or in the presence of CQ (lane 12). Large and small arrow heads indicate duplex RNA and CQ peptide complex and duplex RNAs, respectively.

F i g u r e 1 3 . Inhibition of transcription from a CMV early promoter-driven template by mini 11G-31 and authentic TAR RNA in an extract of HeLa nuclei. A, The template containing the early promoter of CMV was transcribed in the absence (lane 1) and in the presence (lanes 3 and 4) of 100 pmole of TAR RNA, in the presence of 100 pmole of tRNA (total tRNA from yeast; lane 2), or in the presence of 100 pmole of mini 11G-31. Single-stranded DNA markers were loaded in lane M. The newly synthesized transcript is indicated by an arrow. B, The relative level of transcript (364 nt) synthesized in vitro

H. The effects of mini 11G-31 RNA in a cell-free transcription assay As demonstrated above, authentic TAR RNA inhibited the transcription of the CMV template in transcription assay in vitro . In order to examine the effect of the

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Yamamoto et al. Tat Aptamer was quantitated in three independent experiments (experimental variations are indicated by error bars).

392A; Applied Biosystems, USA). In the presence of the reverse primer 5'-GGGTTCCCTAGTTAGCCAGA-3', singlestranded DNA oligonucleotides were converted to doublestranded DNA (dsDNA) byTaq DNA polymerase (Nippon Gene, Japan). Each reaction was carried out in a 100-µl mixture that contained 10 mM Tris-HCl (pH 8.8), 50 mM KCl, 1.5 mM MgCl 2 , 0.1% Triton X-100, 0.2 mM dNTPs, 100 pmole of reverse primer, 78 pmole of DNA oligonucleotide and 2.5 units of Taq DNA polymerase (Takara, Japan). The reaction mixture was subjected to cycles of 94 °C for 1 min, 45 °C for 1 min and 68 °C for 2 min, until a product of the desired size was obtained. The resulting dsDNA template was precipitated in ethanol and transcribed by T7 RNA polymerase to generate TAR RNA or a mutant TAR RNA. Transcription in vitro was completed during incubations at 37 °C for 2 hours using a T7 Ampliscribe kit (Epicentre Technologies, USA). After the synthesis of RNAs and treatment with DNase I, reaction mixtures were fractionated by electrophoresis on a 10% denaturing polyacrylamide gel (PAGE). RNAs were extracted and recovered from the gel after ethanol precipitation.

isolated aptamer (mini 11G-31) on transcription of unrelated templates, we performed further transcription assays in extracts of HeLa cell nuclei (F i g . 13A). Addition of exogenous authentic TAR RNA (100 pmole) inhibited the transcription of the CMV-derived template by about 50-60%, as mentioned above (F i g . 13A, lanes 3 and 4). Transcription from the CMV promoter in the absence (F i g . 1 3 A , lane 1) or in the presence of 100 pmole of tRNA (total tRNA from yeast); (F i g . 13A, lane 2), and in the presence of 100 pmole of mini 11G-31 RNA (F i g . 1 3 A , lane 5) was unaffected or only marginally affected. Quantification of the results of three independent transcription experiments revealed that only TAR RNA inhibited transcription to a significant extent (F i g . 1 3 B ). Thus, the isolated aptamer bound to Tat peptides with high affinity as compared to authentic TAR RNA and it had no negative effect on the transcription of unrelated genes, as judged from the results of transcription in vitro. Therefore, it seems that mini 11G-31 RNA might be very useful as a Tat-specific decoy.

TAR-4 RNA was synthesized chemically. The functional groups were deprotected by established protocols (ABI manual) and the RNA was purified on a 15% PAGE.

B. Transcription assays in vitro in the presence of TAR RNA and its variants

III. Conclusion

In order to investigate the effects of TAR RNA on the cellular machinery at the transcriptional level, we used the cytomegalovirus (CMV) immediate early promoter that either contained or lacked enhancer elements. We chose CMV DNA as the template, as an example, for evaluation of the effect of TAR RNA on the LTR-independent transcription of a template. The CMV early-promoter region (from nt -238 to 364) was amplified by Taq DNA polymerase with specific primers [5'TTAGTCATCGCTATTACCATGG-3' and 5'-AGGCCT GGATTCACAGGACGGGTG-3'] by PCR [94 °C for 3 min, 50 °C for 1.15 min, and 72 °C for 3 min; 30 cycles]. The resulting product of PCR (602 nt) was recovered by ethanol precipitation and used in the transcription assay. The transcription reaction was carried out with an extract of HeLa cell nuclei (Promega, USA) in the presence of [#-32 P]CTP. Initially, 13 units of the nuclear extract, 3 mM MgCl2 , 0.4

The results obtained in this study demonstrate clearly, for the first time, that authentic TAR RNA inhibits transcription from a template under control of the CMV early promoter in a manner that is not related to the Tat/TAR interaction. By analyzing variants of TAR RNA, we identified potential sites in the RNA that were responsible for the inhibition of transcription, in addition to demonstrating the importance of the integrity of authentic TAR RNA for the interactions with several cellular factors in human cell lines. Using a strategy of genetic selection in vitro, we isolated an RNA aptamer, 11G-31, with high affinity to the Tat protein of HIV-1. Both full-length 11G-31 RNA and mini 11G-31 appeared to bind to the Tat peptides with a similar efficiency. The isolated aptamer had two TAR-like RNA motifs opposite one another which were found to assist in the high-affinity binding of the aptamer to Tat peptides. The absence of inhibitory effects on transcription by the aptamer, as compared to the inhibition by authentic TAR RNA, makes the mini 11G-31 RNA an attractive molecule for further analysis as a potential Tat decoy in infections by HIV-1. The next challenge is to test how efficiently the selected aptamer traps the Tat protein in a model system in human cell lines before we proceed to studies with live virus.

mM each ATP, GTP and UTP and 16 µM CTP plus 10 µCi [ #32 P]CTP (3,000 Ci/mmole; Amersham, U.K.) were combined in buffer [20 mM HEPES (pH 7.9), 100 mM KCl, 0.2 mM EDTA, 0.5 mM DTT and 20% glycerol], mixed with 100 pmole of TAR RNA, of a variant or of total tRNA from yeast (Boehringer Mannheim, Germany) and allowed to equilibrate for 15 min at 30 °C. This reaction mixture was supplemented with 100 ng of template DNA for PCR to give a final reaction volume of 25 µl and incubation was continued at 30 °C for a further 45 min. The reaction was terminated by addition of 175 µl of stop solution [0.3 M Tris-HCl (pH 7.4), 0.3 M sodium acetate, 0.5% SDS, 2 mM EDTA and 3 mg/ml of tRNA] and the products were extracted once with phenol and chloroform before precipitation in ethanol. The newly synthesized RNAs were denatured in loading buffer (25 mM EDTA and 4.5 M urea) at 90 °C for 5 min, loaded on a 6% polyacrylamide gel that contained 7 M urea and fractionated by electrophoresis. Bands on the gel were quantitated with an image analyzer (BAS 2000; Fuji Film, Japan).

IV. Experimental Procedures A. Preparation of TAR and mutant TAR RNAs Oligodeoxyribonucleotide templates containing the T7 promoter and sequences that corresponded to the RNAs shown in Figure 1 were synthesized with a DNA synthesizer (model

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C. Synthesis of Tat peptides and gel-shift assays Two Tat peptides that spanned the arginine-rich region of Tat protein were synthesized chemically: CQ (amino acids 3772, CFTTKALGISYGRKKRRQRRRPPQGSQTHQVSL SKQ, 36 mer); and RE (amino acids 49-86, RKKRRQRR RPPQGSQTHQVSLSKQPTSQSRGDPTGPKE, 38 mer). These peptides were purified by HPLC and their compositions were confirmed, after hydrolysis, by reverse-phase HPLC. CQ peptide was titrated against 5'-labeled TAR or a variant in an 8-µl binding reaction [10 mM Tris-HCl (pH 8.0), 70 mM NaCl, 2 mM EDTA, 40 nM total tRNA from yeast (Boehringer Mannheim) and 0.01% Nonidet P-40 (Shell Chemicals, USA)]. Initially, each labeled TAR RNA was denatured at 94 °C for 2 min and allowed to equilibrate at room temperature for 10 min before mixing with various concentrations of the peptide. The mixtures were incubated at 30 °C for 1 h and the complex and free RNAs were separated on a 15% non-denaturing polyacrylamide gel. The amount of each complex on the gel was quantitated with the image analyzer.

RNAs were allowed to compete additionally with another specific pool of competitor RNAs [a selected pool (12-18 N pool, with Tat-binding ability of about 5%)]. For the last two cycles, the concentration of Tat protein was reduced significantly. The binding buffer consisted of 50 mM TrisHCl (pH 7.8) and 50 mM KCl. Pool 0 RNA was pre-filtered through a prewetted nitrocellulose acetate filter (HAWP filter, 0.45 µm, 13.0 mm diameter; Millipore, USA) in a "Pop-top" filter holder (Nucleopore, USA) to select against RNAs that bound selectively to the filter. This pre-filtering was performed after each additional cycle. The Tat-RNA complexes were collected on a filter after each cycle of selection by washing with 1 ml of binding buffer. Bound RNAs were eluted from filters with 0.4 M sodium acetate, 5 mM EDTA and 7 M urea (pH 5.5) at 90 °C over the course of 5 min. After ethanol precipitation, reverse transcription and amplification by PCR were performed with AMV reverse transcriptase (Seikagaku, Japan) and Taq DNA polymerase (Nippon Gene), respectively, as described elsewhere (Urvil et al., 1997). In addition, a mutagenic PCR protocol (Leung et al., 1989) was also employed during the ninth, tenth and eleventh cycles. In these cycles, half of the cDNA reaction mixture was amplified as described above, while the remaining half was amplified in 100 µl of a reaction mixture for PCR that contained 67 mM Tris-HCl (pH 8.8), 16.6 mM (NH 4 )2 SO 4 , 6.1 mM MgCl2 , 6.7 mM EDTA (pH 8.0), 0.17 mg/ml BSA, 10 mM b-mercaptoethanol, 1% DMSO, 0.2 mM dATP, 1 mM each of dCTP, dGTP and dTTP, 0.5 mM MnCl 2 , 5 U of Taq DNA polymerase and 0.4 mM of each primer. The reaction mixture was cycled at 94 °C for 1.15 min, at 50 °C for 1.15 min and at 72 °C for 2.15 min for as many cycles as were needed to produce a band of a product of the appropriate size. The product from this PCR (ca. 0.25 µg) was combined with the product of the standard PCR (ca. 1.0 µg) prior to transcription with T7 RNA polymerase.

D. Tat protein and the RNA pool The Tat protein of HIV-1 that we used for selections was purchased from RepliGen (USA). Initially this Tat protein was tested for LTR-dependent trans-activation in a cell-free transcription assay with an extract of HeLa nuclei and it was demonstrated that the supplied Tat efficiently supported transactivation. The Tat was also analyzed for efficient binding to TAR. From its properties, we reasoned that the supplied protein contained active Tat protein of high purity (>90%). The RNA pool (169.1) for use in selections was described initially by Ellington and Szostak (1992). The RNAs in the pool contained 120 nt (N) random core region that was flanked by two constant regions for amplification, as follows: 5'GGGAGAATTCCGACCAGAAGCTT--120N--CATATGTGCG TCTACATGGATCCTCA-3'. The primers used for amplification of the pool were 5'-AGTAATACGACTCAC TATAGGGAGAATTCCGACCAGAAG-3' (designated 39.169) and 5'-TGAGGATCCATGTAGACGCACATA-3' (designated 24.169). In the selection cycles, yeast tRNA (Boehringer Mannheim) was used as a non-specific competitor.

After the eleventh cycle of selection, the product of PCR was ligated directly into the pCRII vector (Invitrogen, USA) in accordance with the protocol provided by Invitrogen. DNA was isolated from individual clones by the alkalinelysis method and sequenced with a Dye Terminator Sequencing Kit [Applied Biosystems Inc. (ABI)] on a DNA sequencer (model 373A; ABI).

F. Filter Binding Assay E. Selection in vitro

For evaluation of the binding activities of pool RNAs from different selection cycles, as well as those of individual aptamers, internally labeled RNAs were prepared using 0.5 µCi/ml [#-32 P]CTP. Conditions for binding and transcription in vitro were similar to those used for selection except that the molar ratio of RNA to Tat was 1:1 (330 nM : 330 nM). The filters were washed with 1 ml of binding buffer, air-dried, and radioactivity on filters was quantitated with the image analyzer (BAS2000). To ensure that the binding was specific, we added a ten-fold molar excess of tRNA as a nonspecific competitor to each binding reaction.

The protocol that we followed for selection in vitro resembled that reported by Urvil et al. (1997). The first cycle of selection was carried out in binding buffer that contained 5.0 µM (final concentration) RNA (representing approximately 4 x 10 13 RNA sequences) and 0.5 µM Tat protein (HIV-1). Before mixing of Tat protein and pool RNAs, initially, the RNAs in the pool were denatured in binding buffer at 90 °C for 2 min and allowed to cool at room temperature for 10 min to facilitate the equilibration of different conformers. The reaction mixture was incubated for 1 h and filtered as discribed elsewhere (Urvil et al., 1997). After each of the next five cycles, concentrations of pool RNAs were manipulated and RNAs were allowed to compete for binding to Tat in the presence of increasing concentrations of both non-specific RNA (E. coli tRNA) and specific competitor RNA (TAR RNA containing nts +18 through +44) up to the sixth cycle. From the seventh to the eleventh cycle, the pool

G. Gel-shift competitive binding assay The 5'-end labeled mini 11G-31 RNA and TAR RNA were mixed initially at a molar ratio of one to one. Various ratios of TAR to mini 11G-31 RNA, ranging from 1:1 to 1:200, were prepared after adjustment of concentrations with non-labeled

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Yamamoto et al. Tat Aptamer mini 11G-31 and TAR RNA (40 nM mini 11G-31 and increasing concentrations of TAR RNA, from 40-8,000 nM). Both RNAs were denatured at 94 째C for 2 min and then allowed to equilibrate at ambient temperature. The RNA samples were then allowed to bind to 80 nM RE peptide at 30 째C for 12 h, to allow quantitation of the RNA-protein complex at the equilibrium point. The reaction products were separated on a non-denaturing gel and the amounts of complexes formed by the two RNAs were analyzed.

Berkhout, B., and Jeang, K.T. (1 9 9 1 ). Detailed mutational analysis of TAR RNA: critical spacing between the bulge and loop recognition domains. N u c l e i c Acids R e s . 19, 6169-6176. Bohjanen, P.R., Colvin, R.A., Puttaraju, M., Been, M.D., and Garcia-Blanco, M.A. (1 9 9 6 ). A small circular TAR RNA decoy specifically inhibits Tat-activated HIV-1 transcription. N u c l e i c A c i d s R e s . 24, 3733-3738.

To characterize the individual aptamers from the eleventh selection cycle, we performed similar competitive binding assays, in which individual RNAs (10-100 nM) were allowed to compete with authentic TAR in the presence of 100 nM CQ peptide.

Calnan, B.J., Biancalana, S., Hudson, D., and Frankel, A.D. (1 9 9 1 a). Analysis of arginine-rich peptides from the HIV Tat protein reveals unusual features of RNA-protein recognition. G e n e s D e v . 5, 201-210. Calnan, B.J., Tidor,B., Biancalana, S., Hudson, D., and Frankel, A.D. (1 9 9 1 b). Arginine-mediated RNA recognition: the arginine fork. S c i e n c e 252, 11671171.

H. Synthesis of mini 11G-31 RNA duplexes In order to establish the importance of double TAR-like motif of 11G-31 for efficient binding to the Tat peptide (CQ), four strands of oligoribonucleotides were chemically synthesized to prepare four duplex RNAs after deleting the loop sequences from mini11G-31 RNA and their sequences are as follows: 5' RNA oligo (5'-ACGAAGCUUGAUCCCGAGAC3'), 3' RNA oligo (5'-GUCUCGGUCGAUCGCUUCGU-3'), 5'!UU RNA oligo (5-ACGAAGCGAUCCCGAGAC-3'), 3'!UC RNA oligo (5'-GUCUCGGGAUCGCUUCGU-3'). These oligo's functional groups were deprotected by established protocols (ABI manual) and purified on a 20% polyacrylamide gel.

Chang, Y.-N., and Jeang, K.-T. (1 9 9 2 ). The basic RNAbinding domain of HIV-2 Tat contributes to preferential trans-activation of a TAR-2-containing LTR. N u c l e i c A c i d s R e s . 20, 5465-5472. Churcher, M.J., Lmont, C., Hamy, F., Dingwell, C., Greem, S.M., Lowe, A.D., Butler, P.D.G., Gait, M.J., and Karn, J. (1 9 9 3 ). High-affinity binding of TAR RNA by the human immunodeficiency virus Tat protein requires amino acid residues flanking the basic domain and base pairs in the RNA stem. J . M o l . B i o l . 230, 90-110. Cordingly, M.G., La Femina, R.L., Callahan, P.L., Condra, J.H., Sardana, V.V., Graham, D.J., Nguyen, T.M., Le Grow, K., Gotlib, L., Schlabach, A.J., and Colonno, R.J. (1 9 9 0 ). Sequence-specific interaction of Tat protein and Tat peptides with the transactivation-responsive sequence element of human immunodeficiency virus type 1 in vitro. P r o c . N a t l . A c a d . S c i . U . S . A . 87, 8985-8989.

I. Transcription assay in vitro in the presence of mini 11G-31 RNA In order to determine whether or not the isolated aptamer mini 11G-31 RNA could interfere with transcription of unrelated templates, we performed transcription assays in vitro with extracts of HeLa cell nuclei as described above for studies of TAR.

Cullen, B.R. (1 9 8 6 ). Trans-activation of human immunodeficiency virus occurs via a bimodal mechanism. C e l l 46, 973-982. Cullen, B.R. (1 9 9 2 ). Mechanism of action of regulatory proteins encoded by complex retroviruses. M i c r o b i o l . R e v . 56, 375-394.

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Jones, K.A., and Peterlin, B.M. (1 9 9 4 ). Control of RNA initiation and elongation at the HIV-1 promoter. Annu. R e v . B i o c h e m . 63, 717-743. Kubota, S., Endo, S.-I., Maki, M., and Hatanaka, M. (1 9 8 8 ). Role of cysteine-rich region of HIV Tat protein in its trans-activation ability. Virus Genes 2, 113-118.

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Garcia-Martinez, L.F., Ivanov, D., and Gaynor, B. (1 9 9 7 ). Association of Tat with purified HIV-1 and HIV-2 transcription preinitiation complexes. J . B i o l . C h e m . 272, 6951-6958.

Lisziewicz, J., Sun D., Smythe, Lusso, P., Lori, F., Louie A., Markham, P., Rossi, J., Reitz, M., and Gallo, R.C. (1 9 9 3 ). Inhibition of human immunodeficiency virus type I replication by regulated expression of a polymeric Tat activation response RNA decoy as a strategy for gene therapy in AIDS. P r o c . N a t l . A c a d . S c i . U . S . A . 90, 8000-8004.

Gatignol, A., Buckler-White, A., Berkhout, B., and Jeang K.T. (1 9 9 1 ). Characterization of a human TAR RNAbinding protein that activates the HIV-1 LTR. S c i e n c e 251, 1597-1600. Gaynor, R. (1 9 9 2 ). Cellular transcription factors involved in the regulation of HIV-1 gene expression. AIDS 6, 347363.

Long, K.S., and Crothers, D.M. (1 9 9 5 ). Interaction of human immunodeficiency virus type 1 Tat-derived peptides with TAR RNA. B i o c h e m i s t r y 34, 8885-8895.

Gold, L., Polisky, B., Uhlenbeck, O., and Yarus, M. (1 9 9 5 ). Diversity of oligonucleotide functions. Annu. R e v . B i o c h e m . 64, 763-797.

Marciniak, R.A., Calnan, B.J., Frankel, A.D., and Sharp, P.A. (1 9 9 0 ). HIV-1 Tat protein trans-activates transcription in vitro. C e l l 63, 791-802.

Graham, G.J., and Maio, J.J. (1 9 9 0 ). RNA transcription of the human immunodeficiency virus trans-activation response element can inhibit action of the viral transactivator. P r o c . N a t l . A c a d . S c i . U . S . A . 87, 58175821.

Osborne, S.E., and Ellington, A.D. (1 9 9 7 ). Nucleic acid selection and the challenge of combinatorial chemistry. C h e m . R e v . 97, 349-370. Peterline, B.M., Luciw, P.A., Barr, P.J., and Walker, M.D. (1 9 8 6 ). Elevated levels of mRNA can account for the trans-activation of human immunodeficiency virus. P r o c . N a t l . A c a d . S c i . U . S . A . 83, 9734-9738.

Harrich, D., Hsu, C., Race, E., and Gaynor, R.B. (1 9 9 4 ). Differential growth kinetics are exhibited by human immunodeficiency virus type 1 TAR mutants. J . V i r o l . 68, 5899-5910.

Puglisi, J.D., Tan, R., Calnan B.J., Frankel, A.D., and Williamson, J.R. (1 9 9 2 ). Conformation of the TAR RNA-arginine complex by NMR spectroscopy. S c i e n c e 257, 76-80.

Harrich, D., Ulich, C., Garcia-Martinez, L.F., and Gaynor, R.B. (1 9 9 7 ). Tat is required for efficient HIV-1 reverse transcription. EMBO J. 16, 1224-1235.

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Urvil, P.T., Kakiuchi, N., Zhou, D.-M., Shimotohno, K., Kumar, P.K.R., and Nishikawa, S. (1 9 9 7 ). Selection of RNA aptamers that bind specifically to the NS3 protease of hepatitis C virus. E u r . J . B i o c h e m . 248, 130-138.

Rice, A.P., and Mathews, M.B. (1 9 8 8 ). Transcriptional but not translational regulation of HIV-1 by the Tat gene product. Nature 332, 551-553.

Vaishav, Y.N., and Wong-Staal, F. (1 9 9 1 ). The biochemistry of AIDS. A n n u . R e v . B i o c h e m . 60, 577-630.

Rosen, C.A., Sodroski J.G., and Haseltine W.A. (1 9 8 5 ). The location of cis-acting regulatory sequences in human T cell lymphotropic virus type III. C e l l 41, 813-823.

Verhoef, K., Tijms., and Berkhout, B. (1 9 9 7 ). Optimal Tatmediated activation of the HIV-1 LTR promoter requires a full-length TAR RNA hairpin. N u c l e i c A c i d s R e s . 19, 6169-6176.

Rounseville, M.P., and Kumar, A. (1 9 9 2 ). Binding of a host cell nuclear protein to the stem region of human immunodeficiency virus type 1 trans-activation-response RNA. J . V i r o l . 66, 1688-1694.

Weeks, K.M., Ampe, C., Schults, S.C., Steitz, T.A., and Crothers, D.M. (1 9 9 0 ). Fragments of the HIV-1 Tat protein specifically bind to TAR RNA. S c i e n c e 249, 1281-1285.

Rounseville, M.P., Lin, H.-C., Agbottah E., Shukla, R.R., Rabson, A.B., and Kumar, A. (1 9 9 6 ). Inhibition of HIV1 replication in viral mutants with altered TAR RNA stem structures. V i r o l . 216, 411-417.

Weeks, K.M., and Crothers, D.M. (1 9 9 1 ). RNA recognition by Tat-derived peptides: interaction in the major groove? C e l l 66, 577-588. Wu-Baer, F., Lane, W.S., and Gaynor R.B. (1 9 9 5 ). The cellular factor TRP-185 regulates RNA polymerase II binding to HIV-1 TAR RNA. EMBO J. 14, 5995-6009.

Roy, S., Delling, U., Chen, C. H., Rosen, C.A., and Sonenberg, N. (1 9 9 0 ). A bulge structure in HIV-1 TAR RNA is required for Tat binding and Tat-mediated transactivation. G e n e s D e v . 4, 1365-1373.

Wu-Baer, F., Lane, W.S., and Gaynor, R.B. (1 9 9 6 ). Identification of a group of cellular factors that stimulates the binding of RNA polymerase II and TRP-185 to human immunodeficiency virus 1 TAR RNA. J . B i o l . C h e m . 271, 4201-4208.

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Yamada. O., Kraus, G., Luznik, L., Yu, M., and Wong-Staal, F. (1 9 9 6 ). A chimeric human immunodeficiency virus type 1 minimal rev responsive element-ribozyme molecules exhibit dual antiviral function and inhibits cell-cell transmission. J . V i r o l . 70, 1596-1601.

Sodroski, J., Patarca, R. Rosen, C., Wong-Staal, F., and Haseltine, W.A. (1 9 8 5 ). Location of the trans-acting region on the genome of human T-cell lymphotropic virus type III. S c i e n c e 229, 74-77. Sullenger, B.A., Gallardo, H.F., Ungers, G.E., and Gilboa, E. (1 9 9 0 ). Overexpression of TAR sequences renders cells resistant to human immunodeficiency virus replication. C e l l 63, 601-608.

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Belongs to Table I a. The binding assays were performed either in the presence (P) or absence (NP) of Tat protein. Both Tat and individually labeled RNA pools from different cycles were incubated together and then filtered under similar conditions to those used during selection in vitro in the presence of a 10-fold excess a non-specific competitor (tRNA) in 100-Âľl binding buffer [50 mM TrisHCl (pH 7.5), 50 mM KCl]. # Specific RNA competitor (see Experimental Procedures)

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Gene Therapy and Molecular Biology, 1997, Vol. 1, page 467 Gene Ther Mol Biol Vol 1, 467-474. March, 1998.

Sequence-specific control of gene expression by antigene and clamp oligonucleotides Claude Hélène, Thérèse Garestier, Carine Giovannangeli, and Jian-Sheng Sun Laboratoire de Biophysique, Muséum National d'Histoire Naturelle, INSERM U 201 - CNRS URA 481, 43 Rue Cuvier 75231 Paris cédex 05, France.

_________________________________________________________________________________ Correspondence: Claude Hélène, Tel: (1) 40 79 37 08; Fax: (1) 40 79 37 05; E-mail biophy@mnhn.fr

Summary Gene expression can be artificially controlled by synthetic oligonucleotides that bind either to the gene itself or to its messenger RNA. Binding of an antigene oligonucleotide to the major groove of DNA involves the formation of a triple helix. Covalent attachment of an intercalating agent to the third strand oligonucleotide strongly stabilizes the triple-helical complex. Oligonucleotide-intercalator conjugates inhibit transcription factor binding and transcription initiation. Introduction of N3' P5' linkages into the oligonucleotide leads to stronger complexes that are able to arrest the transcription machinery at the elongation step. Cellular DNA is accessible to oligonucleotides within the chromatin structure of the cell nucleus as demonstrated for HIV proviral DNA in chronically infected cells. A triple helix can be formed on a single-stranded nucleic acid by an oligonucleotide made of two portions : one binds to the target sequence by forming Watson-Crick base pairs, the second one binds to this double-helical region to form a triple-helical complex. These clamp oligonucleotides are able to arrest replication of a single-stranded DNA or reverse transcription of a viral RNA. The antigene and clamp strategies can be adapted to a gene therapy protocol. A DNA expression vector is used to obtain a RNA transcript that can bind to DNA (antigene RNA) and block transcription.

to form a local triple helix (Thuong and Hélène, 1993). Alternatively an oligonucleotide may inhibit transcription by strand invasion of a double-helical template, as observed with PNAs (Peptide Nucleic Acids) (Nielsen et al., 1994). The targeted sequence may be located in the promoter or enhancer region of the gene or within the transcribed portion. A triple helix can also be formed on a single-stranded nucleic acid by clamp (Giovannangeli et al., 1991, 1993) or circular oligonucleotides (Kool, 1991; Wang and Kool, 1995). If the target is a RNA, these oligonucleotides are expected to inhibit translation of a messenger RNA or reverse transcription of a viral RNA. This review will deal with triple helix-forming oligonucleotides and their gene regulatory activities at both the transcriptional and translational levels.

I. Introduction Tissue-specific regulation of gene expression is achieved through binding of sequence-specific transactivating factors to DNA sequences upstream of the transcription start site. Gene expression can be artificially controlled with oligonucleotides according to several strategies (for review see Hélène, 1994). Antisense oligonucleotides bind to complementary sequences on messenger RNAs and inhibit translation of the message into the coded protein. Ribozymes are also targeted to messenger RNAs (or viral RNAs) and induce a catalytic cleavage of the recognized RNA, thereby inhibiting translation of the mRNA (or expression of the viral RNA). An oligonucleotide decoy can be used to sequester a transcription factor and control the expression of genes that are regulated by this transcription factor. Several genes are expected to respond to the oligonucleotide decoy due to the involment of each transcription factor in the regulation of gene families. Oligonucleotide aptamers can be targeted to proteins involved at any step of gene control and expression . Control of gene transcription can be achieved with antigene oligonucleotides that bind to double-helical DNA

II. The antigene strategy A. Sequence specific triple-helix formation on double-helical DNA Triple helix formation involves the recognition of Watson-Crick base pairs by hydrogen bonding interactions within the major groove of the double helix (Thuong and Hélène, 1993). Oligonucleotides and oligonucleotide 467

Hélène et al: Control of gene expression by antigene and clamp oligonucleotides analogues can wind around the double helix ; their orientation is dependent on base sequence (Figure 1). Recognition of the purines in T.A and C.G base pairs may be achieved by T and protonated C (C+ ), respectively, by forming Hoogsteen hydrogen bonds (as originally described by Hoogsteen). Pyrimidic oligonucle-

for (C,T)- containing oligonucleotides. However triple helices can be observed at pH7 if most cytosines have thymine and no cytosine neighbors. Alternatively, the purines of T.A and C.G base pairs can be recognized by A and G, respectively. A purinic oligonucleotide binds in an antiparallel orientation with respect to the target oligopurine sequence. The two base triplets T. A x A and C. G x G are not isomorphous ; therefore an adjustement of the backbone conformation is required to form a triple helix. The parallel orientation of (T, C) - containing oligonucleotides and the antiparallel one of (A, G) containing oligonucleotides assumes that all nucleotides adopt an anti conformation (Beal and Dervan, 1991). Such orientations have been experimentally observed in all experiments reported to date. A syn conformation of the nucleosides would lead to a reverse orientation. It should be noted that T and C+ can form base triplets with T. A and C. G base pairs, respectively, in a reverse Hoogsteen configuration that should lead to an antiparallel orientation of the (T, C) - containing third strand. This has never been observed with natural oligonucleotides, because T.A x T and C. G x C+ base triplets are isomorphous and the free energy of base triplet formation and stacking may be higher in the Hoogsteen as compared to the reverse Hoogsteen configuration. Oligonucleotides synthesized with G's and T's can also form triple helices with an oligopyrimidine•oligopurine sequence of double-helical DNA. The orientation of the (G,T)-containing oligonucleotide depends on base composition (number of 5' GpT 3' and 5' TpG 3' steps, length of G and T tracts). Parallel and antiparallel orientations involve Hoogsteen and reverse Hoogsteen configuration of the C•GxG and T•AxT base triplets, respectively (Sun et al., 1996). In order for the third strand oligonucleotide to wind smoothly around the major groove of DNA, all purines of the target sequence must be on the same strand of the double helix. Otherwise the backbone of the third strand would have to cross the major groove at the site where a pyrimidine interrupts the oligopurine tract. However it is possible to recognize a pyrimidine within an oligopurine sequence by a base forming a single hydrogen bond with the pyrimidine base. This possibility has been exemplified by introducing a guanine in a (C,T)-oligonucleotide to recognize a thymine in an oligopurine sequence, forming a non-canonical A•TxG base triplet. The energetics of this interaction depends on the flanking base triplets (Kiessling et al., 1992). It is also possible to enhance the binding energy by attaching an intercalating agent at the site facing

Figure 1: Base triplets formed by natural nucleosides with Watson-Crick T.A and C.G base pairs. The column on the lefthand side corresponds to the Hoogsteen configuration. The third strand runs parallel to the oligopurine target. The column on the right-hand side corresponds to the reverse Hoogsteen configuration. The third strand is antiparallel to the oligopurine strand of the double-helical target (Sun et al., 1996).

otides adopt a parallel orientation with respect to the oligopurine sequence. The two base triplets T. A x T and C. G x C + are isomorphous, i.e., the oligopyrimidine winds without any distortion of its backbone around the targeted double-helical sequence. The requirement for cytosine protonation to form a stable C. G x C+ base triplet makes the stability of triple helices pH-dependent 468

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Figure 2: Location of binding sites of triplex-forming oligonucleotides on genomic DNA. In A the oligonucleotide compete with the binding of transcriptional activators, in B with the basal transcription machinery ; in C the oligonucleotide may arrest the transcription machinery during the elongation step.

vitro transcription system (Cooney et al., 1988). Binding of an oligonucleotide to a transcription factor binding site competes with the binding of the regulatory protein and, thereby, modulates transcription initiation (Cooney et al., 1988, Maher et al., 1992, Grigoriev et al., 1992, Ing et al., 1993). Several in vitro transcription systems have been used to demonstrate this competitive inhibition. However the elongation process is much more difficult to inhibit because the stability of the triple-helical complex is usually not sufficient to arrest the transcription machinery once it is launched on its double-helical template. Two strategies have been described to achieve such a transcription arrest : i) the oligonucleotide can be covalently attached to an intercalating agent (Figure 3). The oligonucleotideintercalator conjugate binds more tightly to its target DNA due to the additional binding energy provided by intercalation at the triplex-duplex junction or within the triple-helical region (Sun et al., 1989 ; Giovannangeli et al., 1996 ; Silver et al., 1997) ; ii) chemical modifications of the oligonucleotide may provide the analogue with a tighter binding affinity. PNAs do bind tightly to double-helical DNA but they involve a strand-displacement reaction where two PNA molecules binds to an oligopurine sequence on one strand of the

the interruption of the oligopurine sequence (Zhou et al., 1995). The recognition of two oligopurine sequences alternating on the two strands of the DNA double helix can be achieved by two oligonucleotides linked to each other by a linker whose length and nature depends on the bases in the third strand and the site (5' PuPy 3' or 5' PyPu 3') where the third strand crosses the major groove (see Sun, 1995, for review).

B. Transcription inhibition by triple helixforming oligonucleotides The specificity of recognition of a double-helical target by an oligonucleotide provides the basis of the so-called "antigene" strategy to inhibit gene expression at the transcriptional level (HĂŠlĂ¨ne, 1991, 1994). When the target is located within the control region (promoter, enhancer), the bound oligonucleotide may inhibit transcription factor binding. When the oligonucleotide binds downstream of the transcription start site it may inhibit the elongation step of the transcription process (Figure 2). The possibility of inhibiting transcription by a (G,T)containing oligonucleotide was first described in an in 469

Hélène et al: Control of gene expression by antigene and clamp oligonucleotides double helix, forming a local triple helix, whereas the second strand (an oligopyrimidine sequence) remains single-stranded (Nielsen et al., 1994). Several other chemical modifications have been tested for their ability to form triple-helices (Escudé et al., 1993). 2'-O methyl pyrimidine oligonucleotides form more stable complexes than DNA and RNA oligonucleotides. A (C,T)-containing RNA binds more tightly than the corresponding DNA oligonucleotide to a DNA double helix. In contrast neither

2'-O-methyl nor RNA purine oligonucleotides form stable triple helices when compared to a DNA oligonucleotide. Among all chemical modifications tested so far, N3' ! P5' phosphoramidate linkages confer upon pyrimidine oligonucleotides a tighter binding than that of isosequential phosphodiester oligomers, even at pH 7 (Escudé et al., 1996). Purine oligophosphoramidates do not appear to form stable triple helices.

Figure 3 : Left : Schematic representation of a triple-helical complex where an oligonucleotide (black ribbon) wraps around the major groove of the double helix. The oligonucleotide can be covalently attached (star) to an intercalating agent that i) stabilizes the triplex (Sun et al., 1989), ii) induces chemical cleavage of the target site (François et al., 1989), iii) photo-induces cleavage of the target double helix (Perrouault et al., 1990), iv) can be used to cross-link the two strands of DNA under UV irradiation (Takasugi et al., 1991 ; Giovannangeli et al., 1997).

#B and SRF). When a pyrimidine oligonucleotide tethered to an intercalating agent binds to this sequence the triplehelical complex inhibits the binding of the transcription factors in vitro (Grigoriev et al., 1992). After transfection of the plasmid into lymphocytic cell lines, the oligonucleotide-intercalator conjugate inhibits transcription of a reporter gene (CAT) placed downstream of the IL2R" promoter. A mutant of the target sequence, where three pyrimidines interrupt the oligopurine target sequence, is unable to form a triple-helical complex with the oligonucleotide-intercalator conjugate but does not prevent transcription factor binding. The oligonucleotideintercalator conjugate does not exhibit any inhibitory effect on transcription from the mutated IL2R" promoter in contrast to the wild-type sequence (Grigoriev et al., 1993). This experiment demonstrates that the effect of the oligonucleotide-intercalator conjugate is indeed due to binding to the targeted DNA sequence in the IL2R" promoter and not to another cellular component (e.g, a transcription factor) involved in controlling transcription from this promoter.

C. Sequence specificity of transcription inhibition by antigene oligonucleotides Oligonucleotide-intercalator conjugates, PNA and oligophosphoramidates have been shown to inhibit transcription in vitro in a sequence-specific manner. The data available within cells are more scarce. Very few studies provide evidence that the effect observed on gene expression is due to oligonucleotide binding to DNA to form a triple-helical complex. In some cases the observed effect on transcription might be due to binding of the oligonucleotide to a transcription factor rather than to DNA. When the gene of interest is carried by a plasmidic vector it is possible to provide evidence for the involvement of triple helix formation in the inhibi tion of gene transcription by introducing mutations in the target sequence. For example in the promoter region of the asubunit of the interleukin-2 receptor (IL2R") there is a 15 base pair oligopyrimidine•oligopurine sequence which overlaps the binding sites of two transcription factors (NF-

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Hélène et al: Control of gene expression by antigene and clamp oligonucleotides helix formation. They clearly indicate that the target site has been reached by the oligonucleotide within cells. However, until now the target sites have been limited to plasmidic vectors and not to endogenous genes. The yield of mutations reflects only a fraction of the cross-linked sites since it is expected that DNA repair systems remove part of the cross-links to restore the original sequence of DNA (Wang et al., 1995, Sandor and Bredberg, 1994, Raha et al., 1996).

In most cases, however, especially for an endogenous gene, it is difficult to construct a mutant of the target sequence. The control experiments rely upon changes of the oligonucleotide sequence with all the problems associated with this type of control since all potential interactions of the oligonucleotide (including self associaiton) may change upon sequence alterations.

D. Accessibility of DNA in cell nuclei to triple helix-forming oligonucleotides

III. Clamp oligonucleotides

One of the main questions raised by the development of the antigene strategy in vivo deals with the accessibiity of the target sequence within the chromatin structure in the cell nucleus. In order to answer this question we have developed a strategy based upon using oligonucleotidepsoralen conjugates. When such a conjugate forms a triple-helical complex with DNA, the psoralen moiety can be cross-linked to one or both strands of the double helix upon UV irradiation (Takasugi et al., 1991). The crosslink arrests DNA replication when a restriction fragment containing the target sequence is used as a template for exponential (PCR) or linear amplification using primers flanking the target sequence. In linear amplification using a single primer a truncated product is obtained when replication is arrested at the cross-linked site. If PCR is used the inhibitory effect of the cross-link can be quantitated by using quantitative PCR methods. Alternatively if the site of triple helix formation and crosslinking overlaps a restriction site it is possible to reveal the inhibition of restriction enzyme cleavage at this particular site by using probes that overlap the targeted DNA region. The absence of inhibition at other restriction sites for the same enzyme provides an internal control of the sequence specificity of the cross-linking reaction and, therefore, of triple helix formation. The first strategy (linear amplification) has been used for a plasmidic vector carrying the IL2R" promoter sequence (Guieysse et al., 1996). The second (PCR) and third (cleavage inhibition) strategies have been used to demonstrate the accessibility of the proviral HIV sequence in chronically-infected cells (Giovannangeli et al., 1997). The results presently available show that the DNA sequences that have been used as targets for oligonucleotide-psoralen conjugates are indeed accessible within the chromatin structure of cell nuclei. This might not be true of all targeted sequences due to the nucleosomal structure of chromatin. If the oligonucleotide interacts with a sequence where transcription factors bind to activate transcription it is likely that the oligonucleotide may have access to its target sequence as do transcription factors. Kinetic parameters might play an important role inasmuch as some triple-helical complexes exhibit a slow rate of formation as compared to protein binding (Maher et al., 1990, Rougée et al., 1992). Oligonucleotide-psoralen conjugates have been used to induce site-specific mutations on plasmids. These mutations are located at the specific site where psoralen cross-linking is induced by UV irradiation after triple

An oligopurine sequence on a single-stranded nucleic acid can be recognized by a complementary (antisense) oligonucleotide. The short double helix with an oligopyrimidine•oligopurine sequence can, in turn, be recognized by a third strand oligonucleotide to form a triple helix. The two oligonucleotides can be linked to each other to form a unique molecule that can clamp the target sequence on the single-stranded template (Giovannangeli et al., 1991) (Figure 4). The nature of the third strand (oligopyrimidine, oligopurine or (G,T)oligonucleotide) determines its orientation with respect to the oligopurine target sequence. Therefore the linker between the antisense and the "antigene" portions will join a 3'- to a 3'-end or a 5'- to a 5'-end (for an antiparallel orientation of the third strand) or a 3'- to a 5'-end (for a parallel orientation). In the last case a circular oligonucleotide can also be synthesized (Kool, 1991). For (mostly) entropic reasons the circular oligonucleotide will bind more tightly than the clamp oligonucleotide which in turn binds much more tightly than two separate oligonucleotides (at least in the micromolar range of concentrations). Clamp oligonucleotides have been shown to inhibit primer extension by DNA polymerase on a single-stranded template under conditions where antisense oligonucleotides are devoid of any inhibitory activity (Giovannangeli et al., 1993). They can also arrest reverse transcription on a single-stranded RNA template. We have recently shown (C. Giovannangeli et al.,, unpublished results) that a clamp oligonucleotide is able to block HIV infection of CD4-positive cells at an early step after infection, most likely reverse transcription. No proviral DNA is detected after viral infection. Control experiments were carried out with a modified sequence of the clamp oligonucleotide and, more importantly (see above for antigene oligonucleotides), with a mutated version of the target sequence using the same clamp oligonucleotide. In both cases no inhibition of viral infection was observed indicating that the inhibitory effect on the wild-type sequence is likely due to clamp formation. An antisense oligonucleotide targeted to the same sequence exhibited no inhibition. Clamp oligonucleotides can be covalently attached to an intercalating agent. If the antisense portion is made a little longer than the third strand portion, intercalation can lock the complex in place on the single-stranded target (Giovannangeli et al., 1993). 471

HĂŠlĂ¨ne et al: Control of gene expression by antigene and clamp oligonucleotides

Figure 4: Oligonucleotide clamps formed of two portions connected by a loop ; they form both Watson-Crick and Hoogsteen hydrogen bonds with the single-stranded target. Attachment of an intercalator to one end of the clamp oligonucleotide stabilizes the complex (provided the Watson-Crick portion is made longer than the Hoogsteen portion) (Giovannangeli et al., 1991, 1993).

potential of a rat glioblastoma cell line (C6) in nude mice and in syngenic rats. As observed with episomal DNA vectors expressing an antisense RNA, inhibition of IGF1 or IGF1-R expression induces an immune response in syngenic rats which leads to a shrinkage of grafted tumors (Trojan et al., 1993). Further experiments are presently under way on chemically-induced hepatocarcinoma in rats (Frayssinet et al., 1997). The antigene strategy could form the basis of a novel gene therapy approach to control the expression of specific genes, as does the antisense strategy.

IV. Gene therapy protocols based on the antigene strategy Antisense RNAs can be generated in cells by transcription of appropriate DNA vectors. It is conceivable to use a DNA vector to express an RNA that could form a triple helix with a targeted sequence on cellular DNA. Recent experiments described by Judith and Joseph ILAN's group suggest that a potential triple helix-forming RNA can be obtained from an episomal vector. They observed the biological responses expected for transcription inhibition. The target gene was either IGF1 (Shevelev et al., 1997) or its receptor IGF1-R (Rininsland et al., 1997). In both cases an oligopyrimidine-oligopurine sequence was used as a target, in the 5'-untranslated region of the IGF1 gene or in the 3'-untranslated region of the IGF1-R gene. Only the vector expressing the oligopurine third strand RNA sequence was shown to inhibit transcription of either gene. This oligopurine sequence was inserted in a much longer RNA transcript whose folding might play an important role in the biological effect. No evidence has been provided yet for the formation of a triple helix by the RNA transcript and the target gene. However several control sequences did not exhibit any activity. For example the RNA transcript inhibiting IGF1 did not show any activity on IGF1-R transcription. Inhibition of IGF1 or IGF1-R by a potential antigene (triple helix) mechanism inhibits the tumorigenic

V. Conclusion Triple helix formation represents an alternative to antisense oligonucleotides to control gene function. Antigene oligonucleotides targeted to the DNA double helix can inhibit transcription. Clamp oligonucleotides targeted to a viral sequence can inhibit reverse transcription. They might also inhibit translation of a messenger RNA (even though there is no data yet available on translation inhibition). Circular oligonucleotides forming a triple helix with a singlestranded template might also be useful in both approaches. Strand displacement reactions as observed with PNAs (which involve triple helix formation by two PNAs on one of the two DNA strands) might represent an alternative to antigene oligonucleotides which bind to DNA without any opening of the double helix. Further

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Hélène et al: Control of gene expression by antigene and clamp oligonucleotides molecular modeling studies. Nucleic Acids Res. 21, 55475553.

experiments with oligonucleotide analogues that form stable triple helices (e.g., oligophosphoramidates) will tell us whether the antigene or clamp strategies can be applied to biologically-relevant in vivo situations. Antisense oligonucleotides have reached the stage of clinical trials in several pathological disorders. The information gained on bioavailability, pharmacokinetics, delivery, routes of administration... will be useful in any development of antigene oligonucleotides. Whether there is any advantage in targeting the gene rather than its messenger RNA (or pre-mRNA) remains to be determined in each particular case. Nuclease-resistant analogues (such as N3' ! P5' oligophosphoramidates) could have long-lasting effects on gene transcription. The lower number of targets (two alleles for each gene) as compared to messenger RNAs might be an obvious advantage, especially for oligonucleotide analogues, such as oligophospho-ramidates, that do not bind strongly to cellular proteins. This should allow us to obtain a biological response at rather low oligonucleotide concentrations provided the target sequence is accessible within the chromatin structure of cell nuclei. Recent experiments have indeed shown this to be the case (Guieysse-Peugeot et al., 1996 ; Giovannangeli et al., 1997). In addition there might be proteins within cells that bind strongly to triple-helical complexes formed upon binding of antigene oligonucleotides to their target DNA sequence. Proteins with these charcteristics have been recently described (Kiyama et al., 1991 ; Guieysse-Peugeot et al., 1997). Target sequences for antigene oligonucleotides remain limited to oligopyrimidine•oligopurine tracts of double-helical DNA. The design of nucleoside analogues or modifications of oligonucleotides involving, e.g., the insertion of intercalating agents should allow us to extent the range of triple helixforming DNA sequences. Together with minor groovebinding ligands with an increased range of sequencespecific recognition (Gottesfeld et al., 1997), major groove-specific ligands such as antigene oligonucleotides provide a new way of controlling gene transcription in vivo. The parallel development of a gene therapy approach based on triple helix formation opens new possibilities to control gene expression in pathological disorders.

Escudé C., C. Giovannangeli, J.S. Sun, D.H. Lloyd, J.K. Chen, S. Gryaznov, T. Garestier & C. Hélène (1996) Stable triple-helices formed by oligonucleotide N3' ! P5' phosphoramidates inhibit transcription elongation. Proc. Natl. Acad. Sci. USA 93, 4365-4369. François J.C., T. Saison-Behmoaras, C. Barbier, M. Chassignol, N.T. Thuong & C. Hélène (1989) Sequencespecific recognition and cleavage of duplex DNA via triple-helix formation by oligonucleotides covalently linked to a phenanthroline-copper chelate. Proc. Natl. Acad. Sci. USA 86, 9702-9706. Giovannangeli C., T. Montenay-Garestier, M. Rougée, M. Chassignol, N.T. Thuong & C. Hélène (1991) Singlestranded DNA as a target for triple helix formation. J. Am. Chem. Soc. 113, 7775-7777. Giovannangeli C., N.T. Thuong & C. Hélène (1993) Oligonucleotide clamps arrest DNA synthesis on a singlestranded DNA target. Proc. Natl. Acad. Sci. USA 90, 10013-10017. Giovannangeli C., L. Perrouault, C. Escudé, N.T. Thuong & C. Hélène (1996) Specific inhibition of in vitro transcription elongation by triplex-forming oligonucleotide-intercalator conjugates targeted to HIV proviral DNA. Biochemistry 35, 10539-10548. Giovannangeli C., S. Diviacco, V. Labrousse, S.M. Gryaznov, P. Charneau & C. Hélène (1997) Accessibility of nuclear DNA to triplex-forming oligonucleotides : the integrated HIV1 provirus as target. Proc. Natl. Acad. Sci. USA 94, 79-84. Gottesfeld J.M., L. Neely, J.W. Trauger, E.E. Baird, P.B. Dervan (1997) Regulation of gene expression by small molecules. Nature 287, 202-205. Grigoriev M., D. Praseuth, P. Robin, A. Hemar, T. SaisonBehmoaras, A. Dautry-Varsat, N.T. Thuong, C. Hélène & A. Harel-Bellan (1992) A triple helix-forming oligonucleotide-intercalator conjugate acts as a transcriptional repressor via inhibition of NF KB binding to IL-2 receptor " regulatory sequence. J. Biol. Chem. 267, 3389-3395. Grigoriev M., D. Praseuth, A.L. Guieysse, P. Robin, N.T. Thuong, C. Hélène & A. Harel-Bellan (1993) Inhibition of interleukin-2 receptor "-subunit gene expression by oligonucleotide-directed triple helix formation. C. R. Acad. Sci. Paris, Sciences de la vie/Life Sciences 316, 492-495. Guieysse A.L., D. Praseuth, C. Hélène (1997) Identification of a triplex DNA-binding protein from human cells. J. Mol. Biol. 267, 289-298.

References Beal P.A & P.B. Dervan (1991) Second structural motif for recognition of DNA by oligonucleotide-directed triplehelix formation. Science 251, 1360-1363.

Guieysse A.L., D. Praseuth, M. Grigoriev, A. Harel-Bellan, C. Hélène (1996) Detection of covalent triplex inside human cells. Nucleic Acids Res. 24, 4210-4216.

Cooney M., G. Czernuszewicz, E.H. Postel, S.J. Flint & M.E. Hogan (1988) Site-specific oligonucleotide binding represses transcription of the human c-myc gene in vitro. Science 241, 456-459.

Hélène C. (1991) The anti-gene strategy : control of gene expression by triplex-forming-oligonucleotides. Anticancer Drug Design 6, 569-584.

Escudé C., J.C. François, J.S. Sun, G. Ott, M. Sprinzl, T. Garestier & C. Hélène (1993) Stability of triple helices containing RNA and DNA strands : experimental and

Hélène C. (1994) Control of oncogene expression by antisense nucleic acids. Europ. J. Cancer 30A, 1721-1726.

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Ing N.H., J.M. Beekman, D.J. Kessler, M. Murphy, K. Jayaraman, J.G. Zendegui, M.E. Hogan, B.W. O'Malley & M.J. Tsai (1993) In vivo transcription of a progesteroneresponsive gene is specifically inhibited by a triplexforming oligonucleotide. Nucleic Acids Res. 21, 27892796.

Silver G.C., J.S. Sun, C.H. Nguyen, A.S. Boutorine, E. Bisagni, C. Hélène (1997) Stable triple-helical DNA complexes formed by benzopyridoindole- and benzopyridoquinoxaline- oligonucleotide conjugates. J. Am. Chem. Soc. 119, 263-268.

Kiessling L.L., L.C. Griffin & P.B. Dervan (1992) Flanking sequence effects within the pyrimidine triple-helix motif characterized by affinity cleaving. Biochemistry 31, 28292834.

Sun J.S., J.C. François, T. Montenay-Garestier, T. SaisonBehmoaras, V. Roig, N.T. Thuong, C. Hélène (1989) Sequence-specific intercalating agents : intercalation at specific sequences on duplex DNA via major groove recognition by oligonucleotide-intercalator conjugates. Proc. Natl. Acad. Sci. USA 86, 9198-9202.

Kiyama R. & R.D. Camerini-Otero (1991) A triplex DNAbinding protein from human cells : purification and characterization. Proc. Natl. Acad. Sci. USA 88, 1045010454.

Sun J.S. (1995) Rational design of switched triple helixforming oligonucleotides: extension of sequences for triple helix formation. In "Modelling of Bimolecular Structures and Mechanisms, A. Pullman et al. (Eds), Kluwer Academic Publishers, Netherlands, pp. 267-288.

Kool E.T. (1991) Molecular recognition by circular oligonucleotides : increasing the selectivity of DNA binding. J. Am. Chem. Soc. 113, 6265-6266. Lafarge-Frayssinet C., H.T. Duc, C. Frayssinet, A. Sarasin, D. Anthony, Y. Guo, J. Trojan (1997) Antisense IGF I transfer into a rat hepatoma cell line inhibits tumorigenesis by modulating MHC-I cell surface expression. Cancer Gene Therapy 4, 276-285.

Sun J.S. T. Garestier & C. Hélène (1996) Oligonucleotide directed triple helix formation. Curr Opin Struct Biol 6, 327-333. Takasugi M., A. Guendouz, M. Chassignol, J.L. Decout, J. Lhomme, N.T. Thuong & C. Hélène (1991) Sequencespecific photo-induced cross-linking of the two strands of double-helical DNA by a psoralen covalently linked to a triple helix-forming oligonucleotide. Proc. Natl. Acad. Sci. USA 88, 5602-5606.

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Rininsland F., T.R. Johnson, C.L. Chernicky, E. Schulze, P. Burfeind, J. Ilan, J. Ilan (1997) Suppression of insulin-like growth factor type I receptor by triple-helix strategy inhibits IGF-I transcription and tumorigenic potential of rat C6 glioblastoma cells. Proc. Natl. Acad. Sci. USA 94, 5854-5859. Rougée M., B. Faucon, J.L. Mergny, F. Barcelo, C. Giovannangeli, T. Montenay-Garestier & C. Hélène (1992) Kinetics and thermodynamics of triple helix formation : effects of ionic strength and mismatches. Biochemistry 31, 9269-9278. Sandor Z. & A. Bredberg (1994) Repair of triple helix directed psoralen adducts in human cells. Nucleic Acids Res. 22, 2051-2056. Shevelev A., P. Burfeind, E. Schulze, F. Rininsland, T.R. Johnson, J. Trojan, C.L. Chernicky, C. Hélène, J. Ilan, J. Ilan (1997) Potential triple helix-mediated inhibition of IGF-I gene expression significantly reduces tumorigenicity

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Physical approaches to the study of chromatin fibers Kensal van Holde* , Sanford H. Leuba* and Jordanka Zlatanova*,# *Department of Biochemistry and Biophysics, Oregon State University, Corvallis, OR 97331-7305 USA and #Institute of Genetics, Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria __________________________________________________________________________________________________ Correspondence to: Prof. Jordanka Zlatanova, Department of Biochemistry and Biophysics, Oregon State University Corvallis, OR 97331-7305, USA, Phone: (541) 737 4581, Fax: (541) 737 0481, E-mail: zlatanoj@ucs.orst.edu

Summary Investigations of the structures of complex macromolecular assemblies (like chromatin fibers, microtubules, etc.) have traditionally utilized two approaches, which we term macroscopic and microscopic. The macroscopic methods include hydrodynamic and radiation-scattering techniques. While applicable to molecules in solution, they present results which are only averages over the molecules in the sample. If, as is usually the case, these structures are heterogeneous, interpretation can become hopelessly ambiguous. At the other extreme, the traditional highresolution microscopic techniques (transmission electron microscopy and its numerous variants) while sensitive to even local variations in structure, impose often devastatingly harsh conditions on delicate biological structures. Recently, two kinds of microscopic methods have been developed which hold great promise for studies of macromolecular assemblies. The first is cryo-electron microscopy, which allows preservation of much of solution structures. Second, and potentially even more promising, are the various scanning probe microscopic methods, especially scanning force microscopy. In its present stage of development, this technique allows detailed structural studies under relatively mild conditions. Together, cryo-electron microscopy and scanning force microscopy have already provided new insights into the static structure of chromatin. Even more exciting are the prospects for imaging in liquid media, now under development. These hold promise for study of not only the statics, but dynamics as well, for functionally important structures like chromatin fibers.

study may be much more complex than this, often requiring the presence of ongoing metabolic processes. Two general approaches have been taken toward the physical study of such structures. In one class of techniques, which we shall call macroscopic, a solution of the macromolecular structures, in a defined solvent medium, is examined by one or another of various hydrodynamic methods or by the scattering of radiation (light, X-rays, neutrons). These techniques have the advantage that the medium can be made to at least approximate the in vivo environment, and can be varied at will. On the other hand, these methods suffer from the major disadvantage that only average properties of a highly heterogeneous molecular population are observed. In the second class of techniques, which we call microscopic, individual macromolecular complexes are observed by one or another of several microscopic methods capable of resolution to a few nanometers or better. This allows, in principle, the observation of local variations in the fiber, or perturbations in its structure, without the disadvantage of averaging. Until very recently, however, all microscopic methods capable of the requisite resolution required conditions of fixation,

I. Introduction Elucidation of the fine structures of complex, irregular, and asymmetric macromolecular assemblies has always presented an especially difficult problem for molecular biologists. As molecular biology ascends toward the cellular level, the fact that structures which exhibit neither regular periodicity nor symmetry clearly constitute a major class of higher order subcellular organization makes the problem of increasing importance. An example which has attracted much attention in recent years is the interphase chromatin fiber. Despite efforts to impose regularity in terms of various kinds of model fiber folding, it is clear even from studies of composition that the fiber must be heterogeneous along its length. It also must be subjected to major perturbations in vivo as processes like transcription and replication occur. The problems presented by heterogeneity are further complicated by the fact that we wish to study objects like the chromatin fiber in an environment as close as possible to the physiological one. This means, at a minimum, that certain ionic conditions should be maintained in the surrounding medium; in actuality the proper conditions for

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van Holde et al: Physical approaches to the study of chromatin fibers staining, and/or dehydration wholly incompatible with the native environment of macromolecular assemblies. It is the purpose of this review to briefly describe the advantages and limitations of each kind of technique, and to attempt to point out the new directions which methodology is taking in attempts to circumvent the restrictions described above. Although we will concentrate, for an example, on the chromatin fiber, much of what will be described is applicable to any of the giant, irregular macromolecular assemblies which form the functional components of cells.

II. Macroscopic techniques The classical methods for the study of the structures of macromolecular assemblies in solution can be divided into hydrodynamic and scattering methods. These are all old techniques; the basic principles were elucidated in the period between 1920 and 1940; the subsequent advances have been mainly in applications and instrumentation.

A. Hydrodynamic studies of chromatin fibers. Sedimentation and diffusion measurements have found their principal application to study of chromatin fibers by virtue of the fact that both the sedimentation coefficient (S) and the diffusion coefficient (D) are related to the frictional coefficient (f). That is

S = M(1-! " #)/Nf (1) D = RT/Nf (2) where M denotes molecular mass, ! " partial specific volume, # solution density, N Avogadro's number, R the gas constant and T the absolute temperature. The frictional coefficient is a measure of the resistance offered Figure 1. Two different models for a dodecameric oligonucleosome that predict almost exactly the same sedimentation coefficient. In Panel A is shown one possible conformation of "beads-on-a-string" model, in which the absence of linker histones has led to partial unwrapping of DNA from histone cores, and a random-coil arrangement of nucleosomes. In Panel B is a planar zig-zag model, with fixed linker lengths, 1.75 turns of DNA per core particle, and 90Ë&#x161; linker-linker angles. Both, using the Kirkwood theory for frictional coefficients (van Holde, 1985), predict about 29S for the sedimentation coefficient of a dodecamer of 11S subunits.

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by the surrounding solution to motion of a macromolecule. It depends upon the size and shape of the particle, but in a manner so complex that it is difficult or impossible to extract quantitative structural information on complex structures from a measure of f. However, changes in the frictional coefficient can be readily measured and can often be interpreted, in at least a semi-quantitative way, in terms of changes in fiber structure. To take one example: the extensive studies of Thomas and coworkers (Butler and Thomas, 1980; Thomas and Butler, 1980), using carefully purified chromatin fractions, exhibit clear evidence for condensation and expansion of chromatin fibers in response to changes in the ionic environment. If sedimentation measurements are to be interpreted in terms of f, one must ascertain in some manner that association of fibers (which would change M) is not involved. Often, such methods are most powerful when applied to simplified systems. An example is found in the study of "chromatin" formed by reconstitution of histone octamers onto dodecameric repeats of a sea urchin 5S gene. Each repeating unit of the construct forms, upon addition of histone octamers, a quite accurately positioned nucleosome, so that the dodecameric repeat contains twelve nearly equally spaced nucleosomes. With such reconstitutes, one has both homogeneity and nearregularity of structure, and it has been possible in such cases to predict the frictional coefficient (or sedimentation coefficient) expected for various possible foldings of the fiber (Hansen et al., 1989). Because the molecules are homogeneous in size, it was easy to eliminate the possibility of aggregation by sedimentation equilibrium studies. Unfortunately, even in such idealized cases a residuum of ambiguity remains, for there exists a range of different structures which can provide the same value of the sedimentation coefficient (Figure 1). Thus, no unique solution with respect to structure is provided.

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recognized by other techniques. This illustrates a common failing of scattering methods; except for extremely regular

The major disadvantage of such methods, obviously, is that a single average quantity is measured, and variations in that quantity can be interpreted in multiple ways. Only in the very simplest systems does interpretation become unambiguous. Thus, for example, the fact that the sedimentation coefficient of dinucleosomes does not change with salt concentration under conditions where the value for trinucleosomes does argues strongly against linker bending as salt increases (Butler and Thomas, 1980; van Holde and Zlatanova, 1996). In a similar vein we note that although early hydrodynamic studies of chromatin fibers did not suggest nucleosomal structure, such experiments were very important in the initial characterization of the nucleosomes themselves (Sahasrabuddhe and van Holde, 1974). Other hydrodynamic methods, which involve rotational diffusion of macromolecules (electric dichroism and electric birefringence, for example) have been employed infrequently in chromatin studies (see van Holde, 1988, for details). The measurement of rotational diffusion is exceedingly sensitive to changes in molecular asymmetry, which is an advantage (van Holde, 1985). However, it is also very sensitive to heterogeneity, and chromatin fiber fragments are rarely of even approximately uniform size. It is our opinion that the era in which populationaveraging hydrodynamic methods provided important information about macromolecular structures is drawing to a close, except for application to very special problems. Too little information is gained from time-consuming experiments that require, in most cases, relatively large quantities of sample.

Figure 2. The kinds of information that can be obtained from low-angle scattering. One way to treat the data is shown here, where the log of scattering intensity at angle ($) is plotted versus sin 2 $. The intercept (A) at $=0 gives the mass of the scattering particles, the initial slope (B) gives the radius of gyration, and the pattern of maxima and minima at higher angles is sensitive to internal structure of the particles. For long, rod-like particles like chromatin fibers, a different analysis of the same kind of data can yield the mass per unit length and cross-section radius of gyration.

structures (such as crystals) scattering measurements do not define structures. Rather, like hydrodynamic methods, they can provide measures of specific average quantities (mass per unit length, cross-section radius of gyration, etc.). The much less intense scattering observed at higher angles provides the possibility of extracting further information (see Figure 2), but even so, the problem usually reduces to one of fitting one or another model to the scattering curve. Finally, unless samples can be highly oriented (and chromatin fibers have been notoriously difficult to orient) all measurements involve an averaging over rotationally random particles, which necessarily loses information. Nevertheless, once the nucleosomal repeating structure of chromatin was deduced, a variety of scattering methods were quickly applied to provide further information about the nucleosomes and the polynucleosomal fiber. These included neutron scattering studies of the nucleosomal core particle (Pardon et al., 1975; Suau et al., 1977) which provided the first physical evidence that the DNA was coiled outside a histone core, in addition to light scattering (Campbell et al., 1978), low angle X-ray (Finch and Klug, 1976) and neutron scattering (Suau et al., 1979) studies of the fibers. The fiber studies were particularly useful in corroborating the early evidence on chromatin fiber structure from electron microscopy, demonstrating that the

B. Scattering methods. Studies of the scattering of various kinds of radiation (light, X-rays, neutrons) from solution can provide information concerning the dimensions, and in some cases, internal structure of dissolved macromolecular assemblies. This is basically because the way in which scattered intensity varies with angle reflects constructive and destructive interference between photons scattered from various parts of the structure (Figure 2). The shorter the wavelength, the finer the detail that can be probed. The use of the scattering of radiation to investigate chromatin fiber structure has a long history. Indeed, soon after the remarkable successes obtained with X-ray scattering from DNA fibers, attempts were made to carry out similar studies with fibers of chromatin (see, for example, Wilkins et al., 1959; Pardon et al., 1967). However, it has proved difficult to obtain well-oriented fibers of chromatin, so others turned to low-angle X-ray scattering of unoriented samples in solution (see Bram and Ris, 1971, for an early example). None of these early scattering studies was interpreted in a manner to suggest the kind of repeating nucleosomal structure soon to be

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van Holde et al: Physical approaches to the study of chromatin fibers dimensions such as the average cross-section radius of gyration and the average mass per unit length observed in aqueous solution were at least broadly commensurate with those observed under the extreme conditions of dehydration required for electron microscopy (see below). The possibility of using contrast-variation (see van Holde, 1985), gives neutron scattering special advantages in the study of chromatin fibers. Because the DNA and protein components of the chromatin fiber have distinctly different neutron scattering power, it has been possible to provide some evidence concerning the distribution of mass in the fibers. Further, the fact that deuterated proteins scatter neutrons differently than proteins containing only 1H has allowed investigation of the (again averaged) position of linker histones within the fiber; to that end, chromatin fibers were depleted of linker histones and then reconstituted with deuterated ones (Graziano et al., 1994). In comparison with hydrodynamic techniques, the scattering methods are clearly capable of providing more detailed information about fiber structure; the curve of scattering intensity vs angle (Figure 2) contains much more information concerning shape and internal structure than does a single hydrodynamic parameter. This is especially true of neutron scattering, although this technique suffers from the limitation that it can be effectively carried out in only a few laboratories. Lowangle X-ray scattering also shows future promise, especially if either better methods for aligning fibers are obtained, or ultra-high intensity sources become available for fast dynamic experiments.

III. Macroscopic vs microscopic techniques There is an enormous gulf dividing the macroscopic methods we have described above from the microscopic techniques that can look at individual fragments of chromatin. The macroscopic methods yield numbers (sedimentation coefficients, repeat distances, radii of gyration, etc.) which are typically averages over an immense number of individual objects which themselves are locally heterogeneous in structure. Therefore, attempts to use such experiments to answer biological questions are frequently frustrating. What, for example, can the sedimentation coefficient or mean square cross-section radius of gyration tell us about the structure of transcriptionally active chromatin when the sample contains an unknown mix of "active", "potentially active" and "inactive" chromatin? Unless some kind of fractionation is used (and such methods are notoriously inefficient to date) each macroscopic technique averages (in one of several possible ways) over the whole spectrum of fiber structures in the sample. Probably the least rewarding experience in chromatin studies over the past three decades have been the attempts to separate "active" from "inactive" chromatin on a scale allowing meaningful macroscopic examination.

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There is one potential way out of this dilemma. It is now possible to reconstitute at least some features of the chromatin structure in vitro. This might allow us, in principle, to make the kinds of fiber structure we believe to be representative of repressed or active chromatin. Unfortunately, we do not know the ground rules. If we want to make something like active chromatin structure in vivo, what are the ingredients? How much histone acetylation do we need? How much linker histone? How much non-histone chromosomal protein, and of what varieties? One can envision an essentially infinite research project, ringing all the possible variations on these themes. It is a contention of this paper that the future advances in biochemistry and molecular biology are going to come not from macroscopic studies of immense numbers of molecules, but from microscopic studies conducted in solvent media, frequently at the single-molecule level. The events that occur in processes like transcription and replication are simply too delicate to be studied with the blunt tools of classical physical biochemistry. We shall be examining, more and more, the interaction of one enzyme with one substrate molecule, the movement of one kinesin molecule along one microtubule, the passage of one polymerase along one DNA sequence. For such measurements to be meaningful, to be assured that we are not simply observing one aberrant event in the molecular chaos, we must substitute repetition in time for repetition in numbers. Consequently, we predict that a forthcoming advance in "single-molecule-chemistry" will be the development of various kinds of automation of experiments to allow repetitive observation of a given process.

IV. Microscopic methods In an attempt to avoid the informational limitation inherent in macroscopic methods, researchers have turned more and more toward microscopic methods. Indeed, the possibility to examine in detail the structures of long chromatin fibers provides the possibility to seek out local heterogeneities which can at least delineate the limits of fiber structures and may be clues to function. However, the microscopic techniques have traditionally been beset by other problems equally serious to those facing macroscopic methods. We can best describe these by first presenting a brief overview of the applications of electron microscopy to the study of chromatin fibers. We shall then, in the final section, describe some new microscopic techniques, and how we see their potential for chromatin fiber analysis.

A. Electron microscopy (EM) of chromatin fibers. The first useful studies of structures like the chromatin fiber at the nanometer level utilized transmission electron microscopy. Indeed, it was the application of the

Gene Therapy and Molecular Biology Vol 1, page 479 spreading technique of Miller and Beatty (1969) that allowed Olins and Olins (1973,1974) and Woodcock (1973) to obtain the first indications of a repeating structure in chromatin. Over the subsequent years, many careful and sophisticated studies have been carried out by this method, and many of our current ideas about the fiber structure are based upon these (see, for examples Finch and Klug, 1976; Thoma et al., 1979; Woodcock et al., 1984; Williams et al., 1986). Nonetheless, conventional transmission EM requires such serious abuse of the sample that practitioners have been concerned from the very first. Samples must in most cases be fixed, stained or shadowed, and then subjected to dehydration in a hard vacuum. To what extent are observed structural features artifacts of such treatment? In the past decade, a new EM technique has emerged which goes a long way toward circumventing these difficulties. This is cryo-EM, in which samples are quickly frozen in a film of vitreous ice and then examined by transmission EM (see Dubochet et al., 1992, for a review). Fixation is unnecessary, and image enhancement techniques can circumvent staining. If sublimation can be limited, the fiber is in essentially the solution (albeit frozen) in which it was prepared, and no substantial dehydration should have occurred. Studies of chromatin fibers (Bednar et al., 1995; Woodcock and Horowitz, 1995) by this technique exhibit nucleosomes in irregular, helix-like structures, which bear only limited resemblances to the picture that has been derived from conventional EM. Although cryo-EM represents, in our opinion, a major step toward the unbiased examination of supramolecular organization, it still suffers from some potential problems and major limitations. It is difficult to be certain that the

process of freezing to very low temperatures, rapid as it may be, does not induce some changes in the structure. Furthermore, the necessary thinness of the ice film (~100 nm) creates the possibility that larger macromolecular fibers be distorted by this confinement (see Dubochet et al., 1992, for discussion). A limitation lies in the fact that the sample is frozen; therefore, any direct investigations of the dynamics of macromolecular function are precluded, although fast freezing can provide serial sampling.

B. Scanning probe microscopy. In the past 15 years, a group of entirely new techniques have emerged which show promise of revolutionizing our approach to many biological problems. There are a number of techniques going under the general term scanning probe microscopy; all share the characteristic that a finely pointed probe traverses the sample, and senses, in one way or another, surface features. A compendium of recent references to all of these methods is provided by Bottomley et al. (1996). Some of these techniques are probably unsuitable for biological studies but two have had considerable impact—scanning tunneling microscopy and scanning force microscopy—SFM (also called atomic force microscopy—AFM). This latter method has found the widest application in biology to date, and will be discussed here. In SFM a probe, with tip curvature of the order of 10 nm or less, is mounted atthe end of a very flexible cantilever arm. Deflections of the arm as the tip traverses the sample are amplified by an optical lever (Figure 3). The sample is usually deposited on atomically flat mica or glass surface.

Figure 3. Schematic drawing of a scanning force microscope. In the tapping mode the cantilevered tip is caused to oscillate, and the sample is scanned by piezoelectric deflection of the sample stage. The tip oscillations are detected by an optical lever, a laser beam reflected off the cantilever onto a split photodiode. A feedback circuit to the piezoelectric crystal raises or lowers the sample stage so as to keep the amplitude of oscillation constant. This signal measures the height at each point in the sample.

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Figure 4. SFM image of unfixed chicken erythrocyte chromatin fibers on glass substrate, deposited from low ionic strength (5 mM triethanolamine) buffer. Individual nucleosomes can be resolved. The height of each nucleosome above the surface is indicated by the shading; highest ones are lightest. Even at this low ionic strength, the fiber is not fully extended or flattened, but forms an irregular, helix-like structure.

envisioned from conventional EM studiesâ&#x20AC;&#x201D;although one more commensurate with some earlier scattering experiments. At low salt, the fibers are rather open, irregular helix-like structures, a kind of conformation which can be predicted from simple assumptions about linker DNA behavior (see Leuba et al., 1994; Yang et al., 1994; Woodcock and Horowitz, 1995; van Holde and Zlatanova, 1996). In further studies, it has been possible to exploit the ease of quantitation of such digitized images to explore the effects on structure of proteolytic removal of portions of histone molecule from the chromatin fiber (Leuba et al., submitted). There remain serious limitations to the kind of studies described above. To date, we have not been able to resolve nucleosomes in the more closely packed condensed fibers formed at higher ionic strength. Most important is the fact that air drying, although avoiding the ravages of total dehydration, may still represent a static, non-native condition. For this reason, there is currently enormous interest in the newest development in this techniqueâ&#x20AC;&#x201D;the possibility of operating tapping mode SFM under liquid. If this can be accomplished with resolution comparable to that obtained by tapping mode in air, a whole new world of experimentation will be opened up. It should then be possible to observe directly the changes in chromatin structure accompanying changes in protein composition or ionic medium, or covalent modification of the histones themselves. Direct, real-time observation of the

The scanning force microscope can operate in a number of modes. In the contact mode, the probe tip is simply drawn across the surface. In the tapping mode, the tip is made to oscillate, and thus "taps" its way across the surface of the sample. In general, the tapping mode is found to produce less distortion to biological samples (see Bustamante and Keller, 1995; Shao and Yang, 1995). With currently available instrumentation, resolution of a few nanometers is readily available. This technique has been employed by us (Leuba et al., 1994; Yang et al., 1994) and by others (Allen et al., 1993) to the study of chromatin fiber structure. The advantages over conventional transmission EM are several: staining is never required, and in some cases even fixation can be dispensed with (see Figure 4). Samples can be studied "in air", or in some cases under liquid. Even the "in air" samples, though dried at moderate relative humidity, are not desiccated as in the high vacuum of the EM, and retain water of hydration. At low ionic strength, individual nucleosomes in the fiber are easily resolved and the image shows, in terms of shading, the relative height of each nucleosome above the surface. Thus, the threedimensional coordinates of each nucleosome can be measured, and distribution of nucleosome-nucleosome distances, internucleosome angles and fiber heights easily accumulated (Figure 5). The investigations reveal a somewhat different picture of the chromatin fiber at low ionic strength than had been 480

Gene Therapy and Molecular Biology Vol 1, page 481 buffer, by Kasas et al. (1997). It seems likely that these techniques can be extended, in the very near future, to the investigation of the structure and dynamics of the chromatin fiber. The major problems remaining may be largely concerned with how to appropriately attach the fibers to the surface to be studied. Too weak a fixation will result in release, or at least excessive Brownian motion of fiber segments during observation, leading to loss of resolution. On the other hand, too firm a fixation may prevent the required conformational changes in response to alterations in the environment or interaction with other molecules. These seem, however, to be technical problems of the sort that are ultimately resolved by skilled experimenters. When this is done, the investigation of dynamic processes in chromatin will at last be possible.

References Allen MJ, Dong XF, O'Neill TE, Yao P, Kowalczykowski SC, Gatewood J, Balhorn R and Bradbury EM (1993) Atomic force microscope measurements of nucleosome cores assembled along defined DNA sequences. Biochemistry 32, 8390-8396. Bednar J, Horowitz RA, Dubochet J and Woodcock CL (1995) Chromatin conformation and salt-induced compaction: Three dimensional structural information from cryoelectron microscopy. J. Cell Biol. 131, 1365-1376. Bottomley LA, Coury JE and First PN (1996) Scanning probe microscopy. Anal. Chem. 68, 185R-230R. Bram S and Ris H (1971) On the structure of nucleohistone. J. Molec. Biol. 55, 325-336. Butler PJG and Thomas JO (1980) Changes in chromatin folding in solution. J. Mol. Biol. 140, 505-529. Bustamante C and Keller D (1995) Scanning force microscopy in biology. Physics Today 48, 32-38. Campbell AM, Cotter RI and Pardon JF (1978) Light scattering measurements supporting helical structures for chromatin in solution. Nucleic Acids Res. 5, 1571-1580. Dubochet J, Adrian M, Dustin I, Furrer P and Stasiak A (1992) Cryoelectron microscopy of DNA molecules in solution. In Methods in Enzymology, DMJ Lilley and JE Dahlberg, Eds., Vol. 211, pp. 507-518. Finch JT and Klug A (1976) Solenoidal model for superstructure in chromatin. Proc. Natl. Acad. Sci. USA 73, 1897-1901.

Figure 5. Quantitative data on chicken chromatin fiber structure from SFM experiments. By measuring several thousand nucleosomes, in a number of fibers, both average values and distributions of (A) center-center distances, (B) internucleosome angles, and (C) heights of nucleosomes above substrate could be determined.

Graziano V, Gerchman SE, Schneider DK and Ramakrishnan V (1994) Histone H1 is located in the interior of the 30 nm filament. Nature 368, 351-354. Hansen JC, Ausio J, Stanik V and van Holde KE (1989) Homogeneous reconstituted oligonucleosomes: Evidence for salt-dependent folding in the absence of histone H1. Biochemistry 28, 9129-9136.

interaction of enzymes and transcription factors with the chromatin fiber should also be possible. There has already been considerable success in studying DNA, and some DNA-protein interactions in buffer. For example, Lyubchenko and Shlyakhtenko (1997) have studied the conformational changes of supercoiled DNA in response to different ionic strengths in just this way. The dynamic interaction of E. coli RNA polymerase with DNA has been demonstrated, under

Kasas S, Thompson NH, Smith RL, Hansma HG, Zhu X, Guthold M, Bustamante C, Kool ET, Kashlev M and Hansma PK (1997) Escherichia coli RNA polymerase activity observed using atomic force microscopy. Biochemistry 36, 461-468.

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van Holde et al: Physical approaches to the study of chromatin fibers Leuba SH, Yang G, Robert C, Samori B, van Holde K, Zlatanova J and Bustamante C (1994) Three-dimensional structure of extended chromatin fibers as revealed by tapping-mode scanning force microscopy. Proc. Natl. Acad. Sci. USA 91, 11621-11625.

Woodcock CL and Horowitz RA (1995) Chromatin organization re-viewed. Trends Cell Biol. 5, 272-277.

Lyubchenko YL and Shlyakhtenko LS (1997) Visualization of supercoiled DNA with atomic force microscopy in situ. Proc. Natl. Acad. Sci. USA 94, 496-501.

Yang G, Leuba S, Bustamante C, Zlatanova J and van Holde K (1994) Role of linker histones in extended chromatin fiber structure. Nature Str. Biol. 1, 761-763.

Miller OL Jr and Beatty BR (1969) Visualization of nucleolar genes. Science 164, 955-957. Olins AL and Olins DE (1973) Spheroid chromatin units ("bodies). J. Cell Biol. 59, 2529. Olins AL and Olins DE (1974) Spheroid chromatin units ("bodies). Science 183, 330-332. Pardon JF, Wilkins MHF and Richards BM (1967) Super-helical model for nucleohistone. Nature 215, 508-509. Pardon JF, Worcester DL, Wooley JC, Tatchell K, van Holde KE and Richards BM (1975) Low-angle neutron scattering from chromatin subunit particles. Nucleic Acids Res. 2, 21632175. Sahasrabuddhe C and van Holde KE (1974) The effect of trypsin on nuclease-resistant chromatin fragments. J. Biol. Chem. 249, 152-156. Shao Z and Yang J (1995) Progress in high resolution atomic force microscopy in biology. Quart. Rev. Biophys. 28, 195251. Suau P, Kneale GG, Braddock GW, Baldwin JP and Bradbury EM (1977) A low resolution model for the chromatin core particle by neutron scattering. Nucleic Acids Res. 4, 37693786. Suau P, Bradbury EM and Baldwin JP (1979) Higher-order structures of chromatin in solution. Eur. J. Biochem. 97, 593-602. Thoma F, Koller T and Klug A (1979) Involvement of histone H1 in the organization of the nucleosome and of the saltdependent superstructures of chromatin. J. Cell Biol. 83, 403-427. Thomas JO and Butler PJG ( 1980) Size-dependence of a stable higher-order structure of chromatin. J. Mol. Biol. 144, 8993. van Holde KE (1985) Physical Biochemistry (2nd Ed.), Prentice Hall, Englewood Cliffs, NJ. van Holde KE (1988) Chromatin, Springer Verlag, New York, Berlin. van Holde KE and Zlatanova J (1996) What determines the folding of the chromatin fiber? Proc. Natl. Acad. Sci. USA 93, 10548-10555. Wilkins MHF, Zubay G and Wilson HR (1959) X-ray diffraction studies of the molecular structure of nucleohistone and chromosomes. J. Mol. Biol. 1, 179-185. Williams SP, Athey BD, Muglia LJ, Schappe RS, Gough AH and Langmore JP (1986) Chromatin fibers are left-handed double helices with diameter and mass per unit length that depend on linker length. Biophys. J. 49, 233-248. Woodcock CL (1973) Ultrastructure of inactive chromatin. J. Cell Biol. 59, 368a.

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Woodcock CL, Frado L-LY and Rattner JB (1984) The higherorder structure of chromatin: Evidence for a helical ribbon arrangement. J. Cell Biol. 99, 42-52.

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Gene Ther Mol Biol Vol 1, 483-494. March, 1998.

Activation at a distance: involvement of nucleoprotein complexes that remodel chromatin Emery H. Bresnick, Wayne K. Versaw, Lloyd T. Lam, E. Camilla Forsberg, and Helene C. Eisenman University of Wisconsin Medical School, 387 Medical Science, 1300 University Ave., Madison, WI 53706 ______________________________________________________________________________________________________ Correspondence: Emery H Bresnick Tel: 608-265-6446, Fax: 608-262-1257, E-mail: ehbresni@facstaff.wisc.edu

Summary Large-scale sequencing of the human genome has confirmed that genes are often spread over several thousand base pairs on a chromosome. The cis-acting regulatory elements that control gene transcription can be located at a considerable distance from the respective gene. Significant advances in understanding the mechanism of eukaryotic gene transcription have revealed a great deal about how the RNA polymerase initiation complex assembles on the promoter region of genes. However, the fundamental problem of how distant genetic regulatory elements, such as enhancers and locus control regions, communicate with proximal elements t o confer cell- and tissue-specific patterns of t r a n s c r i p t i o n r e m a i n s l a r g e l y u n s o l v e d . T h e p r i n c i p l e f o c u s o f t h i s c h a p t e r w i l l b e o n t h e r o l e of chromatin structure in transcriptional regulation by RNA polymerase II, with an emphasis on longrange activation.

I. Introduction to the problem The levels of cellular proteins are regulated by changes in the concentration of the respective mRNAs. Changes in steady-state concentrations of mRNA result from altered synthesis or stability of the mRNA, with alterations in transcription being common. Eukaryotic gene transcription requires a core promoter element, which is the site of assembly of the preinitiation complex, consisting of “basal transcription factors” and RNA polymerase II (Orphanides et al., 1996). Multiple layers of regulation converge to modulate initiation complex assembly and thus alter gene activity. Initial studies on the mechanism of transcription initiation suggested that preinitiation complex assembly occurred by binding of the basal factor TFIID to DNA, followed by the sequential binding of other basal factors (Buratowski et al., 1989). However, more recent evidence supports a model in which a preassembled RNA polymerase II holoenzyme is recruited to the DNA as a unit (Greenblatt, 1997). The holoenzyme is a multiprotein complex consisting of the catalytic subunit of RNA polymerase and basal factors including TFIID, TFIIB and others. Macromolecular interactions between basal factors and RNA polymerase provide an important level of regulation of preinitiation complex assembly. A second level of regulation is conferred by transcription factors, distinct from

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the basal factors, which bind to recognition sites on the promoter. These sequence-specific DNA binding proteins engage in protein-protein interactions with components of the holoenzyme, resulting in enhanced transcription initiation. Furthermore, preinitiation complex assembly can be regulated by transcription factors bound at distant regulatory sites such as enhancers and locus control regions (LCRs). Thus, communication between distal regulatory elements and promoters or “action at a distance” provides a third way to modulate preinitiation complex assembly. The factors functioning through distal sites can be identical to factors that bind directly to promoters. A wealth of data support models of long-range activation involving disruption of chromatin structure, which increases access of the promoter to the transcription machinery. The process by which trans-acting factors induce chromatin transitions, such as the localized disruption of a nucleosome or the unfolding of a broad region of condensed chromatin, is poorly understood. In this chapter, the major emphasis will be on the functional role of chromatin transitions in transcription initiation. A simple model will be developed, which incorporates both looping and chromatin-disruption, to explain “action at a distance”. We will begin to address the influences of chromatin on longrange activation by reviewing fundamental aspects of chromatin structure.

Bresnick et al. Activation and remodeling of chromatin

Figure 1. Functional consequences of nucleosomal organization of factor binding sites.

II. Influence of chromatin structure on transcription At the simplest level, 146 base pairs of DNA duplex are wrapped around an octamer of histones (two H2A-H2B dimers and one H3-H4 tetramer) to form the nucleosome, the basic repeating unit of chromatin (Wolffe, 1995). The DNA between nucleosomes, the linker DNA, averages approximately fifty base pairs. The linker histones H1 and H5 interact asymmetrically with the nucleosome near the entry and exit points of the DNA (Hayes, 1996; Pruss et al., 1996). One role of the linker histones is to facilitate higherorder packaging of the 10 nm â&#x20AC;&#x153;beads on a stringâ&#x20AC;? nucleosomal filament to form the condensed 30 nm chromatin fiber. An additional level of packaging is believed to lead to the formation of loop domains, which, in turn are condensed into a chromosome. The histone octamer can occlude recognition sites on DNA for certain sequence-specific DNA binding proteins that control DNA replication, transcription, and recombination (Hager et al., 1993) (F i g . 1 ). Consequently, the pattern of accessible sites on naked DNA and chromatin templates can differ considerably. However, the nucleosomal organization of DNA does not always negatively affect protein-DNA recognition, as certain proteins can form stable complexes with sites on the surface of a nucleosome (Adams and Workman, 1995; Archer et al., 1991; Li et al., 1994; Pina et al., 1990; Steger and Workman, 1997; Taylor et al., 1991) (F i g . 1). Thus, chromatin organization can also be permissive for transcription. The wrapping of DNA around a histone octamer can bring together distant regions of DNA, facilitating protein-protein interactions between bound regulatory factors (F i g . 1). In this scenario, the nucleosome would serve as an architectural element, enhancing the efficiency of transcription initiation (Quivy and Becker, 1996; Schild et al., 1993; Thomas and Elgin,

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1988). By either allowing or precluding protein-DNA interactions, chromatin structure plays an active role in transcription initiation. The specific positioning of the histone octamer on the DNA can be a critical determinant of recognition site accessibility (Wolffe, 1994). Two distinct types of positioning need to be considered. First, rotational positioning refers to the orientation of the DNA helix on the octamer core of the nucleosome. Rotational positioning refers to which face of the DNA is oriented toward or away from the octamer core. The prevalence of rotationally positioned nucleosomes in intact cells is unclear. Second, translational positioning refers to whether sequences reside within the core or the linker DNA. Preferred translational positioning has been observed in several systems and has important regulatory implications. However, examination of nucleosome positioning at base-pair resolution on the mouse mammary tumor virus (MMTV) promoter has revealed microheterogeneity of translational positions (Fragoso et al., 1995), despite the appearance of rigid translational constraints from lower resolution analysis. Further studies are required to determine whether microheterogeneity is common and functionally important. Nucleosome positioning can be modulated by both DNA binding proteins (Pazin et al., 1997; Roth et al., 1992) and the physicochemical properties of DNA (Muyldermans and Travers, 1994; Satchwell et al., 1986). Beyond the level of regulation conferred by nucleosome positioning, the association of linker histones with chromatin also has significant consequences for transcription (Shen and Gorovsky, 1996). As transcription factor binding sites are often positioned in linker regions (Bresnick et al., 1992; Thomas and Elgin, 1988), the linker histone association can modulate factor access. This could occur through a direct competitive mechanism or by facilitation of chromatin condensation (Graziano et al., 1988; Shen et al., 1995) and

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occlusion of recognition sequences. Linker histones can also affect protein-DNA interactions by modulating mobility of the octamer core on DNA (Pennings et al., 1994; Ura et al., 1995). One consequence would be to ensure that certain recognition sites remain within the octamer core. Condensation of the 10 nm nucleosomal filament into the 30 nm fiber may mask binding sites that remain accessible on nucleosomal DNA. It is difficult to envision a protein-DNA interaction, which requires precise contacts with the major or minor groove, occurring on the condensed 30 nm fiber. However, recent evidence suggests that the 30 nm fiber is a dynamic structure (Ericsson et al., 1990), which could allow a window of opportunity for a proteinDNA recognition event to occur. As assembly of the initiation complex on a promoter requires sequence-specific recognition of the TATA box or the initiator element on the DNA by basal transcription factors, it is not surprising that multiple levels of chromatin structure affect initiation. The principles of how chromatin influences transcription initiation are also likely to be relevant to elongation. RNA polymerases can elongate through arrays of nucleosomes without completely displacing histones from the DNA (Ericsson et al., 1990). However, RNA polymerase pausing is enhanced on nucleosomal templates (Izban and Luse, 1991). Polymerase pausing is a functionally relevant step of the elongation process (Krumm et al., 1995; Rasmussen and Lis, 1995; Yankulov et al., 1994). Studies with a defined in vitro system using phage SP6 polymerase and reconstituted chromatin led to a detailed model for how RNA polymerase negotiates a nucleosomal template. As the polymerase approaches a nucleosome, the nucleosome is transferred to the DNA behind the polymerase via an intramolecular reaction (Clark and Felsenfeld, 1992; Studitsky et al., 1994). One can envision a variety of ways to modulate this transfer reaction to generate a highly regulated process. Additional studies are required to determine whether this mechanism is applicable to eukaryotic RNA polymerases transcribing chromosomal templates.

III. Regulatory elements that function over long distances on chromosomes A. Enhancers DNA regulatory elements that increase the transcriptional activity of promoters in a distance-and orientation-independent manner are called enhancers (Muller et al., 1988). The defining criterion of distance-independence is not entirely accurate, as the activity of certain enhancers can be significantly reduced when moved several thousand base pairs away from a promoter. In addition, the intervening DNA between an enhancer and promoter is not inert, and therefore, different DNA sequences differ in permissiveness for activation (Schreiber and Schaffner, 1989). Nevertheless, there is usually flexibility in the proximity of an enhancer to a promoter as well as enhancer location. Enhancers are normally located upstream or

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downstream of genes and even in introns. Several classes of enhancers exist, depending on the nature of the binding proteins. A common feature of enhancers is the presence of clustered cis-acting elements that bind transcription factors. Differential distribution or modifications of these factors give rise to distinct classes of enhancers. Enhancers can have cellspecific, tissue-specific, or ubiquitous activity depending on the distribution of the factors. Furthermore, posttranslational modifications can modulate the activity of the enhancer binding proteins (Goldman et al., 1997; Hill and Treisman, 1995; Karin et al., 1997). Thus, enhancers can be constitutively active or induced by environmental cues such as hormones and nutrients.

B. Locus control regions A second class of positive-acting genetic elements exists that shares certain features with enhancers, but appears to have a distinct activity. These locus control regions (LCRs) confer copy number-dependent and positionindependent expression of a linked gene when integrated into chromosomal DNA (Bresnick, 1997). When an exogenous gene is stably integrated into a chromosome, the activity of the gene is often activated or repressed, depending on the DNA sequences flanking the integration site (Wilson et al., 1990). Integration of the gene next to an enhancer can elevate promoter activity. By contrast, integration into condensed chromatin or near a silencer, a genetic element that represses transcription (Hanna-Rose and Hansen, 1996), can reduce promoter activity. By overcoming the integration site position effects, LCRs generate an autonomously regulated gene or gene cluster (Forrester et al., 1987; Grosveld et al., 1987). Copy-number dependence simply refers to the fact that the expression level of a gene bears a linear relationship with the number of integrated gene copies. Analogous to enhancers, LCRs have a strong activating function (Tuan et al., 1989) and function over long distances on chromosomes. A distinction between LCRs and enhancers is that certain enhancers are incapable of supporting copy number-dependent and position-independent expression of integrated genes (Trudel and Costantini, 1987). It is unknown whether this reflects a distinct mechanism for LCRs and enhancers or if simply a maximal quantum of enhancer activity is required to overcome position effects, and the mechanism is identical.

C. Activation mechanisms of enhancers and LCRs Three classes of mechanisms are commonly invoked to explain â&#x20AC;&#x153;action at a distanceâ&#x20AC;? by enhancers and LCRs looping, tracking, and chromatin disruption. First, analogous to well-characterized prokaryotic systems, enhancer-bound transcription factors physically interact with factors that bind to promoters. As the intervening DNA between distal and proximal sites forms a loop (Mastrangelo et al., 1991; Su et al., 1991), this type of model is called the

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looping or protein-protein interaction model (Schleif, 1992). Physical interactions between distal- and proximal-bound transcription factors increase the local concentration of activators at the promoter, resulting in recruitment of the holoenzyme to the promoter or, potentially, the stabilization of a preexisting complex. Both actions would enhance transcription initiation. Parameters that affect the efficiency of activation include DNA binding affinity, the affinity of the protein-protein interaction, and potentially factors that modulate DNA bending. It seems intuitive that the fundamental mechanism of transcription would be conserved, and therefore a looping mechanism would also be used in eukaryotes. However, the chromatin organization of the eukaryotic nucleus provides a formidable impediment to DNA looping, demanding additional regulatory mechanisms to contend with chromatin structure. Tracking mechanisms assume that regulatory factors bound at distal sites relative to a promoter processively move or “track” toward the promoter (Ouhammouch et al., 1997). The upstream factors could recruit RNA polymerase at the distal site, and migrate toward the promoter in conjunction with the polymerase. On the other hand, the upstream factors could track toward the promoter and then recruit polymerase at the promoter. Similar consequences of chromatin organization would be predicted for looping and tracking models, i.e., DNA recognition by regulatory factors would be modulated. Chromatin would likely affect tracking in an analogous manner to its effects on transcriptional elongation. As discussed in detail below, the remodeling of chromatin by multiprotein complexes is a critical step in eukaryotic transcriptional regulation. It is easy to envision how regulatory factors could alter local histone-DNA contacts, leading to nucleosome disruption. However, it is more difficult to conceptualize how the chromatin structure of an entire domain could be modulated by LCRs.

IV. Long-range activation by locus control regions - a role for multiprotein complexes that remodel chromatin? Since the initial description of the !-globin LCR (Forrester et al., 1987; Grosveld et al., 1987), a variety of genes have been reported to have LCRs (Carson and Wiles, 1993; Chauveau and Cogne, 1996; Dang and Taylor, 1996; Ess et al., 1995; Jones et al., 1995; LadekjaerMikkelsen et al., 1996; Lang et al., 1991; Madisen and Groudine, 1994; Montoliu et al., 1996; Talbot et al., 1994). Thus, it appears that inclusion of an LCR within a chromosomal domain may be a common means of establishing and/or maintaining an active chromatin structure. We expect that many more LCRs will be identified as the expression of more genes is analyzed in vivo. The transient transfection assay, which is often used to characterize promoter and enhancer activity, would not be expected to reveal an LCR requirement. The activation mechanism of LCRs involves decondensation of chromatin structure, and transiently transfected DNA is neither replicating nor assembled into organized chromatin.

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Although several LCRs have been described, much of what is known about the activation mechanism of LCRs is derived from the !-globin system.

A. Structure and function of the -globin LCR The human !-globin genes ", G# , A# , $, and ! exist in a cluster on chromosome 11 (F i g . 2) and are differentially expressed in an erythroid-specific manner during development (Baron, 1997).

Figure 2 . Organization of the human !-globin gene domain.

All of the genes share a common regulatory element, the !-globin LCR, which consists of four erythroid-specific DNaseI hypersensitive sites (HS1 - HS4), at the 5’ end of the locus (Forrester et al., 1986; Tuan et al., 1985). HSs are regions of chromatin, typically encompassing about 200 base pairs of DNA, which are characterized by strong susceptibility to nuclease cleavage. This structural discontinuity of the chromatin fiber is a hallmark of a nucleoprotein complex (Becker, 1994). The !-globin LCR is crucial for establishing an erythroid-specific chromosomal domain. Thus, the activation property of the LCR can be shared among multiple genes on a chromosome (Bresnick and Felsenfeld, 1994; Furukawa et al., 1994; Milot et al., 1996). However, the LCR does not appear to be involved in determining which globin gene is active at a particular stage of development. Evidence for lack of involvement of the LCR in globin gene switching comes from studies in transgenic mice whereby normal switching occurs with transgenes lacking the LCR (Starck et al., 1994). Despite this compelling observation, the neutral role of the LCR in globin gene switching remains controversial (Engel, 1993), as most transgenes lacking an LCR are subject to position effects. There is no question, however, that the LCR induces a decondensation of the chromatin structure of the !-globin locus, which is required for generation of a transcriptionallycompetent !-globin domain. The physiological significance of the !-globin LCR is illustrated by a human genetic disease, Hispanic thalassemia. This disease is characterized by deletion of a portion of the LCR, resulting in formation of condensed chromatin throughout the locus and silencing of the globin genes

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(Forrester et al., 1990). The analysis of the global activity of the LCR to decondense chromatin has been complicated by the fact that no single factor mediates this activity (Caterina et al., 1994).

B. Protein components of the -globin LCR Each of the HSs contains multiple recognition sequences for both ubiquitous and erythroid-specific transcription factors (Bresnick and Felsenfeld, 1993; Caterina et al., 1991; Elnitski et al., 1997; Lam and Bresnick, 1996; Philipsen et al., 1993; Talbot et al., 1990; Yant et al., 1995). Considering that the HSs only exist in erythroid cells, one would expect the erythroid-specific factors to be crucial for formation and/or maintenance of the HSs. In this regard, the requirements for formation of HS4 have been studied (Stamatoyannopoulos et al., 1995). The recognition sites for two erythroid-specific factors, GATA1 and NF-E2, were important for formation of HS4. By contrast, a site that binds several ubiquitous factors (CACCC/Sp1) was not important. However, this does not exclude a role for ubiquitous transcription factors in LCR function, as these proteins could modulate the activity of the LCR, rather than being necessary for formation of a stable complex with chromatin. A common thread among the tissue-specific binding proteins of the LCR (GATA1, NF-E2, and TAL1) is that they are critical for hematopoiesis (Orkin, 1996). In vitro protein-DNA interaction studies reveal whether a factor binds with specificity and high affinity to a recognition site. However, not all high-affinity interactions are physiologically relevant. In certain cases, without requisite posttranslational modifications or accessory factors, a factor may bind to a site with low affinity in vitro, in contrast to the intact cell. Of course, consideration of whether a site is conserved throughout evolution is suggestive of functional significance (Gumucio et al., 1992). The HSs of the LCR are each characterized by multiple conserved recognition sequences. In vivo footprinting studies support the notion that numerous binding sites within a single HS are occupied simultaneously by factors (Ikuta and Kan, 1991; Reddy et al., 1994; Strauss et al., 1992), suggesting that they function as an integrated complex. The identification of proteins that function through cisacting elements is often more complex than deduction based on the DNA sequence. More often than not, multiple factors interact with an identical or highly-related recognition sequence. Even if one can conclude that a site is important based on evolutionary considerations, the functional factor remains unknown. The NF-E2 binding site within HS2 is a good example of this scenario, as multiple proteins bind tightly to this sequence [NF-E2 (Andrews et al., 1993), AP1 (Lee et al., 1987), NRF1 (Caterina et al., 1994; Chan et al., 1993), and NRF2 (Moi et al., 1994)]. Complex methodologies based on immunoprecipitating nucleoprotein complexes with specific factor antibodies have the potential

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to determine which factor is bound to a site in intact cells (Orlando, 1997; Boyd and Farnham, 1997; Bresnick et al., 1992). Another enigmatic issue is why the LCR consists of four distinct HSs, rather than a single cluster of cis-acting elements. Multiple HSs are required for the long-range activation property of the LCR (Bresnick and Tze, 1997; Bungert et al., 1995). Individual HSs, such as HS2 and HS3, can strongly activate transcription when positioned near a promoter but are incompetent for long-range activation (Bresnick and Tze, 1997). In addition, the ability of the LCR to confer position-independent gene expression appears to require multiple HSs. We postulated that multiple HSs may be required to form a stable nucleoprotein structure that reassembles with high fidelity after each round of DNA replication (Bresnick, 1997; Bresnick and Tze, 1997). An alternative possibility is that different sites recruit distinct coactivators, which could function synergistically. We also postulated that chromatin modifying enzymes could be recruited through protein-protein interactions with LCR-bound factors (F i g . 3 ). These enzymes could mediate the long-range effects of the LCR on chromatin structure and transcription (Bresnick, 1997; Bresnick and Tze, 1997). It was recently shown that the p45 subunit of the heterodimeric factor NF-E2 physically interacts with the transcriptional coactivator, CBP (Cheng et al., 1997) (F i g . 4 ), which is a histone acetyltransferase (HAT) (Ogryzko et al., 1996). CBP, in turn, physically interacts with another HAT, PCAF (Yang et al., 1996). PCAF is present in a large macromolecular complex in human K562 erythroleukemia cells (Forsberg et al., 1997), in which the LCR is active. An understanding of long-range activation by the LCR requires knowledge of the nuclear machinery that mediate chromatin structure transitions.

V. Regulatory complexes that unfold chromatin A. Nuclear signaling enzymes that acetylate histones A common posttranslational modification of core histones is the acetylation of conserved lysine residues (Wade et al., 1997). The amino terminal â&#x20AC;&#x153;tailsâ&#x20AC;? of the core histones are believed to physically interact via electrostatic forces with the negatively charged phosphodiester backbone of DNA (Lee and Hayes, 1997). Several conserved lysine residues within the tails are subject to acetylation on their epsilon amino group (Gershey et al., 1968). Neutralization of the positive charge of the lysine would be expected to reduce the affinity of the tail for the DNA backbone, thus increasing the accessibility of DNA sequences within the nucleosome. Consistent with this idea, histone acetylation can enhance the binding of transcription factors to nucleosomal recognition sites (Lee et al., 1993; VetteseDadey et al., 1996). Genetic studies have verified the

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Figure 3. Model of chromatin modifying enzyme involvement in domain opening by the !-globin locus control region.

importance of the lysine residues that are acetylated (Durrin et al., 1991). The acetylation reaction is carried out by a class of nuclear enzymes termed HATs, which mediate chromatin remodeling (Wolffe, 1995). At least one HAT, GCN5, is conserved from yeast to man (Candau et al., 1996), emphasizing the universal importance of histone acetylation. GCN5 exists in a large macromolecular complex in yeast (Grant et al., 1997; Marcus et al., 1994; Saleh et al., 1997) and human (Forsberg et al., 1997) cells. A paradoxical question is how do HATs regulate specific gene expression when they can acetylate histones globally? The answer is to employ a targeting mechanism to recruit HATs to specific genetic loci. A seminal discovery that shed light on the specificity of histone acetylation was recently made by David Allis and colleagues. A Tetrahymena HAT was cloned, revealing that it was homologous to a yeast transcriptional coactivator, GCN5 (Brownell et al., 1996). Once it was determined that GCN5 had HAT activity, the picture rapidly unfolded. It was known that GCN5 was necessary for transcriptional activation by the transcription factor GCN4 (Georgakopoulos and Thireos, 1992). As GCN4 physically interacts with GCN5, a simple model emerged in which GCN4 binds to DNA and recruits GCN5 through a specific protein-protein interaction. Thus, a solution to the problem of how HATs are targeted is that protein-protein interactions between the HAT and DNA binding proteins mediate gene-specific recruitment. Many unsolved issues remain, such as once the HAT is recruited, does it modify chromatin by processively moving on the chromosomal template? What are the mechanisms that regulate the extent of the chromatin modification and terminate the activating signal? How can a limited number of HATs engage in protein-protein interactions with diverse DNA binding proteins? Does a common protein domain mediate interactions with HATs, or alternatively, do HATs have a moldable domain, analogous to chaperonins, which can interact with structurally diverse proteins? While the answers to these questions are

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unknown, it is clear that several HATs exist, with potentially distinct functions. A human GCN5-related HAT, PCAF, was cloned, which does not appear to have a yeast homolog (Yang et al., 1996). PCAF shares a common carboxy-terminal domain with GCN5, but has a unique amino terminus. Analogous to GCN5, PCAF also exists in a large macromolecular complex (Forsberg et al., 1997; Yang et al., 1996; Grant et al., 1997). Two other proteins, CBP (Yang et al., 1996) and ACTR (Chen et al., 1997), directly interact with PCAF. Surprisingly, both CBP and ACTR have intrinsic HAT activity. This raises yet another question, i.e., why would multiple HATs be present in the same heteromeric complex? This could be explained by a model in which individual HATs have unique specificities and coordinately function to generate a specific pattern of acetylated histones in chromatin. Another possibility is that HATs might have substrates other than the core histones. In this regard, acetylation of the DNA binding tumor suppressor protein p53 recently was shown to stimulate sequence-specific DNA binding by p53 (Gu and Roeder, 1997). Further studies on HAT structure and function should reveal principles for how posttranslation modifications of chromosomal proteins regulate transcription. The identification of protein-protein interactions between HATs and nuclear proteins will likely provide important clues to their functional roles. Indeed, as indicated above, CBP physically interacts with the p45 subunit of NF-E2, which is a key regulator of the !-globin LCR.

B. Involvement of histone acetyltransferases in LCR function. Considering that NF-E2 can interact with CBP, and CBP is present in a complex with PCAF (F i g . 4 ), it is reasonable to propose that the CBP-PCAF HAT complex is recruited to the !-globin locus by the !-globin LCR. The implications of this are significant, as HATs could mediate the long-range chromatin decondensing activity of the LCR.

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A relevant observation is that analysis of the acetylated state of histones of the chicken !-globin locus revealed acetylated histones distributed throughout the locus (Hebbes et al., 1994). Thus, sites of acetylation were not restricted to regulatory sequences, such as promoters or enhancers, or coding regions of the globin genes. This observation is consistent with a role for acetylation in establishment and/or maintenance of active domains, rather than exclusive functions to determine promoter accessibility or to regulate transcriptional elongation. Besides HATs, other multiprotein chromatin remodeling complexes could also be important in long-range activation.

F i g u r e 4 . Recruitement of CBP- and PCAF-containing HAT complex by NF-E2.

C. Other chromatin remodeling complexes - SWI/SNF, NURF, and RSC Genetic studies in yeast revealed a series of proteins (SWI - yeast mating type switching)/SNF - sucrose nonfermenting) that were critical for transcription of various genes. The SWI/SNF proteins (Winston and Carlson, 1992) were distinct from transcription factors and components of RNA polymerase. The proteins form a multimeric complex (Cairns et al., 1994; Peterson et al., 1994) and appear to function by modulating chromatin structure (Kruger et al., 1995) to increase the accessibility of cis-acting elements. The SWI/SNF complex isolated from yeast and human cells consists of at least twelve stably associated proteins and is estimated to be approximately two megadaltons (Cairns et al., 1994; Peterson et al., 1994; Wang et al., 1996). SWI/SNF may be physically associated with the RNA polymerase II holoenzyme (Wilson et al., 1996), although this remains debatable (Cairns et al., 1996). In vitro proteinDNA interaction studies have shown that SWI/SNF facilitates factor binding to nucleosomal templates (Cote et al., 1994; Imbalzano et al., 1994; Kwon et al., 1994; OwenHughes et al., 1996), suggesting that SWI/SNF disrupts the

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association of histones with DNA; the fate of the histones is unclear. At least one component of SWI/SNF, SWI2/SNF2, has homology with DNA helicases (Khavari et al., 1993), which unravel double-stranded DNA (Lohman and Bjornson, 1996). The helicase homology may be an important clue to how SWI/SNF modulates chromatin structure and transcription. DNA helicases move processively on DNA in a way that is fueled by ATP hydrolysis (Lohman and Bjornson, 1996). SWI2/SNF2 hydrolyzes ATP similar to helicases. It is unknown, however, whether nucleotide hydrolysis confers upon SWI/SNF the ability to track on DNA. The nucleotide hydrolyzing activity is necessary to facilitate factor binding, but the permissive chromatin transition induced by SWI/SNF is stable without further nucleotide hydrolysis (Imbalzano et al., 1996). The current data are consistent with the hypothesis that SWI/SNF can track on DNA and disrupt nucleosomes, thus promoting factor binding. It seems reasonable to propose that SWI/SNF may play a role in decondensation of higher-order chromatin structure. A combination of histone modification by HATs and chromatin disruption by SWI/SNF would ensure that a domain resides in an uncondensed state and is thus transcriptionally-competent (F i g . 3). Despite major advances in understanding how HATs are targeted to a chromosomal template, studies on the targeting of SWI/SNF are in their infancy. The recruitment of SWI/SNF to chromatin may occur through a protein-protein interaction mechanism analogous to HATs. It was recently shown that binding of the glucocorticoid receptor to DNA stimulates the nucleosome disruption activity of SWI/SNF (Ostlund Farrants et al., 1997). Another DNA-bound transactivator, NF1, did not affect SWI/SNF activity. Thus, SWI/SNF may mediate the well-characterized activity of the glucocorticoid receptor to disrupt chromatin (Hager et al., 1993) by a protein-protein interaction, recruitment mechanism. In addition to targeting mechanisms based on proteinprotein interactions, HATs and SWI/SNF may function coordinately, and SWI/SNF may recognize features of active chromatin induced by HATs. A related chromatinremodeling complex in Drosophila, NURF (nucleosome remodeling factor) (Tsukiyama and Wu, 1995), was recently shown to interact with amino terminal tails of core histones (Georgel et al., 1997). NURF shares certain features with SWI/SNF, such as a polypeptide related to SWI2/SNF2 containing helicase homology and ATPase activity (Tsukiyama et al., 1995), the ability to disrupt chromatin structure and facilitation of factor binding. Thus, analogous to NURF, SWI/SNF may interact with histone tails, and this interaction could be modulated by acetylation. As multiple HATs are present within a cell, it is of interest to ask whether multiple chromatin disrupting complexes like SWI/SNF exist. Yet another chromatin remodeling enzyme complex, RSC (remodels structure of chromatin), was recently isolated from yeast (Cairns et al.,

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1996). RSC shares certain features with SWI/SNF, such as a component similar to SNF2/SWI2. However, differences between RSC and SWI/SNF include an approximately tenfold greater abundance of RSC and a requirement of RSC for mitotic growth.

VI. Concluding remarks A great deal of knowledge is emerging rapidly on how multiprotein complexes mediate chromatin transitions. Intuitively, it is easy to conceptualize how factors binding to a promoter lead to nucleosome disruption and enhance transcription by facilitating preinitiation complex assembly. A more challenging intellectual puzzle, however, is to unravel the determinants of how distant regulatory elements function. Is long-range activity mediated by the same chromatin-remodeling enzymes necessary for nucleosome disruption on promoters? As identical transcription factors can function through upstream elements and promoters, the chromatin remodeling machinery may be identical. In addition, the requirement for multiple polypeptides within the regulatory enzyme complexes needs to be explained. These components could enhance the functionality of the complexes by modulating enzymatic activity, targeting the complex to genes, or regulating the subcellular localization or stability of the active component. Based on evolutionary conservation of the transcription machinery, it is likely that looping is a fundamental step in eukaryotic transcripional activation. To incorporate the additional level of regulation demanded by chromatin, one can propose a bimodal activation mechanism. Disruption of chromatin structure would establish a transcriptionallycompetent domain or local chromosomal region, in which promoters are accessible to the transcription machinery. Protein-protein interactions and DNA looping would stabilize and/or facilitate assembly of a bona fide preinitiation complex. If the promoter is rendered accessible by chromatin disruption, the initiation complex could assemble without protein-protein interactions with upstream activators, albeit at a low frequency. The assumption is that assembly of the preinitiation complex occurs in a stochastic or all-or-none fashion. The level of gene transcription in a cell population would therefore depend on the number of engaged initiation complexes. Analysis of gene expression in single cells has yielded data consistent with a stochastic activation mechanism (Fiering et al., 1990; Ko et al., 1990; Walters et al., 1995; Weintraub, 1988).

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Wade, P. A., Pruss, D., and Wolffe, A. P. (1 9 9 7 ). Histone acetylation: chromatin in action. T r e n d s B i o c h e m . S c i . 22, 128-132. Walters, M. C., Fiering, S., Eidemiller, J., Magis, W., Groudine, M., and Martin, D. I. (1 9 9 5 ). Enhancers increase the probability but not the level of gene expression. P r o c . N a t l . A c a d . S c i . U . S . A . 92, 7125-7129. Wang, W., Cote, J., Xue, Y., Zhou, S., Khavari, P. A., Biggar, S. R., Muchardt, C., Kalpana, G. V., Goff, S. P., Yaniv, M., Workman, J. L., and Crabtree, G. R. (1 9 9 6 ). Purification and biochemical heterogeneity of the mammalian SWI/SNF complex. EMBO J. 15, 5370-5382. Weintraub, H. (1 9 8 8 ). Formation of stable transcription complexes as assayed by analysis of individual templates. P r o c . N a t l . A c a d . S c i . U . S . A . 85, 5819-5823. Wilson, C., Bellen, H. J., and Gehring, W. J. (1 9 9 0 ). Position effects on eukaryotic gene expression. A n n u . R e v . C e l l B i o l . 6, 679-714. Wilson, C. J., Chao, D. M., Imbalzano, A. N., Schnitzler, G. R., Kingston, R. E., and Young, R. A. (1 9 9 6 ). RNA polymerase II holoenzyme contains SWI/SNF regulators involved in chromatin remodeling. C e l l 84, 235-244. Winston, F., and Carlson, M. (1 9 9 2 ). Yeast SNF/SWI transcriptional activators and the SPT/SIN chromatin connection. Trends Genet. 8, 387-391. Wolffe, A. (1 9 9 5 ). Chromatin: Academic Press. Wolffe, A. P. (1 9 9 4 ). Nucleosome positioning and modification: chromatin structures that potentiate transcription. Trends Biochem Sci 19, 240-244. Yang, X. J., Ogryzko, V. V., Nishikawa, J., Howard, B. H., and Nakatani, Y. (1 9 9 6 ). A p300/CBP-associated factor that competes with the adenoviral oncoprotein E1A. Nature 382, 319-324. Yang, X. J., Ogryzko, V. V., Nishizawa, J., Howard, B. H., and Nakatani, Y. (1 9 9 6 ). A p300/CBP-associated factor that competes with the adenoviral oncoprotein E1A. Nature, 319-324. Yankulov, K., Blau, J., Purton, T., Roberts, S., and Bentley, D. L. (1 9 9 4 ). Transcriptional elongation by RNA polymerase II is stimulated by transactivators. C e l l 77, 749-759. Yant, S. R., Zhu, W., Millinoff, D., Slightom, J. L., Goodman, M., and Gumucio, D. L. (1 9 9 5 ). High affinity YY1 binding motifs: identification of two types (ACAT and CCAT) and distribution of potential binding sites within the human beta globin cluster. N u c l e i c A c i d s R e s . 23, 4353-4362.

Iborra et al: Nuclear compartments Gene Ther Mol Biol Vol 1, 495-508. March, 1998.

Dedicated sites of gene expression in the nuclei of mammalian cells. Fransisco Iborra, Ana Pombo and Dean A Jackson. CRC Nuclear Structure and Function Research Group, Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford, OX1 3RE, UK. ___________________________________________________________________________________________________ Co r r e spo nde nc e to: Dean. A. Jackson, Tel: +44- 1865-275527, Fax: +44- 1865-275501, E-Mail: Jackson@Path.OX.AC.UK. Key Words: Nuclear compartments, chromatin domains, transcription factories, nuclear structure.

Summary E s t a b l i s h i n g s i t e s o f t r a n s c r i p t i o n i n t h e n u c l e i o f higher eukaryotic c e l l s i s a very complex process. Before transcription can begin, a series of transcription factors must associate with their recognition motifs, within promoters and more remote activating sequences. Once bound, these factors and associated proteins are believed to form a complex that positions the RNA polymerase holoenzyme s o that transcription can commence. A s a consequence, active genes assume a specialized chromatin state across regions that define functional domains. Global nuclear architecture appears to stabilize these active domains by providing local environments dedicated to gene expression. As the spatial organization of these sites is unaffected by the removal of most chromatin they must be associated with a structural network. This nucleoskeleton, the associated transcription 'factories' and chromatin loops that arise as DNA binds proteins within factories are fundamental features o f nuclear structure i n higher eukaryotes. We argue that concentrating proteins needed t o perform different steps o f RNA synthesis within specialized nuclear compartments will be important in orchestrating events required for efficient gene expression.

Over recent years, remarkable progress has been made in understanding a number of extremely complex functions performed by our genetic material - DNA. For example, we now have a reasonably clear picture of the basic elements required to initiate gene expression and understand the principles - if not the details - that control gene expression in different cell types. It is clear how different sequence motifs in DNA operate as binding sites to position specialized expression activating 'transcription factors' within gene promoters and how different, though often related, motifs might be located within more distant 'enhancer' elements. In some cases, even more remote 'locus control region' (LCR) elements have been show to exert a dominant effect in establishing chromatin domains competent for gene expression.

factors bound to these different DNA motifs (Tjian and Maniatis, 1994). Proteins bound at the different sites are then though to associate forming a tertiary complex which, in the presence of secondary transcription factors acting as adaptors, provides a protein surface that first interacts with RNA polymerase and then positions the polymerase on the promoter (Goodrich et al., 1996) so that transcription can begin (Zawel and Reinberg, 1995; Aso et al., 1995). Though the complexity of factors involved in this process appears daunting, it now seems likely that this process will be simplified by the use of preformed sub-assemblies. For example, in addition to the synthetic machinery, RNA polymerase II complexes can contain elongation factors, RNA processing components, enzymes required for DNA repair and chromatin remodelling proteins (Koleske and Young, 1995; Maldonado et al., 1996; McCracken et al., 1997; Kim et al., 1997).

To state matters rather simply, the fundamental process of gene expression involves the combined action of protein

In some genes, promoter-bound transcription factors are sufficient to direct efficient expression. Often, however,

I. Introduction

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Iborra et al: Nuclear compartments remote enhancer sequences are required to establish appropriate levels of gene expression. Like promoters, enhancers contain complex arrays of factor recognition motifs that bind appropriate factors. Complex that includes factors bound within both promoters and enhancers probably contribute to the activation process. While there are many possible mechanisms for enhancer function the fact that they work when linked to promoters on catenated DNA molecules, suggests that protein complexes assembled on the promoter and enhancer cooperate during the activation process. Enhancers with different efficiencies have been shown to drive uniform rates of transcription from active genes (Boyes and Felsenfeld, 1996; Osheim et al., 1996); differences in transcription rate reflect the number of active genes in a population and not different polymerase densities on individual genes. A similar mode of action has been proposed for locus control regions (Milot et al., 1996). Promoters appear to determine the rate of initiation while these distal sequence motifs control switching between active and inactive states. Activating expression in individual cells is only part of an even more complex story. Mammalian genomes are estimated to have some 75,000 different genes. Only a minority of these are expressed in different cells. In rough terms, ~1/3rd genes perform house-keeping functions, ~1/3rd specialized functions in ~250 different cell types throughout the body and the remaining ~1/3rd specialized functions in the brain. The activity of highly active genes in expressing tissues and the same gene in non-expressing tissues can vary by up to 108 fold. This remarkable difference in levels emphasises the stability of the inactive and active states and confirms the efficiency of mechanisms that define regions of the genome that are competent for gene expression.

II. Gene expression and chromosomal position effects Factors that influence gene expression are clearly very complex. Even when different genes are introduced together into cells and expressed transiently from plasmids their activity can be influenced drammatically by factors such as their spatial organization (Emerman and Temin, 1984). When genes are integrated into the genome 'position effects' add an additional complexity that commonly results in the eventual extinction of the ectopic gene (Palmiter and Brinster, 1986). Some genomic sites are non-permissive for expression. This phenomenon was first characterized in Drosophila when it was observed that genes translocated close to heterochromatin were commonly switched off (Singh, 1994, for early references). The dominant suppressive properties of constitutive heterochromatin (e.g. centromeres) can spread over 1 Mbp or more; cell to cell

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variations in suppression result in variegation'.

'position effect

In Drosophila, heterochromatin protein 1 (HP1) and the related Polycomb (Pc) proteins maintain a repressed chromatin state. The critical chromatin organization modifier (chromo) domain identified in HP1 and Pc is found in many proteins, including human homologues. Pc is found in large, multi-subunit protein complexes (~5 MDa) and controls expression from homeotic genes during Drosophila development (Franke et al., 1992). Though chromatin status is clearly the target, Pc-repressed chromatin is not resistant to digestion by restriction endonucleases and so is not subjected to generalized condensation. Modification of chromatin stability (stable chromatin is likely to prevent transcription factors access), sequestration into an inactive nuclear compartment and reduced chromatin flexibility may account for these observations (McCall and Bender, 1996, for discussion). Importantly, this system provides a means of developing chromatin compartments or domains with no absolute requirement for chromatin binding to a nucleoskeleton.

III. Chromatin domains as units of gene expression The structure of active and inactive chromatin is clearly different (Edmondson and Roth, 1996). Much inactive chromatin is condensed and forms heterochromatic chromatin clumps inside the cell (Figure 1). Active chromatin must be accessible to proteins involved in gene expression and is consequently relatively open or dispersed. The ease with which chromatin is cut by nucleases provides the best indicator of activity status (Wolffe, 1995). The most accessible, 'hypersensitive' sites highlight regions of functional importance - promoters, enhancers, locus control regions and sites of nuclear matrix attachment. Hypersensitive sites arise as a consequence of structural changes that appear when the repetitive nature of nucleosomal chromatin is disrupted by proteins such as transcription factors. These interactions are clearly stable during successive rounds of transcription but appear to be displaced during replication. In addition, transcriptional status correlates with a generalized nuclease 'sensitivity' that results from an open 10nm - chromatin fibre. However, as sensitivity often extends many kbp outside established expression units, it is clear that other factors must be involved, implying that remote sequences determine the boundaries of functional domains. Changes in chromatin structure that accompany the transition from an inactive to active state are believed to be controlled by products of the SWI/SNF genes, first described in Saccharomyces cerevisiae (Kingston et al.,

Iborra et al: Nuclear compartments Figure 1. Morphology of the mammalian cell nucleus. Growing HeLa cells were processed by standard EM techniques and thin sections stained. Inside the nucleus (n), the most prominent feature is the nucleolus (nu), with many fibrillar centres (fc) surrounded by dense fibrillar components (dfc) and a dispersed granular component (gc). The nucleoplasm contains dense patches of condensed chromatin (hc) that stand out against the relatively amorphous nuclear interior. The nucleus is separated from the organelle-rich cytoplasm (c) by a nuclear membrane (nm). Nuclear pores allow molecules to pass between the nucleus and cytoplasm. Bar, 1 Âľm.

contrast to the relatively amorphous nuclear interior. Within the interior, specialized staining techniques allow different features to be recognized (Monneron and Bernard, 1969). EDTA regressive staining, for example, confirms that roughly half of this region is occupied by dispersed chromatin. The remaining, interchromatin space, is rich in hnRNPs and contains characteristic structures such as perichromatin fibrils, perichromatin granules, interchromatin granules and interchromatin granule clusters. Though the precise functions of these structures remain unresolved, they are believed to play different roles in RNA metabolism (Fakan, 1994).

1996). The SWI genes were shown to be required for mating type switching and subsequently, to form a complex capable of disrupting chromatin structure. Interestingly, the SWI/SNF complex has been shown to co-purify with the yeast RNA polymerase II holoenzyme, suggesting that it might operate to disrupt euchromatin, during RNA synthesis (Koleske and Young, 1995)

IV. Nuclear compartmentalization The influence of gene position on expressional status emphasises the importance of higher-order chromatin structure in establishing patterns of gene expression. Protecting such structures in vivo, through successive rounds of transcription and replication, will be crucial to the maintenance of a cell's expression programme.

Recently, the use of immuno-staining as a routine analytical tool has emphasized the structural complexity within eukaryotic nuclei (Spector, 1993; Strouboulis and Wolffe, 1996; Jackson and Cook, 1996). Autoantibodies to protein components of snRNPs were first used to demonstrate that these proteins concentrate in 20-50 nuclear speckles in mammalian cells. These major sites were later shown to be inter-connected by a fibro-granular 'network' of minor sites (Figure 2). Like splicing components, many other proteins involved in gene expres-

The cytoplasm of a typical mammalian cell is highly structured, containing many classes of membranated organelles with specialized roles. The nucleus contains no equivalent structures and appears ill-organized, in comparison. A section of a human cell emphasizes this impression (Figure 1). The nuclear membrane, nucleolus and regions rich in condensed heterochromatin stand out in

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Figure 2. Nuclear compartments rich in splicing proteins and nascent transcripts. HeLa cells were permeabilized with saponin in an isotonic buffer and sites of RNA synthesis labelled for 15 min with Br-UTP (A-D) or biotin-CTP (E-H). Sm (B) or SC35 (F) antigens and Br-RNA (C) or biotin-RNA (G) were indirectly immunolabelled and DNA stained with TOTO-3 (A,E). Optical sections (~700 nm) show a classical distribution of Sm and SC35 antigens, with major sites 'speckles' and dispersed minor foci; nucleoli are blank. In the nucleoplasm, most sites of transcription lie adjacent to the minor foci (D and H, merges of B,C and F,G respectively). Note that in (G), anti-biotin antibodies label an extensive mitochondrial network. This indicated the preservation of cellular structure under conditions used. Loss of structure generally correlates with the appearance of mitochondrial transcription, not seen here (C). Bar, 2.5 Âľm. See Pombo and Cook (1996) for details.

CTP); under these condition it is a simple matter to control the rate of transcription and so ensure that only sites of synthesis are labelled. Cells labelled with Br-UTP and examined by light microscopy, after immuno-labelling sites of incorporation (F i g u r e 2 ), demonstrate that the majority of labelled transcripts are concentrated within a limited number of nuclear sites, and not diffusely spread throughout chromatin (Jackson et al., 1993: Wansink et al., 1993). Labelling under in vivo conditions (microinjected Br-UTP or cells grown in medium supplemented with Br-U) gives the same impression if short labelling periods (~5 min) are used (Fay et al., 1997); after longer labelling intervals staining patterns are complicated by the presence of labelled RNA in transit to the cytoplasm.

sion give intriguingly punctate staining patterns (van Driel et al., 1996). Understandably, such images are assumed to demonstrate that different proteins accumulate at functionally important sites. In some cases, however, the complexity of staining is greater than anticipated, implying that proteins might accumulate at nonfunctional, storage, sites.

V. Sites of transcription In some cases, the inability of antibodies to proteins involved in different nuclear functions to distinguish active and inactive sites can be overcome by labelling active sites directly. For example, sites of transcription in mammalian cells can be labelled using 3[H]-uridine (Fakan and Puvion, 1980). High resolution analyses have identified perichromatin fibrils as the features most closely associated with transcription sites. Under these conditions, however, scatter of emitted irradiation (50% of gains lie more than 200 nm from their source using standard EM autoradiography) prevents the unambiguous identification of transcription sites. Recently, resolution has been improved by labelling transcription sites in permeabilized cells using modified RNA precursors (Br-UTP or biotin-

The ability to label sites of transcription in this way allows the active sites of RNA synthesis to be compared with the organization of other components involved in gene expression, by immuo-fluorescence (van Driel et al., 1996; Huang and Spector, 1996; Pombo and Cook, 1996; Grande et al., 1997).

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Figure 3. Genes in action. Chromatin templates and associated transcripts can be visualized after 'spreading' nuclear contents. Such 'Miller spreads' are commonly prepared from amphibian oocytes where the nuclear membrane can be removed manually and nuclear contents dispersed in water. As mammalian nuclei cannot be manipulated in this way, HeLa cells (A,B) must be dispersed in a 0.33% solution of a commercial detergent (Joy), during spreading. Ribosomal RNA genes usually appear clustered and are rich in nascent transcripts (A). In contrast, extra-nucleolar transcription unit, in dispersed chromatin, have very few associated transcripts (B). Bar, 1 Âľm. See Miller and Bakken (1972) for details.

~200 nm across (F i g u r e 1 ). As the nascent transcripts mature they pass into the granular component (gc) where they become associated with the ribosomal proteins.

VI. The nucleolus - a dedicated site of ribosomal RNA biosynthesis The specialized site of transcription that has been studied most intensively in mammalian cells is the nucleolus (Fischer et al., 1991; Shaw and Jordan, 1995). Each diploid human cell has an estimated 300-400 copies of the gene needed to make ribosomal RNA. These are located on chromosomes 13, 14, 15, 21 and 22 where the ~40 kbp repeats are grouped into clusters usually with 3-5 genes in each unit. At any time, ~1/3 of the rRNA genes are active. These are expressed within specialized sub-nuclear organelles called nucleoli (Figure 1). Most diploid human cells have 1 or 2 nucleoli but cells in culture can have more; nucleolar morphology is a marker for growth status and can be used as one indicator in the diagnosis of malignancy.

The high demand for ribosomal RNA molecules means that these genes are the most active in human cells - a proliferating cell must produce ~5x106 ribosomes during each cell cycle of ~24 hours. Nucleolar structure ensures that the components required for RNA production are organized to optimize efficiency. This is borne out by the appearance of the active genes and their associated nascent transcripts (F i g u r e 3 ). When nucleoli are disrupted and spread, each active gene is seen to have between 100-150 engaged RNAs. The polymerases are loaded with such efficiency (~1/120 bp) that the active transcription units remain devoid of histones (Miller and Bakken, 1972).

VII. Specialized sites of transcription in the nucleoplasm

The anatomy of nucleoli is well documented (Figure 1). Fibrillar centres (fcs) are rich in the synthetic machinery (RNA polymerase I). Fcs are surround by a zone called the dense fibrillar component (dfc) where the nascent transcripts accumulate. An individual fc is probably coated with the genes and nascent products from a single active rDNA cluster (3-5 genes); in cross section, the products of a single gene appear as densely staining areas measuring

While early experiments analysed by LM supported the existence of nucleoplasmic transcription centres more detailed studies were required to assess if nucleoli are a paradigm for transcription site organization (HozĂĄk et al.,

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F i g u r e 4 . Distribution of transcription sites. HeLa cells were grown for 5 min in medium supplemented with Br-uridine. EM sections were prepared and sites of RNA synthesis immunolabelled with 9 nm gold particles. Within the nucleus (n), most particles lie in clusters (arrowheads). The bulk of the nuclear interior, nuclear membrane (nm) and cytoplasm (c) are unlabelled. Parts of two nucleoli (nu) are seen in this section. Bar, 250 nm. See Iborra et al. (1996) for details.

any moment (Cox, 1976). To confirm this crucial point, we have shown recently (Jackson et al., submitted) that HeLa cells with ~2,500 nucleoplasmic sites have ~75,000 active nucleoplasmic RNA polymerases. Unlike nucleoli, however, genes transcribed in the nucleoplasm are known to have rather low densities of associated transcripts. When HeLa nuclei are disrupted by hypotonic treatment and spreading dispersed chromatin fibres rarely have more than a few putative transcripts (Figure 3). Even cells infected with adenovirus, at their peak of transcription, have only 1 transcript/7.5 kbp adenovirus DNA (Beyer et al., 1981; Wolgemuth and Hsu, 1981). Using HeLa cells disrupted with sarkosyl - under conditions that retain all engaged RNA polymerases - we have confirmed that most active genes have very few (usually 1-3) transcripts. This suggests, perhaps remarkably, that an average transcription site contains the machinery to simultaneously synthesise and process 30 transcripts associated with ~20 different genes. Because the nascent transcripts only occupy 0.5% of a HeLa cell nucleus (Iborra et al., 1996) these cannot contain all the active chromatin. It seems probable, therefore, that active genes will surround the synthetic sites, as indicated above (Figure 5B). Note, however, that despite their small size and evident complexity, the

1994). Electron microscopy of HeLa cells labelled with RNA precursor analogues (Iborra et al., 1996) both in vivo and in vitro has shown a typical cell to have ~2500 distinct transcription compartments (Figure 4), with labelled zones measuring 50-150 nm (mean is 80 nm) across. The labelled sites often lie along the borders of nuclear regions rich in condensed chromatin and usually lie adjacent to or are surrounded by 'clouds' of dispersed chromatin (Figure 5B). The nascent sites also contain the synthetic machinery (Figure 5A) which quite often appears concentrated towards one sub-region of the labelled zone. The sites also contain transcription factors, splicing factors and others proteins involved in RNA processing. Transcription sites often lie close to, but never within, interchromatin granule clusters (Figure 5C) that are rich in many splicing factors and appear as 'speckles' by LM when stained with antibodies to splicing components such as Sm and SC35 proteins (Figure 2). The main difference between nucleolar and nucleoplasmic transcription sites concerns the organization of the active genes. Like nucleoli, nucleoplasmic transcription sites must contain many active polymerases. This can be inferred from the number of sites and estimates of the number of active polymerases and genes in a cell at

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Gene Therapy and Molecular Biology Vol 1, page 501 nucleoplasmic sites have less than half the RNA density of equivalent sites in nucleoli.

transcription sites contain predominantly (if not exclusively) RNA polymerase III transcription units. In addition, we (AP) have characterized the organization of a number of genes that are activated by the transcription factors PTF and OCT-1 in association with a PTF-rich sub-nuclear compartment.

As many genes appear to be transcribed from individual sites, that are also capable of performing a range of downstream processing events, we have called these sites transcription 'factories'. In view of the complexity of these factories, it is interesting to consider whether different genes with common requirements (e.g. activating transcription factors) might accumulate at individual sites. Though the chromosomal arrangement of genes will be dominant in determining the composition of individual transcription sites, circumstances could arise where related genes, on different chromosomes, are transcribed within the same compartment. Preliminary experiments have hinted that this might be so. For example, we (AP and DAJ) have shown that a fraction (~1/10th) of nucleoplasmic

VIII. The spatial organization of transcription sites Another remarkable feature of the structure of transcription factories is that the spatial organization of labelled nascent RNA persist in nuclei when almost all chromatin is removed (F i g u r e 6 ; Jackson et al., 1993; Wansink et al., 1993: Iborra et al 1996). The nascent RNA, unlike most mRNA en route to the nuclear periphery (Verheijen et al., 1988), is tightly associated with the nuclear matrix (Figure 7), confirming that interactions within transcription factories are stable under a range of conditions, independently of chromatin. This emphasises the important point that the transcription factories are structures in their own right and do not arise passively as a consequence of the organization of other nuclear components. This view of global nuclear organization supports the idea that active RNA polymerases are 'fixed' and is incompatible with transcription complexes that track along the chromatin during RNA synthesis. In addition, transcription factors, polymerases and other proteins involved in gene expression will perform critical structural roles, binding chromatin at functionally important sites that become spatially restricted through their indirect association with the nucleoskeleton. While such an arrangement will have profound organizational consequences in higher eukaryotes, it is worth noting that such an arrangement has been shown to exist within virus particles (Prasad et al., 1996). The in vivo organization of transcription sites has also been analyzed using an approach that determines the spatial distribution of active genes and their products using fluorescent in situ hybridization (FISH). Though the processing required for hybridization leads to loss of morphological detail, this approach has provided data on the relative organization of active genes and local processing compartments (Xing et al., 1993) and has suggested interesting mechanisms by which mature mRNA molecules might be transported to the cytoplasm (Rosbach and Singer, 1993).

Figure 5. The architecture of transcription sites. HeLa cells were permeabilized and sites of RNA synthesis labelled with biotin-CTP for 15 minutes. Sites of synthesis were immunolabelled with 9 nm gold (A-C). Most sites also contained RNA polymerase, immunolabelled with 15 nm gold (A); note that the 9 and 15 nm gold particles are generally subcompartmentalized within transcription sites. Transcription sites commonly appeared to be surrounded a halo of chromatin clouds, visualized after EDTA regressive staining (B). Bismuth binds phosphoproteins (such as RNA polymerase II) and stains transcriptions sites (C), though adjacent regions were often unstained. Interchromatin granule clusters stained with bismuth but never contained labelled transcripts. Bar, 100 nm. See Iborra et al. (1996) for details.

IX. Sites of pre-mRNA splicing While it is clear that components involved in major nuclear functions are compartmentalized, the extent to which these compartments correlate with sites of function

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Figure 6. nucleoskeleton.

The

Encapsulated HeLa cells were permeabilized, chromatin cut with nucleases, ~90% DNA removed and a 500 nm resinless section prepared. Agarose (A) surrounds the cytoplasmic (C) and nuclear remnants that are separated by the nuclear lamina (L). The nucleoskeleton, a diffuse network of coated filaments (arrowheads) connects regions of the nuclear interior such as the nucleolus (NU) replication factories (F) and many dense sites (D), some of which arise from transcription sites. Bar, 1 Âľm. See Jackson and Cook (1988) for details.

demand/turnover but could also be sites of storage and/or assembly that are subsequently dispatched elsewhere. The dynamic features of these compartments have been assessed using an essential splicing factor (SF2/ASF) tagged with green fluorescent protein Misteli et al., 1997).

is not always easy to assess. This is most apparent when function is difficult to measure directly. Understanding the organization of sites of pre-mRNA splicing serves to emphasise this point (Figure 2). A series of sophisticated analyses have shown that many genes lie close to the splicing speckles (Moen et al., 1995). As in situ hybridization shows these sites to be rich in polyA+ nuclear RNA and much, but not all, splicing occurs cotranscriptionally it appears that the speckles represent predominant sites of active splicing. Other observations refute this view. First, cells grown in 3[H]uridine for short times show little or no labelling within IGCs/speckles; even after long incorporations these structures remain poorly labelled (Puvion and Puvion-Dutilleul, 1996). Second, high resolution analyses of nascent transcripts confirm that while many lie close to IGCs no transcription occurs within them (Figure 5; Iborra et al., 1996; Puvion and Puvion-Dutilleul, 1996; Pombo and Cook, 1996). Finally, in situ hybridization techniques designed to analyze the sites of splicing indicate that speckles are not the predominant active sites (Zhang et al., 1994).

X. RNA transport pathways In the majority of cases, activities present within transcription factories, adjacent to the site of RNA synthesis, will ensure that mature mRNA molecules leave factories. Though the majority of splicing occurs at the time of transcription, some RNAs containing introns may move into the downstream transport pathways. Details of events that control RNA transport and export are complex and have been reviewed extensively (Gorlich and Mattaj, 1996). It is not appropriate to discuss an extensive literature here, though we would like to consider one interesting aspect of transport that has arisen from our own studies. It is usually assumed that nuclear RNAs and their associated proteins move inside the nucleus as independent entities (Dreyfuss et al., 1993). Movement is probably diffusion driven in inter-chromatin channels. A detailed analysis of large (200S) RNP particles has demonstrated that RNA molecules are associated with protein according to size: RNA from 1.5 to 35 kb is found in particles of similar mass (Spann et al., 1989; Sperling et al., 1997). This observation is believed to reflect the high protein

Possible explanations of these controversies range from the obvious limitations, technical capabilities and resolution of different approaches used to cell-specific differences. Perhaps these observations indicate that the major (speckles) and minor sites represent 2 parts of the same population that is able to respond to demand - is dynamic - performing splicing as and when required. The major sites might represent regions of particularly high 502

Gene Therapy and Molecular Biology Vol 1, page 503 content of the particles, that contain a modular structure thought to be a remnant of earlier splicing events. These particles are stable even though ~95% RNA present is fully spliced mRNA. In our analysis of the movement of nuclear RNA labelled with bromouridine in vivo, we have shown that the majority of RNA transport occurs in association with structures equivalent to these 200S particles. The particles contain Br-RNA and are rich in SR-proteins, a family of proteins involved in splicing (Manley and Tacke, 1996). Interestingly, the size distribution and numbers of labelled particles in transit suggest that each contains many mRNAs.

XI. Global nuclear structure Many lines of evidence indicate the functional importance of global nuclear structure. We know, for example, that chromosomes occupy discrete nuclear domains during interphase and that these often assume preferred though never precise nuclear locations (Cremer et al., 1993). Preferred orientations are not uncommon (probably a vestige of mitosis) and may differ at different stages of the cell cycle; even at this level nuclear structure must be dynamic (Ferguson and Ward, 1992). Though chromosomes in interphase are decondensed relative to their familiar mitotic counterparts, it is clear from in situ hybridization analyses that individual domains remain locally restricted and that the linear arrangement of genes, together with local structural features, closely reflect those seen in mitosis (Yokota et al., 1995). Apparently dramatic differences in the organization of chromosomes during mitosis and corresponding structures during interphase probably reflect the dispersal of chromatin domains through elongation of the chromosome axis, with relatively minor changes in chromatin condensation. As the nucleus reforms after mitosis, chromosome decondensation will generate inter-chromatin channels, both between and within chromosomes, allowing access of different nuclear components to all functionally important parts of the genome.

F i g u r e 7 . Transcription at the nuclear matrix.

HeLa cells with nascent transcript containing Br-UMP (Figure 4 ) were extracted with 2 M NaCl and Br-RNA immunolabelled with 5 nm gold particles. Samples were then embedded and sections prepared (A,B). A typical nuclear region (A) shows numerous dense areas separated by the fibrogranular 'nuclear matrix'. Many of the dense areas are rich in Br-RNA (B; higher magnification of area indicated in A); these are clearly remnant transcription sites. Note that immunolabelling before embedding gives much higher particle densities because label is not restricted to the detection of antigens on a section surface. This increases sensitivity but does not alter the number of transcription sites seen. Bars, 100 nm.

XII. The nuclear matrix and nucleoskeleton By their very nature, nuclear functions such as transcription and replication must be dynamic processes. It was surprising, therefore, when the products of these processes were shown to retain their spatial organization in extracted nuclei from which almost all chromatin had been removed. To explain such observations, it was argued that the active enzymes were associated with and organized by a structural 'nuclear matrix'. Subsequently, the nuclear matrix of extracted cells has been shown to participate in

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Iborra et al: Nuclear compartments almost all aspects of nuclear function, in higher eukaryotes (reviewed in Berezney et al., 1996; Nickerson et al., 1996; Stein et al., 1996).

scs may be important determinants of chromosome structure (Zhao et al., 1995; Strick and Laemmli, 1995).

A detailed analysis of functional sites in cells extracted under 'physiological' conditions (Jackson and Cook, 1996) has ruled out the possibility that this organization arises during matrix preparation and confirmed that active transcription sites maintain their spatial disposition when almost all chromatin, representing about 40% of nuclear mass, is removed. This crucial observation shows that the active sites are intimately associated with an organizational solid phase, probably the major structural feature within eukaryotic nuclei (Figures 6 and 7 ). At the heart of this structure lies a network of intermediate filament-like core filaments that provides important structural continuity by connecting sites of functional importance to the nuclear periphery. In situ, this structure is coated and stabilized by hnRNP complexes, many of which will be en route to the cytoplasm. Though structural, this nucleoskeleton must be dynamic - dramatic changes accompany mitosis and more subtle ones result from changes in growth.

XIV. Global organization and dedicated sites of gene expression

XIII. Chromatin domains and loops The nucleoskeleton and functional compartments that it binds are fundamental features of internal nuclear architecture. As these structures are reasonably stable they are likely to be a major source of protein-DNA interactions that form DNA loops and chromatin domains inside the nucleus. Once again a complex literature addresses this issue (Laemmli et al., 1992; Bode et al., 1996). In brief, scaffold and matrix attached sequences (SARs and MARs) are commonly AT-rich sequences with many topoisomerase II binding motifs. Various other proteins have been shown to bind strong S/MAR DNA sites. It is not clear, however, how the organization of these extracted structures reflects that existing inside the cell (Jacks and Eggert, 1992). In contrast, in domains analyzed under isotonic conditions, most sequences responsible for binding chromatin to the nucleoskeleton were of functional importance (Jackson and Cook, 1993; Jackson et al., 1996). It appears that different extraction protocols must accentuate different classes of interactions existingin vivo. Probable chromatin domain boundaries have been characterized in the fruit fly, Drosophila. 'Specialized chromatin structures' (scs) flanking two heat shock genes (HSP70) were shown to correlate with the boundaries of an ~15 kbp active domain - at 87A7 on polytene chromosomes - following heat treatment (Schedl and Grosveld, 1995). These elements contain pairs of very strong DNAse hypersensitive sites flanking a nuclease resistant sequence of ~300 bp. Boundary element attachment factors and their recognition motifs within the

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A priori, we might assume that genes expressed from their natural chromosomal sites operate with the required efficiency. Wide variations in the expression of identical ectopic sequences, at different chromosomal sites, implies that local factors can influence gene expression (Allen et al., 1988; Bonnerot et al., 1990). Different factors must contribute to the range of expression seen. It is clear how chromosomal position, through the suppressive influence of local heterochromatin, might extinguish expression. Partial activities, in contrast, must reflect transcription from sites with different combination of factors that together determine the frequency with which transcription occurs. Other lines of evidence imply that nuclear organization can influence gene function. For example, RNA molecules transcribed from chimeric genes - with RNA polymerase III promoters and polymerase II coding sequences - introduced into mammalian tissue culture cells by transfection, produced normal pre-mRNA but were unable to splice or form mature polyA+ tails and hence mature mRNA (Sisodia et al., 1987). This suggests that splicing and polyadenylation pathways are coupled to transcription by RNA polymerase II and that different pathways are spatially independent. Rather surprising observations suggest that different steps in the expression pathway cooperate to produce mature mRNA. For example, the _-globin promoter was shown to drive synthesis of transcription from an introncontaining gene but not the same sequence from which the non-coding DNA had been removed (Neuberger and Williams, 1988). Remarkably, the same intron-less transcription unit was expressed from CMV or heat-shock promoters. Such observations suggest that nuclei are assembled so that the local organization of components required for gene expression will reproduce a level of activity that reflects that organization. In addition to establishing the desired level of synthesis, this nuclear 'set-up' appears to direct the transcription product onto an appropriate pathway that couples synthesis to the desired combination of postsynthetic events - RNA processing, export and perhaps even cytoplasmic location and function.

XV. Conclusion It is now clear that different layers of organization contribute to the complex processes required for gene expression. Simple recognition motifs in chromatin first

Gene Therapy and Molecular Biology Vol 1, page 505 bind transcription factors and set the activation process in motion. Multi-component pre-initiation complexes then facilitate the binding of the RNA polymerase complex to the promoter (Goodrich, et al., 1996) so that elongation can proceed (Aso et al., 1995; Zawel and Reinberg, 1995). Intellectually, these steps are easy to describe in molecular detail.

present a time averaged image of nuclear order and give no indication of either spatial or temporal dynamics. But even if factors can scan for binding sites, it may be that they then serve to deliver and subsequently confine genes to appropriate nuclear compartments. Once established, such complexes could have a major influence on higher-order chromatin structure, throughout interphase and mitosis.

These processes must occur in the context of systems established to maintain active and inactive chromatin states. Though we know surprisingly little about the signals that define chromatin domains it is clear the certain LCR elements can 'open' chromatin and establish domains that are permissive for transcription (Schedl and Grosveld, 1995). The partial activation of expression from an incomplete LCR together with equivalent experiments on enhancer elements are consistent with the view that these remote elements make vital contributions to the active promoter complex (Wijgerde et al., 1995). The behaviour of genes with incomplete LCRs, situated close to heterochromatin, indicates that the complete LCR maintains transcription activity by ensuring that transcription occurs in all cells, at all times, but does not directly control the rate of transcription (Milot et al., 1996).

Another advantage of dedicated nuclear compartments is that by concentrating components required to perform coupled functions in a limited number of sites, it will be possible to execute these functions with optimal efficiency. If components were released into a 'soluble' nucleoplasm between each activity, a dramatic fall in efficiency might be expected. It is clear that active chromatin domains must maintain their active configuration under circumstances where the majority of chromatin is inert. Though different mechanisms will tend to stabilise these forms, it is important that they are both sufficiently stable to maintain expression and sufficiently unstable to allow reprogramming. Interestingly, proteins capable of remodelling chromatin have been shown to form an integral part of the yeast RNA polymerase II holoenzyme (Koleske and Young, 1995). Restricting the spatial distribution of the complex in this way will tend to preserve existing chromatin states. In addition, compartments rich in transcription factors would also help to re-establish active sites following replication. Elongating DNA polymerases are known to displace certain transcription factor complexes and these must be reestablished if expression is to be maintained. This must occur, however, in the presence of large histone pools that could alter promoter activity by competing for the same sites. Replicating active genes early in S-phase within compartments that contain the components required to reestablish an active chromatin configuration provides the best opportunity of protecting existing patterns of gene expression within eukaryotic cells.

In vivo, additional organizational features appear to influence gene expression. Over recent years a significant body of literature has described the compartmentalization of different nuclear functions. Rather than being dispersed uniformly throughout the nuclear interior, different functions required for expression occur at specialized, dedicated sites. In mammalian cells the density of transcription sites is far lower than expected from estimates of active transcripts and transcription unit complexity, implying than multiple transcription units are active in each site. Morphological analyses together with immunostaining indicate that these sites have a zonal organization, with different regions performing specialized roles. Furthermore, as the transcript containing regions occupy only 0.5% of the volume of a HeLa nucleus this arrangement is inconsistent with the view that transcripts will be uniformly dispersed throughout euchromatin (occupying ~10% of the nucleus) bound to tracking RNA polymerases. Images like Figure 5B support the idea that many chromatin clouds are served by a single, active transcription compartment. This ordered view of transcript synthesis and maturation has interesting functional consequences. Before any genes can be expressed, transcription factor complexes must assemble on its promoter (Goodrich et al., 1996). It is commonly assumed that these factors scan chromatin for potential binding sites. The observation that significant fractions of many factors are nuclear matrix associated complicates this view, implying that factors are part of some nuclear 'solid phase'. Of course such impressions 505

Finally, it is worth considering how nuclear organization could influence the behaviour of particular genes in differentiated cells. With the exception of nucleoli, we known very little about the constellations of genes that contribute to individual transcription sites or whether individual sites can be totally or partially dedicated to the synthesis of particular classes of transcripts. While groups of genes on particular chromosomes will dominate local organization of transcription sites, some contribution from active genes on adjacent chromosomes might be expected. This could be especially prevalent in nonproliferative cells where long periods without the disruptive forces of the cell cycle might allow genes with similar activation requirements to occupy particular sites. Such an arrangement could influence the frequency of common translocations during the development of malignancy. Specialized transcription sites could also

Iborra et al: Nuclear compartments influence the performance of non-chromosomal genes. If cells have a limited number of sites that are competent for the synthesis of specific gene products, understanding how to access these sites would be important in gene therapy. Acknowledgements We thank the Cancer Research Campaign (DAJ), The Wellcome Trust (FI) and the Junta Nacional de Investigaç_o Científica e Tecnológica (Portugal, Program PRAXIS XXI; AP) for support.

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