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Gene Therapy & Molecular Biology FROM BASIC MECHANISMS TO CLINICAL APPLICATIONS

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Gene Therapy and Molecular Biology Vol 3

Table of contents Gene Ther & Mol Biol Vol 3, August 1999

Pages

Type of Article

Article title

Authors (corresponding author is in boldface)

1-14

Review Article

Routes of vector application for brain tumor gene therapy

Nikolai G. Rainov, Xandra O. Breakefield, Christof M. Kramm

15-23

Research Article

Efficient in vivo expression of a reporter gene in rat brain after injection of recombinant replicationdeficient Semliki Forest virus

Kenneth Lundstrom, J. Grayson Richards, J. Richard Pink, and Francois Jenck

25-33

Research Article

Establishment of an assay to determine adenovirus-induced endosome rupture required for receptor-mediated gene delivery

Daniela Schober, Nora Bayer, Robert F. Murphy, Ernst Wagner and Renate Fuchs

35-44

Review Article

Gene regulation in Herpesvirus saimiri and its implications for the development of a novel gene therapy vector

Adrian Whitehouse and Alex J. Stevenson

45-56

Review Article

Regulation of papillomavirus transcription and replication; insights for the design of extrachromosomal vectors

Alison A. McBride

57-65

Review Article

Gene transfer with adeno-associated virus 2 vectors: the growth factor receptor connection

Cathryn Mah, Keyun Qing, Jonathan Hansen, Benjawan Khuntirat, Mervin C. Yoder, and Arun Srivastava

67-74

Research Article

Hepatocyte-specific gene expression by a recombinant adeno-associated virus vector carrying the apolipoprotein E enhancer and !1antitrypsin promoter

Torayuki Okuyama, Motomichi Kosuga, Satori Takahashi, Kyoko Sasaki, and Masao Yamada

75-78

Minireview

Human cytomegalovirus (HCMV) nuclease: implications for new strategies in gene therapy

Elke Bogner


Gene Therapy and Molecular Biology Vol 3

79-89

Review Article

Application of recombinant Herpes Simplex Virus-1 (HSV-1) for the treatment of malignancies outside the central nervous system

George Coukos, Stephen C. Rubin, and Katherine L Molnar-Kimber

91-101

Review Article

Transcriptional repression in cancer gene therapy: targeting HER-2/neu overexpression as an example

Mien-Chie Hung and Shao-Chun Wang

103-112

Review Article

Gene therapy targeting p53

John Nemunaitis

113-121

Research Article

Targeted therapy of CEA-producing cells by combination of E. coli cd/HSV1-tk fusion gene and radiation

Dao-song Xu, Xin-yao Wu, Yun-fei Xia, Ling-hua Wu, Chao-quan Luo, Yin-hao Yang, Lu-qi Zhong, and Bin Huang

123-131

Research Article

Efficacy of antiherpetic drugs in combined gene/chemotherapy of cancer is not affected by a specific nuclear or cytoplasmic compartmentation of herpes thymidine kinases

Bart Degrève, Erik De Clercq, Anna Karlsson, and Jan Balzarini

133-148

Review Article

Glioblastoma multiforme: molecular biology and new perspectives for therapy

Giorgio Palù, Luisa Barzon, and Roberta Bonaguro

149-155

Review Article

Gene-based vaccine strategies against cancer

Daniel Lee, Ken Wang, Liesl K. Nottingham, Jim Oh, David B. Weiner, and Jong J. Kim

157-165

Review Article

Rational vaccine design through the use of molecular adjuvants

Jong J. Kim, Liesl K. Nottingham, Jim Oh, Daniel Lee, Ken Wang, Mera Choi, Tzvete Dentchev, Darren Wilson, Devin M. Cunning, Ara A. Chalian, Jean Boyer, Jeong I. Sin, and David B. Weiner

167-177

Review Article

In vivo production of therapeutic antibodies by engineered cells for immunotherapy of cancer and viral diseases

Mireia Pelegrin, Danièle Noël, Mariana Marin, Estanislao Bachrach, Robert M. Saller, Brian Salmons, and Marc Piechaczyk

179-187

Research Article

Use of DNA priming and vaccinia virus boosting to trigger an efficient immune response to HIV-1 gp120

Dolores Rodríguez, Juan Ramón Rodríguez, Mercedes Llorente, Pilar Lucas, Mariano Esteban, Carlos Martínez-A. and Gustavo del Real

189-196

Review Article

Gene therapy approaches to the treatment of hemoglobinopathies

Linda Gorman and Ryszard Kole


Gene Therapy and Molecular Biology Vol 3

197-206

Research Article

Intramuscular injection of plasmid DNA encoding intracellular or secreted glutamic acid decarboxylase causes decreased insulitis in the nonobese diabetic mouse

Jingxue Liu, Maria Filippova, Omar Fagoaga, Sandra Nehlsen-Cannarella, and Alan Escher

207-221

Review Article

Muscle-based tissue engineering for the musculoskeletal system

DS Musgrave and Johnny Huard

223-232

Review Article

Helper-dependent adenoviral vectors as gene delivery vehicles

Manal A. Morsy, Diane M. Harvey, and C. Thomas Caskey

233-241

Review Article

Gene transfer into muscle for the treatment of muscular dystrophy and haemophilia

Geoffrey Goldspink, Maria Skarli and Paul Fields

243-248

Review Article

Gene therapy for arthritis

Sherry Thornton and Raphael Hirsch

249-256

Review Article

Antisense gene therapy in the longterm control of hypertension

Craig H. Gelband, Michael J. Katovich, Mohan K. Raizada

257-269

Research Article

Construction and deployment of triple Ling Ren, Shani L. Schalles, Weihua Pan, Corinne E. Isom, Sarah E. Loy, Jiaribozymes targeted to multicatalytic Hai Lee, Catharine M. Benedict, Mary T. proteinase subunits C3 and C9 Pickering, James S. Norris, and Gary A. Clawson

271-280

Review Article

Development of hammerhead ribozymes for HIV-1 gene therapy: principles and progress

A. Ramezani and Sadhna Joshi

281-291

Review Article

Use of antisense oligonucleotides to study homeobox gene function

Olubunmi Afonja, Takashi Shimamoto, John E. Smith, Jr., Long Cui, and Kenichi Takeshita

293-300

Research Article

Potential application of dominant negative retinoic acid receptor genes for ex vivo expansion of hematopoietic stem cells

Yoji Ogasawara, Yutaka Hanazono, Hiroshi Kodaira, Masashi Urabe, Hiroyuki Mano, Akira Kakizuka, Akihiro Kume, Keiya Ozawa

301-310

Research Article

Optimized expression of serotonin receptors in mammalian cells using inducible expression systems

Peter Vanhoenacker, Walter Gommeren, Walter H.M.L. Luyten, JosĂŠe E. Leysen and Guy Haegeman


Gene Therapy and Molecular Biology Vol 3

311-325

Research Article

Identification of a negative regulatory mechanism for the repair of U5 long terminal repeat DNA by the human immunodeficiency virus type 1 integrase DNA polymerase

Brian E. Udashkin, Andrea Acel, Avi Shtvi, Benjamin Alt, Henry Triller, Mark A. Wainberg and Emmanuel A. Faust

327-345

Review Article

Brian A. Lenzmeier and Jennifer K. Nyborg

347-354

Review Article

Molecular mechanisms of viral transcription and cellular deregulation associated with the HTLV-1 Tax protein What does acetylcholinesterase do in hematopoietic cells?

355-371

Review Article

The ETS-domain transcription factors: lessons from the TCF subfamily

Shen-Hsi Yang, Paula R. Yates, Yi Mo and Andrew D. Sharrocks

373-378

Roxanne Y.Y. Chan and Bernard J. Jasmin

D.A. Spandidos, G. Sourvinos, S. Transcriptional activation of the ras oncogenes and implications of BRCA1 Miyakis in the cell cycle regulation through p53 checkpoint

379-385

Review Article

Nuclear receptor coactivators as potential therapeutical targets: the HATs on the mouse trap

Arndt Benecke and Hinrich Gronemeyer

387-395

Review Article

High mobility group protein HMGIC: a molecular target in solid tumor formation

Erik Jansen, Marleen M.R. Petit, Eric F.P.M. Schoenmakers, Torik A.Y. Ayoubi, and Wim J.M. Van de Ven

397-412

Review Article

Replication of simple DNA repeats

Maria M. Krasilnikova, George M. Samadashwily and Sergei M. Mirkin

413-422

Research Article

Separation of the DNA replication and transactivation activities of EBNA1, the origin binding protein of Epstein-Barr virus

Derek F.J. Ceccarelli and Lori Frappier

423-435

Research Article

The activation of the lysozyme locus in development is a cooperative process

Matthias C. Huber and Constanze Bonifer

437-445

Review Article

Mechanisms involved in regulation of the estrogen-responsive pS2 gene

Ann M. Nardulli, Jongsook Kim, Jennifer R. Wood, and Lorene E. Romine


Gene Therapy and Molecular Biology Vol 3

447-453

Review Article

Biological function of the USF family of transcription factors

Michèle Sawadogo, Xu Luo, Mario Sirito, Tao Lu, Preeti M. Ismail, Yibing Qyang, and Marilyn N. Szentirmay

455-464

Review Article

The role of chromatin in the establishment of enhancer function during early mouse development

Luca Rastelli and Sadhan Majumder

465-474

Review Article

Molecular mechanisms that regulate hyaluronan synthesis

Paraskevi Heldin


Gene Therapy and Molecular Biology Vol 3, page 1 Gene Ther Mol Biol Vol 3, 1-14. August 1999.

Routes of vector application for brain tumor gene therapy Review Article

Nikolai G. Rainov1, Xandra O. Breakefield2, Christof M. Kramm3 1

Department of Neurosurgery, Faculty of Medicine, Martin-Luther-University, Halle, Germany, 2 Molecular Neurogenetics Unit, Department of Neurology, Massachusetts General Hospital, and Neurosciences Program, Harvard Medical School, Boston, MA, USA University Children’s Hospital, Heinrich-Heine-University Medical Center, Duesseldorf, Germany.

__________________________________________________________________________________ Corresponding author: Nikolai G. Rainov, M.D., Martin-Luther-University Halle, Dept. Neurosurgery, Magdeburger Str. 16, D-06097 Halle, Germany. Tel: +49 345 5571399; Fax: +49 345 5571412; E-mail: nikolai.rainov@medizin.uni-halle.de Key words: adenovirus, brain neoplasms, gene therapy, gene transfer, herpes simplex virus, intra-carotid delivery, liposomes, plasmids, retrovirus. Received: 25 September 1998; accepted: 5 October 1998

Summary The development of highly efficient virus and non-virus vector systems for gene transfer to and gene therapy of brain tumors has advanced to the stage of clinical trials, but has still not successfully addressed some major limiting factors, such as the inability of a single delivery modality or therapeutic transgene to target a maximum number of tumor cells in diffuse or multifocal tumors, such as human glioblastoma, and to confer eradicating cytotoxicity to the whole neoplastic mass. Moreover, the choice of vectors and the route of their administration dramatically affect both the efficiency of tumor transduction and its spatial distribution, as well as the extent of transgene expression within a brain tumor and outside it, in the surrounding tumor cell-infiltrated tissue. Three main routes of vector delivery to experimental brain tumors are reviewed in this paper: stereotactic or direct intratumoral inoculation; intrathecal and intraventricular injection; and intravascular infusion with or without modification of the blood-brain-tumor-barrier. The pros and cons of all these modes of application are discussed in respect to the specific and unique features of tumors in the central nervous system. We conclude that, at the present time, there is no ideal vector or unconditionally efficient application mode, and so the successful approaches to brain tumor gene therapy need to combine different application routes with different vectors and therapeutic genes designed to address the individual features of different tumor types. The intravascular vector delivery route, although at an early stage of development, seems to be the most pervasive and demonstrates the greatest therapeutic potential in animal experiments, but for human use it should be combined either with direct intratumoral vector injections or with CSF vector delivery.

transduced cells surrounding transgene-expressing cells, still the transgene-bearing vector must be delivered to a substantial number of tumor cells (1-10%) throughout the tumor (Moolten, 1996; for review see Kramm et al., 1995, and Spear et al., 1998). The choice of vectors and the route of their administration have been demonstrated to affect both tumor transduction efficiency and spatial distribution, as well as the extent and stability of transgene expression within a tumor, in invasive tumor cells, and in the surrounding normal brain (Zlokovic and Apuzzo, 1997).

I. Introduction The advancement of gene therapy for brain tumors through the stage of animal models into clinical trials has not succeeded in eliminating major limiting factors, such as the inability of a single vector or delivery mode to target a pool of tumor cells large enough to confer cytotoxicity to the whole tumor (Ram et al., 1997). Even with the bystander effect elicited by many therapeutic genes, which is responsible for the killing of non-

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Gene Therapy and Molecular Biology Vol 3, page 2 Three main modes of vector delivery to experimental brain tumors have been extensively studied and compared in animal models and, in some cases, in clinical trials: stereotactic intratumoral inoculation of virus suspension or vector-producing cells (VPC) (Badie et al., 1994; Boviatsis et al., 1994a and 1994b; Bramson et al., 1997; Culver et al., 1992; Eck et al., 1996; Izquierdo et al., 1995; Kramm et al., 1997; Mineta et al., 1994: Oldfield et al., 1993; Rainov et al., 1996; Ram et al., 1993 and 1997); intrathecal and intraventricular injection of virus or VPC (Bajocchi et al., 1993; Kramm et al., 1995, 1996, 1997; Oldfield et al., 1995; Oshiro et al., 1995; Ram et al., 1994; Rosenfeld, 1997; Vincent et al., 1996a); and more recently, intravascular application of virus vectors (Barnett et al., 1998; Chauvet et al., 1998; Doran et al., 1995; Kroll and Neuwelt, 1998; Muldoon et al., 1997; Neuwelt et al., 1991; Nilaver et al., 1995; Rainov et al., 1995 and 1998). The study of the modes of application and the factors which limit vector distribution and propagation in a brain tumor is of great importance to the improvement of present gene therapy strategies and the development of more efficient approaches. Therefore, the present paper will review the routes and methods for delivery of gene therapy vectors to malignant brain tumors, and will focus on strategies which may have the potential of improving the efficiency of gene transfer to brain tumors in vivo.

1991; Mineta et al., 1995); adenovirus (AV) (Badie et al., 1994 and 1998; Boviatsis et al., 1994a; Chen et al., 1994; Eck et al., 1996; Izquierdo et al., 1996; Le Gal La Salle et al., 1993; Maron et al., 1996; Perez-Cruet et al., 1994, Puumalainen et al., 1998); adeno-associated virus (AAV) (Mizuno et al., 1998; Okada et al., 1996); and liposomeDNA complexes (lipoDNA) (Gennuso et al., 1993; Yagi et al., 1994; Zerrouqi et al., 1996; Zhu et al., 1996); or for implantation of retrovirus (RV) producing cells (VPC) (Rainov et al., 1996; Ram et al., 1993; Culver et al., 1992; Takamiya et al., 1993; Tamiya et al., 1995). Since the life cycle of replication-conditional HSV and AV vectors is lytic or damaging to the host cell, there have been no available cell-based vector producer systems for in vivo use of these viruses (Kramm et al., 1995).

A. Direct intratumoral injection of vectors Stereotactic surgery methods combined with 3D computer reconstruction and imaging databases provide powerful options for tumor gene therapy (Kelly, 1997). Volumetric stereotactic procedures can be modified for individually planned delivery of viruses or VPC to single or multiple tumor foci. Despite the potential for high spatial accuracy, direct intra- or peritumoral injections have several disadvantages, such as limited vector distribution to a few millimeters surrounding the injection site (Boviatsis et al., 1994a; Rainov et al., 1996; Lal et al., 1994), and the need of multiple injections of either virus or VPC suspension even with large volumes of inoculum of up to 0.5 ml per injection site (Muldoon et al., 1997; Ram et al., 1997) (Fig. 1A and B). Since the number of stereotactic injection sites is limited for practical reasons by length of surgery and increasing risk of hemorrhage with every new intracerebral puncture track, this mode of application can only provide vector delivery to small intracerebral foci or limited tumor areas (Spear et al., 1998).

II. Intratumoral delivery of vectors The earliest and most straightforward approach to delivery of gene therapy vectors to brain tumors is the stereotactic intratumoral injection (Short et al., 1990; Boviatsis et al., 1994a) or the direct injection after open surgery for brain tumor removal (Ram et al., 1997). It offers the advantages of low systemic toxicity, reduced vector loss, and high local vector concentrations, and can be employed either for application of concentrated vector suspension, as in the case of herpes-simplex-virus type 1 (HSV) (Andreansky et al., 1993; Boviatsis et al., 1994b; Kaplitt et al., 1994; Kramm et al., 1997; Martuza et al.,

Fig. 1: Microscopic appearance of 9L tumors in syngeneic Fischer rats. A. Photomicrograph of tumor tissue in an animal from the TK/GCV group, 7 days after intratumoral grafting of retrovirus-packaging cells (CRIP-MFG-TK) in a 1:5 ratio of producer cells to tumor cells. This section was stained immunohistochemically for HSV-TK. Note the high number of HSV-TK-positive cells (darkbrown, arrows) in the tumor (magnification 200 x, 20 µm frozen section, counterstained with hematoxylin). B. Photomicrograph of tumor tissue 12 days after grafting of CRIP-MFG-TK cells and 5 days after start of GCV application. Necroses (arrowheads) are visible inside the tumor area. HSV-TK-positive cells (arrows) are still detectable, but numbers are much lower (magnification 200 x, 20 µm frozen section, counterstained with hematoxylin). C. X-gal staining 10 days after implantation of 9L cells and 6 days after intrathecal injection of replication-conditional HSV vector bearing the lacZ gene demonstrates widespread distribution of vector in leptomeningial 9L tumor cells (blue, arrows) directly contacting the CSF. In the tumor parenchyma (T), only a few cells display X-gal staining, B = normal brain (75x magnification, 20 µm frozen section, counterstained with hematoxylin and eosin, H&E). D. Frontal tumor (T) in an animal 7 days after intracerebral and intrathecal implantation of 9L cells and 2 days after intrathecal inoculation with HSV vector used in C. Extensive X-gal staining (blue, arrows) is seen in tumor areas which have broken into the lateral ventricle, while other parts of the tumor (T) with no CSF contact show essentially no staining. Normal brain (B) is not affected by intrathecal HSV application (100x magnification, 20 µm frozen section, counterstained with H&E). E. Photomicrograph of intracerebral tumor in the BK/HSV group 24

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Gene Therapy and Molecular Biology Vol 3, page 3 hours after ipsilateral intra-carotid virus injection in the presence of bradykinin (BK). This section was double-stained for ß-gal (blue cells, arrows) and HSV-TK (dark-brown, arrowheads). Note the higher number of stained tumor cells at the tumor/brain border (T = tumor, B = brain) and the absence of transgene protein staining in normal brain (magnification 200x, 20 µm frozen section, counterstained with H&E). F. Photomicrograph of 9L gliosarcoma in the BK/HSV group 48 hours after virus injection in the presence of BK. This section was double-stained for ß-gal (arrows) and HSV-TK (arrowheads). Note the higher intensity of tumor staining, probably due to secondary spread of replication-conditional HSV vector and infection of neighboring tumor cells. Staining is limited to the tumor and does not extend into surrounding normal brain (T = tumor, B = brain) (magnification 200x, 20 µm frozen section, counterstained with H&E).

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Gene Therapy and Molecular Biology Vol 3, page 4 area. Infusion time did not affect distribution, and the volume infused was closely related to the size of the distribution area (Kroll et al., 1996). The same group (Muldoon et al., 1995) infused AV or HSV into normal rat brain for 2 hours at a rate of 0.2 Âľl/min and found widespread infection in tissue volumes of 40 mm3 (replication-defective AV) and up to 200 mm3 (replication-conditional HSV). When applied to rat brain tumors, this technique was able to mediate delivery of virus particles to tumors with an approximate volume of 100 mm3, and also beyond the tumor borders into the surrounding brain tissue (Nilaver et al., 1995). Virus vectors, however, do not travel in the extracellular space of the brain solely by diffusion, since they bind to receptors and are taken up by cells, and because they are very large (e.g. HSV diameter = 150 nm). Tumor or brain cells near the injection or infusion site may take up many more virus particles than cells distant to it, which reduces the particle numbers of the suspension that diffuses further.

Although several reports have previously demonstrated that this mode of vector delivery may be efficient in rodents (Badie et al., 1994; Boviatsis et al., 1994c; Bramson et al., 1997; Culver et al., 1992; Izquierdo et al., 1995; Mineta et al., 1994; Rainov et al., 1996, Ram et al., 1994; Tamiya et al., 1995), it does not reach the same degree of efficiency in humans (Raffel et al., 1994; Ram et al., 1997). Part of the problem seems to be that human glioblastomas (GBM) are much larger, more randomly shaped, and more diffusely infiltrating than the rodent glioma models (Izquierdo et al., 1997; Kramm et al., 1995; Zlokovic and Apuzzo, 1997). Further, they have a lower fraction of dividing tumor cells which limits on site propagation of replication-conditional HSV and integration of RV (Ram et al., 1997, Harsh et al., in preparation, Puumalainen et al., 1998), and, since most vectors derive from common pathogens, the immune system may block infection of tumor cells. Herrlinger et al. (1998) have investigated the role of the immune system in HSV-mediated gene transfer and found that rats preimmunized to HSV had dramatic decrease in transduction efficiency to brain tumors.

The above data demonstrate that convection-enhanced vector delivery is sufficient for targeting a relatively small and circumscribed rodent brain tumor implant. If this technique should be applied to human GBM, much larger tumor volumes have to be targeted and, since there is no selectivity in the delivery mode itself, normal brain tissue may be overloaded with vectors leaking out of the tumor mass.

Direct intratumoral injections into the walls of the tumor resection cavity, although they can be performed under direct visual control and with multiple vector depots very close to each other, have the same basic limitations as stereotactic procedures. Moreover, the depth of injection is limited to 10-15 mm from the resection border, which seems to be insufficient to reach tumor cells migrating away from the main tumor mass. Thus, both stereotactic and "free-hand" injection techniques are inefficient in cases of multiple tumor foci and diffusely infiltrating tumors.

III. Intrathecal and intraventricular vector application The intrathecal gene therapy approach is attractive because access to the cerebrospinal fluid (CSF) is minimally invasive and distribution of virus vectors and VPC may be facilitated by CSF circulation, thus overcoming distribution barriers in solid tumors. Intrathecal delivery seems to be best suited for treatment of leptomeningial tumor manifestations. These are found in adults in secondary intracerebral tumors of carcinomas and lymphomas. The most frequent primary brain tumor in children, the medulloblastoma, often spreads from its primary location in the cerebellum and the fourth ventricle via the CSF pathways along the entire spinal cord down to the cauda equina. Moreover, leukemic leptomeningiomatosis is a frequent site of relapse of acute lymphoblastic leukemia, the most frequent pediatric cancer.

B. Bulk convection-enhanced flow methods An alternative method for efficient and widespread delivery of macromolecules and particles to tumors is convection-enhanced infusion, which is used to supplement simple diffusion and to improve vector distribution by bulk flow inside and outside the tumor (Bobo et al., 1994; Lieberman et al., 1985; Muldoon et al., 1997). Stereotactic injection and subsequent infusion by maintaining a positive pressure gradient is able to improve the distribution of large molecules in animal models (Lieberman et al., 1995). The volume of distribution seems to increase linearly with the infusion volume, if relative small molecules are used (Bobo et al., 1994). Kroll et al. (1996) used convection for delivery of MION, superparamagnetic iron oxide nanoparticles with a size comparable to that of viruses (Shen et al., 1993), to normal rat brain and found out that the concentration of the agent is of primary importance for the size of the distribution

Retroviral vectors, as well as AV and HSV vectors, have been investigated for their use for gene therapy of leptomeningial tumors after intrathecal administration. Ram et al. (1994) implanted retrovirus producer cells into the leptomeningial space of rats, which have been intrathecally challenged with syngeneic tumor cells some 4


Gene Therapy and Molecular Biology Vol 3, page 5 days prior. Prolonged survival was achieved by subsequent GCV treatment. Gene transfer was demonstrated in tumor foci growing in the cistern magna, the injection site of the VPC. Toxicity and gene transfer into normal cells was also evaluated in rats and nonhuman primates without tumors after single and repeated intrathecal application of retrovirus producer cells (Oshiro et al., 1995). Only choroid plexus cells, and no other normal CNS structures, showed transgene expression. Magnetic resonance imaging of brains of non-human primates revealed no pathological changes. In total, no significant toxicity was observed either in rats or in nonhuman primates, even after repeated intrathecal application of retrovirus producer cells with or without subsequent GCV treatment. Interestingly, measurable titers of retroviral particles were detected in lumbar, as well as in cisternal, CSF samples indicating an effective circulation of vector particles within the CSF. According to a former study, CSF does not inactivate retroviruses or lyse vector producer cells, as it occurs when these cells are incubated with serum of the same species (Russell et al. 1995). However, significant CSF retroviral titers could only be detected in vivo over 24 h (Oshiro et al., 1995). The retroviral studies in rats and non-human-primates by Ram et al. (1994) and Oshiro et al. (1995) were performed as preclinical studies for a clinical trial aiming to treat leptomeningial carcinomatosis by intrathecal application of retrovirus producer cells liberating retroviral particles bearing the HSV-tk gene for sensitization of transduced tumor cells towards subsequent GCV treatment (Oldfield et al., 1995). Despite the encouraging data in animals, this clinical trial was closed prematurely after toxic side effects occurred in the first patient (Anderson et al., 1995).

gene transfer into ependymal and leptomeningial cells, as well as into cerebral blood vessels. No marked toxicity was observed in these studies. This was also true for two studies which investigated gene transfer into rodent leptomeningial tumor masses by replication-deficient AV vectors administered intrathecally. Viola et al. (1995) demonstrated AV-mediated gene transfer into the main tumor mass at the intrathecal injection site. Some limited gene transfer was also noted in tumor manifestations in the cauda equina and along the nerve roots emerging from the spinal cord. Vincent et al. (1996a) observed gene transfer into tumor cells along the entire neural axis after intrathecal administration of AV vectors. These authors achieved a significantly longer survival of treated animals by combining intrathecal delivery of AV vectors carrying the HSV-tk transgene with subsequent GCV treatment, but had no long-term survivors. In contrast to this, when replication-conditional herpes vectors with the HSV-tk gene were injected intrathecally in a similar model of rodent leptomeningial neoplasia as above, long-term survival was achieved in approximately 90% of the animals treated with GCV (Kramm et al., 1996b). One reason for these diverging results may be that replication-conditional HSV vectors replicate in dividing tumor cells, and not in non-dividing normal cells, thereby producing and releasing new vector particles on site which move freely through the CSF. After intrathecal application of replication-conditional herpes vectors, Kramm et al. (1996a) also showed extensive gene transfer into leptomeningial tumors along the entire spinal axis (Fig. 1C ), as well as into parenchymal brain tumors (Fig. 1D). Additionally, ependymal and endothelial cells, as well as neurons projecting to the ventricles, showed marked transgene expression during the first two days after injection of herpes vectors. Five and more days after vector application, normal cells no longer showed transgene expression. However, there was a high degree of toxicity to animals, probably due to inflammatory reaction to the virus, which was apparently absent after intrathecal application of retroviral and adenoviral vectors in rodents (Ram et al., 1994; Viola et al., 1995), but has been noted in humans (Ram et al., 1997; Eck, 1997, personal communication). The viral genesis of symptoms in the HSV study in rats is strongly suggested by the fact that GCV treatment, which blocks virus replication, significantly improved and curtailed this toxicity (Kramm et al., 1996b). The ambivalent potential of intrathecal delivery is emphasized by studies with a new generation of replication-conditional HSV vectors (Kramm et al., 1997, Mineta et al., 1995). The prototype of this new generation herpes vector was designed to be safer than the preceding vectors (ribonucleotide reductase-deleted) by deletion of viral neurovirulence genes (gamma 34.5). Application of this new vector intrathecally in

An interesting alternative to retroviral gene transfer was demonstrated by Vrionis et al. (1996a and b) who showed that therapeutic efficiency can also be achieved in a rat model of leptomeningial neoplasia by co-mixture of native and HSV-tk transduced cells in the same tumor with subsequent GCV treatment. This therapeutic approach relies mainly on the bystander effect describing that close proximity of non-transduced tumor cells with TK-positive tumor cells sensitizes non-transduced cells to GCV treatment. Vrionis et al. (1996a) demonstrated that an HSV-TK/GCV system which exploits the bystander effect at a relative low effector-to-target cell ratio (1:1) is more effective for treatment of leptomeningial neoplasia than the gene therapy approach with intrathecal application of retrovirus producer cells. Intrathecal application of adenoviral vectors has also been used for gene therapy. Bajocchi et al. (1993) showed gene transfer into ependymal cells following direct injection into the ventricles. Ooboshi et al. (1995 and 1997) injected replication-deficient AV (1x109 pfu) intrathecally and demonstrated Ă&#x;-galactosidase (Ă&#x;-gal) 5


Gene Therapy and Molecular Biology Vol 3, page 6 combination with GCV in the same rodent model of leptomeningial neoplasia as described above, was associated with no apparent toxicity or mortality, but also with no significant prolongation of survival of treated animals (Kramm et al., 1997).

terms of selective entry for CNS neoplasms (Rainov et al., 1995). In addition to the BTB, some other factors limit intravascular vector delivery to brain tumors. In order to infect a maximum number of tumor cells, virus vectors must be delivered in sufficiently high titers and should not be inactivated by serum factors (Muldoon et al., 1997). High interstitial fluid pressure within tumors also acts to decrease entry of macromolecules and particles (Jain, 1987 and 1994). Larger tumors generally have a higher interstitial pressure than smaller tumor foci (Leunig et al., 1992), which theoretically limits efficiency of vector delivery to human brain tumors (Boucher et al., 1996). Human glioblastomas also have a variable degree of vascularization, and their microvasculature and hemodynamics vary considerably (Warnke et al., 1987). These obstacles for brain tumor vector delivery call for alternative strategies to circumvent them in order to make delivery of vectors more efficient by additional penetration-enhancing or barrier-modulating techniques.

Rosenfeld et al. (1995) used an adeno-associated virus (AAV) vector to transduce medulloblastoma cells in a nude rat model of leptomeningial disease. After intrathecal application, tumor cells transduced with the marker gene Ă&#x;-galactosidase were detected in tumors, as well as in ependymal and subependymal cells, but not in normal brain parenchyma. No evidence of virus toxicity was noted during the course of the experiment. In conclusion, leptomeningial neoplasia, which represents a main problem in the management of primary and secondary brain tumors, especially in children, is a good target for future gene therapy approaches for intrathecal delivery of therapeutic genes.

IV. Intravascular vector application Intravascular methods of vector application make use of a natural and ubiquitously distributed network of arteries, veins and capillaries, which is present in every normal tissue and is even denser in malignant tumors. Intravascular applications, intra-arterial injection of virus vectors in particular, appear to have the greatest potential to date for delivering a vector to the largest proportion of tumor cells and surrounding tissues without afflicting mechanical injury to normal brain tissue or having other toxic consequences (Spear et al., 1998; Muldoon et al., 1997). Intra-arterial vector application with or without disruption of the blood-brain-barrier (BBB) or the bloodtumor-barrier (BTB) seems to offer a solution to the difficulties of vector distribution by employing the extensive tumor neovasculature for transgene delivery to all vascularized tumor foci (Muldoon et al., 1997). In contrast to the normal BBB, which consists of endothelial cells bound together with tight junctions and wrapped by astrocytic processes, and which limits the entry of substances into the interstitial and intracellular space of the brain, the brain tumor neovasculature has a somewhat more permeable barrier (Cox et al., 1976; Inamura and Black, 1994; Long, 1979; Yamada et al., 1982). Although the BTB may be more or less leaky, it still limits delivery of high molecular weight substances to tumor tissue and to immediately adjacent, partially tumor-infiltrated areas of the brain (Groothuis et al., 1991). The varying permeability found throughout the BTB in the majority of malignant brain tumors (BergstrĂśm et al., 1983; Burger et al., 1988) may restrict vector penetration, especially those with a larger size (HSV = 150 nm in diameter, AV = 70100 nm). On the other hand, the existence of a tight BBB throughout the normal brain provides an advantage in

A. Intravascular vector delivery without BTB modulation The average size of a HSV particle is about 150 nm and that of an AV particle is 70-100 nm, and because they are so large, their penetration through normal brain capillaries or tumor neocapillaries is poor (Rainov et al., 1995). There are only a few studies being done with virus or non-virus vectors injected intravascularly without modulation of the BTB or the BBB. Chauvet et al. (1998) injected AV vectors into the middle carotid artery (MCA) of a dog with a benign intracranial meningioma and were able to achieve a high percentage of transduced tumor cells without any concomitant toxic effects to the CNS. Meningioma, however, unlike astrocytoma or glioblastoma, have excessive fenestration and leakiness of tumor capillaries, which probably facilitate virus vector entry (McDermott and Wilson, 1996). Our studies (Rainov et al., 1995) and those of other investigators (Neuwelt et al., 1991; Nilaver et al., 1995) have demonstrated increased transduction rates of tumor cells by HSV or AV after osmotic or pharmacologic barrier disruption, as compared to delivery of vectors across the intact BBB and BTB. Delivery studies of HSV particles and MION across the unmodified BBB and the BTB have shown that there is a small percentage (2-5%) of 9L gliosarcoma cells which can be targeted with HSV, presumably through the somewhat leaky BTB in 9L tumors, or by secondary spread from infected endothelial cells in neocapillaries producing replication-conditional vectors (Rainov et al., 1995). While HSV particles tend to infect cells in the periphery of the tumor and at the tumor/brain border, MION are delivered throughout the tumor and accumulate to some extent in the tumor center

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Gene Therapy and Molecular Biology Vol 3, page 7

Fig. 2: Visual comparison of ß-gal expression in large and small 9L tumor foci in syngeneic Fischer rats injected with replicationdeficient AV and liposome-DNA complexes (lipoDNA) bearing the lacZ gene with and without blood-tumor-barrier disruption by bradykinin (BK). A. Photomicrograph of a small intracerebral tumor focus (< 0.5 mm) in the AV group 48 hours after ipsilateral intracarotid virus injection in the absence of BK. Note the relatively high number of X-gal stained cells (blue, arrows) in the tumor periphery and, to a certain extent, in the tumor center (T), B = tumor-infiltrated surrounding brain (magnification 300x, 20 µm frozen section, counterstained with hematoxylin). B. Photomicrograph of an intracerebral tumor focus in the AV group 48 hours after intra-carotid BK infusion and AV vector injection. An increased number of stained cells is distributed somewhat more evenly throughout the tumor (T), B = normal brain (magnification 200x, 20 µm frozen section, counterstained with hematoxylin). C. Photomicrograph of a tumor in the lipoDNA group 48 hours after vector injection in the absence of BK. Note the high number of X-gal stained cells (blue, arrowheads) throughout the tumor (T). Endothelial cells in capillaries (V) near the tumor/brain border are also stained positively (arrows), B = normal brain (magnification 200x, 20 µm frozen section, counterstained with NeutralRed). D. Photomicrograph of a tumor in the lipoDNA group 48 hours after intra-carotid BK infusion and vector injection. The number of X-gal stained cells (blue, arrows) throughout the tumor (T) is somewhat higher than in the absence of BK, V = tumor vessel, B = normal brain (magnification 200x, 20 µm frozen section, counterstained with NeutralRed).

products in this tumor model is particularly high in small tumor foci (<0.5 mm) away from the main tumor mass. In these foci, almost half of all tumor cells are transduced. A few endothelial cells in normal brain capillaries are also transduced by AV-mediated gene transfer. Intracarotid delivery of non-viral vectors, such as liposome-plasmid

(Rainov et al., 1995). Intra-carotid delivery of AV vector to 9L rat gliosarcoma without BTB disruption results in transgene expression in 3-10% of tumor cells, predominantly located at the tumor-brain border, as well as randomly distributed throughout the tumor (Fig. 2A) (Rainov et al., in press). Virus-mediated expression of marker gene 7


Gene Therapy and Molecular Biology Vol 3, page 8 DNA-complexes (lipoDNA), without BTB disruption renders more than 30% of the tumor cells positive for the marker gene (Rainov et al., in press). The pattern of distribution is homogenous throughout the tumor, with a slightly higher transduction rate in the tumor periphery (Fig. 2C). Although lipoDNA-mediated gene transfer without barrier modification has increased efficacy as compared to HSV- and AV-mediated gene transfer, it is less tumor-specific, since a considerable number of endothelial and glial cells also express the respective transgene. LipoDNA complexes represent alternative vehicles for gene transfer and avoid some of the unwanted features of virus vectors (Hug and Sleight, 1991). The route of in vivo administration may affect dramatically the uptake of liposomes by normal and tumor cells. Intravenously injected liposomes are taken up mainly by the reticulo-endothelial system (RES), particularly in the liver and spleen (Hug and Sleight, 1991). Intra-arterial application of lipoDNA to tumors has not been investigated extensively, and little is known about transduction efficiency in brain tumors in vivo (Gennuso et al., 1993).

high efficiency of osmotic barrier disruption. HSV and AV vector delivery to brain and intracerebral tumors was increased up to four fold by hypertonic mannitol (Neuwelt et al., 1991b; Nilaver et al., 1995). When virus was administered intra-arterially without barrier modification, virtually no infection was detected of either tissue type. MION can penetrate efficiently through the disrupted BBB in rats, and have the advantage of being imageable by MRI. After intra-arterial mannitol infusion, glial cells were predominantly infected by AV, while HSV and MION targeted neurons more efficiently (Muldoon et al., 1998). The degree of barrier opening correlated with the transduction efficiency of glial and neuronal cells (Doran et al., 1995). Osmotic BBB disruption in combination with intra-arterial administration of viral vectors may offer a method of global delivery to treat disseminated brain tumors (Nilaver et al., 1995), although its specificity is far from optimal.

C. Intravascular vector delivery with pharmacological BBB and BTB disruption Vasoactive agents for modification of the BBB and BTB have been identified through studies of peritumoral brain edema and effects on systemic capillaries (Black, 1992; Chan et al., 1983; Cloughesy and Black, 1995). The BTB can develop transient increases in permeability with the intra-arterial delivery of vasoactive agents, while the normal BBB resists the effects of these compounds because of additional biochemical and physical barriers (Inamura and Black, 1994). Vasoactive compounds, including leukotrienes (Black and Chio, 1992; Chio et al., 1992), bradykinin (BK) and its analog RMP-7 (Barnett et al., 1998; Black et al., 1997; Doctrow et al., 1994; Elliott et al., 1996a and 1996b; Inamura et al., 1994a; Matsukado et al., 1996; Nakano et al., 1996; Rainov et al., 1995; Rainov et al., 1998), histamine (Inamura et al., 1994b; Nomura et al., 1994), and calcium antagonists (Matsukado et al., 1994) appear to selectively increase permeability in abnormal brain tumor capillaries.

B. Intravascular vector delivery with osmotic BBB and BTB disruption BBB and BTB can be manipulated to increase permeability for gene therapy vectors, such as viruses or non-viral particles. Several studies have focused on transient osmotic disruption of the BBB and the BTB, and this technique has been well characterized in animal models and in humans as an enhancer of chemotherapeutic drugs and vector delivery to brain tumors (Doran et al., 1995; Neuwelt et al., 1987; Neuwelt et al., 1991b; Nilaver et al., 1995; Z端nkeler et al., 1996). The mechanism of osmotic disruption of the barrier includes shrinkage of endothelial cells with subsequent opening of the capillary tight junctions, which is achieved by application of hypertonic solutions of sugars or salts into the arterial system (Rapoport and Robinson, 1986). Infusion of mannitol is most commonly used because of its relatively low toxicity and the applicability to humans (Muldoon et al., 1998). Mannitol offers the possibility of global delivery of drugs and virus vectors throughout the vasculature, which can reach even infiltrating tumor foci distant to the main mass (Neuwelt and Hill, 1987; Neuwelt et al., 1991a and b). With mannitol disruption of the BBB and BTB, however, delivery and uptake of therapeutic agents is less specific to the tumor and tends to spread the toxic agents throughout the whole affected hemisphere, which may increase toxicity to normal brain tissue (Z端nkeler et al., 1996).

BK, a nonapeptide hormone with peripheral vasodilatation effect, permeabilizes the vascular endothelium in brain capillaries at low concentrations (10 g/kg/min), when delivered intra-arterially in rodents, and its barriermodifying effects are specific to brain tumor neocapillaries (Inamura and Black, 1994). BK exerts its effects by interaction with specific B2 receptors (Hess et al., 1992) on endothelial cells, which mediate contraction of the endothelial cell cytoskeleton with subsequent temporary opening of the tight junctions (Doctrow et al., 1994; Inamura et al., 1994a; Sanovich et al., 1995) and may also increase the rate of pinocytosis/transcytosis in endothelial cells (Raymond et al., 1986). It has also been

Studies of delivery of virus and non-virus vector particles across the BBB and BTB have demonstrated the

8


Gene Therapy and Molecular Biology Vol 3, page 9 demonstrated that BK and RMP-7 increase intracellular free calcium levels (Doctrow et al., 1994) and stimulate a nitric oxide-mediated pathway in tumor vasculature and/or in tumor cells itself (Nakano et al., 1996).

transduce brain tumor cells, and that BTB modification by BK further increases the number of transgene-expressing tumor cells without apparent adverse effects (Rainov et al., in press). With AV and lipoDNA, it remains to be determined whether the increase in transduction following BK infusion will result in long-term survival in experimental brain tumor models.

BTB disruption by low-dose BK can facilitate selective uptake of HSV vectors administered through the carotid artery to single or multiple tumor foci in the rodent brain, with essentially no infection of normal neurons and glia (Rainov et al., 1995). Transgene expression after intra-arterial BK infusion and HSV vector bolus injection is particularly intense in the periphery of the tumor, a zone with distinct biological and biomechanical properties such as high mitotic rate, angiogenesis, parenchymal invasion, and low interstitial pressure (Boucher et al., 1996). Up to 25% of tumor cells in this region express transgene proteins after BK/HSV administration, as compared to less than 0.1% of cells in normal brain tissue (Fig. 1E and F). In contrast, MION uptake is increased by BK predominantly in the tumor center and has less effect at the infiltrating edge (Rainov et al., 1995). Furthermore, this study demonstrated HSV infection of multiple bilateral tumor nodules by unilateral BK infusion and HSV injection, which suggests that BK may have generalized effects beyond the site of infusion in rat brain with extensive collateralization.

To replace BK with a new, longer and more selectively acting derivative suitable for human studies, the synthetic nonapeptide RMP-7, H-Arg 1-Pro2-HydroxyPro3-Gly4-Thi5Ser6-Pro7-Tyr(Me)-!-(CH2NH)8-Arg9-OH, a BK analog with three amino acid substitutions, was designed by Alkermes, Inc. (Cambridge, MA). It is more resistant to angiotensin-converting enzyme (ACE) due to the replacement of alanine with 2-thienyl-alanine (Thi 5) and to neutral endopeptidase and carboxypeptidase I, due to replacement of phenylalanine with the Tyr(Me)- !(CH2NH)8 group (Elliot et al, 1996a). Replacement of proline with hydroxyproline (HydroxyPro3) removes the undesirable action on the B1 receptor. RMP-7 has a 5-10 times longer half-life in the blood circulation than BK, is 100-fold more potent in mice, and acts more selectively on endothelial cells by binding to the B2 receptor only, without undesirable blood pressure drops (Elliot et al., 1996a and b). RMP-7 has been FDA-approved for human studies.

The increased rate of tumor infection by HSV after BK infusion has been exploited for eradication of intracerebral 9L tumors in syngeneic rats. In this model, virus vector concentration appears to influence survival rates in a dosedependent fashion when GCV is given systemically starting three days after BK/HSV application and continued for 14 days (Rainov et al., 1998). A concentration of 1x1010 pfu HSV was able to eradicate tumors in 80% of the treated animals, while 1x109 pfu eliminated tumors in 40% of the rats, and 1x108 pfu was sufficient for prolonged survival, but not for permanent tumor cures. No apparent complications of intra-arterial HSV injection were encountered in this study (Rainov et al., 1998).

RMP-7 was tested extensively as an adjunctive therapeutic agent for primary and recurrent malignant gliomas. In humans, intra-carotid or intravenous infusion of low-dose RMP-7 (0.1 g/kg/min for 15 min) was able to increase the delivery to brain tumors of intravenously injected low and high molecular weight tracers, such as aminoisobutyric acid or dextrane, and of cytotoxic agents, such as methotrexate and carboplatin (Elliott et al., 1996b; Matsukado et al., 1996; Muldoon et al., 1998). There are data to suggest that RMP-7 is at least equivalent to BK in terms of enhancement of virus and non-viral particles to brain tumors (Barnett et al., 1998). This study compared BK and RMP-7 and, among other findings, demonstrated no significant difference in the enhancement of HSV delivery across the BTB, which confirms the potential of RMP-7 for application for gene therapy of human brain tumors (Barnett et al., 1998).

In another study, intra-carotid delivery of AV and lipoDNA to 9L rat gliosarcoma with and without BKmediated BTB modification was compared (Rainov et al., in press). For AV-mediated gene transfer, BK infusion increased the amount of transgene-expressing tumor cells from 5 to 19 % (Fig. 2B) and enhanced expression in the center of larger tumor foci. BK infusion prior to lipoDNA injection was able to increase the number of transduced tumor cells from 30% to more than 50%, and to produce a more homogeneous pattern of transgene distribution in the tumor (Fig. 2D). The relatively low tumor specificity of lipoDNA transfer remains unchanged by BK application, with extensive delivery to normal tissue as well. These findings indicate that intra-carotid application of virus and non-virus vectors can preferentially and effectively

In conclusion, the issues of delivery of gene therapy vectors to tumors in the brain seem still to be underappreciated in the literature. Unfortunately, the development of new and more specific virus and non-virus vectors does not address the difficulties of accessing the maximum number of tumor cells in diffuse or invasive and multifocal tumors, such as human GBM. Since there are no ideal vectors or unconditionally efficient application modes or delivery routes, in the future, gene therapeutic approaches for brain tumors will be a combination of different application routes, vectors and transgene 9


Gene Therapy and Molecular Biology Vol 3, page 10 Black KL, Cloughesy T, Huang SC, Govin YP, Zhou Y, Grous J, Nelson G, Fraahari K, Hoh CK, and Phelps M (1997) Intracarotid infusion of RMP-7, a bradykinin analog and transport of gallium-68 ethylenediamine tetra-acetic acid into human gliomas. J Neurosurg 86, 603-609.

combinations, designed to account for the individual features of different tumor types. The intravascular route of vector delivery should gain a much higher popularity and will be aided either by direct intratumoral injections, as in the case of adult multifocal GBM, or by widespread CSF vector delivery as in the case of pediatric brain tumors.

Bobo RH, Laske DW, Akbasak A, Morrison PF, Dedrick RL, and Oldfield EH (1994) Convection-enhanced delivery of macromolecules in the brain. Proc Natl Acad Sci USA 91, 2076-2080.

Acknowledgements

Boucher Y, Leunig M, and Jain RK (1996) Tumor angiogenesis and interstitial hypertension. Cancer Res 56, 4264-4266.

These studies were supported in part by grant 015VE1997 from the State Ministry of Culture and Education of Saxony-Anhalt, Germany, to NGR, and NCI grant CA69246 to XOB

Boviatsis EJ, Chase M, Wei M, Tamiya T, Hurford RK Jr, Kowall NW, Tepper RI, Breakefield XO, and Chiocca EA (1994a) Gene transfer into experimental brain tumors mediated by adenovirus, herpes-simplex-virus (HSV), and retrovirus vectors. Hum Gene Ther 5, 183-191.

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Gene Therapy and Molecular Biology Vol 3, page 14 Vincent AJ, Esandi MD, van Someren G, Noteboom JL, Avezaat CJ, Vecht C, Smitt PA, van Bekkum DW, Valerio D, Hoogerbrugge PM, and Bout A (1996a) Treatment of leptomeningial metastases in a rat model using a recombinant adenovirus containing the HSV-tk gene. J Neurosurg 85, 648-654.

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Vincent AJ, Vogels R, Someren GV, Esandi MC, Noteboom JL, Avezaat CJ, Vecht C, Bekkum DW, Valerio D, Bout A, and Hoogerbrugge PM (1996b) Herpes simplex virus thymidine kinase gene therapy for rat malignant brain tumors. Hum Gene Ther 7, 197-205. Viola JJ, Ram Z, Wallbridge S, Oshiro EM, Trapnell B, TaoCheng JH, and Oldfield EH (1995) Adenovirally mediated gene transfer into experimental solid brain tumors and leptomeningial cancer cells. J Neurosurg 82, 70-76. Vrionis FD, Wu JK, Qi P, cano WG, Cherington V (1996a) Preservation of the bystander cytocidal effect of irradiated herpes simplex virus thymidine kinase (HSV-tk) modified tumor cells. J Neurooncol 30:225-236 Vrionis FD, Wu JK, Qi P, Cano WG, Cherington V (1996b) Tumor cells expressing the herpes simplex virus-thymidine kinase gene in the treatment of Walker 256 meningeal neoplasia in rats. J Neurosurg 84:250-257 Warnke PC, Friedman HS, Bigner DD, and Groothuis DR (1987) Simultaneous measurements of blood flow and blood-totissue transport in xenotrasnplanted medulloblastomas. Cancer Res 47, 1687-1690. Wei MX, Tamiya T, Chase M, Boviatsis EJ, Chang TK, Kowall NW, Hochberg FH, Waxman DJ, Breakefield XO, and Chiocca EA (1994) Experimental tumor therapy in mice using the cyclophosphamide-activating cytochrome P450 2B1 gene. Hum Gene Ther 5, 969-978. Yagi K, Hayashi Y, Ishida N, Ohbayashi M, Ohishi N, Mizuno M, and Yoshida J (1994) Interferon-beta endogenously produced by intratumoral injection of cationic liposomeencapsulated gene: cytocidal effect on glioma transplanted into nude mouse brain. Biochem Mol Biol Int 32, 167-171. Yamada K, Ushio Y, Hayakawa T, Kato A, Yamada N, and Mogami H (1982) Quantitative autoradiographic measurements of blood-brain barrier permeability in the rat glioma model. J Neurosurg 57, 394-398. Zerrouqi A, Rixe O, Ghoumari AM, Yarovoi SV, Mouawad R, Khayat D, and Soubrane C (1996) Liposomal delivery of the herpes simplex virus thymidine kinase gene in glioma: improvement of cell sensitization to ganciclovir. Cancer Gene Ther 3, 385-392. Zhu J, Zhang L, Hanisch UK, Felgner PL, and Reszka R (1996) A continuous intracerebral gene delivery system for in vivo liposome-mediated gene therapy. Gene Ther 3, 472-476. Zlokovic BV, and Apuzzo ML (1997) Cellular and molecular neurosurgery: pathways from concept to reality - part II: vector systems and delivery methodologies for gene therapy of the central nervous system. Neurosurgery 40, 805-813. Z端nkeler B, Carson RE, Olson J, Blasberg RG, DeVroom H, Lutz RJ, Saris SC, Wright DC, Kammerer W, Patronas NJ,

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Gene Therapy and Molecular Biology Vol 3, page 15 Gene Ther Mol Biol Vol 3, 15-23. August 1999.

Efficient in vivo expression of a reporter gene in rat brain after injection of recombinant replicationdeficient Semliki Forest virus Research Article

Kenneth Lundstrom, J. Grayson Richards, J. Richard Pink, and Francois Jenck F. Hoffmann-La Roche, Research Laboratories, CH-4070 Basel, Switzerland __________________________________________________________________________________________________ Correspondence: Kenneth Lundstrom, Ph.D. Tel: 41-61-687 8653, Fax: 41-61-688 4575, E-mail: kenneth.lundstrom@roche.com Keywords: Semliki Forest virus; in vivo expression; rat brain; !-galactosidase Received: 13 October 1998; accepted: 20 October 1998

Summary Recombinant replication-deficient Semliki Forest virus (SFV) expressing bacterial -galactosidase was injected into the amygdala and striatum of male Wistar rats. Reporter gene expression was detected up to 28 days post-injection. The maximal expression levels were obtained 1-2 days postinjection. I n s i t u hybridization studies demonstrated high expression of LacZ mRNA until day 2, but no signal was detected 4 days post-injection. N o significant change i n body weight and temperature, exploratory locomotor behavior and forced motor performances were observed after SFV-LacZ injections. The neuronal gene transfer with SFV vectors did not trigger any major cell toxicity.

al., 1990). The drawback with retroviruses is that only relatively low virus titers can be achieved and only dividing cells are infected. Adenovirus vectors are capable of infecting non-dividing as well as dividing cells and their transduction frequency is generally high (Haffe et al., 1992). The duration of expression is, however, limited due to cellular and humoral immune responses induced by the virus infection (Yang et al., 1994). Adeno-associated virus (AAV) are replication-deficient parvoviruses. They are nonpathogenic and nonimmunogenic, but can replicate in cell culture only in the presence of adenovirus or helper virus (Clark et al., 1995). AAV have only a limited packaging capacity of foreign DNA (<4.5 kb), but can integrate into the host genome. Herpes simplex virus (HSV) offers very good infectivity and allows large inserts of foreign DNA to be introduced (~30 kb). Virus infection can be maintained indefinitely in a latent state, but HSV infections generally show severe cytoxicity to cells. This effect has been reduced by deletion of some viral genes,

I. Introduction A multitude of different methods and vehicles have been developed to increase the efficiency of delivery of recombinant genes in vivo for gene function and gene therapy applications. The non-viral delivery vehicles include naked DNA and a variety of liposome-DNA complexes consisting of cationic lipids (Filion and Phillips, 1997). Naked DNA is highly sensitive to degradation with a half-life of only 5 min when injected intravenously (Lew et al., 1995), whereas the lipid structures can offer an increased protection. However, the low delivery efficiency is a considerable drawback using these vectors (Boulikas 1996). Viral vectors have offered the possibility to achieve higher transfection frequencies. Retroviral vectors are capable of very high transduction rates and even retrovirus producer cell lines can be used for gene delivery (Markowitz et al., 1990). The retrovirus delivered transgene can be stably integrated into the host genome to provide long-term gene expression (Miller et 15


Lundstrom et al: Semliki Forest virus in expression of LacZ gene in rat brain like ICP27 and ICP34.5 from the HSV genome (Howard et al., 1998). Lentiviruses offer good infectivity and longterm expression, and are therefore potential candidates as vectos for gene therapy (Verma and Somia, 1997). Despite the variety of gene delivery methods available, there are still needs for improvements and modifications of existing vectors as well as development of new vector technology. Recently, Sindbis virus, a member of the Alphavirus family, was used for successful high level delivery and expression of !-galactosidase in mouse nucleus caudata/putamen and nucleus accumbens septi (AltmanHamandzic et al., 1997). The goal of our study was to examine the ability of Semliki Forest virus (SFV) vectors (Liljestrรถm and Garoff, 1991), a closely related Alphavirus, to infect neuronal cells in vivo by direct delivery to a desired location in rat brain. SFV has an extremely broad host range which allows efficient infection of many cell types, including post-mitotic cells. In vivo packaging results in high titer (up to 1010 infective particles/ml) replication-deficient recombinant SFV particles. Recombinant SFV-LacZ virus particles were injected into the amygdala and striatum of rat brain. These regions play important roles in controlling motor functions and in regulating emotional states, respectively. We have investigated the neuropathological consequences of the SFV inoculation at different time points. Macroscopical analyses were carried out using neurological and behavioral parameters and the microscopical studies were performed on fixed brain tissue.

II. Results and Discussion A. Injection of SFV-LacZ into rat brain High-titer recombinant SFV-LacZ virus was generated as described in the Experimental procedures and as schematically illustrated in F i g . 1 . The infectivity of the SFV-LacZ virus was tested by infection of BHK cells in 6well plate cultures followed by X-gal staining. 100% infectivity was achieved with a multiplicity of infection (MOI) of 4. To enable the infection of a reasonable large population of cells, 1 x 105 SFV-LacZ particles were injected into the amygdala and striatum of male Wistar rats (F i g . 2 ), respectively, as described in the Experimental procedures.

B. Behavioral studies To study the effect of reporter gene expression based on virus vector delivery, rats receiving SFV-LacZ and control animals injected with sterile culture medium were subjected to behavioral studies. No significant change in body weight and temperature, exploratory behavior and

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F i g . 1 . S e m l i k i Forest virus v e c t o r s for i n v i v o g e n e d e l i v e r y . In vitro transcribed RNA from pSFV3-LacZ and pSFV-Helper2 were cotransfected into BHK cells to in vivo package recombinant SFV-LacZ particles. These were injected into the amygdala and striatum of rat brain.

forced motor performances was observed between the two groups at any time post-injection. Physical and behavioral parameters recorded 1, 7 and 14 days post-injection are described in Table 1. Body weights did not differ significantly and a similar gradual increase was recorded for both SFV-LacZ injected rats and control animals, which is indicative of a good general condition of the animals (normal food and water intake, healthy metabolism). No infection-induced hyperthermia was detected as the body


Gene Therapy and Molecular Biology Vol 3, page 17

Table 1.

Fig. 2. Stereotaxic localization of injection sites. Injection cannulae were lowered into the right striatum (top) and left amygdala (bottom) for local delivery of SFV-LacZ virus or vehicle. Stereotaxic coordinates are from G. Paxinos and C. Watson (The Rat Brain in stereotaxic coordinates, Academic Press, 1997).

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Lundstrom et al: Semliki Forest virus in expression of LacZ gene in rat brain be delivered to the caudate putamen and central amygdala for local expression in the infected cells, mainly neurons, and does not appear to spread into neighbouring regions, except via the ventricular system into ependymal cells of the lateral third and fourth ventricles. Modifications of the injection procedure (i.e. decrease in injection speed, volume or virus concentration) might further reduce or eliminate this spread. Whereas LacZ transcripts were only detected in the first 48 h after injection, !-galactosidase could still be found after 4 weeks. This is most probably due to the high stability of this particular enzyme.

temperature remained normal. No difference between the groups in spontaneous exploration of a novel environment by the animals was recorded in measurement of total distance and vertical activity which is indicative of a state of normal emotional reactivity of the rats. As the animals were still recovering from surgery (1 day post-injection) scores were lower for both SFV-LacZ injected and control animals on day 1 when compared to 7 and 14 days postinjection. No impairment was seen at any post-injection date on forced motor performance; virus-injected animals even performed better (i.e. remained longer on the rotating rod) than control rats on some occasions (1 and 14 days post-injection). No statistically significant differences in muscular strength were detected at any time following injection. Equally, no differences were observed at 2, 4, 21 and 28 days post-injection in groups of smaller size (n = 2, controls; n = 3, virus injections; data not shown). This neurobehavioral evaluation suggests that central injection of recombinant replication-deficient SFV particles has no major consequences on the general health and on regular sensorimotor functions of male Wistar rats.

III. Conclusions Our results clearly demonstrate the feasibility of using SFV vectors for efficient infection of neuronal cells in different regions of the rat brain. We could obtain local expression of !-galactosidase, mostly due to the replication-deficient nature of the recombinant SFV particles. The infection rate at the injection site was extremely high and the duration of the recombinant protein expression at least 28 days. This is comparable to the duration of bacterial !-galactosidase expression obtained with other viral vectors, like adenovirus (Neve 1993) and herpes virus (Fotaki et al., 1997). A further suggestion of the exceptionally high stability of the recombinant !galactosidase came from our in situ hybridization experiments, where we demonstrated that no LacZ mRNA could be detected after 48 h post-injection. Similar observations have been demonstrated in vitro in BHK cells infected with SFV-LacZ virus by RT-PCR techniques, where LacZ mRNA disappears approximately 65 h postinfection (Lundstrom, unpublished data). The kinetics of other recombinant proteins might be different and could result in faster degradation of the gene product. However, the transient nature of the protein expression is evident from our results. Although this will exclude the use of SFV vectors, at least in their present form, for long-term expression, the lack of neuronal cell damage caused by the SFV infection should allow efficient transient gene expression in short term studies. Fast generation of sitespecific knock-in and knock-out gene expression studies should be possible. Our behavioral studies also demonstrated that the SFV injections did not trigger any widespread inflammatory response or extensive cell destruction, although a local inflammatory response was evident at 14 and 28 days post-injection. There were no change in the animalsâ&#x20AC;&#x2122; exploratory locomotor behavior or forced motor performance, further indications of intact neuronal cells, compared to control animals.

C. -galactosidase expression Brain sections from rats injected with SFV-LacZ particles and medium, respectively, were stained with X-gal at the different time points (1, 2, 4 , 7 , 14 and 28 days post-injection) and in situ hybridization with a LacZ gene specific probe was carried out at 1 h, 24 h, 48 h and 4 days post-injection. Twenty-four hours after injection into striatum and amygdala, both LacZ mRNA and !galactosidase were detected at the site of injection as well as in ventricular ependymal cells throughout the brain (F i g . 3 ). The !-galactosidase expression was restricted to the infected cells and their processes and was not observed in other brain regions. LacZ mRNA was restricted to perikarya and no hybridization signal was found in cell processes. Whereas both transcript and recombinant protein were detected at 48 h post-injection (mainly at the injection sites), no mRNA was present at later time points. !galactosidase, on the other hand, was detected, albeit in ever decreasing amounts, 4, 7, 14 (F i g . 4 e ) and even 28 days (F i g . 4 f ) post-injection. In order to determine the presence or not of a toxic effect of the viral infection, adjacent sections were also stained with Toluidine Blue. Using the vehicle-injected animals as controls, virus-induced inflammation (in the form of local glioses) at the injection site could be observed 1-4 weeks after administration. Experiments are in progress with marker protein staining for astroglioses (GFAP) and microglioses (OX-42). Over all, our findings suggested that a reporter gene can

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Gene Therapy and Molecular Biology Vol 3, page 19

Fig. 3. Regional distribution and cellular localization in rat brain of LacZ mRNA and -galactosidase 2 4 h p o s t - i n j e c t i o n . !-galactosidase (blue precipitate) is detected not only at the striatal injection site but also in the ependyma throughout the ventricular system (arrowhead and arrow, respectively in a). Note the expression not only in the neuronal cell bodies (b ) and ependymal cells (c ), but also in presumptive neuronal processes (b ). LacZ mRNA is also detected at the site of injection and in the ependyma (arrowhead and arrow, respectively, in d). The cellular sites of synthesis of LacZ in presumptive neurons and ependymal cells (of the fourth ventricle) are illustrated in e and f , respectively.

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Lundstrom et al: Semliki Forest virus in expression of LacZ gene in rat brain

Fig. 4. Regional distribution and cellular localization in rat brain of LacZ mRNA and -galactosidase 2 , 1 4 a n d 2 8 d a y s p o s t - i n j e c t i o n , r e s p e c t i v e l y . !-galactosidase (blue precipitate) is detected at the striatal and amygdala injection sites (white and black arrowheads, respectively). Note the expression not only in the neuronal cell bodies, but also in their processes (b ). LacZ mRNA is also detected at the striatal and amygdala injection sites (white and black arrowheads, respectively, in c ). The cellular sites of synthesis of LacZ in presumptive neurons are illustrated in d. a-d illustrate 2 days postinjection, e and f the regional distribution of !-galactosidase at 14 and 28 days post-injection, respectively.

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Gene Therapy and Molecular Biology Vol 3, page 21

IV. Experimental procedures C. Behavioral studies

A. Cell cultures and recombinant SFV production

1 . S e n s o r i m o t o r f u n c t i o n was evaluated using a rotarod paradigm in which animals were required to walk on a rotating bar. The bar was 10 cm wide, 5 cm in diameter, 40 cm above the bench and rotated twice per minute. Trained animals were able to follow the slow regular movement of the bar for several minutes. Mild sedation or motor impairment translates into incoordination on the rotating rod and the animals fall off the bar. Time spent on the rotating rod is measured in seconds and maximal cut-off time is 60 s (non-impaired animal).

BHK-21 cells were grown in a mixture of F12-MEM/Iscove (1:1) in 10% FCS (Gibco-BRL) for in vivo packaging of recombinant SFV particles (Lundstrom et al., 1994). Briefly, in vitro transcripts from pSFV3-LacZ (SFV replicase genes + LacZ gene) and pSFV-Helper 2 (SFV structural genes) (Berglund et al., 1993) were co-electroporated into BHK-21 cells (F i g . 1 ). In vivo packaged recombinant SFV particles were collected 24 hours later by harvesting the medium from the cell cultures. The SFV particles were activated with "chymotrypsin and the titer of the virus stocks determined by infection of defined numbers of BHK-21 cells with different dilutions of recombinant SFV-LacZ followed by X-gal staining. The titers were generally in the range of 1 x 10 9 infectious particles / ml. The virus stocks were filter sterilized through 22 µm filter (Millipore) and no further purification or concentration was necessary. The virus stocks were diluted to 1 x 108 infectious particles / ml prior to use.

2 . Muscular c a p a c i t y was evaluated using a grip strength procedure consisting of a quantitative assessment of forelimb grip strength. A triangular bar, 2 mm in diameter, 5 cm wide was connected to a digital strain gauge. This device was used to measure graded changes in the forelimb grip strength of rats. Animals held by the tail grasped the bar and were then gently pulled away from the bar with a smooth steady pull until they released the triangle. The strain gauge remained fixed at its maximum deflection, which was the force required to break the animal’s grip. Three readings were taken for each animal and the maximum of 3 permissible readings was recorded as the grip strength score (in g).

B. Injections of recombinant SFV into rat brain

3 . E x p l o r a t i o n o f a n o v e l e n v i r o n m e n t in a test of free exploratory activity was measured in activity monitors (40 x 40 x 30 cm, Omnitech Electronics) placed in a soundproof room with central light. Locomotion was monitored via a grid of invisible infrared light beams. Horizontal and vertical activity were used in this study to describe the dynamic picture of rats. A vertical sensor monitoring rearing and jumping activity was attached 8 cm above the cage floor. An analyzer constantly collected the beam status information from the activity monitor and activity detected by the horizontal sensors was expressed as total distance run during the 30 min test.

Male Wistar rats (lbm RoRo, SPF, Biological Research Labs Ltd, Switzerland) were housed individually under controlled laboratory conditions (temperature 20 ± 2 o C, relative humidity 50-60%) with ad libitum access to food and water and were maintained on a normal 12 h light-12 h dark cycle (6 am-6 pm). Rats weighed 250-300 g at the time of surgery. They were stereotaxically microinjected under general anesthesia with Ketamine/Xylazine (200/10 mg/kg ip) in physiological saline under thermoregulatory control and oxygen supplementation. Craniotomy was performed using a fine dental drill for injection at one site located over the right striatum (0.2 mm anterior and 2.6 lateral to bregma; Paxinos and Watson, 1997) and at another site located over the left amygdala (2.6 mm posterior and 4.0 mm lateral to bregma). Stainless steel injectors attached to a stereotaxic holder were then lowered 5.0 mm ventral to the skull surface in the striatum and 8.0 mm in the amygdala (F i g . 2 ). These were connected via polyethylene tubing containing viral or control solutions to a 10 µl Hamilton syringe on a microinfusion pump (Harvard PHD 2000). Solutions were infused in a volume of 1 µl over 2 min (0.5 µl/min). The injection needle was left in place for 2 additional min before being slowly withdrawn over 1 min. The wound was then sutured and animals kept warm for 3-4 h after surgery. Post-operative buprenorphine (0.05 mg/kg) analgesic treatment (sc) was given for the next day.

The experimental procedures used in this study received approval by the local ethics committee and were performed in accordance with international and Swiss federal regulations and guidelines on animal experimentation.

D. Histological analyses On each test day (1, 2, 4, 7 , 14, 21 and 28 days postinjection) two to three animals in each group (virus and vehicle) were sacrificed for histological analysis. Directly following CO2 inhalation euthanasia, animals were transcardially perfused (clamped dorsal aorta) with 20 ml of 4% paraformaldehyde (PFA) for fixed brain extraction. Brains were stored for 4 h in the same PFA solution, then cryoprotected in 30% sucrose at 4 o C overnight and stored at -80 o C until sectioned. Free-floating sections were cut on a freezing microtome at 40 µm then reacted for !-galactosidase, as well as LacZ mRNA. Some sections were stained for 0.5-1.0 min in 0.5% Toluidine Blue (Fluka 89640) in 0.2 M acetate buffer pH 4.5.

A group of 21 rats were stereotaxically injected with the SFV-LacZ virus (105 particles/ µl) and 12 control rats received sterile vehicle (culture medium). Animals were carefully evaluated on day 1, 2, 4, 7, 14, 21 and 28 for consequences of viral injections on general health (global appearance, measures of body weight and rectal temperature), sensorimotor coordination, muscular capacity and exploration of a novel environment.

E. Enzyme histochemistry of -galactosidase 21


Lundstrom et al: Semliki Forest virus in expression of LacZ gene in rat brain Oxidation solution was prepared as follows. 80 µl Nonidet P-40 and 0.04 g sodium deoxycholate were added to the oxidation stock solution (40 ml 10x PBS, 360 ml 2x distilled H2 O, 0.65 g potassium ferricyanide (K3 Fe(CN)6 ), 0.84 g potassium ferrocyanide (3 H 2 O) (K 4 Fe(CN)6 3 H2 O), 0.16 g MgCl 2 6 H2 O) and stirred thoroughly and filtered (45 µm). The resulting bright yellow solution was stored at room temperature under light-tight conditions. !-galactosidase was visualized by adding 10 mg of X-gal substrate (Boehringer Mannheim 1680293) to 0.25 ml DMSO. Once dissolved, 10 ml oxidation solution was added with careful mixing to avoid the formation of air bubbles. Tissue sections were rinsed ( 2 x 15 min) in PBS, then reacted with the X-gal solution overnight at 31-33 o C in a dark box. The reacted sections (an insoluble blue indoyl precipitate reaction for !-galactosidase) were then rinsed again (2 x 15 min) in PBS, post-fixed in icecold 4% PFA for 15 min, rinsed in PBS, mounted on precleaned glass slides, counterstained with 1% Neutral Red (Sigma), dehydrated and coverslipped with DePeX.

examined with brightfield optics using a Zeiss Axiophot.

G. Imaging The regional and cellular distribution of X-gal and LacZ mRNA were recorded as digital images using a ProgRes high resolution color camera and Adobe Photoshop software.

Acknowledgements We are grateful to Mr. Andreas Kunz for his help with SFV-LacZ virus production and Ms. Martine Maco and Ms. Martine Kapps for their excellent technical assistance in the neurosurgical injections and behavioral evaluation of rats. Ms. Fabienne Goepfert is acknowledged for X-gal staining of brain sections, Ms. Zaiga Bleuel for help with in situ hybridizations and Mr. Jürg Messer for his assistance in imaging of brain sections.

References

F . In situ hybridization For selected time points we also investigated the regional and cellular expression of LacZ transcripts using a 60-mer oligonucleotide probe (nucleotides 3001-3060) selective for the LacZ gene (Casadaban et al., 1983). The hybridization procedure has been previously described (Saura et al., 1996). Briefly, 12 µm cryostat sections of fresh-frozen rat brains (1 h, 24 h, 48 h and 4 days post-injection) and 30 µm freezingmicrotome sections of perfusion-fixed rat brains (1, 2, 4, 7, 14 and 28 days post-injection) were used. The cryostat sections were mounted on Superfrost Plus# slides then fixed in 4% PFA in PBS, pH 7.4 for 20 min followed by three 5 min washes in PBS. The oligonucleotide was ordered from Genosys Biotechnologies and labeled at the 3’ end with terminal deoxynucleotidyl-transferase (BRL) and [35 S] dATP (New England Nuclear). The labeled probe was separated from unincorporated nucleotides with a Biogel P30 spin column (twice 4 min at 1600 x g, Sorvall SW24). Sections were hybridized with 50 µl of a solution with the following composition: 4 x SSC, 20% dextran sulfate, 0.25 µg/ml herring sperm DNA (denatured) , 50% deionized formamide (BRL), 0.1 M dithiotreitol (DTT) (Fluka), 0.5 x Denhard’s solution and the 35 S-labeled probe (3 x 10 5 cpm). Sections were covered with strips of Fujifilm# and incubated in moist chambers at 43o C overnight. Following removal of the strips, the sections were washed twice in a solution containing 1 x SSC and 10 mM DTT for 15 min at 55o C, then in 0.5 x SSC with 10 mM DTT once for 15 min at room temperature. After a dip in 2 x distilled H 2 O, sections were dehydrated in ethanol, exposed (for up to 4 weeks) to sheet film (Hyperfilm#, !Max, Amersham) or dipped in Ilford K5 nuclear emulsion to reveal the regional and cellular localization of the mRNA, respectively. The film or emulsion was developed in Kodak PL12 or Kodak D19, respectively, then transferred to Kodak Rapid Fix. Nissl- or Neutral Red-counterstained sections were

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Altman-Hamandzic S, Groceclose C, Ma J-X, Hamandzic D, Vrindavanam NS, Middaugh LD, Parratto NP and Sallee FR (1 9 9 7 ) Expression of !-galactosidase in mouse brain: utilization of a novel nonreplicative Sindbis virus vector as a neuronal gene delivery system. Gene Ther 4, 815822. Berglund P, Sjöberg M, Garoff H, Atkins GJ, Sheahan BJ and Liljeström P (1 9 9 3 ) Semliki Forest virus expression system: Production of conditionally infectious recombinant particles. B i o / T e c h n o l o g y 11, 916-920. Boulikas T ( 1 9 9 6 ) Liposome DNA delivery and uptake by cells. O n c o l R e p 3, 989-995. Casadaban MJ, Martinez-Arias A, Shapira SK and Chou J ( 1 9 8 3 ) !-galactosidase gene fusions for analyzing gene expression in Escherichia coli and yeast. Meth E n z y m o l 100, 293-310. Clark KR, Voulgaropoulus F, Fraley DM and Johnson PR ( 1 9 9 5 ) Cell lines for the production of recombinant adeno-associated virus. Hum Gene Ther 6, 1329-1341. Filion MC and Phillips NC ( 1 9 9 7 ) Toxicity and immunomodulatory activity of liposomal vectors formulated with cationic lipids toward immune effector cells. B i o c h e m B i o p h y s A c t a 1329, 345-356. Fotaki ME, Pink JR and Mous J ( 1 9 9 7 ) Tetracyclineresponsive gene expression in mouse brain after amplicon-mediated gene transfer. Gene Ther 4, 901908. Haffe HA, Danel C, Longenecker G, Metzger M, Setoguchi Y et al., ( 1 9 9 2 ) Adenovirus-mediated in vivo gene transfer and expression in normal rat liver. Nature Genet 1, 372-378. Howard MK, Kershaw T, Gibb B, Storey N, MacLean AR, Zeng B-Y, Tel BC, Jenner P, Brown SM, Woolf CJ, Anderson PN, Coffin RS and Latchman DS ( 1 9 9 8 ) High efficiency


Gene Therapy and Molecular Biology Vol 3, page 23 gene transfer to the central nervous system of rodents and primates using herpes virus vectors lacking functional ICP27 and ICP34.5. Gene Ther 5, 1137-1147. Lew D, Parker SE, Latimer T, Abai AM, Kuwahara-Rundell A, Doh SG, Yang Z-Y, Laface D, Gromkowski SH, Nabel GJ, Manthorpe M and Norman J ( 1 9 9 5 ) Cancer gene therapy using plasmid DNA: pharmacokinetic study of DNA following injection in mice. Hum Gene Ther 6, 553564. Liljestrom P and Garoff H (1 9 9 1 ) A new generation of animal cell expression vectors based on the Semliki Forest virus replicon. B i o / T e c h n o l o g y 9, 1356-1361. Lundstrom K, Mills A, Buell G, Allet E, Adami N and Liljestrรถm P (1 9 9 4 ) High-level expression of the human neurokinin-1 receptor in mammalian cell lines using the Semliki Forest virus expression system. Eur J Biochem 224, 917-921. Markowitz S, Hesdorffer C, Ward M, Goff S and Bank A ( 1 9 9 0 ) Retroviral gene transfer using safe and efficient packaging cell lines. Ann NY Acad Sci 612, 407-414. Miller DG, Adam MA and Miller AD ( 1 9 9 0 ) Gene transfer by retrovirus vectors occurs only in cells that are actively replicating at the time of infection. M o l C e l l B i o l 10, 4239-4242. Neve RL ( 1 9 9 3 ) Adenovirus vectors enter the brain. Trends Neurosci 16, 251-253. Paxinos G and Watson C ( 1 9 9 7 ) in The Rat Brain in stereotaxic coordinates, Academic Press. Saura J, Bleuel Z, Ulrich J, Mendelowitsch A, Chen K., Shih JC, Malherbe P, Da Prada M and Richards JG ( 1 9 9 6 ) Molecular neuroanatomy of human monoamine oxidases A and B revealed by quantitative enzyme radioautography and in situ hybridization histochemistry. N e u r o s c i e n c e 70, 755-774. Verma IM and Somia N ( 1 9 9 7 ) Gene therapy - promises, problems and prospects. Nature 389, 239-242. Yang Y, Nunes FA, Berencsi K, Furth EE, Gonczol E and Wilson JM ( 1 9 9 4 ) Cellular immunity to viral antigens limitsE1-deleted adenoviruses for gene therapy in cystic fibrosis. Nature Genet 7, 362-369.

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Gene Therapy and Molecular Biology Vol 3, page 25 Gene Ther Mol Biol Vol 3, 25-33. August 1999.

Establishment of an assay to determine adenovirusinduced endosome rupture required for receptormediated gene delivery Research Article

Daniela Schober, Nora Bayer, Robert F. Murphy1, Ernst Wagner2 and Renate Fuchs Department of General and Experimental Pathology, University of Vienna, A-1090 Vienna, Austria 1 Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, USA 2 Boehringer Ingelheim R&D Vienna, A-1121 Vienna, Austria __________________________________________________________________________________________________ Correspondence: Renate Fuchs, Department of General and Experimental Pathology, University of Vienna, W채hringer G체rtel 18-20, A-1090 Vienna, Austria. Phone: +43-1-40-400-5127; Fax: +43-1-40-400-5130; E-mail: renate.fuchs@akh-wien.ac.at Key words: adenovirus entry, endosome rupture, flow cytometry Received: 9 October 1998; accepted: 19 October 1998

Summary Successful human gene therapy requires methods to transfer recombinant genes to cells efficiently. One p o s s i b i l i t y i s t o use adenoviral-based vectors. The entry route o f adenovirus involves endocytic uptake, penetration of modified viral particles into the cytoplasm by endosome rupture, transport to the nuclear pore complex, disassembly of modified particles and import of the DNA into the nucleus. Since endosome rupture is a rate-limiting step in foreign gene expression, we developed a two-step assay to quantitative virus-mediated membrane rupture. Following endosome labeling of HeLa cells with a pH-sensitive (FITC-dextran) and pH-insensitive (Cy5-dextran) fluidphase marker in the absence or presence of replication-defective adenovirus type 5 (Ad5), first, the pH o f labeled compartments was determined by flow-cytometry o f c e l l suspensions. When compared to control cells, the pH of labeled compartments was elevated by co-internalization of Ad5 indicating endosome l y s i s and penetration o f the marker into the pH-neutral cytoplasm. Second, single-organelle flow analysis (SOFA) of cell homogenates of the same cells was applied t o q u a n t i t a t e t h e a m o u n t o f l a b e l e d a s w e l l a s u n l a b e l e d v e s i c l e s i n t h e p r e s e n c e o f Ad5. Our results demonstrate that adenovirus internalized for 10 min into HeLa cells destroys about 30% of endosomal compartments. This assay can be applied t o rapidly screen various gene delivery systems for their ability to disrupt endosomal membranes and to enter the cytoplasm.

proteins. In contrast to other DNA viruses, adenovirus infects non-dividing cells and replicates in the nucleus but rarely integrates into the host genome. Expression of foreign genes can be achieved by direct DNA insertion into the viral genome or conjugation to the virus. Moreover, replication deficient adenoviruses have been generated that can be propagated in cell lines expressing the complementing viral proteins (Graham et al., 1977; Jones and Shenk, 1979). Recombinant adenoviruses have also been used for gene transfer (Fujita et al., 1995).

I. Introduction Adenovirus (Ad) is widely used as a vehicle to deliver foreign DNA into the cell of interest. This is due to the broad expression of adenoviral receptors on human cells and the particular mechanism of uncoating of this virus. 47 serotypes of adenoviruses have been characterized and classified into 6 subgroups (A - F). Adenovirus is a nonenveloped virus with an icosahedral capsid shell and protruding fibers anchored to the penton base proteins. The linear double stranded DNA is attached to four core 25


Schober et al: Adenovirus-induced endosome rupture So far, the internalization pathway and mechanism of uncoating of adenoviruses of subgroup C (e.g. Ad2 and Ad5) are fairly well characterized, although not fully understood at the molecular level. After binding to primary cellular receptors (MHC-class I complex and coxsackievirus-adenovirus receptor) via the distal portion of its fiber protein (Bai et al., 1994; Bergelson et al., 1997; Hong et al., 1997), subsequent internalization of the virus requires binding of five RGD motifs in the penton base to ! v"-integrins (Wickham et al., 1993; Nemerow et al., 1994). Following endocytosis via clathrin coated pits and vesicles (Wang et al., 1998), adenovirus undergoes a series of modifications of its capsid proteins in the low pH environment of endosomes that ultimately results in rupture of the endosomal membrane (Greber et al., 1993). The low pH-dependent, virus-induced endosome lysis also requires the presence of ! v"-integrins (Wickham et al., 1994). During endocytosis and penetration into the cytoplasm, capsid proteins are degraded or dissociated and finally the DNA core is freed from the hexon (Greber et al., 1993; Greber et al., 1996). Binding of the DNA core to the nuclear pore complex results in its disassembly and import of the viral genome into the nucleus (Greber and Kasamtsu, 1996; Greber et al., 1997). Thus, the capacity of adenovirus to rupture the endosome and the selective targeting of viral DNA to the nucleus provides a powerful vehicle for gene transfer (Curiel et al., 1991; Wagner et al., 1992; Wagner, 1998).

resolve distinct fluorescent endocytic vesicles in cell homogenates, a method termed single-organelle flow analysis (SOFA) (Murphy, 1985, 1990; Murphy et al., 1989; Wilson and Murphy, 1989). In the present study we co-internalized replicationdefective adenovirus type 5 (Ad5) together with pHsensitive (FITC) and pH-insensitive (Cy5) derivatives of the fluid-phase marker dextran into HeLa cells. The pH of labeled compartments was determined by flow cytometry and, in addition, the integrity of total vesicles and fluorescent endosomes was evaluated by SOFA. Using these techniques, we here demonstrate that Ad5 elevates the pH of labeled compartments, suggesting endosome lysis and access of the dextran into the pH neutral cytoplasm. This conclusion was verified by SOFA, in that a reduction in the number of labeled as well as unlabeled vesicles in the presence of Ad5 was observed. The results confirm the utility of these flow cytometric methods for monitoring adenovirus-induced endosome lysis.

II. Results A. Flow cytometry of HeLa cell endosomes Fluid-phase markers are non-specifically internalized into cells and can therefore be used to label all endocytic vesicles, depending on the internalization conditions applied (time, temperature). Furthermore, fluid-phase markers do not bind to cellular membranes and are released into the cytoplasm when endosomes are lysed (Yoshimura, 1985; Defer et al., 1990). Under control conditions, internalized markers will be exposed to the low pH environment of intact endosomes whereas in the presence of membrane disrupting agents they will be released into the pH-neutral cytoplasm (see F i g . 1 ).

Adenovirus has long been known to increase the permeability of the plasma or endosomal membrane at low pH (Seth et al., 1985; Seth, 1994). In this process, cointernalized macromolecules can gain access to the cytoplasm. Using an in vitro assay (a so-called endosome leakage assay), we have recently shown that adenovirus leads to release of small and large molecules from isolated endosomes when incubated in low pH buffer (Prchla et al., 1995). Although this in vitro assay allows determination of conditions required for endosomal content release, it is laborious and time consuming. Since efficient gene transfer primarily depends on endosome rupture, we sought to establish a rapid assay to determine the endosomolytic activity of adenovirus in vivo. Macromolecules and viruses taken up by endocytosis are exposed to the low pH environment of the endosomes due to the activity of the vacuolar proton ATPase (Mellman et al., 1986; Mukherjee et al., 1997). Therefore, we took advantage of the pH-dependence of FITC-derivatives to selectively label endosomes and measured endosomal pH using flow cytometry and the dual fluorescence (dual fluorochrome) ratio method (Murphy et al., 1984; Cain and Murphy, 1986). Flow cytometry can also be applied to

F i g u r e 1 . Receptor mediated adenovirus entry and virusinduced endosome rupture.

26


Gene Therapy and Molecular Biology Vol 3, page 27 F i g u r e 2 . Experimental set-up for FACS and SOFA analysis of HeLa cells infected with adenovirus type 5 (Ad5) in the presence of FITC- and Cy5-dextran.

We therefore internalized a pH-sensitive (FITC) and insensitive (Cy5) derivative of the fluid-phase marker dextran (MW 70 kD) in the presence or absence of Ad5 (1000 particles / cell) into HeLa cells for 10 min at 34째C, followed by a 10 min chase in marker-free medium (for experimental set-up see F i g . 2). Under this condition primarily late endocytic compartments will be labeled with the marker (Schober et al., 1998). Flow cytometry of cell suspensions was then used to determine the amount of internalized marker (reflected by Cy5 fluorescence) and endosomal pH (reflected by the ratio of FITC and Cy5 fluorescence). The total amount of marker internalized was found to be stimulated by adenovirus by 40% when compared to controls. For pH measurements, a standard curve was generated for each sample by measuring the FITC/Cy5 ratio as a function of external pH. As shown in F i g . 3, increasing the pH of the external medium (in the presence of permeant ions) from 4.5 to 7.5 results in a linear increase in the FITC/Cy5 ratio. Based on this calibration curve, an average endosomal pH of 6.3 +/- 0.1 was obtained when marker was internalized in the absence of Ad5 (F i g . 4 ). When Ad5 was co-internalized with the dextran, the pH of labeled compartments was increased to 7.3 +/- 0.1, suggesting virus-induced endosome rupture and release of internalized marker into the pH neutral cytoplasm.

Figure 3 . Normalized pH calibration curves of FITC/Cy5-dextran labeled endosomes obtained by flow cytometry of cell suspensions and post-nuclear supernatants (PNS), respectively. HeLa cell endosomes were labeled as described in F i g . 2 . Cell suspensions or PNS were incubated with pH buffers containing permeant anions and azide to deplete cellular ATP and nigericin to equilibrate internal with external pH.

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Schober et al: Adenovirus-induced endosome rupture

B. Single-organelle flow analysis (SOFA) of HeLa cells infected with adenovirus

non-fluorescent objects can be detected in this fashion. In order to reproducibly measure endocytic vesicles, a method for choosing a consistent threshold is needed. As described previously (Wilson and Murphy, 1989), we chose a threshold value of SS just above the maximum value observed when sheath fluid without sample was analyzed.

To verify whether the increase of the "endosomal" pH of labeled compartments in the presence of adenovirus is due to loss of internalized fluid-phase marker from acidic organelles such as endosomes, we applied single-organelle flow analysis (SOFA). When a cell homogenate or postnuclear supernatant (PNS) is subjected to SOFA, the following parameters can be analyzed at the same time: forward scatter (FS) and side scatter (SS), both of which are related to size and optical density, and FITC- and Cy5fluorescence (Wilson and Murphy, 1989). Using these parameters the following information can be obtained: (i ) size distribution and number of vesicles of a certain size; (i i ) the degree of co-localization of two distinct fluorescent markers; and (i i i ) the internal pH of individual vesicles. Our goal was to determine (for cells treated and untreated with adenovirus) the number of total endocytic vesicles (unlabeled and fluorescent), the number of fluorescent endosomes, and the average pH of intact endosomes.

As an illustration of the SOFA method, all endocytic compartments (endosomes, lysosomes, recycling vesicles) of HeLa cells were labeled by continuous internalization of FITC-and Cy5-dextran for 2 h at 34째C. Thereafter, cells were rapidly cooled, washed and homogenized with a ballbearing homogenizer ensuring minimal destruction of vesicles during homogenization (Balch and Rothman, 1985). Nuclei and unbroken cells were removed by centrifugation and the resulting PNS was subjected to SOFA (see Materials and Methods). For control purposes, a PNS was prepared from unlabeled cells and also analyzed by SOFA. To differentiate large vesicles (such as late endosomes) from small vesicles, an analysis window was created with a lower FS value just above the maximum observed for sheath fluid alone (as above for SS). As depicted in F i g . 5 and Table 1, about 29% of the total events (objects) detected in both unlabeled (F i g . 5 A ) and labeled (F i g . 5E) samples were in this large vesicle window, while about 61% of all events fell in a corresponding window for small vesicles. In histograms displaying the fluorescence parameters, a region defining events positive for FITC and/or Cy5 fluorescence was created to exclude essentially all events from unlabeled cell homogenates. When either large or small vesicles from F i g . 5 A were depicted in dual fluorescence histograms, a minute number (0.2%) of vesicles from unlabeled cells were detected in the fluorescence-positive region (F i g . 5 B and F). This confirmed that the region was appropriately defined. (F i g . 5 C ). In the PNS of FITC/Cy5-dextran labeled HeLa cells, 64% of all vesicles counted (small and large) were fluorescent (F i g . 5 G , T a b l e 1). However, whereas the majority (83%) of the large vesicles contained FITC/Cy5-dextran (F i g . 5F), only 47% of the small vesicles were fluorescent (F i g . 5 H , Table 1). Thus, nearly all large vesicles can be defined as endocytic vesicles due to labeling with internalized fluorescent fluid-phase marker. Consequently, the number of vesicles in this population was used as one indication of endosome disruption by adenovirus.

When measuring the properties of subcellular organelles by SOFA, a criterion must be chosen to define which objects in an organelle suspension are to be analyzed. This is normally accomplished by using a threshold on a light scattering parameter (FS or SS), since both fluorescent and

Having defined the flow-cytometer settings to analyze endosomes, FITC- and Cy5-dextran was internalized into HeLa cells in the absence or presence of Ad5 (10 min pulse, 10 min chase) as in F i g . 2 . Cell homogenates were prepared, centrifuged at low speed and the resulting supernatant (PNS) was subjected to SOFA. As shown in F i g . 6 A , the total number of large vesicles was reduced by co-internalization of Ad5 to 69% of controls. Further support for endosome lysis due to adenovirus entry is

F i g u r e 4 . Influence of adenovirus on the pH of labeled compartments of HeLa cells. HeLa cell endosomes were labeled with FITC/Cy5-dextran without or with Ad5 as described in F i g . 2 . Cell suspensions were analyzed by flow cytometry and the internal pH was calculated using the pH calibration curve shown in F i g . 3 . Values depicted are the mean +/- SD from 3 independent experiments.

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Gene Therapy and Molecular Biology Vol 3, page 29

F i g u r e 5 . Selection of gates to define small, large and fluorescent vesicles for SOFA. All endocytic compartments in HeLa cells were labeled by continuous internalization with 2 mg / ml FITC-dextran and 0.1 mg / ml Cy5-dextran for 2 h at 37째C. A postnuclear supernatant was prepared as described in Materials and Methods and subjected to SOFA (E-H ). Results for a post-nuclear supernatant from unlabeled HeLa cells are also shown (A-D).

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Schober et al: Adenovirus-induced endosome rupture unlabeled sample

labeled sample

100.000

100.000

events counted

vesicles (% of total)

nonfluorescent (% of total) (% of total) fluorescent

fluorescent (% of total) (% of gated)

large vesicles

27.8

0.2

5.0

24.0

83.0

small vesicles

62.7

0.2

32.0

29.0

47.0

T a b l e 1 . Quantitation of large, small and fluorescent vesicles in post nuclear supernatants analyzed by SOFA. The data presented in F i g . 5 are summarized and expressed as % of total events counted (F i g . 5 B , D , F , H ). In addition, when endosomes had been labeled with FITC/Cy5-dextran (F i g . 5 E , F , H ) fluorescent vesicles were also normalized to the amount observed in the respective FS gate for large and small vesicles. vesicles was determined based on FS and SS histograms (as in F i g . 5 A ). (B ) The number of large fluorescent endosomes in scatter-gated histograms of FITC and Cy5 fluorescence is shown (as in F i g . 5 F ). Data are expressed as percent of the corresponding values obtained in the absence of virus.

F i g u r e 7 . Influence of adenovirus on the internal pH of residual intact endosomes. The internal pH of large fluorescent endosomes for the samples in F i g . 6 B was calculated using the pH calibration curve shown in F i g . 3 .

F i g u r e 6 . Adenovirus internalization reduces the number of large vesicles (A) and fluorescent endosomes (B ). FITC/Cy5-dextran was internalized into HeLa cells in the absence or presence of Ad5 as described in F i g . 2 . A PNS was prepared and analyzed by SOFA. (A) The number of total large

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Gene Therapy and Molecular Biology Vol 3, page 31 provided when bona fide endosomes, i.e. fluorescent vesicles, are analyzed. In Ad5-infected cells, the number of FITC/Cy5-positive endosomes was decreased to 73% of controls (F i g . 6 B ). This demonstrates that quantitation of large vesicles by SOFA can be used to reflect endosome rupture.

lysed) has not been quantitated. Our results show that relatively small amounts of Ad5 (1000 particles / cell) when co-internalized for 10 min with the fluid-phase marker dextran and chased for an additional 10 min (to label late endosomes but not lysosomes (Prchla et al., 1994)) stimulated dextran uptake by 40%. This is in good agreement with published data (Defer et al., 1990). Under the same conditions, the virus destroyed about 30% of all endocytic compartments. Since the number of large vesicles in FS/SS histograms primarily reflect endocytic compartments (F i g . 5 and Table 1), the endosomolytic activity of a given agent can be rapidly analyzed without prior endosome labeling with fluorescent tracers. Thus, this system offers the advantage to rapidly screen DNA-delivery systems such as low pH activated liposomes for their ability to lyse endosomes and to enter the cytosol.

Finally, the influence of adenovirus on the average internal pH of residual intact endosomes was determined. Endosomes maintain their low intravesicular pH after cell homogenization at 4째C for up to 20 h in the absence of highly permeant ions (Fuchs et al., 1989; Wilson and Murphy, 1989). A pH calibration curve similar to that obtained for whole cells was created for endosomes in the PNS analyzed by SOFA (F i g . 3 ). Using this calibration curve, an average internal pH of 6.0 +/- 0.1 was found for large vesicles (endosomes) from control cells (F i g . 7), while co-internalization of Ad5 slightly decreased the pH of residual intact endosomes (5.5 +/- 0.2). This confirms that the increase in pH of labeled compartments observed in cell suspensions by flow cytometry is indeed due to release of internalized marker into the cytoplasm, rather than to alteration of endosomal pH per se.

Using adenovirus as gene delivery system one has to bear in mind that destruction of endosomes will also affect subsequent endocytic uptake of nutrients, hormones, and growth factors as well as signaling events from endosomes. So far, it is unknown how rapidly the endosomal system is regenerated after adenovirus internalization. Presumably, this may depend on whether for the particular cell of interest, transport to lysosomes occurs by endosome maturation or by carrier vesicles (Murphy, 1993; Gruenberg and Maxfield, 1995). We intend to apply SOFA to investigate the recovery of the endocytic system after adenovirus infection.

III. Discussion We here demonstrate that flow cytometry is a rapid and sensitive technique that can be used to analyze the endosome-disrupting potential of adenovirus. The two-step analysis involves 1/ determination of endosomal pH of labeled compartments by flow cytometry of cell suspensions, and 2/ SOFA of cell homogenates of the same cells. The first analysis indicates the potential endosome leakage induced by the virus that it then verified and quantified by SOFA. Our results show that short cointernalization (10 min) of Ad5 and fluid-phase marker results in rupture of about 30 % of endocytic vesicles.

Acknowledgments This work was supported by Austrian Science Foundation grants P-10618-MED and P-12967-GEN to R.F.

IV. Materials and Methods

Adenoviruses of subgroup B and C have been shown to increase the rate of fluid-phase uptake and in addition to permeabilize the plasma or endosomal membrane for small and large molecules (Yoshimura, 1985; Otero and Carrasco, 1987). In particular, adenoviruses type 2 and 5 (subgroup C) are known to enter the cytoplasm by endosome lysis. Comparison of the data presented in this investigation with previous studies are difficult, because in former studies large quantities of adenovirus (2000 - 5000 particles / cell) were used and the internalization conditions applied resulted in labeling of early and late endosomes as well as of lysosomes (Defer et al., 1990). Furthermore, permeabilization of the plasma membrane could not be differentiated from endosome rupture. Adenovirus-mediated enhancement of cytoplasmic delivery has mainly been analyzed using toxins or toxin-conjugates that inhibit protein synthesis (Seth, 1994). So far, adenovirus-induced endosome rupture in vivo (i.e. the number of endosomes

A. Chemicals All chemicals were obtained from Sigma unless specified. Fluorescein isothiocyanate-conjugated (FITC)-dextran (FD 70) was extensively dialyzed against Tris-buffered saline pH 7.4 (TBS) and finally against phosphate buffered saline (PBS) before use. Cy5.18-OSu (Cy5) was obtained from Amersham (UK) and coupled to dextran (M r 70 kD) as described (Rybak and Murphy, 1998).

B. Cell culture and virus propagation HeLa cells (Wisconsin strain, kindly provided by R. Rueckert, University of Wisconsin) were grown in monolayers in MEM-Eagle (GIBCO) containing heat-inactivated 10% fetal calf serum; in suspension culture Joklik's MEM (GIBCO) supplemented with 7% horse serum was used. Adenovirus serotype 5 mutant dl 312 (Ad5), a replication incompetent strain deleted in the E1a region was propagated in 293 cells

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Schober et al: Adenovirus-induced endosome rupture (Graham et al., 1977; Jones and Shenk, 1979).

at pH 7.5. Finally, the average pH of labeled compartments was determined using the pH calibration curve.

C. Endosome labeling for flow cytometry F. Flow cytometry

HeLa suspension cells (2x10 7 ) were preincubated in 2 ml DMEM containing 10% FCS for 30 min at 37°C. For labeling of all endocytic compartments, HeLa cells were incubated in 2 ml fresh medium with serum containing 2 mg / ml FITC-dextran and 0.1 mg / ml Cy5-dextran for 2 h at 37°C. To determine the influence of adenovirus on endosomal pH and endosome integrity, endosomes were labeled by incubation of HeLa cells in 2 ml DMEM containing 10% FCS, 6 mg / ml FITC-dextran, and 1 mg / ml Cy5-dextran without or with Ad5 (MOI of 1000) for 10 min at 34°C followed by a 10 min chase in DMEM in the absence or presence of Ad5. Internalization was halted by addition of ice-cold PBS (pH 7.4), pelleting the cells and washing the pellet twice with 30 ml ice cold PBS. The final cell pellet was resuspended in 2 ml PBS and divided into 7 aliquots. One aliquot (further diluted with PBS to 500 µl) was analyzed immediately by flow cytometry, the remaining aliquots were used for generation of the pH calibration curve (see below).

A dual laser FACS Calibur (Becton Dickinson Immunocytometry Systems) equipped with argon-ion and reddiode lasers was used. FITC-fluorescence (488 nm excitation) was measured using a 530 nm band pass filter (30 nm band width) and Cy5-fluorescence (635 nm excitation) was measured using a 661 nm band pass filter (16 nm band width). Forward light scatter and 90° (side)-scatter, along with both fluorescence values, were collected in list mode using 256channel resolution. For flow cytometry of cell suspensions, data for 10.000 cells were collected, while 100.000 events per sample were collected for SOFA. The following threshold parameters were defined for SOFA (see also Results and F i g . 5 ): 1/ Forward scatter (FS) and side scatter (SS): As described (Wilson and Murphy, 1989), a threshold value of SS just above the maximum value observed when sheath fluid without sample was analyzed was chosen. To differentiate large vesicles (such as late endosomes) from small vesicles, an analysis window was created with a lower FS value just above the maximum observed for sheath fluid alone (as above for SS). 2/ FITC and Cy5 fluorescence: In dual fluorescence histograms, threshold parameters were set after analyzing the PNS of unlabeled cells. Thus, a region was defined for FITC and/or Cy5 fluorescence positive events.

D. Preparation of post-nuclear supernatant (PNS) for SOFA All manipulations were carried out at 4°C. Following endosome labeling, the cells were washed twice with 50 ml PBS and pelleted. The cell pellet was resuspended in 4 vol. PBS and homogenized with a ball-bearing homogenizer (Balch and Rothman, 1985). The resulting homogenate was centrifuged for 15 min at 4300 g (Rotixa/RP, Hettich) to obtain the postnuclear supernatant (PNS). The PNS was diluted 1:5 with PBS (pH 7.4) and immediately subjected to SOFA and generation of the pH standard curve, respectively (Murphy et al., 1989; Wilson and Murphy, 1989; Murphy, 1990).

References Bai, M., Campisi, L. and Freimuth, P. (1 9 9 4 ). Vitronectin receptor antibodies inhibit infection of HeLa and A549 cells by adenovirus type 12 but not by adenovirus type 2. J . V i r o l . 68, 5925-32. Balch, W.E. and Rothman, J.E. (1 9 8 5 ). Characterization of protein transport between successive compartments of the Golgi apparatus: asymmetric properties of donor and acceptor activities in a cell-free system. Arch. B i o c h e m . B i o p h y s . 240, 413-425.

D. Generation of pH standard curves and calculation of internal pH for flow cytometry and SOFA 50 µl aliquots of cells or PNS were resuspended in 250 µl of buffers of various pH. Buffers (pH 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5) were obtained by mixing 50 mM HEPES with 50 mM MES (both containing 50 mM NaCl, 30 mM ammonium acetate, 40 mM sodium azide and 1 µM nigericin) accordingly. The samples were left on ice for 5 min for ATP-depletion and for equilibration of intravesicular pH.

Bergelson, J.M., Cunningham, J.A., Droguett, G., Kurt Jones, E.A., Krithivas, A., Hong, J.S., Horwitz, M.S., Crowell, R.L. and Finberg, R.W. (1 9 9 7 ). Isolation of a common receptor for Coxsackie B viruses and adenoviruses 2 and 5. S c i e n c e . 275, 1320-3. Cain, C.C. and Murphy, R.F. (1 9 8 6 ). Growth inhibition of 3T3 fibroblasts by lysosomotropic amines: correlation with effects on intravesicular pH but not vacuolation. J . C e l l P h y s i o l . 129, 65-70.

E. Calculation of the pH of labeled compartments

Curiel, D., Agarwal, S., Wagner, E. and Cotten, M. (1 9 9 1 ). Adenovirus enhancement of transferrin-polylysinemediated gene delivery. P r o c . N a t l . A c a d . S c i . U S A 88, 8850-8854.

The mean fluorescence value for each fluorochrome of experimental samples (8 parallels) and samples of the pH standard curve (duplicates) was calculated and the corresponding mean autofluorescence of unlabeled cells was subtracted from each. The ratio of the resulting average FITC and Cy5 values was calculated for each condition and normalized to the value obtained for that sample after clamping

Defer, C., Belin, M., Caillet-Boudin, M. and Boulanger, P. (1 9 9 0 ). Human adenovirus-host cell interactions: comparative study with members of subgroups B and C. J . V i r o l . 64, 3661-3673.

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Gene Therapy and Molecular Biology Vol 3, page 33 Fuchs, R., Male, P. and Mellman, I. (1 9 8 9 ). Acidification and ion permeabilities of highly purified rat liver endosomes. J . B i o l . C h e m . 264, 2212-2220.

Bowser, R. (1 9 8 9 ). Determination of the biochemical characteristics of endocytic compartments by flow cytometric and fluorometric analysis of cells and organelles. F l o w C y t o m e t r y : A d v a n c e d Research a n d C l i n i c a l A p p l i c a t i o n s , 221-254 (Yen, A. ed.) CRC Press, Inc., Florida.

Fujita, A., Sakagami, K., Kanegae, Y., Saito, I. and Kobayashi, I. (1 9 9 5 ). Gene targeting with a replicationdefective adenovirus vector. J . V i r o l . 69, 6180-6190.

Nemerow, G., Cheresh, D. and Wickham, T. (1 9 9 4 ). Adenovirus entry into host cells: a role for ! v integrins. T r e n d s C e l l B i o l . 4, 52-55.

Graham, F., Smiley, J., Russell, W. and Nairn, R. (1 9 7 7 ). Characteristics of a human cell line transformed by DNA from human adenovirus type 5. J . G e n . V i r o l . 36, 5974.

Otero, M. and Carrasco, L. (1 9 8 7 ). Proteins are cointernalized with virion particles during early infection. V i r o l o g y 160, 75-80.

Greber, U. and Kasamtsu, H. (1 9 9 6 ). Nuclear targeting of SV40 and adenovirus. T r e n d s C e l l B i o l . 6, 189-195.

Prchla, E., Kuechler, E., Blaas, D. and Fuchs, R. (1 9 9 4 ). Uncoating of human rhinovirus serotype 2 from late endosomes. J . V i r o l . 68, 3713-3723.

Greber, U., Suomalainen, M., Stidwill, R., Boucke, K., Ebersold, M. and Helenius, A. (1 9 9 7 ). The role of the nuclear pore complex in adenovirus DNA entry. EMBO-J. 16, 5998-6007. Greber, U., Webster, P., Weber, J. and Helenius, A. (1 9 9 6 ). The role of the adenovirus protease on virus entry into cells. EMBO-J. 15, 1766-1777.

Prchla, E., Plank, C., Wagner, E., Blaas, D. and Fuchs, R. (1 9 9 5 ). Virus-mediated release of endosomal content in vitro: different behavior of adenovirus and rhinovirus serotype 2. J . C e l l B i o l . 131, 111-123.

Greber, U., Willetts, M., Webster, P. and Helenius, A. (1 9 9 3 ). Stepwise dismantling of adenovirus 2 during entry into cells. C e l l 75, 477-486.

Rybak, S.L. and Murphy, R.F. (1 9 9 8 ). Primary cell cultures from murine kidney and heart differ in endosomal pH. J . C e l l P h y s i o l . 176, 216-22.

Gruenberg, J. and Maxfield, F.R. (1 9 9 5 ). Membrane transport in the endocytic pathway. C u r r . O p i n . C e l l B i o l . 7 , 552-563.

Schober, D., Kronenberger, P., Prchla, E., Blaas, D. and Fuchs, R. (1 9 9 8 ). Major and minor receptor group human rhinoviruses penetrate from endosomes by different mechanisms. J . V i r o l . 72, 1354-1364.

Hong, S.S., Karayan, L., Tournier, J., Curiel, D.T. and Boulanger, P.A. (1 9 9 7 ). Adenovirus type 5 fiber knob binds to MHC class I alpha 2 domain at the surface of human epithelial and B lymphoblastoid cells. EMBO-J. 16, 2294-2306.

Seth, P. (1 9 9 4 ). Mechanism of adenovirus-mediated endosome lysis: role of the intact adenovirus capsid structure. B i o c h e m . B i o p h y s . R e s . C o m m u n . 205, 1318-1324.

Jones, N. and Shenk, T. (1 9 7 9 ). An adenovirus type 5 early gene function regulates expression of other early viral genes. P r o c . N a t l . A c a d . S c i . U S A 76, 3665-3669.

Seth, P., Pastan, I. and Willingham, M. (1 9 8 5 ). Adenovirusdependent increase in cell membrane permeability. J . B i o l . C h e m . 260, 9598-9602.

Mellman, I., Fuchs, R. and Helenius, A. (1 9 8 6 ). Acidification of the endocytic and exocytic pathways. A n n . R e v . B i o c h e m . 55, 663-700.

Wagner, E. (1 9 9 8 ). Effects of membrane-active agents in gene delivery. J . C o n t r o l . R e l . 53, 155-158.

Mukherjee, S., Ghosh, R. and Maxfield, F. Endocytosis. P h y s i o l . R e v . 77, 753-803.

Wagner, E., Zatloukal, K., Cotten, M., Kirlappos, H., Mechtler, K., Curiel, D. and Birnstiel, M. (1 9 9 2 ). Coupling of adenovirus to transferrin-polylysine/DNA complexes greatly enhances receptor-mediated gene delivery and expression of transfected genes. P r o c . N a t l . Acad. Sci. USA 89, 6099-6103.

(1 9 9 7 ).

Murphy, R. F., Powers, S. and Cantor, C. R. (1 9 8 4 ). Endosome pH measured in single cells by dual fluorescence flow cytometry: rapid acidification of insulin to pH 6. J . C e l l B i o l . 98, 1757-1762.

Wang, K., S, H., KapoorMunshi, A. and Nemerow, G. (1 9 9 8 ). Adenovirus internalization and infection require dynamin. J . V i r o l . 72, 3455-3458.

Murphy, R.F. (1 9 8 5 ). Analysis and isolation of endocytic vesicles by flow cytometry and sorting: demonstration of three kinetically distinct compartments involved in fluidphase endocytosis. P r o c . N a t l . A c a d . S c i . U S A 82, 8523-6.

Wickham, T., Filardo, E., Cheresh, D. and Nemerow, G. (1 9 9 4 ). Integrin alpha v beta 5 selectively promotes adenovirus mediated cell membrane permeabilization. J . C e l l B i o l . 127, 257-264.

Murphy, R.F. (1 9 9 0 ). Ligand binding, endocytosis, and processing. F l o w C y t o m e t r y a n d S o r t i n g , 355-366 (Melamed, R. M., Lindmo, T., Mendelssohn, M. L. eds.) Wiley-Liss, Inc., New York.

Wickham, T., Mathias, P., Cheresh, D. and Nemerow, G. (1 9 9 3 ). Integrins alpha v beta 3 and alpha v beta 5 promote adenovirus internalization but not virus attachment. C e l l 73, 309-319.

Murphy, R.F. (1 9 9 3 ). Models of endosome and lysosome traffic. A d v . C e l l M o l . B i o l . M e m b r . 1, 1-7.

Wilson, R.B. and Murphy, R.F. (1 9 8 9 ). Flow-cytometric analysis of endocytic compartments. M e t h o d s C e l l

Murphy, R.F., Roederer, M., Sipe, D.M., Cain, C.C. and

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Struct. Funct. 10, 391-404.

Yoshimura, A. (1 9 8 5 ). Adenovirus-induced leakage of coendocytosed macromolecules into the cytosol. C e l l

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Gene Therapy and Molecular Biology Vol 3, page 35 Gene Ther Mol Biol Vol 3, 35-44. August 1999.

Gene regulation in Herpesvirus saimiri and its implications for the development of a novel gene therapy vector Review Article

Adrian Whitehouse* and Alex J. Stevenson Molecular Medicine Unit, University of Leeds, St James's University Hospital, Leeds, LS9 7TF United Kingdom __________________________________________________________________________________ *Correspondence: A. Whitehouse Tel: +44-113-206 5865; Fax: +44-113-244 4475; E-mail: A.Whitehouse@leeds.ac.uk Received: 7 July, 1998; accepted: 23 July 1998

Summary We have investigated the potential of HVS as a human gene therapy vector and found that it is capable of infecting an extremely broad spectrum of human cell lines and primary cultures with efficiencies that are at least as good as (and in many cases better than) currently available vector s y s t e m s . L i k e o t h e r s w e found that the virus was capable o f stably transferring a functional heterologous gene by virtue o f episomal maintenance. Although transduced clones can be established in all cases, we have also been able to demonstrate low levels of virus production from t h e s e c e l l s . T h i s f i n d i n g n e c e s s i t a t e s t h e d e v e l o p m e n t o f disabled mutants for potential future clinical applications. Fundamental research carried out in this laboratory has identified the interactions between the two known transcriptional regulatory genes encoded by HVS. Overall, these results suggest that ORF50 and ORF57 are ideal essential candidate genes to delete in order to produce a replicationdisabled HVS. This will provide the basis for a novel gene therapy vector which is theoretically capable of addressing the problems faced by current vector systems.

of the immediate early genes. These viruses could only replicate in â&#x20AC;&#x2DC;helperâ&#x20AC;&#x2122; cell lines which provided the IE gene product in trans (DeLuca et al., 1985; DeLuca and Schaffer, 1987). The vectors were engineered to carry heterologous genes in the deleted portions of their genomes, but were often toxic to the cells which they infected (Glorioso et al., 1985; Johnson et al., 1992; Sabel et al., 1995).

I. Herpesviruses as gene therapy vectors Herpesviruses are classified as large DNA viruses having genomes of between 100 and 250 kb. They are divided into alpha, beta and gamma sub-groups on the basis of their biological and genetic properties (Roizman et al., 1981). As a family their advantages as gene therapy vectors relate to an ability to package large DNA insertions and establish lifelong latent infections in which the genomic material exists as a stable episome. Nearly all of the research in this field has focused on the use of Herpes simplex virus (HSV) vectors for gene transfer to the nervous system (Coffin and Latchman, 1996). HSV encodes several proteins which modulate viral and cellular gene expression via a temporal cascade of immediate-early (IE), early and late genes (Honess and Roizman, 1974; DeLuca and Schaffer, 1985). These first generation HSV based vectors were disabled by the deletion of one or more

A second broad category of HSV based vectors are amplicons. These are plasmids containing an HSV lytic replication origin and terminal packaging signals. They can be amplified and packaged into infectious HSV-1 particles in the presence of helper-virus (Spaete and Frenkel, 1982; Kwong and Frenkel, 1984; Geller and Breakefield, 1988; Geller and Freese, 1990). As such they constitute a cloning vehicle which can efficiently carry genetic information between prokaryotic and eukaryotic cells. Amplicons retain many of the characteristics of

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Whitehouse and Stevenson: Herpesvirus saimiri as a gene therapy vector standard HSV vectors but viral stocks tend to have lower titres, making them less useful for gene therapy applications.

homologous sequences are found in approximately equivalent locations and in the same relative orientation. However, conserved gene blocks are separated by unique genes with respect to each virus (Albrect and Fleckenstein, 1990; Nicholas et al., 1992; Russo et al., 1996; Virgin et al., 1997).

A problem shared by all HSV based vector systems is the fact that the genome has no ‘latent origin’ of DNA replication, meaning that a state of episomal maintenance cannot be established in dividing cells. However, Epstein Barr virus (EBV), a member of the gamma-herpesviruses, is capable of establishing a latent state in dividing cells where the viral episome replicates co-ordinately with cell division and is inherited by all progeny cells. Such a vector derived from EBV may be suitable for stem cell gene therapy. However, EBV is associated with a number of human malignancies and lymphoproliferative disorders, necessitating extensive modification of the virus genome to eliminate those genes involved in transformation.

III. Potential of HVS as a gene therapy vector All herpesvirus vector systems which have previously been assessed were based on human herpesviruses and are inevitably likely to be ineffective in the majority of individuals due to the inherent immune response induced by the wild type virus. A herpesvirus of non-human origin, capable of infecting human cells without a cytopathic effect therefore represents an attractive candidate as a gene therapy vector, as there should be no immediate inate immune response in the recipient. Earlier publications have demonstrated that a selectable HVS has the ability to persist in a variety of human cell lines for long periods of time apparently without the production of infectious progeny (Grassmann and Fleckenstein 1989; Simmer et al., 1991).

One example of an alternative strategy, which has been employed with some success in the laboratory, is the construction of HSV amplicons containing EBV sequences that maintain the plasmid as an episome in the infected cell nucleus (Wang and Vos, 1996; Wang et al., 1997). Such developments are bound to lead to improvements in Herpes Simplex virus based vectors and the eventual creation of ‘niche’ gene therapy applications. However, all of the current options suffer from inherent problems and limitations which are far from trivial. This review highlights a potential alternative, Herpesvirus saimiri (HVS), and illustrates the importance of basic research in the quest for better vector systems.

In order to assess the potential of HVS as a possible gene therapy vector, we have generated a HVS recombinant virus (based on a non-transforming strain, A-11) which expresses EGFP (Cormack et al., 1996), and the neomycin resistance gene under the control of distinct promoters. These heterologous genes have been cloned into the repeat regions of the HVS genome, theoretically preventing alteration of the wild type virus phenotype (Figure 1 ). Analysis using this virus, which can be grown to a high titre, has demonstrated infection of a wide variety of human cell lines at approaching 100% efficiency including, A549 (lung carcinoma), HT-29 (colonic adenocarcinoma), MIA-PACA (pancreatic carcinoma), K562 (chronic myelogenous leukaemia), Jurkat (T-cell lymphoma), Molt-4 (T-cell leukaemia) and Raji (Burkitt's lymphoma) cells (Figure 2) (Stevenson et al., 1999). In contrast to previously published work we have detected low levels of virus replication in all of these cell lines at early stages post infection, even in the absence of apparent cytopathic effects. However, the virus DNA is clearly able to establish a latent episomal state within the cell which segregates to the progeny upon division. Figure 3 shows the development of a clone of A549 cells resulting from a single infected cell. The period of this experiment was four weeks, but the clone is still growing (and remains bright green) six months later. This result and similar data generated in other cell lines are extremely encouraging and we believe this system offers enormous potential for the delivery of therapeutic genes to cancerous cells, as well as to bone marrow and stem cells.

II. Herpesvirus saimiri Herpesvirus saimiri is a lymphotrophic rhadinovirus (2 herpesvirus) of squirrel monkeys (Saimiri sciureus), which persistently infects its natural host without causing any obvious disease. However, HVS infection of other species of New World primates results in fulminant polyclonal T-cell lymphomas and lymphoproliferative diseases (Fleckenstein and Desrosiers, 1982). Certain strains of HVS are also capable of transforming human T lymphocytes to continuous growth in vitro (Beisinger et al., 1992). The genome of HVS (strain A11) consists of a unique internal low G+C content DNA segment (L-DNA) of approximately 110 kbp which is flanked by a variable number of 1444 bp high G+C content tandem repetitions (H-DNA) (Albrecht et al., 1992). Analysis indicates it shares significant homology with other herpesviruses: EBV, bovine herpesvirus 4, Kaposi’s sarcoma-associated herpesvirus (KSHV or human herpesvirus 8) and murine gammaherpesvirus 68 (MHV68) (Albrect and Fleckenstein, 1990; Bublot et al., 1992; Gompels et al., 1988a;b; Neipel et al., 1997; Russo et al., 1996; Virgin et al., 1997). The genomes of EBV, KSHV, MHV68 and HVS have been shown to be generally colinear, in that 36


Gene Therapy and Molecular Biology Vol 3, page 37

F i g u r e 1 . Construction of GFP/Neo virus. A recombinant virus was generated by transfection of OMK cells with the recombination vector followed by super-infection with wild type virus. Recombinants were initially selected for by the addition of Geneticin to the culture medium followed by two rounds of plaque purification.

F i g u r e 2 . Examples of human cancer cell lines infected with GFP/Neo virus. (a) SW480 (colonic carcinoma) (b) HT-29 (colonic carcinoma) (c) Miapaca (pancreatic carcinoma).

F i g u r e 3 . Segregation of GFP amongst dividing human lung carcinoma cells (A549). A549 cells were infected with GFP/Neo virus and selected in the presence of Geneticin. The figure shows the development of an individual clone over the period of four weeks.

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Whitehouse and Stevenson: Herpesvirus saimiri as a gene therapy vector

F i g u r e 4 . Diagrammatic representation of the ORF 50 transcripts. ORF 50 produces two transcripts, the first is spliced containing a single intron and is detected at early times during the productive cycle, whereas the second is expressed later and is produced from a promoter within the second exon.

al., 1992), a sequence-specific transactivator (Gruffat et al., 1990). The HVS R gene or ORF 50 produces two transcripts. The first is spliced containing a single intron and is detected at early times during the productive cycle, whereas the second is expressed later and is produced from a promoter within the second exon (Figure 4). The spliced transcript is 5-fold more potent in activating the delayed-early ORF 6 promoter. The function of the nonspliced transcript is unclear (Nicholas et al., 1992; Whitehouse et al., 1997a). Further analysis of ORF 50 indicates it responds to particular DNA sequences specifically contained within the promoters of the genes it transactivates. Deletion and gel retardation analysis have identified a consensus ORF50-recognition sequence, CCN 9GG, required for transactivation by both ORF 50 transcripts (Whitehouse et al., 1997b). The response elements have significant homology to the EBV.R response element consensus sequences, GNCCN9GGNG. It

To develop this virus further as a gene therapy vector and to minimise the risk of pathogenicity, disabled HVS vectors are required. In order to generate a replicationdisabled vector, genes essential for the replication of the virus must be deleted. Ideal candidate genes to disable viruses are those expressed early in the viral replication cycle and which are involved in the regulation of viral gene expression. The following section discusses these genes and the role they play in HVS replication.

IV. Gene regulation in HVS A. The ORF 50 gene products We have recently identified the two major transcriptional regulating genes encoded by HVS. The first transcriptional activator is homologous to the EBV BRLF1 gene product, R (Nicholas et al., 1991; Albrecht et

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Gene Therapy and Molecular Biology Vol 3, page 39 has been shown by guanine methylation studies that the CCN 9GG motif is essential for EBV.R binding, suggesting that the R binds to adjacent major grooves of the DNA (Gruffat et al., 1990; 1992; Gruffat & Sergeant, 1994). The ORF 50 response elements map to within 32 bp which contain a CCN 9GG motif. However, the flanking sequences are significantly different to the EBV.R response elements, suggesting that the ORF 50 gene products have different sequences required for recognition and fixation of the proteins to their target. At present we are unable to determine using gel retardation analysis if the ORF 50 gene products bind directly to the response elements, or whether the retarded complex identified is due to the recruitment of host cell proteins by ORF 50.

target gene promoter sequences and appear to be mediated at the post-transcriptional level. At present we are unable to determine the actual effect of ORF 57 i.e. whether it affects the processing, transport or translational efficiency of mRNA. The more widely studied ORF 57 homologue, ICP27, appears to act post-transcriptionally by affecting mRNA processing suggesting ICP27 regulates usage of polyadenylation sites as a means of controlling gene expression (McGregor et al., 1996; McLauchlan et al., 1992). It has also been demonstrated that a bacterially expressed ICP27 fusion protein specifically binds to the 3’ ends of RNA leading to accumulation and increased half life of the mRNAs (Brown et al., 1995). The RNA binding motif (residues 138 and 152), is similar to an RGG box motif and this is believed to be an RNA binding determinant (Mears and Rice, 1996). Furthermore, it has recently been shown that ICP27 shuttles between the nucleus and cytoplasm. Shuttling occurs only at late stages during infection and is dependent on the coexpression of HSV late mRNAs, suggesting that ICP27 facilitates the export of late mRNAs (Soliman et al., 1997; Phelan et al., 1997). However, not all ICP27 homologues, including ORF 57, contain an homologous RGG box motif. Nevertheless, ORF 57 does encode an arginine-rich amino terminus, which may contain alternative RNA binding determinants. Deletion and mutational analysis of the N-terminal region of ORF 57 may help to clarify its role, if any, in RNA binding.

We believe that ORF 50 probably binds to the response sequences because of its homology with EBV.R protein which has been purified and shown to specifically recognise its response elements (Gruffat & Sergeant, 1994). However, further analysis of the ORF 50 response element by mutagenesis is required as is the production of purified ORF 50 gene products to investigate these hypotheses. The EBV.R protein has been shown to transactivate three promoters. We believe that ORF 50s gene products also transactivate multiple promoters. We have searched the HVS genome for additional ORF 50 response elements using the motif, CCN 9GG and have identified 69 putative response elements. Further characterisation of these putative elements to localise in a promoter region of a defined ORF expressed delayed-early or late in the virus replication cycle, has identified 10 possible promoter regions which may contain ORF 50 responsive elements (Whitehouse et al., 1997b). We are currently examining these genes for possible transactivation by either of the ORF 50 gene products and investigating whether late genes are transactivated by the later ORF 50b transcript. Alternatively, the ORF 50b gene product, which has been shown to transactivate ORF 6 to a lesser extent, may compete with ORF 50a for binding to the response elements, thus acting as a negative regulator of transcription.

In addition to ORF 57’s transactivating capabilities we have demonstrated that it can downregulate gene expression, specifically on intron-containing genes (Whitehouse et al., 1998a;b). In addition, the more widely studied homologue, ICP27, has been shown to be involved in the switch from early to late gene expression (McGregor et al., 1996; McLauchlan et al., 1989; 1992; Rice et al., 1993; Sandri-Goldin et al., 1995) and in the downregulation of viral IE and early genes. It is also required for the expression of late genes (McLauchlan et al., 1989; Rice et al., 1993; Sandri-Goldin et al., 1995; Sandri-Goldin and Mendoza, 1992). Furthermore, ICP27 contributes to the shut off of host cell protein synthesis and contributes to a decrease in cellular mRNA levels during infection, as deletion mutant infections result in increased levels of cellular protein synthesis and mRNA than do wild type infections (Hardwicke and Sandri-Goldin, 1994; Hardy and Sandri-Goldin, 1994; Hibbard and SandriGoldin, 1995; Schroder et al., 1989).

B. The ORF 57 gene product The second transactivator encoded by ORF 57 is homologous to genes identified in all classes of herpesviruses. These include the EBV transactivator encoded by BMLFI, ICP27 of HSV, BICP27 in bovine herpesvirus 1, ORF 4 encoded by varicella-zoster virus, UL69 in human cytomegalovirus, and ICP27 in equine herpesvirus 1 (Davidson and Scott, 1986; Kenney et al., 1989; Nicholas et al., 1988; Perera et al., 1994; Singh et al., 1996; Winkler et al., 1994; Zhao et al., 1995). The ORF 57 gene product has transregulatory functions which, unlike ORF 50’s gene products, are independent of the

HVS contains 76 major open reading frames, of which only 4 contain introns. This suggests that this virus makes limited use of the host cellular splicing machinery. Preliminary experiments have shown that during HVS infection, antigens associated with the small nuclear ribonucleoproteins (snRNPs), which are subunits of splicing complexes (reviewed in Kramer, 1995), are 39


Whitehouse and Stevenson: Herpesvirus saimiri as a gene therapy vector redistributed in the nucleus and become concentrated in specific intranuclear structures (Cooper et al., 1999). This redistribution has also been observed during herpes simplex virus infection (Phelan et al., 1993; Sandri-Goldin et al., 1995). Sequence analysis has shown that ORF 57 is more highly conserved with respect to other members of the ICP27 family at the 3’-terminal region of the gene. We believe that the ORF 57 gene product contains a functional domain within the C-terminus which is required for the repressor function of this protein. It has been demonstrated that the C-terminal domain of ICP27 must remain intact for its inhibitory effect (McMahon and Schaffer, 1990; Sandri-Goldin et al., 1995). This region contains a cysteine-histidine rich region which resembles a single “zinc finger-like” motif or “zinc knuckle” which is conserved in all ICP27 homologs including ORF 57 (histidine residue 383 and cysteine residues 387 and 392 in ORF 57). Similar motifs occur in a number of splicing factors (Sandri-Goldin and Hibbard, 1996). Further studies are been undertaken to determine if this domain is essential for the repressor activity of ORF 57 and to determine which cellular genes interact with ORF 57.

downregulated at a similar time during the replication cycle. This series of events regulating gene expression in HVS differs from other herpesviruses. IE genes in all herpesviruses are defined as those which can be transcribed efficiently in the absence of de novo protein synthesis. Therefore, they mostly encode transcriptional regulators which are required for viral gene expression. However, despite their obvious role in virus replication the major IE genes are not conserved amongst herpesviruses. For example, during HSV replication five IE genes; ICP0, ICP4, ICP22, ICP27, ICP47 are expressed in the absence of viral protein synthesis. The fact that only one of these genes is conserved in HVS (ORF 57 is homologous to ICP27), may be unsurprising as HSV and HVS belong to different subfamilies of the herpesvirus genera. However, EBV, a member of the same subfamily as HVS, also differs from HVS in the IE genes it encodes. Upon reactivation, two major IE genes are expressed which are the key transactivating genes in EBV. The first, the IE BZLF1 gene product, Z, is sufficient to trigger reactivation, when overexpressed in latently infected cells (Buisson et al., 1989; Furnari et al., 1994; Rooney et al., 1989). Z is able to transactivate several promoters containing Z responsive elements, as well as to regulate its own promoter (Furnari et al., 1994; Liebermann et al., 1989; Packman et al., 1990; Rooney et al., 1989). The second IE protein, the BRLF1 gene product, R, is also a sequence specific transactivator. HVS does not encode a Z homologue. However, ORF 50 is homologous to the EBV R protein. Overall, this suggests that the two genes encoded by HVS which are homologous to genes found in other herpesviruses play a critical role in the HVS replication cycle.

C. A novel feedback mechanism which regulates HVS gene expression More recently, we have demonstrated that these two major transcriptional control genes interact to regulate HVS gene expression via a novel feedback mechanism summarised in F i g u r e 5 . (Whitehouse et al., 1998b). The ORF 57 gene is produced at low levels early in the replication cycle until transactivated by the early ORF 50a gene product. Sequences within the ORF 57 promoter contain an ORF 50 response element which are essential for transactivation by the ORF 50a gene product and which result in an increase in RNA levels of the ORF 57 transcript. In addition, ORF 50a transactivates other genes which contain ORF 50 response elements within their promoters, for example the major DNA binding protein (Nicholas et al., 1992; Whitehouse et al., 1997a;b). Once transactivated by ORF 50a the ORF 57 gene product has several functions. As discussed previously, it has been shown to transactivate a range of HVS genes through posttranscriptional modification. Second, it downregulates ORF 50a, due to the presence of the intron within its coding region (Whitehouse et al., 1998a). Therefore we believe a feedback mechanism is in operation involving ORF 50a and ORF 57, which regulates gene expression in HVS, whereby a gene is downregulated by the product of the gene is has previously transactivated. Third, we believe the intron containing ORF 57 gene is responsible for its own downregulation by the same mechanism as that with which it represses ORF 50a, as both genes are

Acknowledgements This work was supported in part from grants from Yorkshire Cancer Research, The Candlelighters Trust, West Riding Medical Trust, Medical Research Council and the Wellcome Trust.

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Whitehouse and Stevenson: Herpesvirus saimiri as a gene therapy vector vivo gene delivery. Gene Ther 4, 1132-1141. Whitehouse, A., Carr, I. M., Griffiths, J. C. and Meredith, D. M. (1 9 9 7 a ). The herpesvirus saimiri ORF 50 gene, encoding a major transcriptional activator homologous to the Epstein-Barr Virus R protein, is transcribed from two distinct promoters of different temporal phases. J . V i r o l . 71, 2550-2554. Whitehouse, A., Cooper, M. and Meredith, D. M. (1 9 9 8 a ). The IE gene product encoded by ORF 57 of herpesvirus saimiri modulates gene expression at a posttranscriptional level. J . V i r o l . 72, 856-861. Whitehouse, A., Cooper, M., Hall, K. and Meredith, D. M. (1 9 9 8 b ). The open reading frame (ORF) 50a gene product regulates ORF 57 gene expression in herpesvirus saimiri. J . V i r o l . 72, 1967-1973. Whitehouse, A., Stevenson, A. J., Cooper, M. and Meredith, D. M. (1 9 9 7 b ). Identification of a cis-acting element within the herpesvirus saimiri ORF6 promoter that is responsive to the HVS.R transactivator. J . G e n . V i r o l . 78, 1411-1415. Winkler, M., Rice, S. A. and Stamminger, T. (1 9 9 4 ). UL69 of human cytomegalovirus, an open reading frame with homology to ICP27 of herpes simplex virus, encodes a transcriptional activator. J . V i r o l . 68, 3943-3954. Zhao, Y., Holden, V. R., Smith, R. H. and Oâ&#x20AC;&#x2122;Callaghan, D. J. (1 9 9 5 ). Regulatory function of the equine herpesvirus 1 ICP27 gene product. J . V i r o l . 69, 2786-2793.

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Gene Therapy and Molecular Biology Vol 3, page 45 Gene Ther Mol Biol Vol 3, 45-56. August 1999.

Regulation of papillomavirus transcription and replication; insights for the design of extrachromosomal vectors Review Article

Alison A. McBride Laboratory of Viral Diseases, National Institutes of Allergy and Infectious Disease, National Institutes of Health, Bethesda, Maryland 20892-0445, Building 4, room 137, 4 CENTER DR MSC 0445, Bethesda, MD 20892-0445 __________________________________________________________________________________ Correspondence: Alison A. McBride, Ph.D., Tel: 301-496-1370; Fax: 301-480-1497; E-mail: alison_mcbride@nih.gov Received: 16 October 1998; accepted: 20 October 1998

Summary The papillomaviruses infect and replicate in the stratified layers of skin and mucosa and give rise t o b en i g n l e si ons c a l l e d wa r t s o r pa pi l l o m as . Th e v i r us i nf e c t s ba sa l ep it he li al ce ll s an d wi th in these persistently infected c e l l s the viral genome i s maintained at l o w l e v e l s as extrachromosomally replicating viral DNA. The genomes of papillomaviruses can also be stably maintained as high copy number extrachromosomal elements in cell lines and within these cells the viral genomes replicate in synchrony with cellular DNA. The E1 and E2 viral proteins regulate viral transcription, initiation of replication and long term episomal maintenance of viral genomes. This review will describe the functions of the E1 and E2 proteins and discuss how these functions can be exploited in the design of extrachromosomal replicating vectors for gene therapy.

episomal maintenance of viral genomes within replicating cells (Piirsoo et al., 1996). Papillomavirus genomes and the E2-TA protein interact with mitotic chromosomes in dividing cells and this association is likely to be important for genome segregation (Skiadopoulos and McBride, 1998; Lehman and Botchan, 1998).

I. Introduction Certain DNA viruses, such as papillomavirus or Epstein-Barr virus, are able to maintain their genomes as stable extrachromosomal elements in the nuclei of infected cells. The papillomaviruses are small DNA viruses that infect basal epithelial cells and replicate in terminally differentiating keratinocytes. These viruses have been isolated from a wide range of vertebrates and they exhibit both host species and tissue specificity. Viral DNA replication has been studied mostly in bovine papillomavirus type 1 (BPV-1) and the human papillomaviruses (HPV), HPV-1, -11, -16 and -18 and -31. The viral E1 and E2 proteins are important for initiation of viral DNA replication and for regulation of viral transcription. The E1 protein is the primary viral replication initiator protein (Ustav and Stenlund, 1991a; Mohr et al., 1990; Yang et al., 1991) and E1 also functions as a transcriptional repressor (Sandler et al., 1993; Le Moal et al., 1994); the viral E2 protein(s) are transcriptional regulatory proteins that regulate the expression of the other viral gene products and, in addition, play an important role in DNA replication. The E2 transactivator protein is also required for long-term

II. The papillomavirus life cycle and function of the viral proteins Papillomaviruses infect and replicate in stratified epithelium and give rise to benign lesions called warts or papillomas. Papillomaviruses infect the lower basal layer of cells of a stratified epithelium (Figure 1). The !6"4 integrin protein, expressed exclusively in this cell layer, acts as a receptor for the virus (Evander et al., 1997). Damage to the superficial layers of the epithelium is probably necessary to allow access of virus to the basal layer. Within basal cells the viral genome is amplified to a low copy number and maintained as an extrachromosomally replicating circle of double stranded DNA (Figure 2). DNA replication in these cells probably requires the viral E1 and E2 replication proteins. The viral E5 protein is also expressed in basal cells. E5 45


McBride: papillomavirus in design of extrachromosomal vectors

F i g u r e 1 . Diagram of differentiating cells in a stratified epithelium and expression of viral functions in a papilloma. Papillomaviruses infect basal skin cells; within these cells the viral genome is replicated extrachromosomally and early gene products are expressed. Viral DNA amplification and late gene expression only occur in differentiating cells.

Figure 2. Circular genomic map of bovine papillomavirus type 1 (BPV-1). The early ORFs (E1-E8) and late ORFs (L1 and L2 are indicated). The LCR (long control region) contains regulatory elements for transcription and DNA replication such as the origin and minichromosome maintenance element (MME). E2 DNA binding sites are represented by red circles and promoters by arrows.

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Gene Therapy and Molecular Biology Vol 3, page 47 (Gilbert and Cohen, 1987; Ravnan et al., 1992). Long term, stable maintenance of papillomavirus-derived plasmids requires expression of the E1 and E2 proteins, the replication origin and a region from the LCR, that has been designated a minichromosome maintenance element (MME) (Piirsoo et al., 1996). This element contains multiple high affinity E2 binding sites. Recent studies have shown that both the BPV1 E2 transactivator protein and BPV genomes are associated with cellular chromosomes at mitosis (Skiadopoulos and McBride, 1998; Lehman and Botchan, 1998). This could be the mechanism by which approximately equal numbers of viral genomes are segregated to daughter cells at cell division to ensure that all basal cells of a papilloma contain viral DNA . The third stage of viral replication is vegetative DNA replication, which is required to generate progeny virus. Vegetative DNA replication only occurs as the basal cells of a papilloma migrate upwards and differentiate in the stratum spinosum layer. Increased expression of the E2 proteins also occurs in the stratum spinosum and may be important for amplification of viral DNA (Burnett et al., 1990). The E2 protein is important for initiation of viral DNA replication but it has also been shown that HPV-31 E2 can arrest cells in S phase (Frattini et al., 1997). Clearly this could be important for vegetative replication by allowing sustained synthesis of viral DNA. There appears to be a switch from bidirectional theta replication in the maintenance stage of replication to a rolling circle mode in the vegetative stage (Flores and Lambert, 1997). Little else is known about vegetative viral DNA replication because of the requirement for terminally differentiating keratinocytes and difficulties in reproducing these conditions in a culture system. However, great advances are being made by replicating papillomaviruses in organotypic raft cultures and in xenografts of mice and these systems are proving to be very useful in studying the entire viral life cycle (reviewed in Meyers and Laimins, 1994).

stimulates the activity of growth factor receptors expressed by the cell and induces cellular proliferation (reviewed in Howley, 1995). Enhanced proliferation of basal cells may be important to increase the population of infected cells and to provide a suitable environment for establishment of a productive viral lesion. As basal cells differentiate and migrate up to the stratum spinosum, expression of the E2 proteins is greatly increased and vegetative DNA replication begins (Burnett et al., 1990; Howley, 1995). The cells in this layer do not normally divide nor express cellular proteins necessary for DNA replication. Therefore, the viral E7 protein is required to induce the differentiated keratinocytes to enter S-phase and synthesize cellular replication proteins by binding to and inactivating the cellular retinoblastoma protein, Rb (reviewed in Jones and Munger, 1996). However, the conflicting signals of cell cycle progression and differentiation induce the p53 protein, which in turn signals cells to undergo apoptosis or growth arrest. The viral E6 protein can inactivate this function of p53 by targeting it for degradation by the ubiquitin-proteasome pathway (reviewed in Kubbutat and Vousden, 1996). The viral E4 protein is also abundant in the more differentiated layers of a papilloma. It has been hypothesized that E4 may function as a nuclear structural protein, an RNA splicing and transport factor, or in release of viral particles from the papilloma (reviewed in Howley, 1995). In the upper differentiated layers of the papilloma, the viral capsid proteins L1 and L2 are synthesized and virions are assembled (reviewed in Howley, 1995).

III. Different modes of DNA replication in the papillomavirus life cycle Three modes of DNA replication take place in the papillomavirus life cycle: initial DNA amplification, maintenance replication and vegetative replication. After initial uptake of the virus, the virion particle is uncoated and the genome transported to the nucleus of the basal cell where it is presumed to be amplified to a low copy number (Zhou et al., 1995). Presumably, a low level of the E1 and E2 proteins must be expressed early after infection since there is no evidence that they are in the viral particle. Most experimental studies have examined transient DNA replication in cultured cells, a system that is probably most analogous to this initial amplification stage and which requires the E1 and E2 proteins and the viral replication origin (Ustav and Stenlund, 1991a; Ustav and Stenlund, 1991a). Stable episomal maintenance is the second stage of papillomavirus DNA replication. In a papilloma, the infected basal cells proliferate and maintain low levels of extrachromosomal viral DNA. The genomes of papillomaviruses can also be stably maintained as high copy number extrachromosomal elements in cell lines (Law et al., 1981) and within these lines the viral genomes replicate in synchrony with cellular DNA. The viral genome copy number remains constant overall but the genomes are replicated by a random choice mechanism

IV.Transcriptional regulation by the viral E1 and E2 proteins Papillomavirus transcription is regulated primarily by the viral E2 gene products. These proteins regulate transcription by binding to specific DNA sites located in the viral genomes (see Figure 2). In bovine papillomavirus type 1 several gene products are expressed from the E2 ORF and they have been shown to function as transcriptional activators and repressors (Figure 3). cDNA species that could potentially encode truncated human papillomavirus E2 repressor proteins have been cloned but, as yet, no such proteins have been identified. Some HPVs may have evolved a mechanism to both activate and repress viral transcription with the full-length E2 protein (see Figure 5, reviewed in McBride and Myers, 1997; Fuchs and Pfister, 1994).

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McBride: papillomavirus in design of extrachromosomal vectors

F i g u r e 3 . A structural and functional map of the BPV-1 E2 proteins.

F i g u r e 4 . A structural and functional map of the BPV-1 E1 proteins.

The full-length E2 protein from all papillomaviruses consists of a 200 amino acid N-terminal transactivation domain linked to a 100 amino acid C-terminal DNA binding and dimerization domain by a flexible hinge region of variable length and sequence (reviewed in McBride and Myers, 1997; McBride et al., 1989; Dostatni et al., 1988). The E2-TA protein activates transcription by binding to specific DNA binding sites that are located within enhancer elements in the viral genome (reviewed in McBride and Myers, 1997). There are seventeen different E2 binding sites in the BPV-1 genome that vary in affinity for the E2 protein over two orders of magnitude (Li et al., 1989) (Figure 2). The well-studied genital-associated HPV genomes contain only four E2 sites in the LCR (Figure 5).

The C-terminal domain of E2 binds specifically to DNA as a dimer. The X-ray crystal structure of the Cterminal 85 amino acids of E2 bound to DNA was the first example of an anti-parallel "-barrel DNA binding structure (Hegde et al., 1992). The DNA binding domain forms an eight-stranded anti-parallel "-barrel made up of four strands from each subunit. A pair of !-helices symmetrically positioned on the outside of the barrel contain the amino acids residues that are required for specific DNA interaction. The DNA binding domain of the Epstein Barr virus EBNA1 protein has a very similar structure to the E2 DNA binding domain despite no sequence similarity (Bochkarev et al., 1995).

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Gene Therapy and Molecular Biology Vol 3, page 49 function both by direct competition with E2-TA for binding to the E2 DNA binding sites and by the formation of inactive heterodimers with the full-length E2-TA protein (Lim et al., 1998; Barsoum et al., 1992) (Figure 5). In several HPVs associated with the anogenital tract, the full length E2 protein appears to repress the promoter located upstream from the E6 gene (reviewed in McBride and Myers, 1996, 1997; Fuchs and Pfister, 1994). This probably occurs when the E2 dimer binds to E2 DNA binding sites that overlap binding sites for the cellular SP1 and TFIID transcription factors. Recent studies have indicated that these proximal E2 binding sites have lower affinity for the E2 protein than the E2 binding sites are located further upstream from the promoter start site. This has led to a model in which low levels of E2 bind to the higher affinity upstream E2 sites and activate transcription, but at high levels of E2 protein the lower affinity proximal E2 sites are occupied leading to transcriptional repression (Figure 5). The situation is probably even more complex in a papilloma as the levels and activities of the E2 proteins and cellular transcription factors are likely modulated by cell cycle and epithelial differentiation.

The 200 amino acid E2 transactivation domain, unlike many other transactivation domains, appears to have a very constrained structure that is easily disrupted by deletion or certain non-conservative point mutations (reviewed in McBride and Myers, 1997). The transactivation domain is also critical for the replication function of the E2 protein and for interaction with the E1 protein (reviewed in McBride and Myers, 1997). The exact mechanism of transactivation has not been elucidated but probably involves interaction with components of the basic transcriptional machinery. BPV1 E2 has been shown to interact with SP1, TBP, TFIIB and a novel cellular protein, AMF-1 (Li et al., 1991; Steger et al., 1995; Rank and Lambert, 1995; Breiding et al., 1997). In BPV-1, the E2 ORF encodes three different polypeptides; the E2-TA transactivator protein is encoded by the entire ORF and two smaller polypeptides, E2-TR and E8/E2, are encoded by the 3' half of the ORF. The shorter polypetides function as transcriptional repressors by antagonizing the function of E2-TA (Hubbert et al., 1988; Lambert et al., 1987; Spalholz et al., 1985; Lambert et al., 1989; Choe et al., 1989). The repressors contain only the DNA binding/dimerization domain and

F i g u r e 5 . Mechanisms of transcriptional regulation by the papillomavirus E2 proteins. A. BPV1 expresses a transcriptional transactivator with a transactivation domain and DNA binding/dimerization domain. Two shorter repressor proteins contain only the DNA binding/dimerization domain and repress E2 transactivation by forming heterodimers with the transactivator and by competing for the binding to the E2 sites in the viral genome. B . In many HPVs the full-length E2 protein can activate transcription by interacting with higher affinity E2 binding sites upstream from the transcriptional start site. At higher levels of E2, the lower affinity sites close to the promoter become occupied. This displaces essential cellular factors, SP1 and TFIID and results in repression of basal promoter activity.

49


McBride: papillomavirus in design of extrachromosomal vectors binding of an E1/E2 complex to the replication origin, which is located just upstream from P89.

V. Initiation of viral DNA replication by the E1 and E2 proteins In addition to the cellâ&#x20AC;&#x2122;s replication machinery, papillomavirus DNA replication requires the full-length E2 transactivator protein, the viral E1 protein and the replication origin (Ustav and Stenlund, 1991a; Ustav et al., 1991b; Ustav and Stenlund, 1991a). The minimal origin of replication consists of an E1 binding site, an E2 binding site and an AT rich region that may facilitate origin unwinding. (Ustav et al., 1991b). The E1 protein has several replication-associated activities such as originspecific binding (Wilson and Ludes-Meyers, 1991) and helicase activities (Yang et al., 1993) and forms a complex with the E2 transactivator (Mohr et al., 1990; Blitz and Laimins, 1991; Seo et al., 1993; Spalholz et al., 1993; Sedman and Stenlund, 1995) (Figure 4). The E1 and E2 sites have relatively low affinity for their respective proteins but together they cooperatively bind to the origin with high affinity (Figure 6). After the initial binding of an E1/E2 complex to the origin, the E1 protein oligomerizes to form a trimer or hexamer that encircles the DNA and E2 dissociates from the origin (Sedman and Stenlund, 1996, 1998). The E1 helicase function of the hexamer then unwinds the DNA at the origin to allow DNA synthesis to begin (Sedman and Stenlund, 1998) (Figure 6). The E2-TA transactivator plays an auxiliary role in replication by enhancing and regulating the functions of the E1 protein. In addition to cooperatively binding to the origin with the E1 protein, E2 alleviates repression of replication by nucleosomes (Li and Botchan, 1994) and interacts with cellular replication proteins such as RPA (Li and Botchan, 1993).

VI. Viral genome plasmid maintenance and genome segregation

F i g u r e 6 . Model of initiation of viral DNA replication. The E1 and E2 proteins initiate DNA replication by cooperatively binding to specific sites in the viral origin of replication. It has been proposed that an E1-alone complex then assembles in a ring-like hexamer structure around the DNA and the helicase activity of E1 unwinds the origin to allow access of the cellular replication machinery.

Rodent cells transformed by BPV-1 maintain approximately 50 to 200 copies of the viral genome indefinitely as extrachromosomal nuclear plasmids (Law et al., 1981). Cell lines derived from cervical carcinomas can also maintain human papillomavirus genomes as extrachromosomal elements (Bedell et al., 1991). Plasmids containing the minimal viral replication origin replicate transiently in cells expressing the E1 and E2 proteins but the replicated DNA is lost with time. Long-term stable maintenance of origin-containing plasmids also requires regions from the LCR that contain multiple high affinity E2 DNA binding sites in addition to the replication origin (Piirsoo et al., 1996). This region has been designated the minichromosome maintenance element (MME) and can be substituted by ten tandem copies of E2 DNA binding sites, suggesting that the E2 protein and the E2 DNA binding sites are important for genome segregation (F i g u r e 7 ). This finding is supported by the observation that the E2TA protein and BPV-1 genomes are associated with

The viral E1 replication protein can also function as a transcriptional repressor. Inactivation of E1 increases the immortalizing or growth transforming potential of HPV16 and BPV-1, respectively (Schiller et al., 1989; Lambert and Howley, 1988; Romanczuk and Howley, 1992) and this correlates well with the frequent disruption of E1 and/or E2 expression found in HPV-associated carcinomas. The E1 protein of BPV-1 can repress E2-mediated transactivation of the viral P89 promoter, which expresses the E6 and E7 gene products (Sandler et al., 1993; Le Moal et al., 1994). This is probably a consequence of 50


Gene Therapy and Molecular Biology Vol 3, page 51

F i g u r e 7 . Requirements for long-term episomal maintenance of papillomavirus genomes.

scaffold or chromosomal periphery. The chromosomal periphery is a region around the condensed chromatids that contains many proteins, some of which form a network of fibrils and granules (Hernandez-Verdun and Gautier, 1994). Several components of the nuclear matrix are found in the perichromosomal region as well as a number of â&#x20AC;&#x153;passengerâ&#x20AC;? proteins from the nucleus and nucleoli. The E2-TA protein (but not the E2-TR or E8/E2 proteins) has been shown to be associated with the nuclear matrix (Hubbert et al., 1988) and it will be interesting to determine whether the same interactions are important for the association with mitotic chromosomes. Nuclear matrix attachment sites have also been identified in the BPV-1 genome (Adom and Richard-Foy, 1991; Adom et al., 1992; Tan et al., 1998) and it is possible that these sites are also important for interaction of the genomes with mitotic chromatin instead of, or in addition to, E2 DNA binding sites. Although the overall viral copy number in a population of BPV-1 transformed cells remains relatively constant, several studies have shown that individual cells contain a wide range of copy numbers (Roberts and Weintraub, 1988; Ravnan et al., 1992; Ravnan and Cohen, 1997). Stewart et al. (1994) also demonstrated that there is significant randomization in replication and/or partitioning. This suggests that segregation does not occur by a very precise mechanism and is consistent with the model that the E2 proteins and viral genomes randomly associate with mitotic chromatin as passenger molecules. This model would also predict that the viral copy number depends on the levels of the E2-TA protein. A similar phenomenon has been observed for EpsteinBarr virus (EBV). EBV infects and immortalizes Blymphocytes and the viral genome is maintained indefinitely as an extrachromosomal element. The EBNA-1

condensed mitotic chromosomes in dividing cells (Skiadopoulos and McBride, 1998; Lehman and Botchan, 1998) (F i g u r e 8 ) and supports a model in which viral genomes are attached to mitotic chromatin indirectly via the E2 protein and E2 DNA binding sites (F i g u r e 9 ). This interaction would ensure that approximately equal numbers of viral genomes are segregated to daughter cells. Viral genomes that replicate as extrachromosomal plasmids may also require a mechanism to ensure that they are not lost from the nucleus during cell division. Association with cellular chromosomes would ensure that viral genomes are enclosed in the nuclear membrane during telophase. The genomes may also interact with some cellular component that ensures that they are in a transcriptionally active region of the nucleus as the cells move into the G1 stage of the cell cycle. The BPV-1 E2-TA protein interacts with mitotic chromatin in the absence of viral genomes. Conversely, the E2-TR and E8/E2 proteins are dispersed throughout the cell during mitosis and are excluded from mitotic chromatin. This indicates that the DNA binding domain of the E2 protein is not sufficient for the interaction with mitotic chromosomes and suggests that the interaction is not mediated by binding to cellular DNA sequences. The finding that a DNA-binding defective E2-TA protein retains the ability to interact with mitotic chromatin also supports this. Furthermore, deletions within the Nterminal domain abrogate the ability of E2 to interact with mitotic chromosomes. These findings indicate that the Nterminal transactivation domain of E2-TA is necessary for the interaction (Skiadopoulos and McBride, 1998). As yet, it is not known what component of mitotic chromatin is important for interaction of the E2 protein with mitotic chromatin. One possibility is that E2 interacts with some constituent of the chromosomal 51


Gene Therapy and Molecular Biology Vol 3, page 52

F i g u r e 8 . Papillomavirus genomes and the E2-TA transactivator protein are associated with cellular chromosomes in mitotic cells. E2 proteins were detected in COS7 cells expressing the E2-TA protein by indirect immunofluorescence using an E2-specific antibody. Panels A and B show COS-7 cells as a control. Panels C and D show COS-7 cells expressing E2-TA. In panels A and C, cellular DNA was detected by propidium iodide staining. In panels B and D, FITC-labeled E2 protein is detected in the same field of cells. BPV DNA was detected by fluorescent in situ hybridization in C127 cells (E and F) and 137 cells (that contain BPV-1) (G and H). In panels E and G cellular DNA was detected by the propidium iodide signal. In panels F and H the same field of cells are shown with the FITC-labeled BPV DNA signal. Mitotic cells are indicated by white arrows.

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Gene Therapy and Molecular Biology Vol 3, page 53 This maintenance requires both E2-TA and the multiple E2 binding sites in the MME element of papillomavirus (Piirsoo et al., 1996) or EBNA-1 and the multiple EBNA1 binding sites in the oriP element of Epstein-Barr virus (Yates et al., 1985). In both cases the viral proteins and genomes are associated with condensed cellular chromosomes during mitosis (Skiadopoulos and McBride, 1998; Lehman and Botchan, 1998; Grogan et al., 1983, Harris et al., 1985). The EBNA-1 protein promotes prolonged nuclear retention of plasmids containing EBNA1 DNA binding sites even in the absence of replication (Krysan et al., 1989; Middleton and Sugden, 1994) and it has been proposed that this is due to the association with mitotic chromosomes.

VIII. Papillomavirus-derived gene therapy vectors Gene therapy vectors that replicate and are retained extrachromosomally have several advantages over those that integrate in a random fashion into the host genome. These vectors will persist in proliferating cells and should not generate mutations by insertion into the cellular chromosomes. Such vectors can be maintained at a high copy number and are not susceptible to positional effects, such as inactivation, that are dependent on the integration site. EBV-based vectors that contain the EBNA-1 gene and the oriP replication origin have been developed and can express foreign gene products in primate and human cells (reviewed in Calos, 1996). Another class of EBV vectors have been developed that only contain the EBNA-1 gene and repeats of the EBNA-1 binding site required for nuclear retention. In these vectors the oriP origin has been replaced with a cellular replication origin and the resulting vectors are able to replicate in a wider range of mammalian cells (Krysan et al., 1989). The EBNA-1 protein also has the advantage that it is not recognized by the cell-mediated immune system (Levitskaya et al., 1995) as it is resistant to the proteasome-mediated degradation that is required for antigen presentation (Levitskaya et al., 1997). However, there is a report that the EBNA-1 protein can cause lymphomas in transgenic mice expressing this protein in B-cells (Wilson et al., 1996). Sarver et al. (1981) first described the use of papillomaviruses as vectors in 1981. In general, these vectors comprised the 69% transforming region of the virus (the genome minus the late region) and the foreign gene to be expressed. A newer vector only contains the LCR and the E1 and E2 genes (Ohe et al., 1995). However, these vectors have a limited host range and, in some cases, insertion of a foreign transcriptionally active foreign gene causes the plasmid to integrate (Waldenstrom et al., 1992). This is probably because the small papillomavirus genomes are very compact and contain multiple overlapping genes and regulatory signals that can be inadvertently disrupted. The presence of an active heterologous enhancer and promoter could interfere with viral replication by transcriptional interference. Using the detailed knowledge of the mechanisms of papillomavirus

Figure 9 . This diagram shows a model in which papillomavirus genomes are linked via the E2-TA protein to condensed mitotic chromosomes.

protein of EBV is a transcriptional transactivator and a replication protein and it is the only viral protein required for replication and maintenance of plasmids containing the oriP origin of replication (which contains a number of repeated EBNA DNA binding sites) (Yates et al., 1985). The EBNA 1 protein and EBV genomes have also been shown to be randomly associated with mitotic chromatin (Grogan et al., 1983; Harris et al., 1985) and it has been suggested that these properties might be important for the genome segregation and nuclear retention function of EBNA-1. The EBNA-1 protein also promotes prolonged nuclear retention of plasmids containing EBNA-1 DNA binding sites but no origin of replication (Krysan et al., 1989; Middleton and Sugden, 1994) and it has been proposed that this is due to the interaction of plasmids with mitotic chromosomes.

VII. Similarities between the papillomavirus E2 and Epstein-Barr virus EBNA-1 protein The Epstein-Barr virus EBNA-1 protein and the papillomavirus E2-TA protein have common roles in the life cycles of their respective viruses (Grossman and Laimins, 1996). Both proteins are transcriptional transactivators that activate transcription by binding to specific binding sites within the viral genomes. Notably, both proteins have dimeric DNA binding. domains with almost identical anti-parallel "-barrel structures, despite no amino acid homology (Bochkarev et al., 1995). Both viruses replicate and maintain their genomes as extrachromosomal elements in persistently infected cells. 53


McBride: papillomavirus in design of extrachromosomal vectors replication and genome maintenance, it should be possible to generate a new class of papillomavirus vectors. These vectors could express the E1 and/or E2 genes from different promoters suitable for a specific cell type and either viral or cellular replication origins could be incorporated, as has been described for EBV-based vectors (Krysan et al., 1989). The addition of repeated E2 binding sites may be sufficient to maintain the vector as an episome when either a viral or cellular replication origin is used.

Dostatni, N., Thierry, F., and Yaniv, M. (1 9 8 8 ). A dimer of BPV-1 E2 containing a protease resistant core interacts with its DNA target. EMBO J. 7, 3807-3816. Evander, M., Frazer, I.H., Payne, E., Qi, Y.M., Hengst, K., and McMillan, N.A. (1 9 9 7 ). Identification of the a6 integrin as a candidate receptor for papillomaviruses. J . V i r o l . 71, 2449-2456. Flores, E.R. and Lambert, P.F. (1 9 9 7 ). Evidence for a switch in the mode of human papillomavirus type 16 DNA replication during the viral life cycle. J . V i r o l . 71, 7167-7179. Frattini, M.G., Hurst, S.D., Lim, H.B., Swaminathan, S., and Laimins, L.A. (1 9 9 7 ). Abrogation of a mitotic checkpoint by E2 proteins from oncogenic human papillomaviruses correlates with increased turnover of the p53 tumor suppressor protein. EMBO J. 16, 318-331.

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Fuchs, P.G. and Pfister, H. (1 9 9 4 ). Transcription of papillomavirus genomes. I n t e r v i r o l o g y 37, 159-167.

Adom, J.N. and Richard-Foy, H. (1 9 9 1 ). A region immediately adjacent to the origin of replication of bovine papilloma virus type 1 interacts in vitro with the nuclear matrix. Biochem. Biophys. Res. Commun. 176, 479-485.

Gilbert, D.M. and Cohen, S.N. (1 9 8 7 ). Bovine papilloma virus plasmids replicate randomly in mouse fibroblasts throughout S phase of the cell cycle. C e l l 50, 59-68. Grogan, E.A., Summers, W.P., Dowling, S., Shedd, D., Gradoville, L., and Miller, G. (1 9 8 3 ). Two Epstein-Barr viral nuclear neoantigens distinguished by gene transfer, serology, and chromosome binding. P r o c . N a t l . A c a d . S c i . U S A 80, 7650-7653.

Barsoum, J., Prakash, S.S., Han, P., and Androphy, E.J. (1 9 9 2 ). Mechanism of action of the papillomavirus E2 repressor: repression in the absence of DNA binding. J . V i r o l . 66, 3941-3945. Bedell, M.A., Hudson, J.B., Golub, T.R., Turyk, M.E., Hosken, M., Wilbanks, G.D., and Laimins, L.A. (1 9 9 1 ). Amplification of human papillomavirus genomes in vitro is dependent on epithelial differentiation. J . V i r o l . 65, 2254-2260.

Grossman, S.R. and Laimins, L.A. (1 9 9 6 ). EBNA1 and E2: a new paradigm for origin-binding proteins? Trends. M i c r o b i o l . 4, 87-89. Harris, A., Young, B.D., and Griffin, B.E. (1 9 8 5 ). Random association of Epstein-Barr virus genomes with host cell metaphase chromosomes in Burkitt's lymphoma-derived cell lines. J . V i r o l . 56, 328-332.

Blitz, I.L. and Laimins, L.A. (1 9 9 1 ). The 68-kilodalton E1 protein of bovine papillomavirus is a DNA binding phosphoprotein which associates with the E2 transcriptional activator in vitro. J . V i r o l . 65, 649656.

Hegde, R.S., Grossman, S.R., Laimins, L.A., and Sigler, P.B. (1 9 9 2 ). Crystal structure at 1.7 A of the bovine papillomavirus-1 E2 DNA- binding domain bound to its DNA target. Nature 359, 505-512.

Bochkarev, A., Barwell, J.A., Pfuetzner, R.A., Furey, W.J., Edwards, A.M., and Frappier, L. (1 9 9 5 ). Crystal structure of the DNA-binding domain of the Epstein-Barr virus origin-binding protein EBNA 1. C e l l 83, 39-46.

Hernandez-Verdun, D. and Gautier, T. (1 9 9 4 ). The chromosome periphery during mitosis. B i o e s s a y s 16, 179-185.

Breiding, D., Sverdrup, F., Grossel, M.J., Moscufo, N., Boonchai, W., and Androphy, E.J. (1 9 9 7 ). Isolation of a BPV1 E2 transactivation domain binding factor required for both transcriptional activation and DNA replication. V i r o l o g y 221, 34-43.

Howley, P.M.(1 9 9 5 ). Papillomavirinae: The viruses and their replication. In V i r o l o g y (Fields, B.N., Knipe, D.M., and Howley, P.M., Eds.) Lippincott-Raven, Philadelphia and New York. 2045-2076. Hubbert, N.L., Schiller, J.T., Lowy, D.R., and Androphy, E.J. (1 9 8 8 ). Bovine papilloma virus-transformed cells contain multiple E2 proteins. P r o c . N a t l . A c a d . S c i . USA 85, 5864-5868.

Burnett, S., Strom, A.C., Jareborg, N., Alderborn, A., Dillner, J., Moreno-Lopez, J, Pettersson, U., and Kiessling, U. (1 9 9 0 ). Induction of bovine papillomavirus E2 gene expression and early region transcription by cell growth arrest: correlation with viral DNA amplification and evidence for differential promoter induction. J . V i r o l . 64, 5529-5541.

Jones, D.L. and Munger, K. (1 9 9 6 ). Interactions of the human papillomavirus E7 protein with cell cycle regulators. S e m i n . C a n c e r B i o l . 7, 327-337.

Calos, M.P. (1 9 9 6 ). The potential of extrachromosomal replicating vectors for gene therapy. Trends. G e n e t . 12, 463-466.

Krysan, P.J., Haase, S.B., and Calos, M.P. (1 9 8 9 ). Isolation of human sequences that replicate autonomously in human cells. M o l . C e l l B i o l . 9, 1026-1033.

Choe, J., Vaillancourt, P., Stenlund, A., and Botchan, M. (1 9 8 9 ). Bovine papillomavirus type 1 encodes two forms of a transcriptional repressor: Structural and functional analysis of new viral cDNAs. J . V i r o l . 63, 1743-1755.

Kubbutat, M.H.G. and Vousden, K.H. (1 9 9 6 ). Role of E6 and E7 oncoproteins in HPV-induced anogenital malignancies. S e m i n . V i r o l . 7, 295-304. Lambert, P.F. and Howley, P.M. (1 9 8 8 ). Bovine papillomavirus type 1 E1 replication-defective mutants

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amino-terminal domain. P r o c . N a t l . A c a d . S c i . U S A 86, 510-514.

Lambert, P.F., Hubbert, N.L., Howley, P.M., and Schiller, J.T. (1 9 8 9 ). Genetic assignment of multiple E2 gene products in bovine papillomavirus-transformed cells. J . V i r o l . 63, 3151-3154.

McBride, A.A. and Myers, G.(1 9 9 6 ). The E2 proteins .in Human P a p i l l o m a v i r u s e s (Myers, G. , Baker, C., Wheeler, C., Halpern, A., McBride, A., and Doorbar, J., Eds.) .Los Alamos National Laboratory, Los Alamos.

Lambert, P.F., Spalholz, B.A., and Howley, P.M. (1 9 8 7 ). A transcriptional repressor encoded by BPV-1 shares a common carboxy-terminal domain with the E2 transactivator. C e l l 50, 69-78.

McBride, A.A. and Myers, G.(1 9 9 7 ). The E2 proteins: an update .in H u m a n P a p i l l o m a v i r u s e s 1 9 9 7 (Myers, G., Baker, C., Munger, K., Sverdrup, F., McBride, A. , and Bernard, H.-U., Eds.) .Los Alamos National Laboratory, Los Alamos. http://hpvweb.lanl.gov/

Law, M.F., Lowy, D.R., Dvoretzky, I., and Howley, P.M. (1 9 8 1 ). Mouse cells transformed by bovine papillomavirus contain only extrachromosomal viral DNA sequences. P r o c . N a t l . Acad. S c i . USA 78, 2727-2731.

Meyers, C. and Laimins, L.A. (1 9 9 4 ). In vitro systems for the study and propagation of human papillomaviruses. C u r r . T o p . M i c r o b i o l . I m m u n o l . 186, 199-215. Middleton, T. and Sugden, B. (1 9 9 4 ). Retention of plasmid DNA in mammalian cells is enhanced by binding of the Epstein-Barr virus replication protein EBNA1. J . V i r o l . 68, 4067-4071.

Le Moal, M.A., Yaniv, M., and Thierry, F. (1 9 9 4 ). The bovine papillomavirus type 1 (BPV1) replication protein E1 modulates transcriptional activation by interacting with BPV1 E2. J . V i r o l . 68, 1085-1093.

Mohr, I.J., Clark, R., Sun, S., Androphy, E.J., MacPherson, P., and Botchan, M.R. (1 9 9 0 ). Targeting the E1 replication protein to the papillomavirus origin of replication by complex formation with the E2 transactivator. S c i e n c e 250, 1694-1699.

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Ohe, Y., Zhao, D., Saijo, N., and Podack, E.R. (1 9 9 5 ). Construction of a novel bovine papillomavirus vector without detectable transforming activity suitable for gene transfer. Hum. Gene Ther. 6, 325-333. Piirsoo, M., Ustav, E., Mandel, T., Stenlund, A., and Ustav, M. (1 9 9 6 ). Cis and trans requirements for stable episomal maintenance of the BPV-1 replicator. EMBO J. 15, 1-11.

Levitskaya, J., Sharipo, A., Leonchiks, A., Ciechanover, A., and Masucci, M.G. (1 9 9 7 ). Inhibition of ubiquitin/proteasome-dependent protein degradation by the Gly-Ala repeat domain of the Epstein-Barr virus nuclear antigen 1. P r o c . N a t l . A c a d . S c i . U S A 94, 12616-12621.

Rank, N.M. and Lambert, P.F. (1 9 9 5 ). Bovine papillomavirus type 1 E2 transcriptional regulators directly bind two cellular transcription factors, TFIID and TFIIB. J . V i r o l . 69, 6323-6334.

Li, R. and Botchan, M.R. (1 9 9 3 ). The acidic transcriptional activation domains of VP16 and p53 bind the cellular replication protein A and stimulate in vitro BPV-1 DNA replication. C e l l 73, 1207-1221.

Ravnan, J.-B. and Cohen, S.N. (1 9 9 7 ). Transformed mouse cell lines that consist predominantly of cells maintaining bovine papillomavirus at high copy number. V i r o l o g y 213, 526-534.

Li, R. and Botchan, M.R. (1 9 9 4 ). Acidic transcription factors alleviate nucleosome-mediated repression of DNA replication of bovine papillomavirus type 1. P r o c . N a t l . A c a d . S c i . U S A 91, 7051-7055.

Ravnan, J.-B., Gilbert, G.M., Ten Hagen, K.G., and Cohen, S.N. (1 9 9 2 ). Random-choice replication of extrachromosomal bovine papillomavirus (BPV) molecules in heterogeneous clonally-derived BPV-infected cell lines. J . V i r o l . 66, 6946-6952.

Li, R., Knight, J., Bream, G., Stenlund, A., and Botchan, M. (1 9 8 9 ). Specific recognition nucleotides and their DNA context determine the affinity of E2 protein for 17 binding sites in the BPV- 1 genome. Genes D e v . 3, 510-526.

Roberts, J.M. and Weintraub, H. (1 9 8 8 ). Cis-acting negative control of DNA replication in eukaryotic cells. C e l l 52, 397-404.

Li, R., Knight, J.D., Jackson, S.P., Tjian, R., and Botchan, M.R. (1 9 9 1 ). Direct interaction between Sp1 and the BPV enhancer E2 protein mediates synergistic activation of transcription. C e l l 65, 493-505.

Romanczuk, H. and Howley, P.M. (1 9 9 2 ). Disruption of either the E1 or the E2 regulatory gene of human papillomavirus type 16 increases viral immortalization capacity. P r o c . N a t l . Acad. S c i . USA 89, 31593163.

Lim, D.A., Gossen, M., Lehman, C.W., and Botchan, M.R. (1 9 9 8 ). Competition for DNA binding sites between the short and long forms of E2 dimers underlies repression in bovine papillomavirus type 1 DNA replication control. J . V i r o l . 72, 1931-1940.

Sandler, A.B., Vande Pol, S.B., and Spalholz, B.A. (1 9 9 3 ). Repression of bovine papillomavirus type 1 transcription by the E1 replication protein. J . V i r o l . 67, 5079-5087. Sarver, N., Gruss, P., Law, M.F., Khoury, G., and Howley, P.M. (1 9 8 1 ). Bovine papilloma virus deoxyribonucleic acid: a novel eucaryotic cloning vector. M o l . C e l l . B i o l . 1, 486-496.

McBride, A.A., Byrne, J.C., and Howley, P.M. (1 9 8 9 ). E2 polypeptides encoded by bovine papillomavirus type 1 form dimers through the common carboxyl-terminal domain: Transactivation is mediated by the conserved

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McBride: papillomavirus in design of extrachromosomal vectors Schiller, J.T., Kleiner, E., Androphy, E.J., Lowy, D.R., and Pfister, H. (1 9 8 9 ). Identification of bovine papillomavirus E1 mutants with increased transforming and transcriptional activity. J . V i r o l . 63, 1775-1782.

Wilson, J.B., Bell, J.L., and Levine, A.J. (1 9 9 6 ). Expression of Epstein-Barr virus nuclear antigen-1 induces B cell neoplasia in transgenic mice. E M B O J . 15, 3117-3126.

Sedman, J. and Stenlund, A. (1 9 9 5 ). Co-operative interaction between the initiator E1 and the transcriptional activator E2 is required for replicator specific DNA replication of bovine papillomavirus in vivo and in vitro. E M B O J . 14, 6218-6228.

Wilson, V.G. and Ludes-Meyers, J. (1 9 9 1 ). A bovine papillomavirus E1-related protein binds specifically to bovine papillomavirus DNA. J . V i r o l . 65, 5314-5322. Yang, L., Li, R., Mohr, I.J., Clark, R., and Botchan, M.R. (1 9 9 1 ). Activation of BPV-1 replication in vitro by the transcription factor E2. Nature 353, 628-632.

Sedman, J. and Stenlund, A. (1 9 9 6 ). The initiator protein E1 binds to the bovine papillomavirus origin of replication as a trimeric ring-like structure. EMBO J . 15, 50855092.

Yang, L., Mohr, I., Fouts, E., Lim, D.A., Nohaile, M., and Botchan, M. (1 9 9 3 ). The E1 protein of bovine papillomavirus 1 is an ATP-dependent DNA helicase. P r o c . N a t l . A c a d . S c i . U S A 90, 5086-5090.

Sedman, J. and Stenlund, A. (1 9 9 8 ). The papillomavirus E1 protein forms a DNA-dependent hexameric complex with ATPase and DNA helicase activities. J . V i r o l . 72, 68936897.

Yates, J.L., Warren, N., and Sugden, B. (1 9 8 5 ). Stable replication of plasmids derived from Epstein-Barr virus in various mammalian cells. Nature 313, 812-815.

Seo, Y.-S., Muller, F., Lusky, M., Gibbs, E., Kim, H.-Y., Phillips, B., and Hurwitz, J. (1 9 9 3 ). Bovine papilloma virus (BPV)-encoded E2 protein enhances binding of E1 protein to the BPV replication origin. P r o c . N a t l . Acad. Sci. USA 90, 2865-2869.

Zhou, J., Gissmann, L., Zentgraf, H., Muller, H., Picken, M., and Muller, M. (1 9 9 5 ). Early phase in the infection of cultured cells with papillomavirus virions. V i r o l o g y 214, 167-176.

Skiadopoulos, M.H. and McBride, A.A. (1 9 9 8 ). BPV1 viral genomes and the E2 transactivator protein are associated with cellular metaphase chromosomes. J V i r o l 72, 2079-2088. Spalholz, B.A., McBride, A.A., Sarafi, T., and Quintero, J. (1 9 9 3 ). Binding of bovine papillomavirus E1 to the origin is not sufficient for DNA replication. V i r o l o g y 193, 201-212. Spalholz, B.A., Yang, Y.C., and Howley, P.M. (1 9 8 5 ). Transactivation of a bovine papilloma virus transcriptional regulatory element by the E2 gene product. C e l l 42, 183-191. Steger, G., Ham, J., Lefebvre, O., and Yaniv, M. (1 9 9 5 ). bovine papillomavirus 1 E2 protein contains activation domains: one that interacts with TBP another that functions after TBP binding. EMBO J. 329-340.

The two and 14,

Stewart, A.-C., Jareborg, N., Simonsson, M., Alderborn, A., and Burnett, S. (1 9 9 4 ). Segregation properties of bovine papillomaviral plasmid DNA. J . M o l . B i o l . 236, 480490. Tan, S.H., Bartsch, D., Schwarz, E., and Bernard, H.U. (1 9 9 8 ). Nuclear matrix attachment regions of human papillomavirus type 16 point toward conservation of these genomic elements in all genital papillomaviruses. J . V i r o l . 72, 3610-3622. Ustav, M. and Stenlund, A. (1 9 9 1 a ). Transient replication of BPV-1 requires two viral polypeptides encoded by the E1 and E2 open reading frames. EMBO J. 10, 449-457. Ustav, M., Ustav, E., Szymanski, P., and Stenlund, A. (1 9 9 1 b ). Identification of the origin of replication of bovine papillomavirus and characterization of the viral origin recognition factor E1. EMBO J. 10, 4321-4329. Waldenstrom, M., Schenstrom, K., Sollerbrant, K., and Hansson, L. (1 9 9 2 ). Replication of bovine papillomavirus vectors in murine cells. Gene 120, 175181.

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Gene Therapy and Molecular Biology Vol 3, page 57 Gene Ther Mol Biol Vol 3, 57-65. August 1999.

Gene transfer with adeno-associated virus 2 vectors: the growth factor receptor connection Review Article

Cathryn Mah 1-3, Keyun Qing1-3, Jonathan Hansen1-3, Benjawan Khuntirat1-3, Mervin C. Yoder4, and Arun Srivastava1-3,5 1

Department of Microbiology & Immunology, 2Walther Oncology Center, 5Division of Hematology/Oncology, Department of Medicine, 4 Herman B Wells Center for Pediatric Research and Department of Biochemistry & Molecular Biology, Indiana University School of Medicine, and 3Walther Cancer Institute, Indianapolis, IN 46202 __________________________________________________________________________________________________ Correspondence: Arun Srivastava, Ph.D., Department of Microbiology & Immunology, Indiana University School of Medicine, 635 Barnhill Drive, Medical Science Building, Room 257, Indianapolis, IN 46202-5120. Phone: (317) 274-2194; Fax: (317) 274-4090; E-mail: asrivast@iupui.edu Received: 30 September 1998; accepted: 10 October 1998

Summary Adeno-associated virus 2 (AAV)-based vectors have gained attention as a potentially useful alternative to the more commonly used retroviral and adenoviral vectors for human gene therapy. However, there are at least two major obstacles that limit high-efficiency transduction by AAV vectors. The first relates to the extent of expression of the cellular receptor for AAV, and the second concerns the rate-limiting step of the viral second-strand DNA synthesis. With reference to the first obstacle, although the ubiquitously expressed cell surface heparan sulfate proteoglycan (HSPG) has been reported to be a receptor AAV, HSPG alone is insufficient for AAV infection, and human fibroblast growth factor receptor 1 (FGFR1) has been identified as a co-receptor for successful viral entry into the host cell. With reference to the second obstacle, a cellular protein, designated the single-stranded D-sequence binding protein (ssD-BP), phosphorylated at tyrosine residues, has been identified which plays a crucial role in viral second-strand DNA synthesis. The ssD-BP is phosphorylated by the protein tyrosine kinase activity of the human epidermal growth factor receptor (EGFR). Thus, both FGFR1 and EGFR are crucial determinants in the life cycle of AAV, and further studies on the interaction between the FGFR and EGFR may yield new insights not only into its role in the host cell but also in the optimal use of AAV vectors in human gene therapy. therapy of cystic fibrosis (Flotte and Carter, 1997). Although AAV possesses a broad host-range that transcends the species barrier (Muzyczka, 1992), the efficiency of AAV-mediated transduction has been reported to vary widely. Recently, the ubiquitously expressed cell surface heparan sulfate proteoglycan (HSPG) was identified as a receptor for AAV (Summerford and Samulski, 1998), it has become increasingly clear that HSPG alone is insufficient for AAV infection. For example, our recent studies have documented a significant donor variation in terms of the ability of AAV vectors to transduce primary human bone marrow-derived CD34+ hematopoietic progenitor cells (Ponnazhagan et al., 1997). In these

I. Introduction The non-pathogenic nature of the adeno-associated virus 2 (AAV), a single-stranded DNA-containing human parvovirus (Srivastava et al., 1983), coupled with the remarkable site-specific integration of the wild-type (wt) AAV genome into the human chromosome 19 (Kotin et al., 1990, Samulski et al., 1991), generated a significant interest in the development of AAV vectors as a potentially useful alternative to the more commonly used retrovirus and adenovirus vectors in human gene therapy (Berns and Giraud, 1996). Indeed, AAV vectors have been successfully used for gene transfer in vitro as well as in vivo, and are currently in Phase II clinical trials for gene 57


Mah et al: AAV vectors and the EGFR connection studies, AAV-mediated transgene expression ranged between 15-80% of infected cells from approximately 50% of normal volunteer donors, whereas AAV failed to bind to CD34+ cells from approximately 50% of donors and consequently, cells from these donors could not be transduced. Similarly, we have reported that the efficiency of AAV transduction in permissive cells does not correlate with the receptor number, and that a cellular protein, designated as the single-stranded D-sequence-binding protein (ssD-BP), phosphorylated at tyrosine residues, plays a crucial role in the viral second-strand DNA synthesis (Qing et al., 1997; 1998), a rate-limiting step in AAV-mediated transgene expression (Fisher et al., 1996; Ferrari et al., 1996). Thus, the two obstacles encountered in attempting to obtain high-efficiency transduction by recombinant AAV vectors will be discussed briefly as follows.

(Bartlett and Samulski, 1998), could not bind FGF. Stable transfection with huFGFR1 cDNA alone allowed for a low-level of FGF binding, the extent of which was significantly higher when M07e were co-transfected with both HSPG and FGFR1 cDNAs. Interestingly, transfection with the muHSPG cDNA alone resulted in significant binding of FGF. These results suggest that M07e cells do indeed express the endogenous FGFR gene. As expected, mock-transfected Raji cells also failed to bind FGF as they lack both HSPG and FGFR. Only low levels of FGF binding were detected in Raji clones stably transfected with either the HSPG or FGFR1 expression plasmids alone, whereas in Raji cells co-expressing both, a significant binding of FGF occurred, further corroborating the requirement of both HSPG and FGFR1 for ligand binding. It was also interesting to note that the binding patterns of AAV to both the M07e and Raji cells coexpressing HSPG+FGFR1 genes closely resembled that of FGF binding. Taken together, these results strongly suggested that cell surface expression of both HSPG and FGFR1 is required for successful binding of AAV to the host cell (Qing et al., 1999).

II. The first obstacle A. Successful infection of cells by AAV requires fibroblast growth factor receptor 1 (FGFR1) as a cell surface co-receptor

In order to determine whether non-permissive cells could be rendered positive for AAV transduction following stable transfection with cDNAs encoding muHSPG, or huFGFR1, or both, individual clonal isolates from both cell types were either mock-infected or infected with a recombinant AAV vector under identical conditions and analyzed for transgene expression by fluorescence-activated cell-sorting (FACS). Whereas little transgene expression was seen in mock-infected M07e cells, as expected, it was evident that Mo7e cells expressing either HSPG alone, or both HSPG and FGFR1, but not FGFR1 alone, could be readily transduced by the recombinant AAV vector. Expression of the exogenous HSPG in M07e cells was sufficient to render the cells permissive to AAV infection because M07e cells express the endogenous FGFR gene. On the other hand, Raji cells failed to be transduced by recombinant AAV if only the exogenous HSPG or FGFR1 genes were expressed, but co-expression of both HSPG and FGFR1 conferred AAV infectivity to these cells, albeit at a relatively low-efficiency. Inclusion of additional individual clonal isolates from both cell types yielded very similar results. These studies establish that co-expression of both HSPG and FGFR1 is required both for binding and also entry of AAV into the host cell (Qing et al., 1999).

It has previously been demonstrated that all cell types which bind AAV can also be infected by AAV (Ponnazhagan et al., 1996, 1997; Qing et al., 1998; Summerford and Samulski. 1998; Bartlett and Samulski, 1998). For example, human cell lines such as HeLa, KB, and 293, which have been shown to be permissive for AAV infection, can bind AAV, whereas non-permissive cells, such as M07e, cannot. Interestingly, however, we noted that murine NIH3T3 cells, which could not be transduced by a recombinant AAV vector, could bind AAV quite efficiently. As NIH3T3 cells are known to express HSPG (Ledoux et al., 1992), this observation suggested that in addition to HSPG as a primary receptor for binding, AAV might require a putative cell surface co-receptor for efficient entry. Since fibroblast growth factor (FGF) has an absolute requirement for HSPG prior to efficient binding to the fibroblast growth factor receptor (FGFR) (Green et al., 1996), we reasoned that FGFR might be a potential candidate. We examined human cell types known to be non-permissive for AAV infection, such as the human megakaryocytic cell line M07e (Ponnazhagan et al., 1996), as well as those that do not express either HSPG or FGFR, such as the human lymphoblastoid cell line Raji (Kiefer et al., 1990; Lebakken and Rapraeger, 1996). These cell types were stably transfected with cDNA expression plasmids containing either the murine HSPG core protein (Syndecan-1) (Saunders et al., 1989), or the human FGFR1 (Johnson et al., 1990), or both, followed by the determination of radiolabeled FGF-binding, radiolabeledAAV binding, and recombinant AAV-mediated transgene expression. M07e cells, known to lack HSPG expression

B. FGFR autophosphorylation is not required for AAV-mediated transduction Since ligand binding to FGFR consequently leads to receptor dimerization followed by ion activation of the FGFR-associated protein tyrosine kinase (PTK), 58


Gene Therapy and Molecular Biology Vol 3, page 59 ultimately resulting in recruitment of intracellular signaling molecules (Rapraeger et al., 1991; Ledoux et al., 1992; Roghani and Moscatelli; 1992, Givol and Yayon, 1992; Kan et al., 1993), it was of interest to investigate whether FGFR PTK activity affected AAV-mediated transgene expression. To this end, cells permissive for AAV infection, human 293 and HeLa cells, were either mock-treated, or first treated with specific inhibitors of FGFR PTK (Mohammadi et al., 1997) followed by infection with a recombinant AAV vector under identical conditions and the extent of transgene expression was determined as described above. These experiments demonstrated that none of the FGFR PTK inhibitors tested had any significant effect on AAV-mediated transgene expression. From these studies, we conclude that FGFR PTK activity is not required for AAV-mediated transgene expression (Qing et al., 1999).

expression in the presence of FGF was not due to phosphorylation of the ssD-BP in 293 cells as these assays carried out with prior treatment with genistein also resulted in similar results (89% inhibition with FGF, 0% inhibition with EGF). Taken together, these results strongly suggest that HSPG-FGFR1 interaction is crucial not only for binding, but also for entry of AAV into the host cell. Based on all available information, we propose a model for the initial step in AAV infection, which is depicted in Figure 1. In this model, co-expression of cell surface HSPG and FGFR1 is required for successful AAV binding followed by viral entry (Panel A), both of which are blocked by FGF (Panel B) (Qing et al., 1999).

III. The second obstacle A. Inhibitors of epidermal growth factor receptor (EGFR) protein tyrosine kinase (PTK) activity increase the transduction efficiency of recombinant AAV

C. FGF treatment perturbs AAV binding to non-permissive as well as permissive cells, and abrogates viral entry into permissive cells

We have previously shown that inhibition of tyrosine phosphorylation of the ssD-BP by genistein, a specific inhibitor of all protein tyrosine kinases (Akiyama et al., 1987; Barnes and Peterson, 1995; Constantinou and Huberman, 1995; Carlo-Stella et al., 1996), increased transduction efficiency by recombinant AAV (Qing et al., 1997). To investigate which kinase may be responsible for tyrosine phosphorylation of the ssD-BP, we studied the effects of various kinase inhibitors such as apigenein (MAP kinase) (Kuo and Yang, 1995), herbimycin A (pp60c-src) (Fukazawa et al., 1991), LY294002 (PI 3kinase) (Vlahos et al., 1994), staurosporine (CaM kinase, MLC kinase, PK-A, PK-C, PK-G) (Couldwell et al., 1994), tyrphostin A48 (EGF-R PTK) (Gazit et al., 1989), wortmannin (MAP kinase, MLC kinase, PI 3-kinase, PI 4-kinase) (Okada et al., 1994), in addition to genistein, on the transduction efficiency of recombinant AAV. Following treatment with these reagents, cells were infected with a recombinant AAV vector, followed by staining with X-gal 48 hrs post-infection. The results indicated that in addition to genistein, treatment with tyrphostin A48, a specific inhibitor for EGFR PTK, caused an increase in the numbers of blue cells. These results suggest that EGF-R PTK may be involved in recombinant AAV-mediated transgene expression (Mah et al., 1998).

The following experiments further supported the contention that FGFR1 acts as a co-receptor for AAV biding and entry. First, we hypothesized that treatment of non-permissive cells such as NIH3T3 cells, and permissive cells such as 293 cells, with large excess of FGF would perturb the ability of AAV to bind to the host cell. Binding studies with NIH3T3 and 293 cells were carried out using radiolabeled AAV in the presence or absence of excess amounts of FGF, with additional controls including wt AAV or heparin (as positive controls) and EGF (as a negative control). The results of these experiments documented that AAV binding to NIH3T3 cells was inhibited by heparin, as expected (Summerford and Samulski, 1998), and FGF also inhibited AAV binding to a significant extent, whereas EGF had no effect under identical conditions. As expected, unlabeled wt AAV significantly inhibited binding of radiolabeled AAV to 293 cells. Likewise, excess FGF was also able to reduce AAV binding to 293 cells. On the other hand, as with the NIH3T3 cells, similar concentrations of EGF had no significant effect on AAV binding to 293 cells. Second, we reasoned that excess amounts of FGF might perturb AAV infection. To this end, equivalent numbers of 293 cells were infected with a recombinant AAV vector either in the absence or presence of excess FGF or EGF, under identical conditions. Forty-eight hrs post-infection, transgene expression was evaluated by X-gal staining as previously described (Ponnazhagan et al., 1996; 1997). The results of these experiments indicated that AAVmediated transduction of 293 cells was inhibited in the presence of FGF by approximately 89%, but in the presence of EGF by only 2%. The lack of transgene

In order to further investigate the role of EGFR PTK in recombinant AAV transduction efficiency, other inhibitors specific for EGFR PTK, tyrphostins 1, 23, 25, 46, 47, 51, 63, and AG1478 (Yaish et al., 1988; Gazit et al., 1989; Lyall et al., 1989; Levitzki, 1990, Levitzki et al., 1991) were tested for their effects on recombinant AAV 59


Mah et al: AAV vectors and the EGFR connection

Figure 1. A possible model for the role of cell surface HSPG and FGFR1 in mediating AAV binding and entry into the host cell. Coexpression of HSPG and FGFR1 is required for successful binding of AAV followed by viral entry into a susceptible cell (Panel A), both of which are perturbed by the ligand, FGF, which also requires HSPG-FGFR1 interaction (Panel B ) (Qing et al., 1999).

increase recombinant AAV transduction efficiency without causing deleterious effects (Mah et al., 1998).

transduction. For controls, tyrphostins specific for tumor necrosis factor ! (TNF-!) production, AG126, TNF-! cytotoxicity, AG1288 (Novogrodsky et al., 1994), platelet-derived growth factor receptor protein tyrosine kinase (PDGFR PTK), AG1295 and AG1296 (Kovalenko et al., 1994), were also used. It was evident that among all the inhibitors tested, treatment with tyrphostin 1 resulted in the greatest increase in recombinant AAV transduction efficiency (without causing significant cytotoxicity) followed by that of tyrphostins 23, 63, 25, 46, then 47. Again, these results emphasize the role EGFR PTK plays in recombinant AAV-mediated transgene expression. As expected, the control tyrphostins AG126, AG1288, AG1295, and AG1296 had no significant effect. In toxicity experiments, with reference to the mock-treated or solvent alone controls, both tyrphostin 1 and tyrphostin 23 are far less toxic than either genistein or hydroxyurea (HU), two reagents that have been previously shown to increase AAV transduction efficiency (Russell et al., 1995; Ferrari et al., 1996; Qing et al., 1997). Therefore, treatment of primary cells with tyrphostin may offer a physiological means to

We have previously demonstrated that recombinant AAV transduction efficiency correlates well with the phosphorylation state of the cellular ssD-BP (Qing et al., 1998). For example, in HeLa cells, the ssD-BP is predominantly in the phosphorylated form, and these cells are not readily transduced by recombinant AAV vectors. 293 cells, on the other hand, are very well transduced by recombinant AAV, and have been demonstrated to contain predominantly the dephosphorylated form of the ssD-BP. Following treatment of HeLa cells, all active tyrphostins caused a significant increase in the amount of dephosphorylated form of the ssD-BP, as determined by electrophoretic mobility-shift assays (EMSAs). Consistent with our previous data (Qing et al., 1998), the amount of dephosphorylated ssD-BP for each treatment corresponded with the level of increase in transduction efficiency for each of the compounds. That is, the greater the amount of dephosphorylated ssD-BP, the greater the increase in AAVmediated transgene expression. When 293 cells, either mock-treated or treated with EGF, were analyzed, the ssD-

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Gene Therapy and Molecular Biology Vol 3, page 61 BP was present mostly in the dephosphorylated form in mock-treated cells as observed previously (Qing et al., 1997, 1998), whereas EGF treatment resulted in a significant increase in the amount of the phosphorylated form of ssD-BP. These results strongly suggest that EGFR PTK plays a direct role in the phosphorylation of the ssD-BP (Mah et al., 1998).

in these cells. To this end, equivalent numbers of A431 and H69 cells were either mock-treated or treated with EGF and analyzed by EMSA. As expected, EGF-treatment had no significant effect on the phosphorylation state of the ssD-BP in either cell type. In A431 cells, the ssD-BP was found to be predominantly in the phosphorylated form due to the high-levels of EGFR PTK expression. On the other hand, both phosphorylated and dephosphorylated forms of the ssD-BP were detected in H69 cells. Interestingly, however, treatment with either tyrphostin or genistein resulted in the conversion from the phosphorylated to the dephosphorylated form of the ssD-BP, consequently resulting in increased transduction in A431 cells. Under identical conditions, however, neither tyrphostin nor genistein treatments had any effect on the phosphorylation state of the ssD-BP in H69 cells, and these cells could not be transduced by AAV as they lack the cell surface receptor for AAV. Although it may not be readily apparent which cellular protein tyrosine kinase is responsible for the phosphorylation of the ssD-BP in H69 cells, these results are in agreement with the conclusion that the phosphorylation of the ssD-BP in A431 cells is catalyzed by the EGFR PTK (Mah et al., 1998).

B. Recombinant AAV transduction efficiency correlates inversely with the EGFR expression If EGFR PTK is responsible for catalyzing phosphorylation of the ssD-BP, then AAV-mediated transgene expression would be expected to be significantly lower in cells which express higher numbers of EGFRs than those which express fewer numbers of EGFRs. Therefore, AAV transduction efficiency would inversely correlate with the extent of EGF-R expression. To further investigate this hypothesis, equivalent numbers of cells known to express very high numbers of EGFRs, A431 cells (Giard et al., 1973), and cells known to express very low numbers, H69 cells (Gamou et al., 1987), in addition to HeLa and 293 cells, were infected with a recombinant AAV vector under identical conditions followed by X-gal staining 48 hrs post-infection. Consistent with previously published data (Qing et al., 1998), the transduction efficiency in HeLa and 293 cells was approximately 4% and 20%, respectively. As expected, the transduction efficiency in A431 cells was less than 1%. Contrary to our hypothesis, very little transduction (<1%) was also noted in the H69 cells. This apparent paradox was addressed by performing radiolabeled EGF and AAV binding assays. EGF binding assays demonstrated that A431 cells bound the greatest amounts of EGF, followed by HeLa, then 293 cells. H69 cells bound negligible amounts of EGF, as expected. It was evident from AAV binding assays that, similar to M07e cells, previously shown to lack AAV receptors (Bartlett and Samulski, 1998), H69 cells also do not express the cellular receptor for AAV. On the other hand, the low transduction efficiency seen in the A431 cells could not be attributed to a lack of expression of AAV receptors as these cells expressed far greater numbers of AAV receptors than HeLa or 293 cells.

C. Stable transfection of EGFR cDNA into 293 cells causes phosphorylation of the ssDBP and results in inhibition of AAVmediated transgene expression As 293 cells can be efficiently transduced by recombinant AAV vectors, since they contain predominantly the dephosphorylated from of the ssD-BP (Qing et al., 1997, 1998), we examined whether the deliberate over-expression of EGFR PTK in these cells would lead to phosphorylation of the ssD-BP, and consequently, result in the inhibition of AAV-mediated transgene expression. 293 cells were transfected with an EGFR cDNA expression plasmid and a number of stably transfected clones were used to determine the ratios of the dephosphorylated to the phosphorylated forms of the ssDBP and compared with that in control, untransfected 293 cells. Replicate cultures were also evaluated for the efficiency of recombinant AAV transduction, with or without pre-treatment with tyrphostin 1. These results indicated that in each of the transfected 293 cell clones, the ratio of dephosphorylated/phosphorylated ssD-BPs was reduced to an average of 0.45 from greater than 3.5 in control cells, which concomitantly led to a significant decrease in AAV transduction efficiency from approximately 18% in control 293 cells to an average of about 2% in the EGF-R-transfected 293 cell clones. These data strongly support the hypothesis that the EGFR-ssDBP interaction plays a crucial role in AAV-mediated transgene expression.

As the EGFR PTK appeared to catalyze the phosphorylation of the ssD-BP, it was next of interest to examine the effects of EGF, as well as tyrphostin- and genistein-treatments on A431 and H69 cells. Due to the high-levels of expression of EGFR in A431 cells, we hypothesized that the ssD-BP would be present in its phosphorylated form and that EGF treatment would have no effect on it's phosphorylation state. Similarly, it would be expected that H69 cells would also fail to respond to EGF treatment since little expression of the EGFR occurs 61


Mah et al: AAV vectors and the EGFR connection

Figure 2. A possible model for the role of the cellular EGFR PTK in AAV-mediated transgene expression. The phosphorylated ssDBP, which is phosphorylated by the EGFR PTK, binds to the single-stranded Dsequence within the AAV-ITR, and blocks the viral second-strand DNA synthesis. Co-infection with adenovirus, or expression of the Ad E4orf6 protein, or treatment with HU, genistein, or tyrphostins, leads to dephosphorylation of the ssD-BP, either via inhibition of the EGFR PTK, or via activation of a hitherto unknown cellular phosphotyrosine phosphatase, leading to some type of conformational change in the ssD-BP which, in turn, allows the viral second-strand DNA synthesis resulting in augmentation in transcription and translation of the transgene (Mah et al., 1998).

EGFR PTK. Based on all the available data, we propose a model for the subsequent steps in AAV-mediated transduction which is shown in Figure 2. In this model, the phosphorylated ssD-BP, which is phosphorylated by the EGFR PTK, binds to the single-stranded D-sequence within the AAV-ITR, and blocks the viral second-strand DNA synthesis. Co-infection with adenovirus, or expression of the Ad E4orf6 protein, or treatment with HU, genistein, or tyrphostins, leads to dephosphorylation of the ssD-BP, either via inhibition of the EGFR PTK, or

Subsequent in vitro phosphorylation assays performed with the commercially available purified EGFR PTK (McGlynn et al., 1992; Weber et al., 1984) and the affinity column-purified dephosphorylated form of the ssD-BP from 293 cells indicated that the ssD-BP was phosphorylated by the EGFR PTK and that this phosphorylation was abrogated in the presence of tyrphostin 1 and tyrphostin 23. These results provide direct evidence that the ssD-BP is a downstream target of the

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Gene Therapy and Molecular Biology Vol 3, page 63 via activation of a hitherto unknown cellular phosphotyrosine phosphatase, leading to some type of conformational change in the ssD-BP which, in turn, allows the viral second-strand DNA synthesis resulting in augmentation in transcription and translation of the transgene (Mah et al., 1998).

The research in authorsâ&#x20AC;&#x2122; laboratory was supported in part by Public Health Service grants (HL-48342, HL53586, HL-58881, and DK-49218, Centers of Excellence in Molecular Hematology) from the National Institutes of Health, and a grant from the Phi Beta Psi Sorority. A.S. was supported by an Established Investigator Award from the American Heart Association.

IV. Conclusions and future prospects

References

The identification of FGFR1 as a co-receptor for AAV is an important step forward (Qing et al., 1999). Interestingly, however, although FGFRs have been shown to be expressed in every organ and tissue examined (Givol and Yayon, 1992), the relative abundance of their expression in skeletal muscle and in neuroblasts and glioblasts in the brain correlates particularly well with the documented high efficiency of AAV-mediated transduction in these tissues in vivo (Fisher et al., 1997; Kaplitt et al., 1994; Kessler et al., 1996; McCown et al., 1996; Xiao and Samulski, 1996). Since there are at least four distinct but related members in the FGFR family, viz FGFR1, FGFR2, FGFR3, and FGFR4 (Ledoux et al., 1992), it should be of interest to now systematically examine, both in vitro and in vivo, the relative involvement of each of these members in facilitating successful infection by AAV.

Akiyama T, Ishida J, Nakagawa S, Ogawara H, Watanabe S, Itoh N, Shibuya M, Fukami Y (1 9 8 7 ). Genistein, a specific inhibitor of tyrosine-specific protein kinases. J B i o l C h e m 262, 5592-5595. Barnes S, Peterson GT (1 9 9 5 ). Biochemical targets of the isoflavone genistein in tumor cell lines. S o c E x p B i o l Med 280, 103-109. Bartlett JS, Samulski RJ (1 9 9 8 ) Fluorescent viral vectors: A new technique for the pharmacological analysis of gene therapy. Nature Med 4, 635-637. Berns KI, Giraud C (1 9 9 6 ) Biology of adeno-associated virus. Curr Top Microbiol Immunol 218, 1-23. Carlo-Stella C, Regazzi E, Garau D, Mangoni L, Rizzo MT, Bonati A, Dotti G, Almici C, Rizzoli V (1 9 9 6 ) Effect of the protein tyrosine kinase inhibitor genistein on normal and leukaemic haemopoietic progenitor cells. Br J Haematol 93, 551-557.

The demonstration that the cellular EGFR PTK catalyzes phosphorylation of the ssD-BP, a crucial player in AAV-mediated transduction, this kinase should be an easy target for inhibition by low-toxicity compounds for their ability to significantly increase recombinant AAV transduction efficiency which may prove to be valuable for gene therapy. Although it is possible that other factors, in addition to the ssD-BP phosphorylation state, act in concert to influence the AAV transduction efficiency, it is noteworthy, however, that skeletal muscle and brain tissues, which have been shown to be extremely welltransduced by recombinant AAV vectors in vivo (Fisher et al., 1997; Kessler et al., 1996; Kaplitt et al., 1994; McCown et al., 1996; Xiao and Samulski, 1996), express little to no EGFR (Lim and Hauschka, 1984; Styren et al., 1993). Further studies on the interaction between FGFR and additional downstream target proteins, and the possible interaction between FGFR and EGFR should allow for a clearer understanding of molecular events involved in highefficiency AAV transduction which, in turn, should lead to improvements in the optimal use of AAV vectors in human gene therapy.

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McGlynn E, Becker M, Mett H, Reutner S, Cozens R, Lydon NB (1 9 9 2 ) Large scale purification and characterization of a recombinant epidermal growth factor receptor protein tyrosine kinase. Eur J Biochem 207, 265-275. Mohammadi M, McManhon G, Sun L, Tang C, Hirth P, Yeh BK Hubbard SR, Schlessinger J (1 9 9 7 ) Structures of the tyrosine kinase domain of fibroblast growth factor receptor in complex with inhibitors. S c i e n c e 276, 955960.

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Summerford C, Samulski RJ (1 9 9 8 ) Membrane-associated heparan sulfate proteoglycan is a receptor for adenoassociated virus type 2 virions. J V i r o l 72, 1438-1445.

Ponnazhagan S, Wang X-S, Woody MJ, Luo F, Kang LY, Nallari ML, Munshi NC, Zhou SZ, Srivastava A (1 9 9 6 ). Differential expression in human cells from the p6 promoter of human parvovirus B19 following plasmid transfection and recombinant adeno-associated virus 2 (AAV) infection: Human megakaryocytic leukaemia cells are non-permissive for AAV infection. J G e n V i r o l 77, 1111-1122.

Vlahos CJ, Matter WF, Hui KY, Brown RF (1 9 9 4 ) A specific inhibitor of phosphatidylinositol 3-kinase, 2-(4morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002) . J B i o l C h e m 269, 5241-5248. Weber W, Bertics PJ, Gill GN (1 9 8 4 ) Immunoaffinity purification of the epidermal growth factor receptor. J B i o l C h e m 259, 14631-14636. Xiao X, Li J, Samulski RJ (1 9 9 6 ) Efficient long-term gene transfer into muscle tissue of immunocompetent mice by adeno-associated virus vector. J V i r o l 70, 8098-8108.

Qing KY, Khuntirat B, Mah C, Kube DM, Wang X-S, Ponnazhagan S, Zhou SZ, Dwarki VJ, Yoder MC, Srivastava A (1 9 9 8 ) Adeno-associated virus type 2mediated gene transfer: Correlation of tyrosine phosphorylation of the cellular single-stranded D sequence-binding protein with transgene expression in human cells in vitro and murine tissues in vivo. J V i r o l 72, 1593-1599.

Yaish P, Gazit A, Gilon C, Levitzki A (1 9 8 8 ) Blocking of EGF-dependent cell proliferation by EGF receptor kinase inhibitors. S c i e n c e 242, 933-935.

Qing KY, Mah C, Hansen J, Zhou SZ, Dwarki VJ, Srivastava A (1 9 9 9 ). Human fibroblast growth factor receptor 1 is a co-receptor for infection by adeno-associated virus 2. Nature Med 5, in press. Qing KY, Wang X-S, Kube DM, Ponnazhagan S, Bajpai A, Srivastava A (1 9 9 7 ). Role of tyrosine phosphorylation of a cellular protein in adeno-associated virus 2-mediated transgene expression. Proc N a tl A c a d S c i USA 94, 10879-10884. Rapraeger AC, Krufka A, Olwin BB (1 9 9 1 ) Requirement of heparan sulfate for bFGF-mediated fibroblast growth and myoblast differentiation. S c i e n c e 252, 1705-1708. Roghani M, Moscatelli D (1 9 9 2 ) Basic fibroblast growth factor is internalized through both receptor-mediated and heparan sulfate-mediated mechanisms. J B i o l Chem 267, 22156-22162. Russell DW, Alexander IE, Miller AD (1 9 9 5 ) DNA synthesis and topoisomerase inhibitors increase transduction by adeno-associated virus vectors. Proc Natl Acad S c i USA 92, 5719-5723. Samulski RJ, Zhu X, Xiao X, Brooke JD, Houseman DE, Epstein N, Hunter LA (1 9 9 1 ) Targeted integration of adeno-associated virus (AAV) into human chromosome 19. EMBO J 10, 3941-3950 Saunders S, Jalkanen M, O'Farrell S, Bernfield M (1 9 8 9 ) Molecular cloning of syndecan, an integral membrane proteoglycan. J C e l l B i o l 1 0 8 , 1547-1556. Srivastava A, Lusby EW, Berns KI (1 9 8 3 ) Nucleotide sequence and organization of the adeno-associated virus 2 genome. J V i r o l 45, 555-564. Styren SD, DeKosky ST, Rogers J, Mufson EJ (1 9 9 3 ) Epidermal growth factor receptor expression in demented elderly: localization to vascular endothelial cells of brain, pituitary, and skin. Brain Res 615, 181-190.

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Gene Therapy and Molecular Biology Vol 3, page 67 Gene Ther Mol Biol Vol 3, 67-74. August 1999.

Hepatocyte-specific gene expression by a recombinant adeno-associated virus vector carrying the apolipoprotein E enhancer and 1-antitrypsin promoter Research Article

Torayuki Okuyama 1,2, Motomichi Kosuga1,2, Satori Takahashi1, Kyoko Sasaki1, and Masao Yamada1 Department of Genetics, National Childrenâ&#x20AC;&#x2122;s Medical Research Center, Setagaya, Tokyo 154-8509 Japan, Department of Pediatrics, Keio University School of Medicine, Tokyo 160-8582 __________________________________________________________________________________ Correspondence: Torayuki Okuyama, M.D., Department of Genetics, National Childrenâ&#x20AC;&#x2122;s Medical Research Center, 3-35-31 Taishido Setagaya-ku Tokyo 154-8509, Japan. Phone: +81-3-3414-8121 ext. 2752; Fax: +81-3-3414-3208; E-mail: tora@nch.go.jp Key words: adeno-associated virus, liver-specific promoter, ! 1 -antitrypsin promoter, apolipoprotein E enhancer, gene therapy Received: 30 October 1998; accepted: 10 November 1998

Summary An adeno-associated virus vector was constructed to express exogenous genes to the liver. The original plasmid construct carried two expression units; a neomycin resistant gene and human 1antitrypsin cDNA under the control o f hepatocyte specific transcription elements. C e l l s were transfected with the constructed plasmid DNA with another packaging plasmid, and recombinant adeno-associated viruses (rAAV) were then recovered after adenovirus infection. Alternatively, rAAV were recovered by transduction of DNAs of the packaging plasmid and adenovirus into preselected cells carrying constructed proviral DNA. When the transducing abilities were evaluated b a s e d o n G 4 1 8 r e s i s t a n t c o l o n y f o r m a t i o n o n H e L a c e l l s , t h e l a t t e r m e t h o d w a s found t o give almost 10-fold more rAAV. We then isolated G418 resistant colonies and established several independent clones for the HeLa and Hepa1A cells infected with the rAAV. All of the eight clones derived from Hepa1A cells produced significant amounts of the human 1-antitrypsin protein. In contrast, none o f the five clones derived from HeLa c e l l s produced a detectable l e v e l o f 1antitrypsin. Our results suggest that liver-specific promoter and enhancer maintain the tissue specificity in the rAAV construct, and that the rAAV vector system would be useful in hepatocyte directed gene therapy.

administration because of the high immunogenicity (Jaffe et al. 1992; Okuyama et al. 1998). Retroviral vectors are also able to transduce an exogenous gene into hepatocytes, and a long term expression of the transduced gene has been identified in several experiments using rat or dog liver (Rettinger et al. 1994; Kay et al. 1992; Kay et al. 1993; Hafenrichter et al. 1994). However, the expression level is

I. Introduction Liver-directed gene therapy could revolutionize treatments for many genetic disorders such as phenylketonuria, familial hypercholesterolemia and hemophilia (Ledley 1993). Adenoviral vectors efficiently transduce a gene into hepatocytes, easily achieve its expression at a therapeutic level for many diseases, but do not allow a long-term expression and repetitive 67


Okuyama et al: Hepatocyte-specific gene expression using the apo E enhancer

II. Results

generally too low for therapeutic treatments of patients because the transducing efficiency is extremely low.

A. Generation of rAAV vector containing a liver-specific promoter and enhancer

We have previously demonstrated that a retroviral vector expressing an exogenous gene under the control of a human apolipoprotein E enhancer and ! 1-antitrypsin promoter as well as an original retroviral LTR promoter dramatically increase the level of protein production after administration into the rat liver (Okuyama et al. 1996). The apolipoprtein E enhancer has been detected through studies on a gene cluster of apoE/C-I/C-II in human chromosome 19. Studies on transgenic mice disclosed that a 154 bp region located 15 kb downstream of the apolipoprotein E gene was responsible for the high level of expression in hepatocytes (Shachter et al. 1993; Simonet et al. 1993). A 420 bp segment of the 5’ flanking region of the human ! 1-antitrypsin gene contains distinct HNF-1 and HNF-2 binding sites, and both sites are responsible for strong and tissue-specific expression of ! 1-antitrypsin (Li et al. 1988). Recently we found that rats administered the retroviral vector expressing human coagulation Factor X under the control of the 420 bp of ! 1-antitrypsin promoter produced a therapeutic level of functional Factor X (Le et al. 1997). These observations suggested that with the retroviral vectors designed to express an exogenous gene under the control of promoter-enhancer complex of apolipoprotein E and ! 1-antitrypsin, one is able to achieve significantly high levels of transgene expression. However, retroviral vectors can transduce foreign genes only into dividing cells, thus inducing the regeneration with partial hepatectomy which is essential for retroviral genetransduction into hepatocytes. Although the mortality for a 70% hepatectomy is relatively low and the procedure could be justified for life threatening genetic deficiencies,alternative methods that circumvent partial hepatectomy are desirable.

The structure of the rAAV vector TRNAEAT containing liver-specific transcriptional elements is shown in F i g . 1 . The vector contains the human ! 1-antitrypsin (hAAT) cDNA as a reporter gene downstream of the 420bp 5’ flanking sequence of its own gene. The enhancer region located in the apoE/C-I/C-II gene locus is necessary for high level expression of the apoE gene in livers of transgenic mice (Schacter et al. 1993). In an attempt to further enhance expression of the hAAT promoter from an rAAV vector, four copies of ApoE enhancer were placed upstream of the hAAT promoter (Simonet et al. 1993). In addition to these 2.8-kb hepatocyte-specific expression units of hAAT, an 1.5 kb expression cassette of neomycin resistant gene under the control of mouse phosphoglycerate kinase promoter (Soriano et al. 1991; Adra et al. 1987) was also introduced into the AAV vector plasmid pTR (+).

B. Comparison of the efficiency of rAAV generation We produced rAAV vectors via two different methods. Method I was a conventional co-transfection method described before (Zolotukhin et al. 1996). Briefly, HeLa cells were co-transfected with the vector plasmid pTRNAEAT and packaging plasmid pIM45 (Peel et al. 1997) at a molar ratio of 1:2 using the calcium-phosphate method, followed by the infection of human adenovirus 5 at MOI.2. For Method II, we established a stable HeLa cell line “HeLa-TRNAEAT” carrying proviral sequences of AAV-TRNAEAT by transfecting HeLa cells with pTRNAET following the selection in G418. HeLaTRNAEAT cells were transfected with pIM45 and infected with Ad5. Then, wild type HeLa cells were infected with the rAAV generated by these two methods and selected with G418 to determine the Neo-resistant titers. The rAAV titer of the viral stock from Method I was 0.8±0.4x104 cfu/ml, while those from Method II was 1.0±0.2x105 cfu/ml (F i g . 2 ). We were able to generate rAAV stocks with 10 fold higher titer using Method II. Moreover, since more consistent and reproducible results were obtainable with the latter method, we used Method II for generating rAAV for further analysis.

Adeno-associated virus is a replication-defective parvovirus that is being developed as a vector for human gene therapy (Laughlin et al. 1986). One advantage of AAV as a vector is that it can transduce genes into postmitotic cells like cells of the Central Nervous System (Kaplitt et al. 1994), lung epithelial cells (Flotte et al. 1993), or muscle fiber cells (Fisher et al. 1996). Since most hepatocytes in vivo are also in the growth arrested state, AAV vectors is expected to be suitable for in vivo hepatocyte-directed gene therapy. However, little is known about the transduction and expression efficiency of rAAV in hepatocytes in vitro as well as in vivo (Flotte et al. 1995; Fisher et al. 1997; Snyder et al. 1997). In this study, we generated a recombinant adeno-associated virus containing hepatocyte-specific expression unit, and evaluated its transduction efficiency, tissue specificity, and level of expression in gene-transduced cells of hepatocyte origin.

C. Testing the rAAV vectors for infectivity to human and rodent hepatoma cell lines We determined the NeoR titers of HepG2 and Hepa1A cells to evaluate the infectivity of the rAAV to cell lines of hepatocyte origin. The NeoR titers for HepG2 and Hepa-

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F i g u r e 1 . Schematic presentation of recombinant adeno-associated virus “TRNAEAT”. Two expression cassettes were introduced in an opposite direction to each other between two terminal inverted repeat sequences of AAV. Expression of neomysin resistant gene is under the control of phophorylgycerate kinase promoter, and expression of human ! 1 -antitrypsin cDNA is expected to be under the control of human ! 1 -antitrypsin promoter and apolipoprotein E enhancer. Tr, inverted terminal repeat sequence of adeno-associated virus; Neo-R, expression cassette for neomycin resistant gene; ApoEEn, 4 copies of apolipoprotein E enhancer; hAATcDNA, cDNA for human ! 1 -antitrypsin; A, polyA signal of SV40

Figure 3. Infectivity of rAAV in different cell lines. NeoR titers of the rAAV prepared with Method II were determined with 4 different cell lines, HeLa (Lane 1), NIH3T3 (Lane 2), HepG2 (Lane 3), and Hepa1A (Lane 4). Data is presented as the average ± SE.

1A cells were 0.8±0.2x105 cfu/ml and 0.6±0.1x105 cfu/ml, respectively. The same viral solution was used to calculate the titer in HeLa cells and NIH3T3 cells (F i g . 3 ). The relative infectivity of the rAAV in HepG2 and Hepa1A cells compared to those of HeLa cells were 0.75 and 0.67, respectively. These observations suggested that it is possible to transduce exogenous genes into cell lines of hepatocyte origin with similar efficiency as into HeLa or NIH3T3 cells using the rAAV gene transfer system.

F i g u r e 2 . Neo R titers of rAAV “TRNAET” in HeLa cells. The viral stocks of rAAV ”TRNAEAT” were prepared by two different methods, Method I and Method II (see details in text), and Neo R titers of each viral stock were determined in HeLa cells. Data is presented as the average ± SE.

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Okuyama et al: Hepatocyte-specific gene expression using the apo E enhancer T a b l e 1 . Comparison of levels of human ! 1 -antitrypsin (hAAT) expression in Hepa1A cells infected with rAAV “TRNAEAT” and retroviral vector “ApoE(-)haat-LTR”. (Data is presented as the average (±SE) Vectors for hAAT gene transduction

hAAT expression (ng/ million cells / 24 h)

rAAV-TRNAEAT

103.8±23.5

retroviral vector “ApoE(-)hAATLTR”

71.0±6.6

D. Cell-type specific expression of cDNA for human 1 -antitrypsin in Hepa1A cells transduced with the rAAV “TRNAEAT” Hepa1A cells and HeLa cells infected with the rAAV were selected in G418 for 14 days, and eight Hepa1A and five HeLa clones were isolated. To verify that the cDNA region of hAAT was introduced into the cells, the 400 bp partial hAAT cDNA was amplified using purified genomic DNA as template. The expected DNA fragments were amplified in all HeLa and HepG2 clones, indicating that the expression unit of hAAT was integrated into the chromosomal DNA of the cells with the rAAV vector (F i g . 4 A and 4B). In HeLa and HepG2 cells, we identified faint 1.8-kb amplified DNA fragments, corresponding to the endogenous human hAAT gene. The hAAT protein secreted into the media of each clone was assayed by ELISA using human-specific antibody for ! 1antitrypsin. All eight clones of Hepa1A cells secreted hAAT into the media. The average amount of hAAT protein secreted from the rAAV-infected Hepa1A cells was 103.8 ng /106cells /24 hours (F i g . 5 A ). This represented 31% of hAAT secreted from one million cells of wild type HepG2, and was similar to the level of hAAT secreted from Hepa1A cells infected with retroviral vector “apoE(-) haat-LTR” carrying an identical liver-specific expression cassette for hAAT (Table 1). On the other hand, none of the HeLa clones obtained by the infection of rAAVTRNAEAT secreted detectable levels of hAAT protein in cultured media, although expression units for hAAT were administered into the host cell chromosome (F i g . 5B). These results suggested that rAAV-TRNAEAT was able to express exogenous genes exclusively in cells of hepatocyte origin.

Figure 4 . Detection of proviral genome sequence in Hepa1A and HeLa cells infected with rAAV ”TRNAET”. A 400 bp DNA region of human ! 1 -antitrypsin was amplified using purified genomic DNA of rAAV-infected cells as templates. (A) PCR results for the eight Hepa 1A clones. Template DNA samples of PCR reactions were as below, pTRNAET (Lane P), wild type Hepa1A (Lane 0), rAAV-infected Hepa1A clones No.1-No.8 (Lane 1-Lane 8), and wild type HepG2 (Lane G). Lane M stands for DNA size markers, HindI II digested Lambda DNA (left) and Sau3AI digested PUC 19 (right). The 400 bp amplified DNA fragments were identified in Lanes 1 to 8, indicating that proviral sequences of the rAAV were introduced into all of the eight clones. This signal was not identified in Lane 0 and Lane G, but a 1.8 kb signal was identified in Lane G instead. This corresponds to the DNA amplified from human genome for ! 1 -antitrypsin. (B ) PCR results for five independent HeLa cell clones. Template DNA samples of PCR reactions were wild type HeLa cells (Lane 0), and rAAVinfected HeLa clones No.1-No.5 (Lane 1-Lane5). Lane P, Lane G, and Lane M were same as for F i g u r e 4 A . PCR of the five HeLa clones and wild type HeLa cells resulted 1.8 kb DNA fragments amplified from human gene for ! 1 -antitrypsin, but only AAV-infected HeLa clones showed 0.4 kb fragments corresponding to the rAAV proviral sequence.

III. Discussion In this report we have shown that a recombinant adenoassociated virus vector is able to transfer an exogenous gene into human or rodent cells of hepatocyte origin with a similar efficiency as in HeLa cells or in NIH3T3 cells, and 70


Gene Therapy and Molecular Biology Vol 3, page 71 that it is possible to obtain hepatocyte specific transgene expression using a liver-specific promoter and enhancer. We previously generated a retroviral vector expressing ! 1-antitrypsin cDNA under the control of the enhancerpromoter complex of apolipoprotein E and ! 1-antitrypsin. This retroviral vector, apoE(-)haat-LTR, showed markedly increased expression of ! 1-antitrypsin in rat liver in vivo (Okuyama et al. 1996). The rAAV vector TRNAEAT contains the same liver-specific expression cassette, and the levels of transgene expression in Hepa1A cells infected with rAAV-TRNAEAT were similar to those in the same cells infected with the retroviral vector apoE(-)haat-LTR. These in vitro results suggest that a high level of expression could be expected in vivo using rAAVTRNAEAT, if similar gene transduction efficiency with retoroviral vectors is obtainable using the rAAV gene transfer system. In order to evaluate the level of expression in rat liver in vivo, however, it is necessary to prepare high titer viral stocks. It is difficult to generate high titer AAV viral particles in large scale with the conventional cotransfection method (Peel et al. 1997). To circumvent this problem, we established a HeLa cell line encoding the proviral genome sequence of the rAAV-TRNAEAT. Using this cell line, it was possible to obtain more than 10 fold higher titer viral stocks easily, and this result was highly reproducible (F i g . 2 ). Flotte et al. (1995) tried a similar approach using 293 cells, and were successful in generating rAAV with a 5 fold higher titer compared with the conventional co-transfection method. One of the potential advantages of rAAV for hepatic gene therapy is that it is possible to transduce genes into non-dividing cells (Podsakoff et al. 1994). Recently Snyder et. al. (1997) reported persistent transgene expression in mouse after a simple intraportal infusion of the rAAV expressing human Factor IX under control of MuLV LTR promoter/enhancer. This result suggests that the rAAV gene transfer system is promising for in vivo liver-directed gene therapy. However, one major disadvantage of in vivo hepatic gene transfer is that it is difficult to restrict gene transduction to hepatocytes, because there are many nonparenchymal cells, such as Kuppfer cells and sinusoidal endothelial cells, in the liver in addition to hepatocytes. Here we demonstrated that cell-type specific transgene expression was achievable by rAAV carrying liver-specific promoter enhancer sequences. The vector system described here has the potential advantage of eliminating the risk of miss-targeting, a problem encountered when rAAV vectors are used as an in vivo gene delivering vehicle.

Figure 5 . Quantification of human ! 1 -antitrypsin secreted from rAAV-infected Hepa 1A and HeLa cells. The amount of human ! 1 -antitrypsin protein secreted into the media was determined by ELISA. (A) The results of ELISA assay in wild type Hepa1A cells (Lane 0), in rAAV-infected Hepa1A clones No.1-No.8 (Lane1-Lane8), and in wild type HepG2 cells (Lane G). (B) The results of ELISA assay in wild type HeLa cells, rAAV-infected HeLa clones No.1-No.5 (Lane 1-Lane 5), and wild type HepG2 (Lane G). Data is presented as the average of three independent assays.

IV. Materals and Methods A. Plasmid construction The plasmid pIM45, encoding rep and cap genes of AAV, and pTR(+) for constructing vector plasmid of rAAV were generous gifts from Dr. Nick Muzyckzucka of the University of Florida. The structure of retroviral vector plasmid pAp(-) hAAT-LTR was described elsewhere (Okuyama et al. 1996). The plasmid pAp(-)hAAT-LTR was linealized at the unique BglII site, and then partially digested with BamHI. The 2.2 kb BglII-BamHI DNA fragment containing 4 copies of the 154 bp

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Okuyama et al: Hepatocyte-specific gene expression using the apo E enhancer apolipoprotein E enhancer region, 400 bp of human ! 1 antitrypsin promoter sequence, and 1.2kb cDNA for human ! 1 antitrypsin was gel-isolated and ligated with BglII-digested pTR(+) generating the plasmid pTRAET.

G418 used for the selection: 400 µg/ml in NIH3T3 cells, 600 µg/ml in Hepa1A cells, and 800 µg/ml in HepG2 cells, respectively.

pTR(+) is a plasmid for constructing rAAV, using E.coli strain JC8111 (Deiss et al. 1990) as host cells for transformation. The plasmid pTRAET was again digested and linealized with BglII, blunt-ended with the Klenow fragment of E.coli DNA polymerase, and ligated with the 1.6 kb expression cassette of the neomycin resistant gene under the control of mouse phosphorylglycerol kinase promoter, isolated from another plasmid, pPGKNeo (Soriano et al. 1991). The plasmid pTRNAEAT was generated based on this cloning process.

D. Isolation of Hepa1A and HeLa cell clones infected with rAAV “TRNAEAT” Hepa 1A cells and HeLa cells were infected with rAAV “TRNAEAT” for 4 hours at MOI. 0.1, and two days after the infection, 600 µ g/ml of G418 was added to the media. About two weeks after the infection, several colonies were picked up, and further propagated. Finally we established eight Hepa1A clones, and five HeLa clones. Purified genomic DNA samples from these cells were used as templates of PCR reactions for detecting the 400bp DNA region of human ! 1 -antitrypsin cDNA. Forward and reverse primer sequences were 5’CACTCAGAAGCCTTCACTGTCA-3’, and 5’-ACCCAGCT GGACAGCTTCTT-3’. Thirty cycles of PCRs were performed at 1 minute of 95°C, 1 minute of 57°C, and 2 minutes of 72°C. Since the forward and reverse primers were synthesized based on the sequence of exon1 and 2 of human ! 1 -antitrypsin gene, this PCR reaction was expected to generate a 1.8kb DNA fragment covering the whole of intron 1 and the part of exon 1 and 2 of the human genomic DNA (Long et al. 1984).

B. Production of rAAV vector “TRNAEAT” HeLa cells were maintained with DMEM (GIBCO BRL, Grand Island, NY) supplemented with 10% fetal bovine serum (Sanko Junyaku Co. Ltd., Tokyo Japan). The rAAV TRNAEAT was generated by two different methods. Method I involved conventional co-transfection. HeLa cells were transfected with pTRNAEAT and pIM45 at a molar ratio of 1:2 using calcium phosphate precipitation method described before (Chen and Okayama 1987). 24 hours after the transfection, the cells were infected with wild type adenovirus Ad5 with MOI.2 for 2 hours. Three days after the transfection, the cells were harvested, lysed by freezing and thawing 5 times, and incubated at 60 °C for one hour to inactivate co-existing adenoviruses. In Method II, we established the stable HeLa cell clone first. HeLa cells were transfected with the plasmid pTRNAEAT and selected with 600µg/ml G418 (GIBCO BRL) for 14 days, and established the HeLa cell clone HeLa-NAEAT carrying the proviral genome sequences in its chromosomes. To produce rAAV, HeLa-NAEAT was transfected with pIM45, and the transfected cells were treated in the same way as for Method I.

E. Quantification of human 1-antitrypsin produced from rAAV-infected HeLa and Hepa1A cells 24 hour-cultured media were used for ELISA assay to quantify the amounts of human ! 1 -antitrypsin secreted from the cells. The assay was performed in 96-well microtitration plates. Goat anti-human ! 1 -antitrypsin antibody, and peroxidase-conjugated goat anti-human ! 1 -antitrypsin antibody were purchased from Cappel (Durham, NC). After 2hour incubation at room temperature with goat anti-human ! 1 antitrypsin antibody (2 mg/well), non-specific binding was blocked by overnight incubation with 200 ml of 3% BSA and 0.02% sodium azide in PBS at 4°C. After rinsing with washing buffer, 200ml of the cultured media or control samples (purified human ! 1 -antitrypsin, Sigma, St. Louis MO) were added. The standard curve was made from 0 to 100 ng/ml. The microtitration plates were incubated for 3 hours at room temperature and washed four times with PBS. Then 200 ml of peroxidase-conjugated goat anti-human ! 1 -antitrypsin antibody (15mg/ml) was added to each well. After incubation for 2 hours at room temperature, the wells were rinsed five times and 200ml of substrate solution containing 10 mg ophenylendiamine hydrochloride (Sigma), 10ml 30% H2 O2 , and 25ml citrate-phosphate buffer pH5 was added. The reaction was stopped by the addition of 50ml of 3 M H2 SO4 .

C. Determination of neo R titers of the rAAV “TRNAEAT” HeLa and HepG2 cells were maintained with DMEM supplemented with 10% fetal bovine serum, NIH3T3 cells were maintained with DMEM with 10% calf serum (Sanko Junyaku Co. Ltd.), and Hepa1A cells, cells from a mouse hepatoma cell line, (Darlington 1987) were maintained with 75% MEM (GIBCO BRL), 25% Waymouth (GIBCO BRL), 10% fetal bovine serum. Serial dilutions of the viral stocks were made with DMEM, and certain amounts were added into the media of the plates culturing HeLa cells for 4 hours. Then cells were washed with PBS twice, and fed with fresh media for two more days. Two days after the infection, 600µg/ml of G418 (GIBCO BRL) was added to the media, and the culture was continued until distinct colonies were identified. The titer of each viral stock solution was calculated by counting the numbers of the G418 resistant colonies on the plates. The neoR titers of the rAAV to NIH3T3 cells, Hepa1A cells, and HepG2 cells were determined in the same way except for the concentrations of

Acknowledgements

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Gene Therapy and Molecular Biology Vol 3, page 73 Kay MA, Rothenberg S, Landen CN, Bellinger DA, Leland F, Toman C, Finegold M, Thompson AR, Read MS, Brinkhous KM, Woo SLC (1 9 9 3 ) In vivo gene therapy of hemophilia B, sustained partial correction in factor IXdeficient dogs. S c i e n c e 262, 117-119

We thank Dr. Nick Muzyczka for recombinant adenoassociated virus construction, and Ms. K. Saito for editorial assistance. This work was supported in part by grants for pediatric research and gene therapy research from the Ministry of Health and Welfare of Japan.

Laughlin CA, Cardellichio CB, Coon HC (1 9 8 6 ). Latent infection of KB cells with adeno-associated virus type 2. J V i r o l 60, 515-524

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Adra CN, Boer PH, McBurney MW (1 9 8 7 ) Cloning and expression of the mouse pgk-1 gene and the nucleotide sequence of its promoter. Gene 60, 65-74 Chen C, Okayama H (1987) High-efficiency transformation of mammalian cells by plasmid DNA Mol Cell Biol 7, 27452752 Darlington GJ (1 9 8 7 ) Liver cell E n z y m o l o g y 151, 19-39

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Ledley FD (1 9 9 3 ) Hepatic gene therapy, present and future. H e p a t o l o g y 18, 1263-1273 Li Y, Shen RF, Tsai SY, Woo SL (1 9 8 8 ) Multiple hepatic trans-acting factors are required for in vitro transcription of the human alpha-1-antitrypsin gene. M o l C e l l B i o l 8, 4362-4369

in

Deiss V, Tratschin JD, Weitz M, Siegl G (1 9 9 0 ) Cloning of the human parvovirus B19 genome and structural analysis of its palindromic termini. V i r o l o g y 175, 247-254

Long GL, Chandra T, Woo SL, Davie EW, Kurachi K (1 9 8 4 ). Complete sequence of the cDNA for human alpha 1antitrypsin and the gene for the S variant. B i o c h e m i s t r y 23, 4828-4837

Fisher KJ, Gao GP, Weitzman MD, DeMatteo R, Burda JF, Wilson JM (1 9 9 6 ) Transduction with recombinant adenoassociated virus for gene therapy is limited by leadingstrand synthesis. J Virol 70, 520-532

Okuyama T, Huber RM, Bowling W, Pearline R, Kennedy SC, Flye MW, Ponder KP (1 9 9 6 ) Liver-directed gene therapy, a retroviral vector with a complete LTR and the ApoE enhancer-alpha 1-antitrypsin promoter dramatically increases expression of human alpha 1-antitrypsin in vivo. Hum Gene Ther 7, 637-645

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 Flotte TR, Afione SA, Conrad C, McGrath SA, Solow R, Oka H, Zeitlin PL, Guggino WB, Carter BJ (1 9 9 3 ) Stable in vivo expression of the cystic fibrosis transmembrane conductance regulator with an adeno-associated virus vector. Proc Natl Acad Sci U S A 90, 10613-10617

Okuyama T, Li X-K, Funeshima N, Fujino M, Sasaki K, Kita Y, Kosuga M, Takahashi M, Saito H, Suzuki S, Yamada M. (1 9 9 8 ) Fas-mediated apoptosis is involved in the elimination of gene-transduced hepatocytes with E1/E3deleted adenoviral vectors. J G a s t r o e n t e r o l H e p a t o l 13, 5113-5118.

Flotte TR, Barraza-Ortiz X, Solow R, Afione SA, Carter BJ, Guggino WB (1 9 9 5 ). An improved system for packaging recombinant adeno-associated virus vectors capable of in vivo transduction. Gene Ther 2, 29-37

Peel AL, Zolotukhin S, Schrimsher GW, Muzyczka N, Reier PJ (1 9 9 7 ). Efficient transduction of green fluorescent protein in spinal cord neurons using adeno-associated virus vectors containing cell type-specific promoters. Gene Ther 4, 16-24

Hafenrichter DG, Wu X, Rettinger SD, Kennedy SC, Flye MW, Ponder KP (1 9 9 4 ) Quantitative evaluation of liverspecific promoters from retroviral vectors after in vivo transduction of hepatocytes. B l o o d 84, 3394-3404

Podsakoff G, Wong KK Jr, Chatterjee S (1 9 9 4 ). Efficient gene transfer into nondividing cells by adeno-associated virus-based vectors. J Virol 68, 5656-5666

Jaffe HA, Danel C, Longenecker G, Metzger M, Setoguchi Y, Rosenfeld MA, Gant TW, Thorgeirsson SS, StratfordPerricaudet LD, Perricaudet M, Pavirani A, Lecocq JP, Crystal RG (1 9 9 2 ) Adenovirus-mediated in vivo gene transfer and expression in normal rat liver. Nat Genet 1, 372-378

Rettinger SD, Kennedy SC, Wu X, Saylors RL, Hafenrichter DG, Flye MW, Ponder KP (1 9 9 4 ) Liver-directed gene therapy, quantitative evaluation of promoter elements by using in vivo retroviral transduction. P r o c N a t l A c a d S c i USA 91, 1460-1464

Kaplitt MG, Leone P, Samulski RJ, Xiao X, Pfaff DW, O'Malley KL, During MJ (1 9 9 4 ) Long-term gene expression and phenotypic correction using adenoassociated virus vectors in the mammalian brain. Nat Genet 8, 148-154

Shachter NS, Zhu Y, Walsh A, Breslow JL, Smith JD (1 9 9 3 ) Localization of a liver-specific enhancer in the apolipoprotein E/C-I/C-II gene locus. J L i p i d R e s 34, 1699-1707 Simonet WS, Bucay N, Lauer SJ, Taylor JM (1 9 9 3 ). A fardownstream hepatocyte-specific control region directs expression of the linked human apolipoprotein E and C-I genes in transgenic mice. J B i o l C h e m 268, 8221-8229

Kay MA, Li Q, Liu TJ, Leland F, Toman C, Finegold M, Woo SL (1 9 9 2 ) Hepatic gene therapy, persistent expression of human alpha 1-antitrypsin in mice after direct gene delivery in vivo. Hum Gene Ther 3, 641-647

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Okuyama et al: Hepatocyte-specific gene expression using the apo E enhancer Snyder RO, Miao CH, Patijn GA, Spratt SK, Danos O, Nagy D, Gown AM, Winther B, Meuse L, Cohen LK, Thompson AR, Kay MA (1 9 9 7 ). Persistent and therapeutic concentrations of human factor IX in mice after hepatic gene transfer of recombinant AAV vectors. N a t G e n e t 16, 270-276 Soriano P, Montgomery C, Geske R, Bradley A (1 9 9 1 ). Targeted disruption of the c-src proto-oncogene leads to osteopetrosis in mice. C e l l 64, 693-702 Zolotukhin S, Potter M, Hauswirth WW, Guy J, Muzyczka N (1 9 9 6 ) A "humanized" green fluorescent protein cDNA adapted for high-level expression in mammalian cells. J V i r o l 70, 4646-4654

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Gene Therapy and Molecular Biology Vol 3, page 75 Gene Ther Mol Biol Vol 3, 75-78. August 1999.

Human cytomegalovirus (HCMV) nuclease: implications for new strategies in gene therapy Minireview

Elke Bogner Institute of Virology, Robert-Koch-Str. 17, 35037 Marburg, Germany __________________________________________________________________________________________________ Correspondence: Phone: +49-6421-285362; Fax: +49-6421-285482; E- mail: bogner@mailer.uni-marburg.de Received 9 October 1998 and in revised version 14 October; accepted 17 October 1998

I. Introduction Human cytomegalovirus (HCMV), one of eight human herpesviruses, can cause serious illness in neonates as well as in immunocompromised adults (Alford and Britt, 1993). Transplant and AIDS patients, e.g. may develop lifethreatening diseases as a consequence of primary infection or reactivation of latent infection. Additionally, HCMV infections are also associated with congenital neurological complications in approximately 7,500 newborns annually (Alford and Britt, 1993). The current drugs are toxic and cause additional complications including drug resistance. Since present therapeutical approaches are limited new strategies are needed that may result from a better understanding of viral molecular biology. The initial step of production of new virions is the packaging of newly synthesized, concatenated viral DNA into procapsids. As a consequence, blocking of this step will prevent production of viral progeny. Recently a new highly conserved gene product of ORF UL56, p130 (pUL56), was identified and partially characterized (Bogner et al., 1993). The homologous proteins of herpes simplex virus 1 (HSV-1), ICP 18.5 (UL28), and of pseudorabies virus (PrV) were reported to play an important role in DNA packaging. Viral mutants failed to cleave concatenated viral DNA which leads to an accumulation of naked nucleocapsid and uncleaved concatenated DNA in the nucleus (Addison et al., 1990; Mettenleiter et al., 1993). These reports suggested that UL56 may also play a role in virus assembly. The mechanism of DNA translocation into the procapsid and that of end formation by concatemer cutting at packaging sites (pac) are not well understood. Recently it was shown that HCMV p130 has the ability to interact with specific HCMV DNA packaging motifs and to cleave DNA bearing this motifs. Possible implications of this finding, its relation to the function of another HCMV 75

protein, pUL89, the so-called terminase, and to the bacteriophage system are discussed in this review.

II. HCMV p130 (pUL56) is a sequence specific nuclease Viral DNA-replication results in the formation of large head-to-tail DNA concatemers (Ben-Porat and Rixon, 1979), and maturation into unit-length molecules involves sitespecific cleavage at sequences (pac motifs) located within the a sequence (Spaete and Mocarski, 1985). Unit-length DNA is encapsidated in the nucleus and the DNAcontaining C-capsids bud into the cytoplasm through the nuclear membranes. The final event is the envelopment in the TGN and the release into the extracellular space. The process where newly synthesized viral DNA is cleaved and packaged into preformed capsids has long been of interest, because this is the initial step in viral assembly. The HCMV genomic a sequence is a short sequence located at both termini of the genome and in inverted orientation at the L-S junction (Mocarski et al., 1987; Tamashiro and Spector, 1986). The a sequence plays a key role in replication as a cis-acting signal for cleavage and packaging (Chou and Roizman, 1985). There is evidence that cleavage and packaging of DNA are linked processes (Ladin et al., 1980). The HCMV a sequence contains two conserved motifs, pac 1 and pac 2. These packaging motifs have an AT-rich core flanked by a GC-rich sequence. In the case of HCMV, pac sites are located on one side of the cleavage site, whereas in other herpesvirus genomes the cleavage site is between these motifs (Marks and Spector, 1988). We sugggest that during the initial step of viral packaging, the capsid-associated protein p130 may bind to the pac sequences and promote cleavage of the concatemer. Electrophoretic mobility shift assays with DNA probes


Bogner: HCMV nuclease in gene therapy spanning the region of the cis-acting pac elements demonstrated that recombinant baculovirus-infected HCMV p130 formed specific DNA-protein complexes. These data suggested that p130, as a putative cleavage and packaging protein, attaches to the pac sequence. Furthermore it is proposed that viral DNA is taken up into the capsid being scanned along the complex from the bound a sequence across the L- and S-component until an a sequence in an identical orientation is found. The final step requires nicking of both strands at signals on opposite sites of the sequence. Interestingly, by using circular plasmid DNA bearing a single a sequence as a substrate, purified baculovirus expressed HCMV p130 has an enzymatic activity that converts supercoiled plasmid DNA into open circular as well as linear molecules (Bogner et al., 1998; F i g . 1 ). This observation is comparable with the notion that HCMV p130 may be involved in cleavage of viral DNA. By using Apyrase, evidence was provided that in contrast to the adeno-associated virus origin-binding protein Rep68 (Im and Muzyczka, 1990) the reaction is independent of ATP. Interestingly, baculovirus-UL28 cell extracts, containing the HSV-1 homolog ICP18.5 (pUL28), also cleaved supercoiled plasmid DNA molecules bearing the a sequence (F i g . 1 ). It was then suggested that p130 and its herpesviral homologs are involved in cleaving of concatenated viral DNA and packaging into procapsids. It is currently under investigation, whether p130 operates with another viral protein, UL89.

III. Comparison with the bacteriophage system Herpesviruses share common features with respect to DNA maturation with dsDNA bacteriophages. DNA

F i g . 1 : Nuclease activity of the p130 homolog of HSV ICP18.5 (pUL28). Nuclease reactions were incubated for 1 h at 37°C and samples were treated with proteinase K for an additional hour at 37°C. Lane 1, plasmid pON205 in the absence of protein; 2, pON205 treated with the restriction enzyme HindIII; 3, pON205 incubated with rp130; 4, pON205 incubated with HSV UL28; 5, pON205 plus wild-type infected extracts; 6, pON205 incubated with mock-infected extracts. The arrows indicate three different plasmid DNA forms: open circular molecules (a); linear forms (b); and supercoiled molecules (c).

76

replication results in high molecular weight concatenated DNA and procapsids are assembled around a protein scaffold (Black, 1989; Murialdo and Becker, 1978). Translocation of the DNA to the procapsid bacteriophages is an ATPdependent process carried out by terminases (Feiss and Becker, 1983). Terminases are ATP-binding proteins which also bind and cleave concatenated DNA at cohesive (cos; e.g. phage ) or packaging (pac; e.g. phage P22) sites (Gold et al., 1983). In the case of bacteriophage T4, procapsids are apparently filled without sequence specificic cleavage by a ”headful mechanism” (Kalinski and Black, 1986; Streisinger et al., 1967). The HCMV UL89 gene product has some homology to the phage T4 gp17 terminase subunit. T4 terminase has two subunits. The large subunit, gp17, contains the ATP binding sites, the small subunit, gp16, is required for packaging of concatenated DNA (Rao and Black, 1988). There is preliminary evidence that baculovirus-UL89 cell extracts exhibit endonuclease activity (F i g . 2 ). HCMV UL89 is also able to nonspecifically cleave DNA in a manner reminiscent of phage T4 terminase (Black, 1986). Terminase can use either concatemeric or monomeric DNA of any sequence and prefers nonspecific ends in monomeric DNA over pac-containing concatemers (Serwer; 1986). Recently, Krosky et al. (1998) and Underwood et al. (1998) reported on a new drug, which is a derivative of benzimidazole ribonucleosides. HCMV mutants were selected by treatment of infected cultures with increasing amount of the drug. The mutations were mapped to the UL56 gene (Krosky et al., 1998) and to the gene product of pUL89 (Underwood et al., 1998). Based on the identical phenotypes of the drug resistant mutants, it is speculated that these viral proteins form the putative HCMV terminase.


Gene Therapy and Molecular Biology Vol 3, page 77

F i g . 2 : Nuclease activity of the T4 terminase homolog HCMV pUL89. Lanes: 1, pON205 alone; 2, pON205 treated with HindIII; 3, pON205 incubated with extract containing p130; 4, pON205 incubated with extracts containing HCMV UL89; 5, pON205 incubated with wild-type infected extracts; 6, pON205 incubated with mockinfected extracts. Open circular DNA molecules (a), linear (b) and supercoiled molecules (c) are indicated.

IV. Questions for the future Considering that the current drugs (ganciclovir, cidofovir and foscarnet) have limited effects and dosedependent toxicity, new antiviral therapeutics are needed. The mechanism of the current drugs is the inhibition of viral replication through an interaction with viral DNA polymerase (Erriksson et al., 1982; Ho et al., 1992; Mar et al., 1985). The inhibition of the cleavage and packaging of the viral DNA by a nuclease inhibitor may offer a potentially alternative therapy. Taken together, the reports by Addison et al. (1990), Tengelsen et al (1993) and Mettenleiter et al. (1993), demonstrating that the HSV-1 ICP18.5 (pUL28) gene product and the PRV homolog are necessary for cleavage and packaging of concatenated viral DNA, and the observation that HCMV p130 (pUL56) can interact with specific DNA packaging motifs and is able to cleave DNA bearing these motifs, provide a basis for understanding the herpesvirus DNA packaging process at the molecular level. Identification of the structure of the proteins involved is needed as a prerequisite for the development of new antivirals. Knowledge of the three dimensional protein structure is pertinent in revealing the catalytic domain for the enzymatic activity prior to anti-viral drug-design. Regarding that mammalian cell DNA replication does not involve cleavage of concatemeric DNA, drugs targeted to the viral nucleases should be safe and selective. Therefore, our findings may help to develop new nontoxic anti-HCMV 77

reagents for treatment of the immunocompromised patient population.

Acknowledgments I thank Fred Homa for kindly providing recombinant baculoviurs expressing HSV-1 UL28, Edward Mocarski for the a sequence containing plasmid pON205 and Mark Underwood for the baculovirus plasmid containing HCMV UL89. I am grateful to Klaus Radsak for critically reading the manuscript and to lab members for helpful comments. This work was supported by Deutsche Forschungsgesellschaft, Sonderforschungsbereich 286, Teilprojekt A3.

References Addison, C., F.J. Rixon and V.G. Preston ( 1 9 9 0 ) Herpes simplex virus type 1 UL28 gene product is important for the formation of mature capsids. J G e n V i r o l 71, 23772384. Alford, C.A. and W.J. Britt ( 1 9 9 3 ) Cytomegalovirus, p 227255. IN B.Roizman, RlJ. Whiteley and C. Lopez et al. (ed.), T h e h u m a n h e r p e s v i r u s e s . Raven Press, Ltd., New York. Ben-Porat, T. and F.J.Rixon ( 1 9 7 9 ) Replication of herpesvirus DNA. IV: Analysis of concatemers. V i r o l o g y 94, 61-70. Black, L.W. ( 1 9 8 6 ) In vitro packaging of bacteriophage T4


Bogner: HCMV nuclease in gene therapy DNA. V i r o l o g y 113, 336-344. Black, L.W. ( 1 9 8 8 ) DNA packaging in dsDNA bacteriophages. In: The B a c t e r i o p h a g e s , ed. R. Calendar, 2, 321-273. New York: Plenum. Black, L.W. ( 1 9 8 9 ) DNA packaging in dsDNA bacteriophages. A n n u R e v M i c r o b i o l 43, 267-292. Bogner, E., M. Reschke, B. Reis, T. Mockenhaupt and K. Radsak ( 1 9 9 3 ) Identification of the gene product encoded by ORF UL56 of human cytomegalovirus genome. V i r o l o g y 196, 290-293. Bogner, E., K. Radsak and M.F. Stinski ( 1 9 9 8 ) The gene product of human cytomegalovirus open reading frame UL56 binds the pac motif and has specific nuclease activity. J Virol 72 , 2259-2264. Chou, J. and B. Roizman ( 1 9 8 5 ) The isomerization of the herpes simplex virus 1 genome: identification of the cisacting and recombination sites within the domain of the a sequence. C e l l 41, 803-811. Erriksson, D., B. Oberg and B. Wahren ( 1 9 8 2 ) Pyrohphosphate analogs as inhibitors of DNA polymerases of cytomegalovirus, herpes simplex virus and cellular origin. B i o c h i m B i o p h y s A c t a 669, 115-123. Feiss, M. and A. Becker ( 1 9 8 3 ) DNA packaging and cutting, p 305-330. IN R.W. Hendrix, J.W. Roberts, F.W. Stahl and R.A. Weisberg (ed.), Lambda II. Cold Spring Harbor, New York. Gold, M. and A. Becker ( 1 9 8 3 ) The baceriophage ! terminase: partial purification and preliminary characterization of properties. J B i o l C h e m 258, 14619-14625. Ho, H.-T., K.L. Woods, J.J. Bronson, H. DeBoeck, J.C. Martin and M.J.M. Hitchcock ( 1 9 9 2 ) Intracellular metabolism of the antiherpes agent (S)-1-[3-hydroxy-2(phosphonylmethoxy)propyl] cytosine. Mol Pharmacol 41, 197-202. Kalinski, A. and L.W. Black ( 1 9 8 6 ) End structure and mechanism of packaging of bacteriophage T4 DNA. J V i r o l 58, 951-954. Krosky, P.M., M.R. Underwood, S.R. Turk, K. W.-H. Feng, R.K. Jain, R.G. Ptak, A.C. Westerman, K.K. Biron, L.B. Townsend and J.C. Drach (1 9 9 8 ) Resistance of human cytomegalovirus to benzimidazole ribonucleosides maps to two open reading frames: UL89 and UL56. J V i r o l 72, 4721-4728. Ladin, B.F., M.L. Blankenship and T. Ben-Porat ( 1 9 8 0 ) Replication of herpesviurs DNA. V. The maturation of concatemeric DNA of pseudorabies virus to genome length is related to capsid formation. J Virol 33, 1151-1164. Mar, E., J. Chiou, Y. Cheng and E. Huang ( 1 9 8 5 ) Inhibition

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of cellular DNA polymerase alpha and human cytomegalovirus-induced DNA polymerase by triphosphates of 9-(2-hydroxymethyl)guanine and 9-(1,3dihydroxy-2-propoxymethyl)guanine. J V i r o l 53, 776780. Marks, J.R. and D.H. Spector ( 1 9 8 8 ) Replication of the murine cytomegalovirus genome: structure and role of the termini the generation and cleavage of concatemers. V i r o l o g y 162, 98-107. Mettenleiter, T.C., A. Saalm端ller and F.Weiland ( 1 9 9 3 ) Pseudorabies virus protein homologous to herpes simplex virus type1 ICP 18.5 is necessary for capsid maturation. J V i r o l 67, 1236-1245. Mocarski, E.S., A.C. Liu and R.R. Spaete ( 1 9 8 7 ) Structure and variability of the a sequence in the genome of human cytomegalovirus (Towne strain). J G e n V i r o l 68, 22232230. Murialdo, H. and A. Becker ( 1 9 7 8 ) Head morphogenesis of complex double-stranded deoxyribonucleic acid bacteriophages. M i c r o b i o l R e v 42, 529-576. Rao, V.B. and L.W. Black ( 1 9 8 8 ) Cloning, overexpression and purification of the terminase proteins gp16 and gp17 of bacteriophage T4: construction of a defined in vitro DNA packaging system using purified terminase proteins. J M o l B i o l 200, 475-488. Serwer, P. ( 1 9 8 6 ) Arrangement of double-stranded DNA packaged in bacteriophage capsids. J M o l B i o l 190, 509-512. Spaete, R.R. and E.S. Mocarski ( 1 9 8 5 ) The a sequence of the cytomegalovirus genome functions as a cleavage/packagung signal for herpes simplex virus defective genomes. J Virol 54, 817-824. Streisinger, G., J. Emrich and M.M. Stahl ( 1 9 6 7 ) Chromosome structure in phage T4. III. Terminal redundancy and lenght determination. Proc Natl Acad Sci USA 57, 292-295. Tamashiro, J.C. and D.H. Spector ( 1 9 8 6 ) Terminal structure and heterogeneity in human cytomegalovirus strain AD 169. J V i r o l 59, 591-604. Tengelsen LA, Pederson NE, Shaver PR, Wathen MW, Homa FL (1 9 9 3 ) Herpes simplex virus type 1 DNA cleavage and encapsidation require the product of the UL28 gene: isolation and characterization of two UL28 deletion mutants. J Virol 67, 3470-3480. Underwood, M.R., R.J. Harvey, S.C. Stanat, M.L. Hemphill, T. Miller, J.C. Drach, L.B. Townsend and K.K. Biron ( 1 9 9 8 ) Inhibition of human cytomegalovirus DNA maturation by a benzimidazole ribonucleoside is mediated through the UL89 gene product. J Virol 72, 717-725.


Gene Therapy and Molecular Biology Vol 3, page 79 Gene Ther Mol Biol Vol 3, 79-89. August 1999.

Application of recombinant Herpes Simplex Virus-1 (HSV-1) for the treatment of malignancies outside the central nervous system Review Article

George Coukos1, Stephen C. Rubin1, and Katherine L Molnar-Kimber2 Division of Gynecologic Oncology, Department of Obstetrics and Gynecology1; and Thoracic Oncology Laboratory, Department of Surgery2, University of Pennsylvania Medical Center, Philadelphia, PA 19104. __________________________________________________________________________________________________ Correspondence: Katherine L. Molnar-Kimber, Ph.D., Dept. of Surgery, 351 Stemmler Hall, 36th and Hamilton Walk, University of Pennsylvania, Philadelphia, PA 19104-6070, USA. Tel. 215-662-7898; Fax 215-573-2001; E-mail: molnark@mail.med.upenn.edu Key words: gene therapy, HSV-1, cancer Received: 19 November 1998; accepted: 20 November 1998

Summary Attenuated HSV-1 mutants are promising novel vectors for human gene therapy of cancer. In addition t o t h e i r e f f i c a c y i n t r e a t m e n t o f e x p e r i m e n t a l C N S t u m o r s , H S V m u t a n t s h a v e shown promise in treatment of extra-CNS tumors including mesothelioma, melanoma, breast cancer, epithelial ovarian carcinoma, colon carcinoma and non small c e l l lung carcinoma i n various animal models. HSV mutants which have been partially attenuated can function as direct oncolytic agents capable of proliferating within three-dimensional tumors and causing tumor cell death. A major advantage of these replication-restricted HSV mutants is that they can selectively replicate in tumor cells and thus, potentially express transgenes i n a higher percentage o f the tumor c e l l s . Alternatively, superattenuated HSV mutants and amplicons can function as efficient vectors for gene therapy and have the ability to host large and multiple transgenes. A multi-pronged strategy for HSV-based anti-tumor therapy is currently emerging, where multi-attenuated viruses or the oncolytic HSV mutants are used as gene therapy vectors for intratumoral delivery of immunomodulatory or chemotherapy sensitizing transgenes. HSV-based tumor therapy has been reported to induce an anti-tumor immune response in some animal models. These findings may be due t o the combination o f co-expression of immunomodulatory molecules, immunogenic properties of the virus, necrosis of the tumor tissue and subsequent tumor antigen presentation. Thus, HSV oncolytic agents and gene therapy vectors show great potential as anti-tumor therapies. Further studies are required to test the efficacy and safety of these agents in extra-CNS malignancies.

gene therapies are designed to deliver specific suicide genes, such as herpes simplex virus thymidine kinase or cytosine deaminase, into tumor cells (Singhal and Kaiser, 1998; Vile, 1998) which are rendered sensitive to the administration of prodrugs. The suicide gene converts the prodrug into toxic metabolites which can induce lysis in rapidly dividing cells. A third strategy involves the expression of immunomodulatory genes which may stimulate an antitumor response by the host immune system. These genes

I. Introduction Therapeutic strategies for the gene therapy of malignancies have been designed along three main pathways: corrective gene therapies entail the delivery of wild-type tumor suppressor genes to tumors which have been shown to display alterations in those genes. This approach can lead to restoration of normal tumor suppressor function and to tumor regression (Favrot et al., 1998). Secondly, suicide

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Coukos et al: HSV-based oncolytic therapy include various cytokine genes (e.g. granulocyte/macrophagecolony stimulating factor (GM-CSF), interleukin (IL)-12, co-stimulatory molecules (e.g. B7.1) and allogeneic transplantation antigens (e.g. HLA-B7) (Pardoll, 1992). Combinations of the aforementioned strategies are also being investigated (Roth and Cristiano 1997).

suicide genes. In addition, these vectors can deliver cytokine genes, or costimulatory molecules to enhance tumor recognition and killing by the immune system. In the second line of investigation, oncolytic HSV mutants have been engineered by deletion of one or more genes to replicate poorly or not at all in normal host epidermal and neuronal tissues but to be able to replicate 30100 fold more efficiently in tumor cells. For example, viruses were initially attenuated by deletion of thymidine kinase or ribonucleotide reductase and were used as oncolytic agents of CNS malignancies. Since deletion of the thymidine kinase gene made the vector insensitive to the current antiherpetic drugs, acyclovir and ganciclovir, which is an important safety mechanism in case of inappropriate HSV spread, other strategies are being pursued. Ribonucleotide reductase deletion mutants have been efficacious in the treatment of malignant gliomas in immunocompromised and immunocompetent mice (Boviatsis et al., 1994). A third generation of viruses lacking both copies of ICP34.5 demonstrated efficacy in the treatment of several CNS tumors (Andreansky et al., 1997; Chambers et al., 1995; Kesari et al., 1995; Kramm et al., 1997; Yazaki et al., 1995). The HSV ICP34.5 mutants selectively replicated in tumor cells (McKie et al., 1996; Randazzo et al., 1997) and exhibited 105-106 fold attenuation in neurovirulence (Chou et al., 1990; MacLean et al., 1991; Valyi-Nagy et al., 1994). Several strategies have been pursued to further augment the efficacy of these mutants. The efficacy for treatment of experimental human glioma by R3616, an ICP 34.5 mutant, was augmented by radiation therapy in an immunodeficient model (Advani et al., 1998) and by co-expression of IL-4 (Andreansky et al., 1998). Concomitant deletions of the ICP34.5 genes and ribonucleotide reductase (Mineta et al., 1994; Mineta et al., 1995) or the uracil DNA glycosylase gene (Pyles et al., 1997) led to further attenuation but preserved oncolytic efficacy in the treatment of various CNS tumors.

Gene delivery remains one of the most important limitations in cancer gene therapy. The first generations of replication-incompetent adenoviral vectors, widely used in clinical trials for cancer gene therapy, may have limited therapeutic efficacy in bulky tumors, most likely due to localized gene delivery in three-dimensional tumors (Sterman et al., 1998). Replication-selective viral vectors may offer a suitable alternative. Among replication-selective vectors, recombinant Herpes Simplex Virus Type-1 (HSV-1) mutants represent potentially powerful tools for the treatment of cancer. HSV has a large genome of 152 Kb (Fink and Glorioso, 1998). It may be able to accommodate more than 30 Kb of transgene inserts, making it a suitable vector for large and/or multiple transgenes (Fink and Glorioso, 1998). Although HSV-1 is a common pathogen in humans, it very rarely induces serious complications. Attenuation of HSV will most likely augment its safety profile. Recombinant viruses have been engineered to lack specific genes necessary for neurovirulence or viral replication in quiescent cells, resulting in replication-restricted viral mutants that selectively or preferentially replicate in and lyse tumor cells. Thus, depending on the degree of attenuation, HSV-1 mutants can be used not only as vectors for gene therapy but also as direct oncolytic agents. HSV-1 mutants have been shown to be efficacious in the treatment of experimental malignancies localized within the central nervous system (CNS) (Andreansky et al., 1997; Chamberset al., 1995; Jia et al., 1994; Kesari et al., 1995; Kramm et al., 1997; Mineta et al., 1995; Pyles et al., 1997; Yazaki et al., 1995). Two main lines of investigation have been followed. In the first discussed strategy, multiattenuated viral vectors were engineered by deletion of multiple genes to be able to undergo at most one or two rounds of replication within cancer cells (Glorioso et al., 1997). Alternatively, HSV amplicons, which have additional deletions of essential HSV genes and require helper virus or complementation of many HSV functions to replicate in any cells, can be used to express various transgenes (Fraefel et al., 1996; Geller, 1993; Geller and Breakefield, 1991; Ho, 1994). Multi-attenuated viral vectors and amplicons were originally engineered for gene therapy of CNS hereditary conditions, such as neurodegenerative and neuromuscular diseases, based on their ability to express the transgene(s) but not HSV proteins in quiescent cells (Fink and Glorioso, 1998; Geller, 1993; Geller and Breakefield, 1991; Glorioso et al., 1997; Ho, 1994; Huard et al., 1997). These vectors may also be suitable for cancer gene therapy for transduction of

Recent evidence suggests that attenuated Herpes Simplex Virus-1 mutants can be also utilized for peripheral malignancies. The present review will offer a brief summary of HSV-1 mechanism of action, will provide the overall rationale for the utilization of mutant HSV-1 for treatment of malignancies in extra-CNS locations and summarize the evidence accumulated to date.

II. HSV-1 replication The replication cycle and epidemiology of HSV have recently been reviewed (Roizman and Sears, 1996; Whitley, 1996). HSV-1 is a DNA virus with a large genome of 152 Kb. To date, 80 HSV genes have been identified, but approximately 30 are non-essential for its replication in vitro in permissive Vero cells (Fink and Glorioso, 1998; 80


Gene Therapy and Molecular Biology Vol 3, page 81 McGeoch et al., 1988). In the immunocompetent human host, wild-type (wt) HSV-1 infects predominantly tissues of epidermal and neuronal origin (Whitley, 1996). Wt HSV infection of epidermal tissues results in a lytic infection and usually is accompanied by the induction of latency in peripheral neurons and the ganglia. Encephalitis, a lytic infection of the central nervous system, occurs only rarely. Briefly, the replication cycle begins with viral attachment to the cells, which is mediated by recognition of specific envelope glycoproteins, such as glycoprotein (g)B and gC to heparan sulfate (Laquerre et al., 1998; Spear et al., 1992). A cellular protein, EXT, can enhance the expression of heparan sulfate and has been shown to confer susceptibility of some cells to HSV infection (McCormick et al., 1998). In addition, gD can specifically bind to cells via the Herpes virus entry mediator (HVEM) protein (Montgomery et al., 1996) and by two additional, recently identified receptors (Geraghty et al., 1998; Whitbeck et al., 1997). Binding is followed by fusion of the viral envelope with the cell membrane of the infected host, partially mediated by viral gB, gD, and gH. The capsid is transported to the nucleus, where the viral DNA is released. During this process, VP16, a protein associated with the tegument, interacts with cellular transcription factors to activate transcription and expression of immediate early (!) genes ICP0, ICP4, ICP22, ICP27 and ICP47 (DeLuca and Schaffer, 1985; Fink and Glorioso, 1998; Honess and Roizman, 1974; Roizman and Sears, 1996). The viral early genes ("1 and "2 genes), which are mainly involved in nucleotide synthesis and viral DNA replication in quiescent cells, are then transcribed and translated. The late genes (# 1 and # 2) are subsequently expressed, resulting in the synthesis of the protein components of the capsid, tegument and viral envelope (Roizman and Sears, 1996; Subak-Sharpe and Dargan, 1998). There are some genes which are transcribed late as well as early and have been termed # 1 genes (Roizman and Sears, 1996 and ref. therein). Finally, the viral DNA is cleaved and packaged into capsids, and the DNA containing capsids appear to be enveloped at the nuclear membrane. The enveloped capsids transit through the cytoplasm in a multistep process still under investigation and get released from the cell. Along this process the infected cell dies (Roizman and Sears, 1996 and ref. therein).

al., 1997a). Although Ito et al. observed no change in frequency of apoptosis in non-activated cultures of T lymphocytes infected with HSV-1 vs. non-infected cells (Ito et al., 1997b), wt HSV-1 has been reported to induce apoptosis in non-activated human peripheral blood mononuclear cells (Tropea et al., 1995) as well as in other tissues (Irie et al., 1998). The HSV genes which induce apoptosis in the infected cell are being investigated. Since HSV-1 can induce apoptosis at several checkpoints (Galvan and Roizman, 1998), it is likely that HSV-1 encodes several genes which can induce apoptosis. HSV encodes early genes that destabilize cellular RNA, disrupt cellular transcription and degrade cellular DNA (Johnson et al., 1992; Kwong et al., 1988; Roizman and Sears, 1996) and are likely candidates. Additional genes, including the genes which are non-essential for its replication in vitro (McGeoch et al., 1988) may also be involved in induction of apoptosis in the infected host. Apoptosis of the HSV-infected cells can also occur in the absence of de novo protein synthesis, suggesting that proteins present in the virion may directly trigger some apoptotic pathways (Galvan and Roizman, 1998; Koyama and Adachi, 1997). Finally, oncolytic replication-restricted HSV-1 mutants lacking ICP34.5 may induce apoptosis (Chou et al., 1994; Chou and Roizman, 1992) due to the loss of the protective effect that ICP34.5 exerts on the premature shut-off of total protein synthesis in the infected host (Cassady et al., 1998a; Cassady et al., 1998b). HSV-1 infection can also inhibit apoptosis such as that induced by cytotoxic T lymphocytes (Jerome et al., 1998), hyperthermia (Galvan and Roizman, 1998; Leopardi and Roizman 1996), sorbitol treatment (Galvan and Roizman 1998; Koyama and Miwa 1997), anti-fas ligand (Galvan and Roizman, 1998), tumor necrosis factor alpha (TNF!) and C2 ceramide (Galvan and Roizman, 1998) in some cells. Wt HSV encodes at least two genes, ICP4 (Leopardi and Roizman, 1996) and Us3 (Leopardi et al., 1997), which have been shown to protect some infected cells from undergoing apoptosis (Koyama and Miwa, 1997). In addition, as mentioned above, ICP34.5 exerts a protective effect on the premature shut-off of total protein synthesis in the infected host (Cassady et al., 1998a; Cassady et al., 1998b). Although bcl-2 expression does not play a major role in regulation of apoptosis in HSV-1 infected activated T lymphocytes in vitro (Ito et al., 1997b), it may play a role in some systems (Geiger et al., 1997). The specific mechanisms by which apoptosis is regulated in the HSVinfected cells is the subject of current investigation.

The mechanism by which HSV infected cells die is still a matter of investigation. Galvan and Roizman (Galvan and Roizman, 1998) recently indicated that some HSV-infected cells undergo apoptosis, while other cells die of nonapoptotic death. The type of cell death was found to be celltype dependent (Galvan and Roizman, 1998). Normal proliferating cells, such as activated peripheral and cord blood derived T-lymphocytes, succumb to apoptosis when infected by wt HSV-1 (Ito et al., 1997a; Ito et al., 1997b) and this process is independent of the Fas/Fas ligand system (Ito et

III. HSV-1 mutants used as vectors for cancer gene therapy HSV-1 vectors have been engineered following two different strategies. Recombinant viral vectors are derived 81


Coukos et al: HSV-based oncolytic therapy directly from wtHSV-1, and contain deletion or insertional mutations in various genes. Many investigators have taken the approach of producing HSV mutants with multiple gene deletions, as a means to increase the insertion capacity of the vector and thus be able to host multiple transgenes (Fink and Glorioso, 1998; Johnson et al., 1994). For example, HSV mutants have been engineered with multiple mutations or deletions in genes which include ICP4, ICP27, ICP8, UL33, UL42 and gB and gH to attenuate viral replication (Breakefield and DeLuca, 1991; Glorioso et al., 1997). For example, HSV mutants with various combinations of deletions of ICP4, ICP22, ICP27 and ICP42 yield viral mutants with minimal cytotoxicity, due to their inability to replicate in normal cells (Huard et al., 1997; Johnson et al., 1992). Nevertheless, these vectors have been shown to achieve expression of transgenes in normal cells, in which the transgene is expressed with minimal expression of HSV genes. Recombinant multi-attenuated vectors have been utilized in experimental cancer gene therapy, and their use for suicide or immune gene therapy of extra-CNS malignancies is recently gaining interest (Glorioso et al., 1995). A multiattenuated HSV vector with alterations in ICP4, ICP22, ICP27 and ICP41 was utilized to transduce several ovarian cancer cell lines with the suicide gene HSV thymidine kinase, and was found to achieve high transduction efficiency (Wang et al., 1998). Further studies are needed to determine whether sufficient cells can be transduced to yield a clinical benefit. Rees et al. (1998) constructed a mutated HSV vector that could undergo a single round of viral replication and express murine granulocyte colony stimulating factor (mGCSF). This vector exhibited efficient transduction and achieved effective immunization in a murine syngeneic renal carcinoma model (Rees et al., 1998).

amplicon vector carrying IL-2 was found to achieve high therapeutic efficacy in treating intraperitoneal metastatic gastric carcinoma in nude mice and to increase the killing activity of splenocytes (Tsuburaya et al., 1998). Furthermore, subcutaneous murine lymphoma nodules were eradicated in approximately 85% of tumor-bearing mice by co-administration of HSV amplicon vectors expressing the chemokine RANTES and the T cell costimulatory ligand B7.1 (Kutubuddin et al., 1998).

IV. HSV-1 mutants used as direct oncolytic agents Molecular alterations in certain genes of the HSV genome have led to the engineering of replication-restricted HSV mutants, which maintain the ability to infect and rapidly kill proliferating cancer cells but still maintain low (or undetectable) replication rates in normal diploid cells. Several genes have been the target of alterations including the thymidine kinase (UL23) (Jia et al., 1994; Martuza et al., 1991; Sanders et al., 1982), the ICP6 gene (UL39) encoding the large subunit of HSV ribonucleotide reductase (RR) (Boviatsis et al., 1994; Idowu et al., 1992; Kramm et al., 1997), the uracil DNA glycosylase (UNG) gene (Pyles et al., 1997) and the ICP34.5 (Chambers et al., 1995; Kesari et al., 1995; Mineta et al., 1995). The thymidine kinase-negative HSV-1 mutant (Jia et al., 1994; Martuza et al., 1991) was shown to efficiently cause tumor growth inhibition after intraneoplastic inoculation of subcutaneously and subrenally implanted experimental human gliomas with minimal toxicity in immunodeficient mice. It may also be effective for treatment of other solid tumors localized in the periphery. Although HSVtk - mutants were sensitive to foscarnet and phosphonormal acid (Jia et al., 1994), a potential disadvantage of these strains relates to their resistance to commonly used anti-herpetic drugs such as acyclovir or ganciclovir and has spurred the engineering of alternate attenuated HSV vectors. HSV mutants lacking the ribonucleotide reductase through a deletion or mutations of ICP6 gene were also shown to be replication-restricted and demonstrated efficacy in CNS malignancies. The HSV-1 ribonucleotide reductase deficient (RR -) mutant hrR3, containing an E-coli LacZ gene insertion in the ICP6 gene, was recently tested in an experimental metastatic colon carcinoma with liver metastases in an immunodeficient mouse model (Carroll et al., 1996). This mutant displayed selectivity only for the intrahepatic tumors in vivo and did not spread to the surrounding normal liver after intrasplenic injection, supporting the notion that it replicated only in dividing cells, which provided RR in complementation (Carroll et al., 1996). HSV oncolytic agents have also been generated by mutations or deletions of the ICP34.5 genes, altering both copies in the HSV genome (Chambers et al.,

A second type of multi-attenuated vectors, the amplicon vectors, are engineered utilizing plasmids carrying the HSV DNA packaging signal, the HSV origin of DNA replication, expression cassettes regulating the transgenes of interest together with an E-coli origin of DNA replication and antibiotic resistance genes (Frenkel et al., 1994; Geller, 1993; Geller and Breakefield, 1991; Ho, 1994). Although propagation of amplicon vectors initially required coinfection with HSV helper virus (Frenkel et al., 1994; Geller, 1993; Geller and Breakefield, 1991; Ho, 1994), amplicons can now be propagated by complementation using plasmids (Fraefel et al., 1996). Amplicon HSV vectors have been utilized to rapidly transduce hepatoma cells from cultured cells or tissue explants with IL-2 or GM-CSF genes (Karpoff et al., 1997; Tung et al., 1996). Administration of these transduced cells into rats or mice, respectively induced an immune response to the hepatomas. Toda et al. (1998a) showed that co-expression of IL-12 by an HSV amplicon in the presence of an oncolytic G207 helper virus augmented the anti-tumor effect. Preliminary data indicated that an HSV

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Gene Therapy and Molecular Biology Vol 3, page 83 1995; MacLean et al., 1991). Its protein product is implicated in the prevention of the protein synthesis premature shut-off in the infected host, through inhibition of the phosphorylation of the eukaryotic translation initiation factor eIF2! ( Cassady et al., 1998b), as well as in viral exit from the cell (Brownet al., 1994). ICP34.5 -/- mutants have proven efficient in treating several types of CNS malignancies in experimental rodent models (Andreansky et al., 1997; Chambers et al., 1995; Kesari et al., 1995; Kramm et al., 1997) and efficiently treat experimental tumors of melanoma (Randazzo et al., 1997) and mesothelioma origin (Kucharczuk et al., 1997). HSV-1716 was efficacious in the treatment of intraperitoneal (i.p.) human malignant mesothelioma in a severe combined immunodeficient (SCID) mouse model (Kucharczuk et al., 1997), reducing tumor burden and prolonging animal survival in a dose-dependent manner. Administration of the HSV-1716 yielded viral replication only within i.p. tumor nodules. There was no evidence of viral antigen (by immunohistochemistry) or DNA (by polymerase chain reaction analysis) in any mouse organs. The same virus was also used to treat experimental subcutaneous melanoma, yielding similar efficiency and minimal toxicity (Randazzo et al., 1997). Since mRNA for HVEM was readily detected in lung tissue (Montgomery et al., 1996), HSV mutants lacking ICP34.5 were investigated and demonstrated efficacy in vitro and in vivo against several human lung carcinoma lines (Abbas et al., 1998).

and did not spread to normal murine CNS but exerted a direct oncolytic activity in vitro and in vivo against human CNS tumor cell lines and brain tumor xenografts. Moreover, this mutant demonstrated a hypersensitivity to the anti-herpetic drug ganciclovir. G207 is also a derivative of the ICP34.5deleted mutant, R3616, in which "-galactosidase is inserted into ICP6 gene, which encodes the large subunit of the ribonucleotide reductase gene (Mineta et al., 1994). This mutant was also found to be efficacious in the treatment of various CNS tumors (Mineta et al., 1994; Mineta et al., 1995; Yazaki et al., 1995). Both these doubly deleted HSV mutants appear promising for extra-CNS applications. G207 demonstrated efficacy against some tumor cell lines of breast origin both in vitro and in vivo (Toda et al., 1998b). In our laboratory, a single i.p. administration of HSV-G207 to SCID mice bearing i.p. human ovarian carcinoma tumors (SKOV3 cell line) led to significant reduction in tumor volume four weeks later (Table 1). Immunostaining of tumors harvested from HSV-treated animals demonstrated the presence of HSV-1 antigens in multiple scattered areas throughout the tumor nodules, demonstrating the ability of the virus to replicate and penetrate in depth within the tumors (not shown). Extensive necrosis was observed adjacent to the areas that were positive for HSV particles. An emerging strategy for engineering replication selective HSV oncolytic agents involves replication-targeted HSV mutants, achieved through the insertion of tissue-specific promoters regulating HSV replication. To demonstrate the feasibility of this system, an expression cassette containing a heterologous eukaryotic promoter (albumin) regulating ICP4 expression was inserted into an ICP4- mutant (Miyatake et al., 1997). The authors observed that these viruses replicated 10-fold better in albumin-expressing hepatomas than in cells which did not express albumin.

A second generation of multi-attenuated viruses were engineered stemming from a parental ICP34.5-deleted virus, R3616, which is based on the wt HSV-F strain (Chambers et al., 1995). R3616UB was generated by interrupting the uracil DNA glycosylase (UNG) gene in the parental HSV-R3616 mutant (Pyles et al., 1997). This viral strain did not show any replication in primary human neuronal cultures in vitro

Tumor Weight

Pre-treatment

Control (Media)

HSV-G207

12.5±4 mg

278±45 mg

48±7 mg *

T a b l e 1 . To assess the efficacy of HSV-G207 in treating epithelial ovarian cancer in vivo, SCID mice (n=10/group) were administered a single intraperitoneal (i.p.) injection of 5x10 6 SKOV3 cells, which led to the establishment of i.p. tumors two weeks later. HSV-G207 was administered directly i.p. to a group of animals at that time. Control animals received media only. Animals from each group were sacrificed four weeks following treatment. A separate group of animals was sacrificed prior to viral administration at two weeks. Tumors were dissected and weighed. Weights are expressed in mg and values are expressed as the mean ± standard error (M±SE). (* =p<0.001 vs. control animals).

the immune response on the efficacy of HSV-based oncolytic or gene therapy in humans is an important issue. To address this issue, the effects of a pre-existing immunity to HSV-1 was tested in a syngeneic rat model. The presence of antiHSV primed immune response was found to dampen but not

V. HSV mutants used in the immune therapy of cancer Since HSV-1 and HSV-2 infections are highly prevalent in the adult human population (Whitley, 1996), the effects of

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Coukos et al: HSV-based oncolytic therapy abolish gene transfer by an HSV vector (Herrlinger et al., 1998). However, it should be noted that the clinical significance of pre-existing immunity is still unknown in viral-based oncolytic or gene therapy. In fact, HSV-1 or HSV-2 recurrences occur commonly following a primary infection in the immunocompetent human (Whitley, 1996). Moreover, adenoviral-mediated gene transfer in a phase-1 clinical trial for the treatment of malignant mesothelioma was not blocked by significant anti-adenoviral neutralizing antibody titers or significant T cell proliferation (MolnarKimber et al., 1998). Thus, the effect of the immune response on the efficacy of viral therapies will have to be determined in clinical studies.

mutants represent suitable vectors for immunotherapy as they can accommodate large and multiple transgene inserts and efficiently deliver interleukin transgenes into tumors. The administration of a defective HSV vector containing tandem repeats of an amplicon plasmid encoding IL-12 together with a multi-attenuated HSV-1 mutant lacking ICP34.5 and RR (HSV G207) was followed by significant reduction in tumor growth in a syngeneic murine colon carcinoma model (Toda et al., 1998a). Importantly, IL-12 was expressed and secreted by infected tumor cells in vitro and in vivo. Unilateral inoculation of the virus and amplicon was accompanied by regression not only of the inoculated tumor but also of non-inoculated controlateral tumors. In addition, tumor reduction was significantly greater in animals receiving the amplicon plasmid encoding IL-12 compared to those receiving a control LacZ -expressing amplicon plasmid together with the HSV G207 helper. This effect was attributed to the enhancement of tumor-specific CTL activity (Toda et al., 1998a). Moreover, a replication-restricted HSV ICP34.5 -/- mutant encoding murine IL-4, but not IL-10, was shown to significantly prolong the survival of gliomabearing mice (Andreansky et al., 1998). Clearly, similar viruses encoding cytokines or immunostimulatory molecules appear very attractive for the treatment of non-CNS tumors as well. Additional support for the potential of HSV-based cytokine-mediated immunotherapy is provided by the observations that amplicons expressing RANTES and B7.1 (Kutubuddin et al., 1998) or IL-2 (Tsuburaya et al., 1998) or a multi-attenuated HSV vector expressing GM-CSF (Rees et al., 1998) were showed to augment the efficacy of treatment of lymphoma, metastatic gastric carcinoma or renal carcinoma, respectively, as mentioned above.

The interaction of the immune system with HSV-based therapeutic agents could potentially become advantageous. In fact, the utilization of HSV mutants as direct oncolytic agents or as vectors could generate or enhance an anti-tumor immune response. Infection of human cells by wild-type HSV induces an orchestrated immune response, which includes a cellular infiltrate, generation of cytotoxic T lymphocytes (CTL), release of cytokines and induction of an antibody response (Whitley, 1996) and ref. therein). Although ICP47 can decrease the expression of class I major histocompatibility antigens on the cell surface (York et al., 1994), tumor cell infection and death following infection by mutant HSV-1 will most likely induce intratumoral infiltration of lymphocytes and antigen-presenting cells and may lead to unmasking of tumor antigens, triggering an antitumor response. This strategy could become particularly advantageous in tumors that down-regulate the immune response or induce a predominant TH2-like response. Recent experimental evidence supports the concept that HSV-based oncolytic therapy may be followed by an adjuvant tumorspecific immune response (Toda et al., 1998a). In fact, intratumoral administration of HSV-G207 in immunocompetent animals bearing syngeneic tumors led to growth inhibition of distant non-inoculated tumors, likely mediated by an immune response (Toda et al., 1998a).

VI. Toxicity considerations Large amount of pre-clinical data has been accumulated in the rodent model on replication-selective attenuated HSV-1 ICP34.5 -/- mutants following intratumoral â&#x20AC;&#x153;stereotacticâ&#x20AC;? inoculations of viral particles within the CNS. In both immunocompetent as well as immunodeficient mice, intracranial administration of viral particles did not lead to encephalitis (Andreansky et al., 1997; Carroll et al., 1996; Chambers et al., 1995; Kaplitt et al., 1994; Kesari et al., 1995; Mineta et al., 1994). HSV-1716 administered intracranially or intraocularly into SCID mice resulted in low or no virulence (Valyi-Nagy et al., 1994). Similarly, HSV1716 administered i.p. was found to be avirulent in SCID mice in contrast to rapid systemic spread of the wt HSV virus and death of the animals (Kucharczuk et al., 1997). No viral spread was detected beyond the tumor tissue (Kucharczuk et al., 1997). Administration of HSV-1716 to normal human skin in a murine xenograft model was accompanied by no toxicity, while administration of a wild-

Cytokines have been shown to enhance the anti-tumor immune response, but their systemic administration has been accompanied by significant side effects. Local administration of cytokines to tumors has led to decreased magnitude of side effects but may be technically challenging (Pardoll 1996). Recent evidence suggests that gene therapy with delivery of cytokine genes into tumors or the generation of cytokine gene-transduced cancer cell vaccines may represent a very powerful tool for augmenting anti-tumor immune responses (Pardoll, 1996). For instance, expression of interferon gamma (INF# ), tumor necrosis factor alpha (TNF!) or GMCSF in the milieu of the tumor has led to arrest of tumor growth in experimental models in vivo through stimulation of local inflammatory and immune responses (Andreansky et al., 1998; Pardoll, 1996; Tepper and Mule, 1994). HSV-1 84


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type HSV-1 led to rapid destruction of the xenograft (Randazzo et al., 1996). The replication selective hR3, a HSV-1 lacking RR expression, administered systemically (intrasplenic injections) was also found to infect only metastatic human colonic adenocarcinoma tumor nodules within the liver but not the surrounding murine normal liver tissue (Carroll et al., 1996). However, HSV-1 ICP34.5deleted mutants maintain their ability to infect ependymal cells in the CNS (Kesari et al., 1998; Markovitz et al., 1997). Severe intra-CNS inflammation was observed in some rodent strains after intracranial administration of HSV1716 which expressed LacZ (McMenamin et al., 1998). It is possible that additional mutations may significantly decrease any potential for HSV-1 neurotoxicity. Administration of G207, an ICP34.5-deleted/RR- mutated virus was found to be safe following administration to HSVsensitive primates (Markert et al., 1998). Sufficient toxicity data on the HSV ICP34.5 mutants, HSV-1716, and G207 (ICP34.5 -/- ,RR - ), has been presented to the regulatory bodies for initiation of phase I clinical trials. Preliminary results from the dose escalation phase I clinical trials employing HSV-1716 (ICP34.5-/- ) or G207 (ICP34.5-/- , RR -) utilizing intra-CNS administration for the treatment of malignant glioma have reported minimal side effects in humans (Brown et al., 1998; Markert et al., 1998).

We thank Ms. Carmen Lord for her editorial help in the preparation of this manuscript.

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Roth, J., Cristiano, R. (1 9 9 7 ). Gene Therapy for Cancer: What have we done and where are we going? J . N a t l . C a n c e r I n s t . 89, 21-39.

Vile, R. (1 9 9 8 ). Gene Therapy. C u r r . B i o l . 29, R73-5. Wang, M., Rancourt, C., Alvarez, R., Siegal, G., Marconi, P., Krisky, D., Glorioso, J., Curiel, D. (1 9 9 8 ). High efficiency of thymidine kinase gene transfer to ovarian cancer cell lines mediated by herpes simplex virus type 1 vector. In 29 th Annual Meeting of Society of Gynecologic Oncologists; Orlando , FL pp A61.

Sanders, P., Wilkie, N., Davison, A. (1 9 8 2 ). Thymidine kinase deletion mutants of herpes simplex virus type 1. J . G e n . V i r o l . 63, 277-95.

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Gene Therapy and Molecular Biology Vol 3, page 89 Whitbeck, J., Peng, C., Lou, H., Xu, R., Willis, S., Ponce de Leon, M., Peng, T., Nicola, A., Montgomery, R., Warner, M., Soulika, A., Spruce, L., Moore, W., Lambris, J., Spear, P., Cohen, G., Eisenberg, R. (1 9 9 7 ). Glycoprotein D of herpes simplex virus (HSV) binds directly to HVEM, a member of the tumor necrosis factor receptor superfamily and a mediator of HSV entry. J . V i r o l . 71, 6083-93. Whitley, R. Herpes Simplex Viruses. In Fields Virology (1996), 3rd. ed. B.Fields, D.M. Knipe, P.M. Howley Philadelphia, PA, Lippincott-Raven Publishers, 2, pp 2297-2342. Yazaki, T., Manz, H., Rabkin, S., Martuza, R. (1 9 9 5 ). Treatment of human malignant meningiomas by G207, a replication competent multimutated herpes simplex virus 1. Cancer Res. 55, 4752-4756. York, I., Roop, C., Andrews, D., Riddell, S., Graham, F., Johnson, D. (1 9 9 4 ). A cytosolic herpes simplex virus protein inhibits antigen presentationto CD8+ T lymphocytes. C e l l 77, 525-535.

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Gene Therapy and Molecular Biology Vol 3, page 91 Gene Ther Mol Biol Vol 3, 91-101. August 1999.

Transcriptional repression in cancer gene therapy: targeting HER-2/neu overexpression as an example Review Article

Mien-Chie Hung* and Shao-Chun Wang University of Texas M. D. Anderson Cancer Center, Department of Cancer Biology, Section of Molecular Cellular Biology, Box 79, 1515 Holcombe Boulevard, Houston, Texas 77030 . __________________________________________________________________________________________________ * To whom correspondence should be addressed. Phone: (713) 792-3668. Fax: (713) 794-4784. E-mail: mchung@odin.mdacc.tmc.edu Received: 16 October 1998; accepted: 9 November 1998

Summary Overexpression of the HER-2/neu oncogene has been well-documented as a frequent event in human cancers. In c l i n i c , overexpression o f HER-2/neu indicates a unfavorable prognosis and highly correlated with the l o w survival rate o f patients associated with breast and ovarian cancers. Downregulation of the HER-2/neu g e n e e x p r e s s i o n i n c a n c e r c e l l s b y attenuating the promoter a c t i v i t y o f t h e g e n e i s t h e r e f o r e a n a t t r a c t i v e s t r a t e g y t o r e v e r s e the transformation phenotype induced by HER-2/neu overexpression. We have identified a number o f cellular and viral transcriptional regulators, including the ets family member PEA3, the SV40 large T antigen, and the adenovirus type 5 E1A, which are able to repress the HER-2/neu gene expression. Expression of these transcriptional regulators resulted in downregulation of the HER-2/neu promoter activity and reversed the malignant phenotype of the transformed cells i n v i t r o . These observations were followed by a series o f studies t o investigate whether these HER-2/neu repressors can act therapeutically as tumor suppressor genes for cancers that overexpress HER-2/neu. T h e g r o w t h o f tumors derived from HER-2/neu-overexpressing cancer cells was inhibited by the transcriptional repressors, accompanied by decreased HER-2/neu e x p r e s s i o n i n t u m o r c e l l s . T h e r e s u l t s o f t h e s e preclinical studies clearly indicate that transcriptional repressors which downregulate HER-2/neu can be a promising regimen for cancer treatment in a gene therapy format.

transduction through all erbB receptor family members (Craus-Porta et al, 1997, Wallasch et al, 1995, Carraway et al, 1994, Sliwkowski et al, 1994, Plowman et al, 1993), due to the preference for the HER2/neu receptor as a heterodimerization partner for all erbB receptors. After ligand binding, EGFR, HER-3 (also known as erbB3), and HER-4 (also known as erbB4) can heterodimerize with HER2/neu, and can lead to the tyrosine phosphorylation of all of these receptors (Craus-Porta et al, 1997, Wallasch et al, 1995, Sliwkowski et al, 1994).

I. Introduction A. HER2/neu overexpression serves as a critical target for cancer gene therapy The HER-2/neu (also known as c-erbB2) gene encodes a receptor tyrosine kinase (p185) with significant structural and functional homology to the epidermal growth factor receptor (EGFR) (Bargmann et al, 1986a, Hung et al, 1986, Yamamoto et al, 1986). Each protein member of the erbB receptor family contains an extracellular domain, a transmembrane domain, and an intracellular domain with intrinsic tyrosine kinase activity. Although the ligand for the HER2/neu receptor has not been identified, the HER2/neu receptor is known to mediate lateral signal

The oncogenic property of the HER-2/neu protooncogene was originally demonstrated in the rat neu oncogene (Hung et al, 1989, Bargmann 1986b, Hung et al, 1986). As a matter of fact, the mutation-activated rat neu

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Hung and Wang: Targeting HER-2/neu overexpression in cancer gene therapy oncogene, which contains a point mutation in the transmembrane domain of the protein resulting in a constitutive tyrosine kinase activity, was originally isolated from rat neuroblastoma due to its ability to transform mouse cells (Hung et al, 1989, Bargmann et al, 1986b, Hung et al, 1986). In human, the HER-2/neu proto-oncogene is frequently amplified or overexpressed in many types of cancers including breast (Gusterson et al, 1992, Toikkanen et al, 1992, Slamon et al, 1989, Slamon et al, 1987), ovarian (Slamon et al, 1989, Burchuck et al, 1991, Burchurk et al, 1990), lung (Shi et al, 1992, Weiner et al, 1990, Schneider et al, 1989), stomach (Yokota et al, 1988, Park et al, 1989), and oral (Xia et al, 1997) cancers, suggesting that HER-2/neu overexpression plays a critical role in the development of human cell malignancy. The overall survival rate of cancer patients whose tumors have HER-2/neu overexpression is significantly shorter than those patients whose tumor do not have HER-2/neu overexpression (Slamon et al, 1989, Slamon et al, 1987, Burchurk et al, 1990, Weiner et al, 1990, Xia et al, 1997). Furthermore, increased expression of the HER-2/neu gene has been shown to correlate with the number of lymph node metastases in breast cancer patients (Slamon et al, 1987), an observation consistent with many studies in that the mutation-activated neu gene induced metastatic potential in mouse 3T3 cells and that overexpression of the normal human HER2/neu gene enhanced metastatic potential in human breast, ovarian, and non-small-cell lung carcinoma (NSCLC) cells by promoting multiple steps in the metastatic cascade (Tan et al, 1997, Yu et al, 1994, Benz et al, 1993, Yu et al, 1992a, Chazin et al, 1992, Yu and Hung 1991a, Slamon et al, 1989, Slamon et al, 1987). In addition to metastasis of cancer cells, it is generally believed that HER-2/neu overexpression is correlated to chemoresistance of cancer cells. High level of HER-2/neu expression in human NSCLC appeared to result in enhanced resistance to a panel of chemotherapeutic agents (Tsai et al, 1995. Tsai et al, 1993). Similarly, overexpression of HER-2/neu in breast cancer cells induced chemoresistance to Taxol (Paclitaxel) (Yu et al, 1998, Yu et al, 1996). However, the expression level of HER-2/neu seems to be critical for the development of chemoresistance since in certain cell lines moderate p185 expression level is not accompanied with significant drug resistance (Pegram et al, 1997). It is likely that the HER-2/neu expression has to be higher than a threshold level to induce significant drug resistance. Furthermore, the chemoresistance developed in those HER2/neu-overexpressing breast cancer cells is limited to Paclitaxal and Taxotere but not to other drugs (Pegram et al, 1997; Yu et al, 1996; Yu, D. and Hung, M. -C., unpublished results), suggesting a selective mechanism of resistance. it is not yet clear why HER-2/neu overexpression-mediated drug resistance behaves differently between lung and breast cancer cells. However, in the case

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of resistance to Paclitaxel by HER-2/neu overexpression in breast cancer cells, a molecular mechanism has recently been suggested (Yu et al, 1998a): upregulation of p21 by HER-2/neu overexpression inhibits cyclin B/cdc2 kinase activity in G2/M phase which is required for Paclitaxel induced apoptosis. This mechanism clearly indicates that HER-2/neu overexpression in breast cancer cells antagonizes Paclitaxel-induced apoptosis. Since the HER2-neu proto-oncogene overexpression significantly contributes to the malignant development of many types of human cancers in different aspects, molecular strategies which aim to down-regulate the HER2/neu gene expression have become highly attractive approaches to fight against human cancer.

B. Transcriptional repression as an effective means to downregulate HER2/neu expression in cancer cells HER-2/neu gene amplification can be detected in majority of breast tumor tissues with overexpression of the HER-2/neu-encoded p185 protein (Slamon et al, 1989). In established breast cancer cell lines, both gene amplification and transcriptional upregulation are common scenario accounting for the increased HER-2/neu gene expression in different breast cancer cells (Kraus et al, 1987, Millar et al, 1994, Bosher et al, 1996). Interestingly, it has been shown that in 10-20% of HER2/neu -overexpressing breast tumors and in virtually all HER2/neu-positive lung cancers the HER2/neu mRNA and protein expression can occur in the absence of increased gene copy number (Kameda et al, 1990, Kern et al, 1990, Slamon et al, 1989, King et al, 1989, Tandon et al, 1989, Berger et al, 1988). It is therefore likely that both gene amplification and transcriptional upregulation are involved in HER-2/neu overexpression in cancer cells. The promoter of the HER-2/neu gene has been well characterized. In the past few years, knowledge about the cis- and trans-acting elements regulating the transcription of the HER-2/neu proto-oncogene have been rapidly accumulated. A number of cis-acting motifs are distributed along the HER-2/neu promoter, including the binding sites of transcription factors Sp1, OTF1, AP2, E4TF1, and PEA3. Another 13-bp sequence in the promoter region has been identified as a positive element for HER-2/neu transactivation (Miller et al, 1994). The corresponding binding transcription factor(s), however, has not yet been identified. AP2 has been shown to be a strong activator of the HER-2/neu gene and is functionally activated in the HER-2/neu-overexpressing breast cancer cell lines such as MDA-MB-361, MDA-MB-175, ZR-75-1, BT-474, and SK-BR-3 (Hollywood et al, 1993). The high activity of AP2 in these cell lines has been correlated with the elevated HER-2/neu gene expression level in these cells.


Gene Therapy and Molecular Biology Vol 3, page 93 On the other hand, the HER-2/neu gene is subject to the negative regulation of a number of cellular or viral factors through different mechanisms. For example, PEA3, a member of the ets family (X. Xing, S. -C. Wang, and M. -C. Hung, unpublished results; Xing et al, 1997), and the retinoblastoma tumor suppressor (RB) (Yu et al, 1992b) can repress the HER-2/neu gene expression. Interestingly, in addition to the cellular factors, the HER-2/neu gene transcription can also be repressed by a number of viral transcription factors such as the simian virus 40 (SV40) large T antigen and the adenovirus type 5 E1A (Yan et al, 1991a, Yu et al, 1991b). These studies have indicated that repression of transcription is an effective way to reverse the malignant transformation mediated by HER-2/neu overexpression, and have demonstrated the potential application of transcriptional repressors as therapeutic agents targeting HER-2/neu-overexpressing cancer cells.

II. Tumor suppression effects of HER2/neu down-regulation mediated by genes encoding transcriptional regulators A. Tumor suppression by viral transcriptional regulators Both E1A and T antigen are viral proteins, and their ability to suppress HER-2/neu-mediated cell transformation is surely a surprising biological phenomenon. The adenovirus genome is about 36 kb in size. Among the proteins encoded by the adenovirus genome, E1A gene products are nuclear-localized phosphoproteins and have special regulatory role in the adenoviral life cycle (Berk 1986). E1A is the first region to be expressed after infection (Tooze 1981). Other late adenoviral genes can then be turned on by E1A proteins through interacting and modifying the host transcriptional apparatus. There are two types of adenovirus E1A. One is the transforming E1A carried by the adenovirus type 12. This type of E1A gene alone can transform normal cell lines (Schrier et al, 1983). The other type of E1A, such as the adenovirus type 2 or type 5 E1A, can not transform cells by itself alone. It is noteworthy that for the purpose of this review E1A refers to the type 5, non-transforming E1A. E1A was classified as an "immortalization oncogene" due to its ability to cooperate with the transforming ras or E1B genes to transform primary embryo cells (Byrd et al, 1988, Montell et al, 1984, Land et al 1983, Ruley 1983). However, expression of the E1A gene itself does not induce transforming phenotypes (Yu et al, 1992a). As a matter of fact, there are a number of studies indicated that E1A is associated with metastasis- or tumor-suppression activities (Pozzatti et al, 1988, Frisch 1991, van Groningen 1996). Recently, E1A has been shown to induce apoptosis under some conditions (Lowe and Ruley 1993, Rao et al, 1992). This property is similar

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to the well-known tumor suppressor gene p53 that also has the ability to induce apoptosis (Subramanian et al, 1995, Symonds et al, 1994). All of these observations indicate that tagging E1A as an oncogene is a misconception. We have first discovered that the adenovirus 5 E1A gene can repress HER-2/neu overexpression through both transient transfection and adenovirus delivery systems (Yu et al, 1991b, Yu et al, 1990). Transfection of the E1A gene into the genomic rat neu oncogene transformed mouse embryo fibroblast cell lines virtually abolishes the tumorigenicity and metastatic potential induced by the HER-2/neu oncogene through repression of HER-2/neu gene expression (Yu et al, 1992a, Yu et al, 1991b). Reexpression of the HER-2/neu-encoded p185 protein in these E1A transfectants by transfection of a HER-2/neu cDNA construct driven by a promoter that cannot be inhibited by E1A recovered virtually all of the transforming phenotypes including tumorigenicity, the ability to grow in soft agar, and higher in vitro growth rate (Yu et al, 1993a, b). Interestingly, the ability to induce experimental metastasis (measured by lung colonization through i. v. injection of the tumor cells) was only partially recovered. The incomplete regeneration of metastatic potential could be accounted for by the fact that E1A inhibits gelatinolytic activity that was critical for invasive activity of metastatic cells. This result indicates that the suppression of metastasis by E1A is through multiple molecular mechanisms in addition to repressing the HER-2/neu gene expression (Yu et al, 1992a). We have also demonstrated that E1A can indeed function as a tumor suppressor in the HER-2/neu-overexpressing human ovarian cancer cell line by down-regulating the expression of the HER-2/neu mRNA and the p185 protein product (Yu et al, 1995, Yu et al, 1993a, b, Yu et al, 1991b, Yu et al, 1990). The E1A-expressing ovarian cancer cell line had reduced malignancy, including a decreased ability to develop tumors in nude mice. Therefore, for the HER2/neu-overexpressing transforming cells including fibroblasts and human cancer cells, E1A can function as tumor suppressor. And transcriptional repression of the HER-2/neu oncogene contributes to the tumor suppression function. However, since E1A is not a DNA-binding protein, the transcriptional repression of HER-2/neu by E1A has to be mediated through the targeting of other transcription factors. This is supported by our recent study demonstrating that E1A can abolish HER-2/neu overexpression by targeting the coactivator p300, which is required for efficient expression of HER-2/neu (Chen and Hung 1997). To further investigate whether the E1A gene can be used as a therapeutic agent for HER-2/neu-overexpressing human breast and ovarian cancers in living host, a tumorbearing mouse model was established and the E1A gene


Hung and Wang: Targeting HER-2/neu overexpression in cancer gene therapy was delivered by the cationic liposome DC-Chol or a recombinant replication-deficient adenovirus. E1A treatment was able to effectively reduce the mortality of tumor-bearing mice and, in 60-80 % of the treated mice, resulted in tumor-free survival, suggesting that E1A gene therapy is a promising therapeutic regimen for cancers that overexpress HER-2/neu . In addition, the number of mice with distant metastases was significantly reduced even though a local treatment protocol by mammary fat pad injection was used in the orthotopic breast cancer model (Lane and Crawford 1979, Zhang et al, 1995). In addition to the breast and ovarian cancer animal models, we also used a lung cancer animal model to test the therapeutic efficiency of E1A (Chang et al, 1996). In this case, the tumor-bearing mice were established through intratracheal inoculation of lung cancer cells and the E1A gene was delivered by an adenovirus vector through intravenous injection. A significant therapeutic efficacy was observed. Therefore the tumor suppression effect of E1A can be demonstrated through two independent gene delivery systems and three different animal models. Based on these results, a phase I clinical trial, using cationic liposome to deliver the E1A gene was initiated at the M. D. Anderson Cancer Center. Preliminary results suggested a downregulation of the HER-2/neu p185 oncoprotein concomitant with the detection of the E1A gene expression in treated breast and ovarian cancer patients. The simian virus 40 (SV40) large T antigen is a multifunctional protein required for the replication of the viral genome and for cell transformation (Lane and Crawford 1979, Linzer and Levine 1979). This viral protein contains transformation domains which can mediate binding to the retinoblastoma protein (pRb) and p53, respectively (Manfredi and Prive 1994). Our previous studies showed that a mutant SV40 large T antigen can repress rat neu transcription in mouse fibroblast NIH 3T3 cells (Matin and Hung 1993). The mutant large T antigen, named K1, contains a single amino acid change within the pRb-binding/transformation domain, which renders the viral protein unable to bind to pRb, and consequently failed to induce cell transformation (Kalderon and Smith 1984, Cherington et al, 1988, DeCaprio et al, 1988). Since the K1 mutant represses HER-2/neu expression as effectively as the wild-type counterpart (Matin and Hung 1993), we further tested whether K1 can function as a tumor suppressor for HER-2/neu-overexpressing ovarian cancer cells. K1 did suppress cancer cell growth, resulting in a significant therapeutic effect on mice with ovarian cancer with about 40% of treated mice were alive after one year (Xing et al, 1996). The autopsies showed that the mice from the control groups had larger volume of ascites and tumors within the peritoneal cavity or diaphragm or metastasis to the lungs. However, the mice that received K1-liposome complex had more locally distributed tumor

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nodules in their peritoneal cavities. This difference indicates that K1 suppressed the growth of HER-2/neuoverexpressing tumor cells so that the tumors developed with longer latency. The K1-treated mice survived for one year were sacrificed and examined for residual tumors, but no tumors were observed in the peritoneal cavity. Our results indicate that both viral transcription factors, E1A and the large T antigen, can suppress tumor cell growth through a HER-2/neu-involved pathway. However, the possibility that E1A can mediate tumor suppression function through a HER-2/neu-independent mechanism should not be excluded.

B. Tumor suppression by cellular DNAbinding transcriptional factor, PEA3 The mouse PEA3 (Polyomavirus Enhancer Activator 3) gene and its human homologue were first cloned from cDNA expression libraries due to the binding ability to the sequence 5â&#x20AC;&#x2122;-AGGAAG-3â&#x20AC;&#x2122; (the PEA3 binding motif) within the polyomavirus enhancer promoter element (Xin et al, 1992, Higashino et al, 1993). The PEA3 protein contains a stretch of about 85 amino acids with extensive sequence homology with the ETS domain, a conserved region shared by all ets family members that characteristically bind as monomers to the consensus core sequence GGAA by their ETS DNA-binding domains (Monte et al, 1994, Brown et al, 1992, Xin et al, 1992, Karim et al, 1990), and regulates the expression of target genes including genes involved in cell growth and differentiation (Ma et al, 1998, Taylor et al, 1997). The ets gene family currently contains at least 30 members present in a diverse spectrum of metazoan organisms (Degnan et al, 1993, Laudet et al, 1993) Subfamilies can be identified based on sequence/structure homology and the association with other accessory proteins for DNA binding. The PEA3 subfamily is composed of three members : PEA3 (Xin et al, 1992), ERM (Nakae et al, 1995, Monte et al, 1994), and ER81 (Brown et al, 1992). In addition to the ETS domain, members of this subfamily share significant sequence similarity at an N-terminal acidic transcriptional activation domain (Nakae et al, 1995, Wasylyk et al, 1993, Macleod et al, 1992, Seth et al, 1992, Karim et al, 1990). Expression of the PEA3 gene is ubiquitous in different species and can be identified in mouse, rat, monkey, and human cells. However, PEA3 RNA expression is tissue-specific with highest level detected in brain, and, to a lower level, in pancreas, lung, and mammary gland (Xin et al, 1992). Most members of the ets family express at high levels in hemotopoietic cells. Unlike other Ets proteins, PEA3 is the only member identified to date that is apparently not expressed in cells with hematopoietic origins (Xin et al, 1992). The significance of this tissue specific distribution is not clear.


Gene Therapy and Molecular Biology Vol 3, page 95 There have been a number of candidate PEA3-regulated genes reported mainly based on the occurrence of putative PEA3 binding motif in their promoter regions. Interestingly, a great portion of these candidates fall in the category of genes encoding matrix metalloproteinases (Higashino et al, 1995), such as collagenase (Gutman et al, 1990), stromelysin (Buttice et al, 1993), and the urokinase-type plasminogen activator (uPA), a serine proteinase (Nerlov et al, 1992). All these enzymes are believed to involve in the regulation of extracelluar proteolysis, both in the normal organisms and in certain pathological conditions including tumor invasion and metastasis (Matrisian 1994). Consistent with this correlation, exogenous expression of PEA3 in the breast cancer cell line MCF-7 resulted in enhanced tumor invasiveness and metastasis (Mitsunori et al, 1996). Caveats should be taken to interpret these results. It is possible that members of the Ets family can have the same specificity required for DNA binding and share the same binding motif (Xin et al, 1992). As a matter of fact, it has been shown that Ets-2, an Ets protein belonging to the Pointed subfamily (Klambt 1993), is critical for the phobol ester (TPA)-mediated induction of the human stromelysine gene expression through the PEA3 binding motif in the promoter (Buttice et al, 1993). A similar conclusion has been drawn for the promoter of the uPA gene (Pankov et al, 1994). In addition, whether the PEA3 protein directly binds to the putative PEA3 motif in the collagenase promoter is not clear due to the lack of appropriate anti-PEA3 antibodies to confirm the identity of the DNA-binding activity detected on the PEA3 motif (Gutman et al, 1990). The occurrence of the PEA3 binding motif is not limited to those genes which potentially can enhance invasion and metastasis. Two consensus PEA3 binding motifs, distal and proximal, have been identified in the upstream regulatory region of the tumor suppressor gene maspin (Zhang et al, 1997a, Zhang et al, 1997b). Both motifs are positive regulatory elements for expression of the gene; the proximal site is the major functioning motif of the gene in mammary epithelial cells while both sites are equally critical for maspin expression in prostate cells. Functional studies have demonstrated that maspin functions as a tumor suppressor by inhibiting tumor invasion, metastasis, as well as tumor growth (Sheng et al, 1995, Zou et al, 1994). Even though it is still not clear if the PEA3 protein binds to the PEA3 motif in the maspin promoter, these observations are consistent with the prospect that tumor metastasis may be the result of imbalance between enhancing and suppressing factors (Liotta et al, 1991). This point is especially noteworthy given the large number of the ets family members and the resemblance of their DNA-binding domains and the DNA sequences of their target DNA motifs.

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It is interesting to investigate the role of PEA3 in HER-2/neu gene expression and HER-2/neu-mediated transformation since a consensus PEA3- binding motif, 5'AGGAAG-3', is present 26 nucleotide upstream from the major mRNA start site in the promoter of the human, rat, and mouse HER-2/neu gene (Tal et al, 1987). It has been reported that PEA3 can mediate induction of the HER2/neu gene expression through the PEA3 binding motif (Benz et al, 1997). These results, however, were derived from the experiment using the COS monkey cell line. As will be mentioned below, this cell line can be characteristically different from other laboratory human breast and ovarian cancer cell lines, for which the investigation of PEA3's functions would be more biologically relevant. Furthermore, the hypothesis of PEA3-mediated HER-2/neu induction would predict a causal relationship between elevated PEA3 expression and HER-2/neu overexpression in cancer cells. However, analysis of PEA3 gene expression in various breast cancer cells dose not support this hypothesis. In fact, decreased PEA3 RNA expression was detected in breast cancer cell lines with HER-2/neu overexpression (such as BT 474, SK-BR-3, MDA-MB-361), while there was no detectable PEA3 mRNA in other HER-2/neu-overexpressing cell lines (such as MDA-MB-453, ZR-75-1, and MDA-MB134-V) (Baert et al, 1997). Nevertheless, these results suggest a negative role of PEA3 in regulating HER-2/neu expression. This prospect was directly tested in our laboratory and the following results demonstrate that PEA3 is indeed a negative transregulator of the proto-oncogene HER-2/neu (Xing et al, 1997). (1) The purified GST-PEA3 fusion protein can specifically recognize and bind to the consensus PEA3 binding motif on the HER-2/neu promoter. (2) Based on the co-transfection experiments performed on HER-2/neu-overexpressing human cancer cell lines, the HER-2/neu promoter activity can be down-regulated by PEA3 in a dose-dependent manner. However, destruction of the PEA3-binding site on the HER-2/neu promoter by site-directed mutagenesis abolished the promoter activity, indicating that PEA3-induced trans-repression of the HER2/neu promoter might involve competition between PEA3 and another ets-related transcriptional activator(s), which contributes to the transformed phenotype of HER-2/neu . (3) PEA3 can suppress the focus forming ability of mouse embryonic fibroblast transformed by the genomic mutation-activated genomic rat neu. (4) Expression of PEA3 can suppress the growth of HER-2/neu-overexpressing human cancer cell lines in vitro, but not cell lines with basal level of HER-2/neu expression. Based on these results, the tumor suppression function


Hung and Wang: Targeting HER-2/neu overexpression in cancer gene therapy of PEA3 is emerging. Trimble et al, have reported that mammary tumors derived from the transgenic mice bearing the rat neu gene under the control of the mouse mammary tumor virus (MMTV) promoter expressed high level of PEA3 mRNA, suggesting that PEA3 may be required for tumorigenesis and metastasis in HER2/neu overexpressing cells (Trimble et al, 1993). However, the data is also consistent with the possibility that there may exist a negative regulatory loop pathway in which the overexpression of HER-2/neu would turn on the expression of PEA3 which then act as a transcriptional repressor of the HER-2/neu gene and resume the homeostatic balance. The rat neu gene in the transgenic mice setting was driven by the heterologous MMTV promoter which is very likely not subject to the negative control by PEA3. Expression levels of both PEA3 and HER-2/neu would be elevated under this situation. In addition to PEA3, other Ets proteins including ERF and Net have been reported to function as transcriptional repressors (Sgouras et al, 1995, Giovan et al, 1994). Other promoters negatively regulated by Ets binding sites have also been reported (Chen and Boxer 1995, Goldberg et al, 1994). Interestingly, the ets family member Ets2 has recently been reported to function as a tumor suppressor by reversing ras-mediated cellular transformation (Foos et al, 1998). To test whether PEA3 can be used as a therapeutic agent in vivo, tumors were induced in nude mice (nu/nu) with SK-OV-3-ip1, an ovarian cancer cell line derived from SK-OV-3 and has higher HER-2/neu expression. For mice treated with PEA3-DC-Chol complex, 50% of the mice were alive and healthy without palpable tumors after 12 months. The mice of the control group, however, developed tumors and ascites, and died within 6 months. The tumor suppression activity of PEA3 is correlated with HER-2/neu expression since another cell line 2774 c-10, an ovarian cancer cell line with basal level of HER-2/neu expressed, did not have response to PEA3 treatment and the mice died of tumor with 5 months. Tumor samples were examined for the expression of HER-2/neu with immunoblot analysis. The results confirmed that PEA3 delivered by the cationic liposome downregulated the expression of p185. The correlation between PEA3 expression and HER-2/neu downregulation was further demonstrated by immunohistochemical staining of the tumor samples obtained from the PEA3-treated, moribund mice. Approximately 30% of the cancer cells in the tumor were positive for PEA3 protein expression, while the p185 staining was negative for about 50% of cells. Similar level of PEA3 expression was observed for PEA3-treated 2774 c-10-derived tumors while no repression of p185 was detected in these tumors. These in vitro and in vivo data clearly demonstrate the tumor suppression activity of PEA3 and indicate the potential clinical application of

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PEA3-cationic liposome targeting overexpressing cancer cells.

the

HER-2/neu

Even though PEA3 as well as the viral proteins E1A and SV40 large T can all suppress HER-2/neu transcription, they are very likely functioning through different mechanisms. Both E1A and SV40 large T may suppress HER-2/neu in an indirect manner. Association of E1A with the transcriptional co-activator CBP/p300 inhibits the p300 transactivation activity, which is required for efficient expression of the HER-2/neu gene (Chen and Hung, 1997). On the other hand, PEA3 down-regulates the HER-2/neu gene by directly binding to its cognate binding sequence on the promoter. This feature makes PEA3 a more attractive target for further molecular manipulation to develop therapeutic molecules with higher binding affinity and enhanced specificity.

III. Conclusions Overexpression of the proto-oncogene HER-2/neu can lead to cell transformation and tightly correlated with the development of malignant tumor growth in many tissue types. There are molecular approaches to target the promoter of HER-2/neu , which can downregulate the gene expression, reverses the malignant phenotype, and retards tumor growth in animal. The results of our in vivo and in vitro experiments demonstrate using viral or cellular transcriptional repressor genes transferred by safe and efficient molecular vehicles can result in significant therapeutic effects on cancer cells. Since gene overexpression is a common mechanism of cancer as well as other types of diseases such as AIDS, the therapeutic strategy discussed here can have a tremendous potential in clinical application. Finally, the studies of E1A- and PEA3-mediated HER-2/neu repression have unveiled new areas in cancer biology which is excitingly more complicated than what we used to expect. Studies of these areas would be critical for our understanding of cancer.

Acknowledgment The authors are supported by NCI RO1 CA 58880 and CA 77858 (to M.C.H.).

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upregulation of p21 cip1 which inhibits p34 cdc2 kinase. M o l e c u l a r C e l l , in press. Yu, D., Liu, B., Jing, T., Sun, D., Price, J. E., Singletary, S. E., Ibrahim, N., Hortobagyi, G. N., and Hung, M. -C. (1 9 9 8 b ). Overexpression of both p185 c-erbB-2 and p170 mdr-1 renders breast cancer cells highly resistant to taxol. O n c o g e n e 16, 2087-2094. Yu, D., Liu, B., Tan, M., Li, J., Wang, S. -S., and Hung, M. C. (1 9 9 6 ). Overexpression of c-erbB-2/neu in breast cancer cells confers increased resistance to Taxol via mdr1-independent mechanisms. O n c o g e n e 13, 1359-1365. Yu, D., Matin, A., Xia, W., Sorgi, F., Huang, L., and Hung, M. -C. (1 9 9 5 ). Liposome-mediated E1A gene transfer as therapy for ovarian cancers that overexpress HER-2/neu. O n c o g e n e 11, 1383-1388. Yu. D, Wang, S. S., Dulski, K. M., Tsai, C. -M., Nicolson, G. L., and Hung, M. -C. (1 9 9 4 ). c-erbB2/neu overexpression enhances metastatic potential of human lung cancer cells by induction of metastasis-associated properties. Cancer Res. 54, 3260-3266. Yu, D., Shi, D., Scanlon, M., and Hung, M. -C. (1 9 9 3 a ). Reexpression of neu-encoded oncoprotein counteracts the tumor-suppressing activity of E1A. Cancer R e s . 53, 5784-5790. Yu, D., Wolf, J. K., Scanlon, M., Price, J. E., and Hung, M. C. (1 9 9 3 b ). Enhanced c-erbB-2/neu expression in human ovarian cancer cells correlates with more severe malignancy that can be suppressed by E1A. Cancer R e s . 53, 891-898. Yu, D., Hamada, J., Zhang, H., Nicolson, G. L., and Hung, M. -C. (1 9 9 2 a ). Mechanisms of c-erbB2/neu oncogeneinduced metastasis and repression of metastatic properties by adenovirus 5 E1A gene products. O n c o g e n e 6, 22632270. Yu, D., Matin, A., and Hung, M. -C. (1 9 9 2 b ). The retinoblastoma gene product suppresses neu oncogeneinduced transformation via transcriptional repression of neu*. J . B i o l . C h e m . 267, 10203-10206. Yu, D., and Hung, M. -C. (1 9 9 1 a ). Expression of activated rat neu oncogene is sufficient to induce experimental metastasis in NIH3T3 cells. O n c o g e n e 6, 1991-1996. Yu, D., Scorsone, K., and Hung, M. -C. (1 9 9 1 b ). Adenovirus Type 5 E1A products acts as transformation suppressors of the neu oncogene. M o l . C e l l B i o l . 11, 1745-1750. Yu, D., Suen T. C., Yan, D. H., Chang, L. S., and Hung, M. -C. (1 9 9 0 ). Transcriptional repression of the neu protooncogene by the Adenovirus 5 E1A gene products. P r o c . N a t l . A c a d . S c i . U S A 87, 4499-4503. Zhang, M., Maass, N., Magit, D., and Sager, R. (1 9 9 7 a ). Transactivation through Ets and Ap1 transcription sites determines the expression of the tumor-suppressing gene maspin. C e l l G r o w t h D i f f e r e n t i a t i o n 8, 179-86. Zhang, M., Magit, D., and Sager, R. (1 9 9 7 b ). Expression of maspin in prostate cells isregulated by a positive Ets


Gene Therapy and Molecular Biology Vol 3, page 101 element and a negative hormonal reponsive element site recognized by androgen receptor. P r o s . N a t l . Acad. S c i . U S A 94, 5673-5678. Zhang, Y. J., Yu, D. H., Xia, W. Y., and Hung, M. C. (1 9 9 5 ). HER-2/neu-targeting cancer therapy via adenovirusmediated E1A delivery in an animal model. O n c o g e n e 10, 1947-1954. Zou, Z., Anisowicz,A., Hendrix, M. J., et al, (1 9 9 4 ). Identification of a novel serpin with tumor suppressing activity in human mammary epithelial cells. S c i e n c e 263, 526-529.

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Gene Therapy and Molecular Biology Vol 3, page 103 Gene Ther Mol Biol Vol 3, 103-112. August 1999.

Gene therapy targeting p53 Review Article

John Nemunaitis1,2 1

PRN Research, Inc., Dallas, Texas. 2Baylor University Medical Center, Dallas, Texas

__________________________________________________________________________________________________ Correspondence: John Nemunaitis, M.D., 3535 Worth Street, Collins Bldg., 5 th Floor, Dallas, Texas 75246. Phone: 214-8208799; Fax: 214-820-8497; E-mail: aepetro@prninc.com Received 13 October 1998; accepted: 17 October 1998

Summary The product of the p53 gene plays a critical role in the regulation of cell growth. Mutations of this gene are associated with transformation to a malignant phenotype. Correction of the gene defect through transfer of a wildtype p53 gene into malignant cells, or targeting malignant cells with oncolytic viruses (ONYX-015) genetically engineered to proliferate in cells containing mutant genes has been identified as a therapeutic approach by preclinical assessment. Initial clinical trials have confirmed functional activity and expression of the transgene product in Adp53-injected malignant tissue and tumor specific viral proliferation have been observed in patients receiving intratumoral injection of ONYX-015.

I. Introduction

II. p53 mutation

The most common genetic abnormality identified in human malignancy with an occurrence of approximately 60% involves the p53 gene, which is a tumor suppressor gene (Baker, 1990) located on chromosome 18. Disruption of p53 protein production or inhibition of its function is associated with abnormal cellular proliferation and differentiation.

Eighty percent of p53 mutations involving solid tumors are point mutations that result in a single amino acid substitution. At first glance, this may not appear to be a significant abnormality, given that the alteration involves less than 1% of the entire molecule. However, many of the aminoacid substitutions result in a charge change (i.e. positive to negative or vice versa), which dramatically alters the three-dimensional structure of the p53 protein. Once altered, receptor-binding affinity is disturbed. As a result, excess p53 protein is produced with accumulation within the nucleus. Normal cells have undetectable levels of p53 protein. Thus, elevated p53 protein expression often indicates the occurrence of a mutated p53 gene although not always (Barnes, 1992; Lehman, 1991).

Specific functions of the p53 gene product include upregulation of p21, which is a protein that inhibits cyclin-dependent kinase (CDK ), and is necessary for the G1 to S-phase transition. P53 protein also upregulates Bax (a positive regulator of apoptosis), MDM-2 (a negative regulator of p53 function), thrombospondin-1 (inhibitor of angiogenesis), GADD45 (role in DNA repair), and IGF-BP3 (growth regulator) (Harper, 1993; Miyashita, 1995; Dameron, 1994). Extensive analysis of tumors showing evidence of p53 gene dysfunction indicate that abnormal function correlates with poor prognosis in patients with malignancy (Drach, 1998; Horio, 1993; Thorlacius, 1993; Preudhomme, 1997; Lai, 1995).

Other molecules may also be produced by malignant cells which inhibit normal p53 function via binding to the p53 protein, enhancing degradation, or disruption of binding sites. One example of inactivation of p53, which may occur by interaction with another cellular protein, involves the murine double-minute-2 (MDM-2) protein which acts as a false binding site (Teoh, 1997). Another example involving induced degradation is seen in cervical cancer of the p53 protein (Caron de Fromentel, 1992; Vogelstein, 1992; Scheffner, 1992). The majority of

The purpose of this chapter is to describe data which identifies novel therapeutic approaches targeting correction of the p53 gene via transfection with a wildtype p53 gene using a replication defective adenoviral vector carrier and approaches utilizing oncolytic virus ONYX-015. 103


Nemunaitis: Gene therapy targeting p53 cervical cancers harbor the human papilloma virus (HPV), which enhance degradation of the p53 protein (Howley, 1991). Cervical cancer cells, which are HPV positive and contain the p53 mutation (less than 20%) are particularly aggressive, and such patients have an even more dismal prognosis.

the E1 and E3 regions provides empty space (~7KB) where the wildtype p53 gene sequence is inserted (Zhang, 1994). Transfection of several NSCLC cell lines and head and neck cancer cell lines reveal high expression of wildtype p53 protein (the transgene product). Optimal expression is observed at a multiplicity of infection (MOI) of 30-50 plaque-forming units (PFU) per cell (Zhang, 1995; Zhang, 1994). Maximal expression was observed 3 days after transfection and rapidly decreased over the next 5 days. Detection of the transgene product was still observed 15 days following transduction. Similar results were shown in vitro and in vivo. Transgene expression and normal function has been shown in cell lines of breast cancer, ovarian cancer, colorectal cancer, prostate cancer, the central nervous system, and bladder cancer (Harris, 1996; Wills, 1994; Lesoon-Wood, 1995; Blagosklonny, 1996; Bartek, 1990).

Poor survival prognosis has been observed in patients with cancer of the lung, colon, liver, breast, stomach, cervix, non-Hodgkinâ&#x20AC;&#x2122;s lymphoma, and multiple myeloma who have elevated p53 protein expression or a p53 DNA mutation detected from tumor samples prior to treatment (Drach, 1998; Horio, 1993; Thorlacius, 1993; Preudhomme, 1997). The development of p53 gene mutations may also involve environmental carcinogenic factors (Vogelstein, 1992). Malignant cells containing p53 mutations have an increased resistance to death in response to chemotherapeutic agents or ionizing radiation (Lee, 1993), and an increase in metastatic spread (Dutta, 1993). Twenty percent of patients with a p53 mutation have also been found to express antibodies to the mutant p53 protein, although it is unclear whether such patients have an altered prognosis (Crawford, 1982; Caron, 1987; Davidoff, 1992; Winter, 1992; Schlichtolz, 1992).

IV. Safety of the Advp53 vector The Adp53 vector is constructed from a serotype 5 adenovirus. A great deal of data has been accumulated suggesting the safety of this virus (Brandt, 1969). Eighty percent of adults have existing antibodies to adenovirus serotype 5 (Nicholson, 1993), but less 15% of exposed patients become clinically symptomatic. The most common symptoms of an adenoviral serotype 5 infection are flu-like in nature and include cough, gastroenteritis, conjunctivitis, cystitis, and rarely pneumonia. However, these symptoms are rarely seen even in immune compromised patients (Hierholzer, 1992). Oral adenoviral vaccines were given to thousands of military recruits in the 1960s without adverse effects or increase in cancer (Takafuji, 1979). Live adenovirus inocula was also given intratumorally and intra-arterially to patients with cervical carcinoma at the National Cancer Institute in the 1950s (Smith, 1956). No significant toxicities, other than transient fever and malaise, were observed even in subsets of patients treated with steroids and in those in which neutralizing adenovirus antibodies were not present.

In conclusion, an understanding of the p53 gene structure and protein function is important in developing therapeutic approaches, and may assist in the understanding of potential activity and toxicity to therapeutic approaches attempting to correct dysfunction of the p53 gene or protein.

III. Adp53 vector Preclinical studies have reported the introduction of the wildtype p53 gene into human tumor cells with a mutant p53 genotype using a variety of delivery methods including the retroviral vectors, lipid complexes, and adenoviral vectors (Harris, 1996; Wills, 1994; LesoonWood, 1995; Xu, 1997; Blagosklonny, 1996; Zhang, 1995; Nielsen, 1997; Nguyen, 1996). Results demonstrate that the expression of the transgene product provides a normal functioning wildtype p53 protein to the malignant cell, which has been shown to induce tumor regression and improve survival in animal models. Preclinical results also reveal enhanced activity when combined with chemotherapy (Nguyen, 1996; Fujuwara, 1994).

Work was conducted in animal models exploring the most significant serious clinical toxicity to live adenovirus (pneumonia). A unique strain of cotton rats (gigmodon hispidus) has been shown to consistently develop pulmonary infection in response to inoculation with adenovirus serotype 5 (Pacini, 1984). Pathogenicity was related to the dose of the viral inoculum. Additional safety testing has been conducted in mice and cotton rats in which high doses of adenovirus were injected locally and systemically. Animals developed minor histopathologic changes in several organs, but no pulmonary toxicity was observed (Pacini, 1984; Ginsberg, 1991). However, inflammatory infiltrates related to p53 have been observed in the lungs of animals given high doses of Adp53 directly

Vectors utilized for adenoviral introduction of the wildtype p53 gene involve wildtype adenovirus containing deletions of the E1 and E3 replication components (Zhang, 1993). Adenoviruses are single-stranded DNA viruses with genomes of approximately 35kB (Takahashi, 1989), which are easily propagated in human cells, and have been associated with minimal pathogenicity. The deletion of 104


Gene Therapy and Molecular Biology Vol 3, page 105 to the bronchial airway (Zhang, 1995; Ghosh-Choudjury, 1985; Englehardt, 1993; Rich, 1993; Ginsberg, 1990). The resulting inflammatory responses were characterized by interstitial infiltration of neutrophils, and monocytes within 1-2 days after exposure (Ginsberg, 1990; Prince, 1993). This early inflammatory process was felt to be mediated by local elaboration of various cytokines such as tumor necrosis factor, IL-1 and IL-6 (Prince, 1993). An additional inflammatory response also occurs within 3-7 days. At this time, peribronchial infiltration of lymphocytes is observed. Direct exposure of the lung with low concentrations of the adenovirus vector does not appear to be associated with pulmonary toxicity (Simon, 1993; Yei, 1994).

competent adenovirus. No replication competent adenovirus was detected, and elevated antibody formation did not inhibit gene expression with repeat injections (Tursz, 1996). Adenoviral vectors with E1 and E3 deletion containing the E-coli cytosine deaminase gene have also been administered to normal individuals to study immune response (Harvey, 1998). Six volunteers received intradermal injections of 106, 10 7, or 10 8 PFU (2 patients per group). Five of the 6 volunteers showed a rapid increase in anti-Ad5 neutralizing antibody titers above baseline. The peak antibody response occurred 2 weeks after vector injection. Erythema occurred at the site of injection with maximum induration of approximately 7mm by Day 3, and complete disappearance of induration by Day 10. Skin biopsies of the erythema revealed T-cell, B-cell and a macrophage infiltrate. Vector DNA was detected in biopsies of patients who received the 108 dose on Day 3, but no evidence of vector DNA was detected on Day 28. No systemic toxicity was observed in any of the normal volunteers (Harvey, 1998).

The possibility of adenoviral replication competency developing after vector injection also appears to be negligible, given the construction design of the vector (Zhang, 1995). However, complete inhibition of DNA replication solely from E1 deletion has not been 100% successful (Englehardt, 1993; Rich, 1993). This necessitates intense monitoring of the Adp53 clinical material for replication competency. Repeat sequencing of the product reveals that the wildtype p53 genotype does not undergo mutation changes during manufacturing. Expression of the transgene product also does not appear to be toxic. Studies performed in vitro looking at Adp53 transfection of non-malignant fibroblasts and human bronchial epithelial cells in comparison to malignant head and neck tumor cells indicate no change in p53 expression in non-malignant cells. These data suggest that normal cellular p53 expression is not altered by transfection with Adp53. The growth rate and morphology of the nonmalignant fibroblasts and bronchial epithelial cells was not altered following transfection with Adp53 (Zhang, 1995). Theoretical concerns regarding oncogenicity of adenoviruses are also unlikely. The life cycle of an adenovirus does not require integration into the host genome, thus, foreign genes delivered by adenoviral vectors are expressed episomally and have low genotoxicity (Zhang, 1995). DNA from thousands of human tumors have been analyzed for the presence of adenovirus DNA and no integrated viral DNA has been isolated from any human tumor (Green, 1979). Long- and short-term safety of adenoviral injection has been shown in several animal models (Lesoon-Wood, 1995; Zhang, 1995; Nielsen, 1997; Englehardt, 1993; Simon, 1993; Yei, 1994; Xu, 1998; Gomez-Foix, 1992; Le Gal La Salle, 1993).

Finally, if serious viral infection does develop, therapeutic approaches are available. Wildtype adenovirus dissemination has been seen in organ transplant recipients, however, in most cases, the viremia has been eliminated with the use of intravenous Ribavirine (Liles, 1979), although occasionally Ribavirine has not been successful (Mirza, 1994).

V. Preclinical studies with Adp53 Early preclinical studies with Adp53 vector in lung cancer initially utilized the H358 cell line. In one study, 50 mice received injections of 2 x 106 H358 cells, which had been previously transfected with Adp53 in vitro. Eighty percent of control animals developed tumors within 2-3 weeks; however, none of the p53 transfected cells evolved into malignant lesions 6 weeks after injection. Other work with the Adp53 vector involved the use of H326 cells which were derived from a highly aggressive squamous cell NSCLC lesion. This cell line contains a p53 point mutation (Zhang, 1994; Georges, 1993). Inoculation of 2 x 106 H326 cells into the trachea of mice followed by inoculation with Adp53 vector, control vector, or control vehicle, reveals that only 2 of the 8 Adp53treated mice developed tumors 6 weeks after treatment with a mean tumor volume of 8mm3, whereas 7 of 10 of the treated mice, and 8 of 10 of the control vector treated mice developed tumors where the mean volume exceeded 30mm3 within 6 weeks after inoculation. Subsequent approaches exploring the use of Adp53 in combination with Cisplatin revealed enhanced activity.

In humans, (-GAL vector injection was administered to patients with endobronchial lung cancer. Evidence of replication competent adenovirus was studied in caretaker staff samples. Specifically, 73 staff provided 78 blood samples, 272 urine samples, and 193 samples to study antibody formation or the presence of replication 105


Nemunaitis: Gene therapy targeting p53 Animal models have been designed to test whether transfection of head and neck cancer cells with Adp53 may alter response to radiation, chemotherapy or have direct effects. In one model, Adp53 was transfected into a radioresistent human cell line GSQ-3 (squamous cell carcinoma of head and neck). Wildtype p53 protein was shown to be expressed in high levels and have functional activity in the transfected cells (Xu, 1998). A dose of 108 PFU was shown to be sufficient to induce tumor regression without evidence of systemic toxicity (Liu, 1994; Yamamoto, 1998). Animal studies in other tumor xenograph models (ovarian, breast, prostate) have also shown activity following Adp53 injection (Sheikh, 1995; Eastham, 1995; Mujoo, 1996).

endothelial cells, airway epithelial cells, and mammary epithelial cells. Wildtype adenovirus showed cytopathic effects at a MOI as low as 0.01 virus particles within 8-10 days, whereas cytopathic effects of ONYX-015 virus were not observed until MOIs of >100 virus particles were achieved. Thus, safety and antitumor activity appear to be related to the dose of virus infused. Several studies involving oncolytic viruses other than ONYX-015 have been performed in vitro and in vivo in human patients without significant toxicity (Kenney, 1994; Russell, 1994; Asada, 1974; Smith, 1956). Unfortunately, the difficulties in characterizing viral load led to inconsistent results and there was no suggestion of efficacy. Preclinical studies with the ONYX-015 virus in vivo were performed to confirm direct tumor cell lysis through local injection and systemic infusion, and to determine whether or not tumor lysis is observed in response to viral replication (Yang, 1994).

VI. ONYX-015 preclinical studies P53 protein mediates cell cycle arrest via apoptosis if foreign DNA synthesis is occurring within a cell from viral replication (Debbas, 1993; Grand, 1994; Lowe, 1997). DNA tumor viruses such as certain adenoviruses, SV40 and human papilloma virus incode proteins which inactivate p53, thereby allowing their own replication (Debbas, 1993; Lechner, 1992; Gannon, 1987). Specifically, a 55dDa protein from the E1B region of adenovirus serotype 5 binds and inactivates p53 (Barker, 1987). Inability to block p53 function with deletion of the E1B region would enable the p53 protein to maintain its function thereby inhibiting viral replication. The ONYX-015 virus is a DNA adenovirus which was constructed with an E1B deleted region so that it no longer produces the 55kDa protein. In this manner, the virus would not be expected to proliferate in normal cells, but it would be expected to have extensive proliferative capacity in tumor cells which are either p53 mutant or have disrupted p53 function (Bischoff, 1996).

In animal human xenographt studies, intratumor injection of ONYX-015 virus has been tested in cervical cancer (C33 cervical carcinoma cells) and head and neck cancer (HLaC laryngeal carcinoma cells), both of which have a p53 functional deficiency (Heise, 1997). Significant tumor growth inhibition was observed compared to controls. Mice achieving a complete response remained disease-free for 4-6 months before sacrifice. U87 glioblastoma tumors, which do not have a p53 mutation, were not affected by injection with the ONYX-015 virus. Evidence of viral proliferation based on histochemical staining for adenovirus exon protein was confirmed in the sensitive tumors, but not in the U-87 tumors. Additional studies comparing vehicle versus chemotherapy (5-FU or Cisplatin), ONYX-015 alone, or ONYX-015 plus chemotherapy, were carried out (Heise, 1997). Median survival in mice receiving ONYX-015 plus 5-FU was further improved compared to control or ONYX-015 alone. Similar results were seen in combination with Cisplatin.

Initial studies testing the ONYX-015 virus involved incubation of virus with RKO human colon cancer cell lines which have normal p53 function and a subcloned line of RKO, which has a mutant p53 gene. The ONYX-015 virus replicated as efficiently as the wildtype adenovirus in the subclone lacking functional p53 protein, however, the cytopathic effects of ONYX-015 are reduced by 2 logs in the parent tumor line harboring normal p53 function (Bischoff, 1996). Cell lines resistant to ONYX-015 have also been made sensitive through transfection and expression of the E1B 55dDa gene (Bischoff, 1996). Cytopathic effects of ONYX-015 have also been shown in other malignant cells, which have abnormal p53 function, involving the breast, cervix, colon, central nervous system, liver, ovary, pancreas and head and neck region (Heise, 1997). Potential infectivity of ONYX-015 was tested against wildtype adenovirus by infecting nonmalignant (normal p53 functioning) human microvascular

Systemic injections of ONYX-015 at a dose of 108 PFU were also injected for 10 days into the tail vein of nude mice implanted with C33-a or HCT116 human xenographt tumors. Tumor growth was significantly inhibited in the C33-a tumors with ONYX-015 treatment by 55% compared with growth in mice injected with vehicle solution (p=0.004). Comparison of intravenous ONYX-015 virus (IV for 5 days) plus 5-FU (IP for 5 days) in mice showed that 6 of 7 mice had complete tumor regression following the combination, whereas only 2 of 7 mice achieved complete tumor regression following 5-FU treatment alone. The median tumor volume on day 40 was 93(L in the mice receiving ONYX-015 plus 5-FU. However, mice receiving 5-FU alone had a median tumor volume of 461(L, compared to ONYX-015 alone with a tumor volume of 671(L, and saline alone with a tumor volume of 748(L. No significant toxicity was observed. 106


Gene Therapy and Molecular Biology Vol 3, page 107 Results suggest that both intratumor and intra-venous infusion of ONYX-015 when combined with chemotherapy was safe and effective in inducing tumor regression and prolonging survival.

response (( 2-fold increase) was shown in 19 of 20 evaluable patients following course 1. Cytopathic effect assays (CPE) also revealed the presence of Adp53 vector in plasma within 30 minutes of intratumor injection in all 16 patients tested. Tumor biopsies collected 3 days posttreatment demonstrated p53 transgene expression by RTPCR in 10 of 17 (58%) patients receiving vector dose levels ( 3 x 1010 PFU, and only 8 of 27 (30%) patients who received the lower dose level. Toxicity attributed specifically to the vector was limited to transient fever and nausea. Cisplatin-related toxicity was not observed in any greater frequency than it would be expected when Adp53 gene vector was not combined with Cisplatin. Four patients fulfilled a definition of partial response (PR) (8%), 33 patients (64%) experienced stable disease for a transient period of time (minimum 1 month), 11 patients (20%) had progressive disease, and 4 patients (8%) were not evaluable for response (Nemunaitis, 1998; Swisher, in preparation; Nemunaitis, 1998). Overall, median survival was 149 days. The difference in survival between the patients who received Cisplatin or Adp53 + Cisplatin did not achieve statistical significance. Six of 12 patients with endobronchial-injected lesions had sufficient tumor regression to open obstructed airways.

VII. Human studies with Adp53 The first trial published to explore gene transduction of the p53 gene via intratumor injection in humans utilized a retroviral vector. In this trial, 9 patients (median age 68) with NSCLC were treated (Roth, 1996). Four received retrovector p53 gene via bronchoscopic injection, and 5 were treated via a percutaneous injection with CT guidance. Eight of the 9 patients treated had a point mutation, and 1 had a frame shift mutation of the p53 gene. Vector transduction was confirmed in 8 patients by PCR analysis, and 6 patients showed induction of apoptosis (TUNEL assay). Three patients showed evidence of tumor regression (all 3 of these patients received endobronchial injections). No toxic effects were attributed to the vector. Retroviral sequences were not detected in non-injected pulmonary tissue analyzed by PCR, and no evidence of replication competent retrovirus was detected. Unfortunately, low transduction efficiency associated with the retroviral vector was a major limiting factor.

The conclusion of this trial is that Adp53 endobronchial or CT-guided injections at a dose of 1011 PFU in patients with NSCLC are safe and well tolerated. The maximum tolerable dose of the vector has not been reached. This therapy can be administered monthly, alone or with Cisplatin with no increase in Cisplatin-related toxicity. Immune response to the Adp53 vector does not limit continued injections, and there is evidence of objective activity and clinical benefit.

Several studies with Adp53 were subsequently initiated. One Phase I trial investigating tolerability of Adp53 in NSCLC was recently completed. Fifty-two patients with advanced NSCLC who had previously failed conventional treatment were entered into trial (Swisher, 1998). Adp53 doses were escalated from 10 6 to 10 11 PFU and injected monthly into a single primary or metastatic tumor by bronchoscopy (12 patients) or computed tomographic (CT) guidance (40 patients). Patients were treated by direct assignment with or without Cisplatin (80mg/m 2) given IV over 2 hours prior to Adp53 injection. Each patient received up to 6 courses of treatment and median follow-up was 9.9 months. Vectorspecific deoxyribonucleic acid (DNA) was detected by PCR, and p53 transgene expression was determined by reverse transcriptase PCR and immunohistochemistry. Vector was present in plasma within 30 minutes of injection, and decreased in the next 60 minutes (Timmons, 1998). No replication competent adenovirus was detected in any body fluids tested. Antibody titers increased in patients receiving at least 2 doses and remained elevated for several months after completion of injections. In patients who received Cisplatin, the apoptotic index increase from 0.124 to 0.034 (p=0.011) when compared to baseline in samples harvested after the first course of Adp53 injection. The TUNEL assay showed an increase in the number of apoptotic cells in 11 of the 15 evaluable patients, a decrease in 2 patients, and no change in 2 patients (Nemunaitis, 1998). Anti-adenoviral type 5 IgG antibody

Additional work exploring the same Adp53 vector was done in head and neck cancer (Clayman, in press). In this trial, patients with recurrent or refractory squamous cell carcinoma of the head and neck region with a performance status of 0-2 were eligible for trial. Results of this trial concluded that repeated intratumoral injections of up 1011 PFU was safe and well tolerated. Transgene expression occurred despite evidence of adenovirus antibody response. Peri- and post-operative Adp53 injection had no adverse effect on surgical morbidity and/or wound healing. Evidence of activity based on tumor regression following injection of Adp53 was observed (1 CR, 2 PRs) (Clayman, in press; Wilson, 1998). Others have explored the use of Adp53 vectors in head and neck cancer and other tumor types such as colon cancer and ovarian cancer. In another Phase I trial using a different Adp53 vector (SCH-58500), 16 patients with head and neck cancer received escalated doses ranging from 7.5 x 109 PFU to 7.5 x 10 12 PFU (charts of patients received single or multiple intratumor injections). The median age of patients entered into this trial was 60.5 years. Ten of 16 107


Nemunaitis: Gene therapy targeting p53 patients had elevated serum IgG to p53 protein following injection, and p53 transgene expression was confirmed in a subset of patients. Toxicity attributed to the vector was limited to Grade 1/2 fever (11 patients) and injection pain (6 patients). One patient achieved a PR which correlated with the induction of apoptosis and transgene expression (Agarvala, 1998).

(Kirn, 1998). Injections were given throughout the perimeter of the tumor, and the volume of the injected medium was normalized to 30% of the target tumor volume. Neutralizing antibodies were found in 10 of 20 Phase II treated patients prior to injection, and the p53 gene sequence was mutated in 7 of 13 patients. There was also a suggestion of increased response in patients with tumor sized of (5cm in diameter. Thirty-seven percent of patients with tumor (5cm achieved a complete response or partial response compared to 0% of patients with tumor >5cm (n=30). The most frequent side effect observed in the Phase II trial was pain at the injection site and it occurred in 32% of patients. Transient fever and chills occurred in 28%, nausea in 8%, and confusion in 4% of patients. Despite these preliminary results and with the trial not yet completed, results are sufficient to determine that the ONYX-015 virus is well tolerated at a dose of 1010 PFU given to 5 consecutive day every 3 weeks. Subsequent studies exploring ONYX-015 virus (1 x 1010 PFU daily x 5 days every 3 weeks) combined with chemotherapy (Cisplatin 100mg/m2, IV on day 1; and 5FU 800-1,000mg/m2 by continuous infusion per day on days 1-5 every 3 weeks) were thus initiated. Patients with recurrent head and neck cancer who had not previously been exposed to chemotherapy or radiotherapy in the recurrent tumor setting were entered into trial. At the time of the preliminary analysis (Kirn, 1998), 10 patients had been treated and 9 of 10 patients achieved a partial response or complete response. Despite being preliminary, the data is very encouraging particularly when compared to expected response rates, in which similar patients receiving chemotherapy without ONYX-015 virus would be expected to achieve a 30-40% partial or complete response rate, and would not be expected to have a median survival >9 months.

Another trial utilizing SCH-58500 was performed in patients with colorectal cancer with liver metastasis. In this trial, 16 patients received hepatic arterial infusion of Adp53 vector. A single dose was administered prior to laparotomy. Patients received escalating dose levels ranging from 7.5 x 109 PFU to 2.5 x 10 12 PFU. Adverse events included fever in 15 of 16 patients, and headache in 3 of 16 patients. Transgene expression was confirmed in normal liver and tumor. No responses specifically attributed to the Adp53 therapy alone were observed, however, 12 patients subsequently received FUDR and 11 achieved a 50% reduction in disease, suggesting the potential for sequential therapeutic approaches to be considered in trial designs utilizing Adp53 (Agarvala, 1998). SCH58500 was also given to 18 patients with advanced NSCLC. Patients received escalating doses ranging from 107 to 10 10 PFU. No serious adverse events were observed. Only one patient required hospitalization for prolonged persistent flu-like symptoms. Transgene expression was confirmed in patients who received higher dose levels. In 4 of the 6 patients who showed evidence of wildtype p53 expression, progression of transient local disease was stabilized following injection with Adp53 (Schuler, 1998).

VIII. Human trials with ONYX-015 virus

These preliminary results suggest that ONYX-015 replicates in recurrent refractory head and neck cancer, and that ONYX-015 is well tolerated following intratumor injection alone, or when combined with chemotherapy.

Several trials with ONYX-015 virus in treatment of head and neck cancer were recently reported. These trials suggested that ONYX-015 is well tolerated except for transient low-grade fever and that antitumor activity is observed.

ONYX-015 is also being explored at escalating dose levels in patients with gastrointestinal tumors metastatic to liver (Bergsland, 1998). Patients with metastatic disease to the liver were administered intratumoral injections through CT guidance. The starting dose level was 1 x 108 PFU. Injections were given one time every 21 days. Patients not showing progressive disease were eligible for continued injections. A total of 16 patients and 29 injections had been administered at the time of this preliminary analysis, and the dose level of 1 x 108 PFU was reached without evidence of dose-limiting toxicity. Minor toxicities such as flu-like symptoms were observed in 11 patients, transient elevation and coagulation times were observed in 7 patients, lymphopenia in 5, and transient liver function enzyme elevations was observed in

Preliminary Phase I studies indicated that intratumor ONYX-015 injections are well tolerated and viral proliferation has been confirmed in malignant cells by electron microscopy. The duration of tumor response appeared to be greater in patients receiving multiple injections compared to a single injection per cycle (every 21 days). The optimal dose suggested for Phase II investigation was 1 x 1010 PFU given for 5 days every 21 days (unpublished results). Phase II studies performed in refractory head and neck patients utilized a dose of 1 x 10 10 PFU of ONYX-015 daily x 5 days every 3 weeks via intratumor injection 108


Gene Therapy and Molecular Biology Vol 3, page 109 4 patients. Response assessment after cycle 1 revealed 2 patients with minor responses, 9 patients with stable disease, and 4 patients with progressive disease. This is an ongoing trial in which patients are continuing to receive injections, and thus far it can be concluded that the treatment is well tolerated, although evidence of activity remains to be determined.

Overall, preliminary results of Phase I studies indicate that the p53 gene transfer through intratumoral injection using replication vectors is well tolerated, associated with antitumor activity at dose levels equal to and above 1 x 109 PFU. Data also suggest that administration of multiple injections and combination with chemotherapy or radiotherapy may enhance the overall antitumor effect. Phase II trials to determine efficacy are ongoing.

Others have also performed Phase I exploration of ONYX-015 in patients with unresectable carcinoma of the pancreas (Mulvihill, 1998). In another trial, escalating doses of ONYX-015 were administered to patients with to patients with unresectable pancreatic cancer. Sixteen patients received a total of 36 injections. At baseline, 5 of 13 tumors assessed contained mutant p53 gene sequences, and 9 of 10 patients had neutralizing antiadenoviral antibodies. All patients showed escalation of antiadenoviral antibodies following injection. One patient developed Grade 3 hyperbility rubimenia following the injection, otherwise no other Grade 3-4 toxicities were observed at dose levels up to 10 10 PFU. Grade 1-2 flu-like symptoms were reported in all patients. Four patients had minor regressions following the initial cycle of treatment with a 35-45% decrease in disease, 7 patients had stable disease, and 3 patients had progressive disease. Two patients reported a decrease in pain following injection. Preliminary conclusions are that the intratumor injection of ONYX-015 was well tolerated. Continued injections are ongoing.

Acknowledgment The author thanks Ana Petrovich for the manuscript preparation.

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IX. Conclusions

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Results of clinical trials performed are encouraging and shown good tolerability to a variety of Adp53 vectors and confirm that the transgene product expressed from the transfected vector is functional and associated antitumor activity in small numbers of patients. Unfortunately, therapy at this time is limited to direct intratumor injection. If immunologic difficulties leading to vector neutralization can be overcome, safety data suggest that systemic infusion of Adp53 vector may be well tolerated. Studies to limit immunoreactivity to the Adp53 vector through inhibition of the immune response or alteration of the vector or other gene transfer vehicles are ongoing. For instance, using a ligand lyposome complex, wildtype p53 gene was efficiently delivered both in vitro and in vivo in murine squamous cell head and neck cancer models. Injection of the ligand/lyposome complex with the wildtype p53 gene was shown to be taken up in both head and neck and prostate tumors. Transfection was higher in malignant tissue than surrounding normal tissue. Furthermore, enhanced activity was shown following treatment with radiotherapy after ligand/lyposome encapsulated wildtype p53 injection or IV infusion (Pirollo, 1998), without significant toxicity (Joshi, 1998).

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Gene Therapy and Molecular Biology Vol 3, page 113 Gene Ther Mol Biol Vol 3, 113-121. August 1999.

Targeted therapy of CEA-producing cells by combination of E. coli cd/HSV1-tk fusion gene and radiation Research Article

Dao-song Xu1 , 2 Xin-yao Wu 1 Yun-fei Xia 3 Ling-hua Wu 3 Chao-quan Luo 1 Yinhao Yang 1 Lu-qi Zhong 4 and Bin Huang4 1

Department of Biochemistry, and 3Department of Radiation of Tumor Hospital, and 4Experimental Animal Center at Sun Yat-sen University of Medical Sciences, Guangzhou, 510089, P. R. China __________________________________________________________________________________________________ 2

Present address: Heinrich-Pette-Institute for Experimental Virology and Immunology at the University of Hamburg, MartinistraĂ&#x;e 52, D-20251 Hamburg, Germany. Tel: 0049-40-4805 1212. E-mail: xu@hpi.uni-hamburg.de Correspondence: Xin-yao Wu, Ph.D. Professor, Department of Biochemistry, Sun Yat-sen University of Medical Sciences, Guangzhou, 510089, P.R. China. E-mail: xyaow@gzsums.edu.cn Received: 5 September 1998; revised and accepted: 23 October 1998

Summary To enhance the specific cytotoxic effects caused by the transfer of the E . c o l i cytosine deaminase (cd) and HSV1-tk to CEA (carcinoembryonic antigen)-producing cells, the expression of the cd-tk fusion gene, driven by the CEA promoter, was investigated followed by treatment with 5-FC and GCV in combination with radiation. The expression vector pCEAcd-tk, based on pcDNA3, was introduced into CEA-producing cells using liposomes. In CEA-producing cells, the CEA promoter could efficiently drive the expression of the fusion suicide gene. The expression activity of the E. coli cd gene driven by the CEA promoter was about three times higher than that driven by the CMV promoter in transfected LoVo cells. A combination of 5-FC and GCV could cause higher c y t o t o x i c i t y t o t h e c e l l s e x p r e s s i n g C D and TK than the use o f a s i n g l e prodrug alone. The cytotoxic effect after combining the two prodrugs with radiation was the highest among all treatments i n v i t r o . I n v i v o , the result of a subrenal capsule assay showed that the inhibition rates for 5-FC (0.5 mg/g) and GCV (0.1 mg/g) to GLC-82 cells transfected with pCEAcd-tk were 18.04% and 55.00%, respectively. A combination of the prodrugs at the same dose resulted in a 152.50% inhibition rate. In addition, the bystander effect exerted by the pCEAcd-tk/5-FC+GCV system in v i t r o was greater than that induced by cd/5-FC or tk/GCV alone.

CD, which can convert the nontoxic prodrugs, ganciclovir (GCV) and 5-fluorocytosine (5-FC), respectively, into metabolites highly toxic to the genetically-modified tumor cells. Experimental results showed that use of the E. coli cd/5-FC or of the HSV1-tk/GCV systems could inhibit the growth of CEA-producing tumor cells in vitro and in vivo (DiMaio et al., 1994; Richards et al., 1995; Lan et al., 1997). However, it has been observed that some tumor cells were resistant to E. coli cd/5-FC or HSV1-tk/GCV (Golumbek et al., 1992; Mullen et al., 1994; Bennedetti et al., 1997).

I. Introduction CEA (carcinoembryonic antigen)-positive tumors are common clinically. At present, there are no efficient therapeutic measures, especially for the patients who are in the mid- or final stages of this disease. Gene therapy may show its strength as an effective method for treating this carcinoma. The herpes simplex virus type I thymidine kinase (HSV1-tk) and the Escherichia coli cytosine deaminase (E. coli cd) genes are commonly used as suicide genes. The expression products of these two nonmammalian genes are two enzymes, HSV1-TK and E. coli 113


Xu et al: cd/tk fusion gene and radiation in cancer eradication The treatment efficiency of the suicide gene/prodrug system mostly depends on the expression efficiency of the introduced suicide gene in the tumor cells. Therefore, the promoter used to drive the expression of a suicide gene is very important. The most commonly used promoters are viral promoters. However, viral promoters are easily inactivated in mammalian cells, resulting in an unstable and low-efficiency expression of a suicide gene. In addition, viral promoters lack the cell-specific acitivities, which could repress expression of a suicide gene in normal cells. Using a retrovirus vector, high expression of the introduced gene was found only in a small subset of the transfected cells; most of the transfected cells did not display the expression product of the introduced gene (Mullen, 1994).

II. Results A. Enzymatic activities of CD and TK Using PCR, a single fragment of about 450-bp was observed on 2% agarose gels (Figure 1A). The same fragment could be amplified from different healthy donors. Sequencing analysis showed that there was only one base mismatch in the CEA promoter fragment of pCEA (Figure 1B), compared with the CEA promoter sequence published by others (Richards, et al., 1993). CEA quantitation in the cell lines was measured by RIA (radioimmuno assay). CEA concentrations were found to be different in different cell lines (Table 1): LoVo and HT-29 cells displayed the highest levels of CEA (581.4 and 316 nm/mg of cellular lysate, respectively). On the contrary, CEA was not detected in BEL-7402 cells. When cdc (E. coli complementary cd gene obtained by PCR, but using the antisense primer but leaving the native stop codon unchanged) was used as an indicator enzyme, the activity of CEA promoter driving its expression was 3.14 times higher than that of CMV promoter in LoVo cells (Table 2).

Over-expression of the CEA gene was a special feature for CEA-positive tumors, and the high level of CEA in physiological fluids has been used for early diagnosis and as a marker of treatment efficiency (Shively & Beatty, 1985; Thomas et al., 1990). Although there was some CEA expression in the normal epithelial cells of the colon, the level was very low (Baranov et al., 1994; Egan et al., 1977). The over-expression of CEA gene resulted from the activated CEA promoter and not from a mutation in the CEA promoter causing its upregulation (Schrewe et al., 1990; Jothy et al., 1993). The CEA promoter occupies a stretch of 420-bp upstream of the translation start site of the CEA gene (Chen et al., 1995; Richards et al., 1995). In CEA-positive cells, specific trans-acting elements are present which activate the CEA promoter. Because of these properties, the CEA promoter could be used to drive the expression of therapeutic genes only in CEA-positive tumor cells; in this context, the E. coli cd and HSV1-tk genes have been used (Osaki et al., 1994; Richards et al., 1995).

Through sequence analysis, our cd sequence was the same as the E. coli cd sequence of the GenBank No s56903 except that of the start and the stop codons which had been changed on purpose. Compared to the HSV1-tk of Genbank No. v00470, one base mismatch leads to the change of the 17th alanine to valine in the tk used in our experiment (data not shown). In transfected BEL-7402 cells, no CD activity was detected. In pCEAcd-tk transfected cells, the activities of CD and TK were measured respectively (T a b l e 3). The results indicate that all CEA-producing cells have higher enzymatic activities than the corresponding parental cells. In non-transfected BEL-7402 cells, there is a low relative activity of TK. It is the activity of cellular TK, not HSV1TK, because 3HdT was used as the substrate.

The mechanism of cell killing by radiation proceeds via damage of the strands of cellular DNA. Cells able to repair the damaged DNA will survive. Because the E. coli cd/5FC and HSV1-tk/GCV systems kill cells through inhibition of DNA synthesis, they could also be used as radiosensitizing agents. It was found that both the E. coli cd/5-FC and HSV1-tk/GCV systems could enhance the sensitivity of cells to radiation (Khil et al., 1996; Rogulski et al., 1997).

Table 1. Concentration of CEA protein in the tumor cell lines Cell line

Here we investigate whether a combination of E. coli cd/5-FC, HSV1-tk/GCV and radiation exert a greater cytotoxic effect to tumor cells, especially to CEAproducing tumor cells. Use of the CEA promoter can limit the expression of the fusion suicide gene in CEAproducing cells. Under these circumstances, treatment with the two prodrugs and application of a low-dose radiation had a much higher cytotoxicity to the tumor cells while minimizing side-effect to normal cells.

LoVo HT-29

Concentration of CEA (ng/mg of cellular lysate) 581.4 316.8

SGC-7901

60.6

GLC-82

80.2

BEL-7402

BT*

The quantity of CEA protein was measured by use of RIA (Radioimmuno assay) method. * below the threshold of 5 ng.

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Xu et al: cd/tk fusion gene and radiation in cancer eradication

F i g u r e 1 . A . Amplification of CEA promoter. The PCR products were separated on 2% agarose gel. Lane 1, DNA molecular weight marker, Lambda DNA/EcoRI+HindIII; lane 2-9, PCR products from peripheral blood cell genomic DNA of healthy individuals. B . Comparion of the CEA promoter sequence with published CEA DNA sequence in the 5´non-translation region. Query: the sequence of CEA promoter used in the experiment; subject: part of the published CEA DNA sequence (Genbank No: z21818).

Table 2 . Enzymatic activities of CD in tumur cells (specific activity) Cell line

Parental

Tansfected with pCEAcdc

Transfected with pcDNA3cdc

LoVo

0

2748

875

HT-29

0

2034

-

SGC-7901

0

670

-

GLC-82

0

714

-

BEL-7402

0

0

-

Specific activity was defined as nmol of cytosine deaminated/min/mg protein. It was measured spectrophotometrically as a decrease in absorbance at 285 nm, in a 1-ml assay mixture containing cell extract in 50 mM Tris-HCl, pH 7.3, 0.5 mM cytosine. The product was estimated using a molar extinction coefficient 1.038 !10 litre/mol/cm. Table 3 . Enzymatic activites of CD and TK in the cells transfected with pCEAcd-tk Enzymatic activity Cell line LoVo

CD* 2516

TK# 238.1

HT-29

1603

149.2

SGC-7901

421

84.7

GLC-82

507

69.4

BEL-7402

0

10.8

*: specific activity; #: ralative activity to TK actvity in the pcDNA3tk-transfected cells.

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Gene Therapy and Molecular Biology Vol 3, page 115

F i g u r e 2 . Additive cytotoxic effect of combined use of 5-FC and GCV to tumor cells expressing cd-tk. SEMs (standard error means) were presented by error bars(n=3).

F i g u r e 3 . In vitro bystander killing effect of pCEAcd-tk/5-FC+GCV. The pCEAcd-tk transfected LoVo cells were mixed with 3 non-transfected LoVo cell in different portions. The mixed cells were seeded on a 96-well plate at a density of 2 ¥10 cells per well. 24 hrs later, the cells were exposed to 0.5µM GCV and 100 µM 5-FC. After the cells were incubated for 72 hrs, the surviving rates were measured by MTT assay. Bars, SEMs (n=3).

treatment with 5-FC plus GCV produces 5.4% surviving rate. When the same dose of 5-FC or GCV was used alone, the survival rates were 40.2% and 56.7%, respectively. The subrenal capsule assay (SRCA) result indicates that the tumor inhibition rate is much higher when using a combination of the two prodrugs in nude mice (Table 5).

B. Cytotoxicity of 5-FC and GCV to tumor cells expressing CD and TK CEA-producing cells transfected with pCEAcd-tk become more sensitive to 5-FC and GCV than parental cells as deduced from growth inhibition in vitro measuring the IC50 (concentration of 50 % growth inhibition) (Table 4). Use of 5-FC in combination with GCV has a remarkable additive cytotoxic effect to CEA-producing cells expressing CD and TK (Figure 2). In addition, the in vitro pCEAcdtk/5-FC+GCV system has a higher bystander effect than cd/5-FC or tk/GCV (Figure 3). In the 20% group,

C. Radiosensitization of pCEAcd-tk/5FC+GCV When 100 µMol 5-FC or 0.5 µMol GCV is added to LoVo cells transfected with pCEAcd-tk, 6.5 Gy and 4.9 115


Gene Therapy and Molecular Biology Vol 3, page 113 Table 4 . The IC50 of tumor cells to 5-FC and GCV I C 5 0 SD( µM ) Cell line LoVo* LoVo HT-29* HT-29 SGC-7901* SGC-7901 GLC-82*

5-FC 67.2±

GCV

26.8

0.75±

9650.34± 563.00 162.70±

0.86±

9580.50± 762.58 287.57±

17.30± 12.83±

143.61

3360.80

58.88

977.56

37.78

51.50

59.12

74.09

1.22

1.15

5.16

890.91±231.73

81.34

GCV

0.24

840.70±125.42

40.48

10865.20± 481.82 232.10±

5-FC

0.16

25250.60±430.85

56.20

Ratio IC50#

7.51

GLC-82

13720.63±2407.38

950.60±124.30

BEL-7402*

9080.85± 375.31

678.59± 35.70

BEL-7402

11070.14±2512.17

780.41±147.20 3

Cells were seeded at a density of 2!10 cells/well on 96-well plates. Different concentrations of 5-FC, GCV were added. After 72hrs, the percentage of growth inhibition was measured by the MTT assay. The results represent mean±SD(n=3). IC50=the concentration of 50% growth inhintory rate. * cells transfected with pCEAcd-tk; # parental cell IC50/transfected cell IC50 to 5-FC or GCV.

Table 5 . In vivo growth inhibition of pCEAcd-tk transfected GLC-82 cells by 5-FC and GCV Drug

Dose (mg/gm)

Schedule

Do

Dn

D n-D o

Inhibition rate(%)

Control

0.04*

1/d!2

40.5±6.26

43.8±10.36

3.3± 9.01

----

5-FC

0.5

1/d!2

39.7±5.93

42.5±12.68

2.7±11.34

18.04

GCV

0.1

1/d!2

36.6±5.55

38.1±11.3

11.5± 9.44

55.00

5-FC+GCV

0.5+0.1

1/d!2

41.9±4.68

40.2±16.6

-1.7±16.91

152.50

The prodrugs were given by the intraperitonal injection. *: ml of 0.9% NaCl per gram; Do: tumor volume before transplantation, Dn: tumor volume after the animal was sucrificed.

difference, 109 A"G (Figure 1B). It was found that the essential part of CEA promoter was located between nucleotides 295-318 (Richards, et al, 1995). Through footprinting, Chen, et al (1995) and Hauck and Stanners (1995) found that there were 5 FP (footprinting) regions in the cis-acting sequence of the CEA promoter, in which FP1-4 represented the positive regulatory elements whereas FP5 (-568 to -560, where +1 is the start of translation) represented the negative regulatory elements. Sp1 and Sp1like factors could bind to Fp1, FP2 and FP3. The protein bound to FP4 was AP4. The only different base in the CEA promoter used in our experiments is in the FP4 region. Although the FP4 region was not an essential part of the CEA promoter, it may affect the activity of the CEA promoter. Using a common Taq DNA polymerase, an active CEA promoter could be obtained (DiMaio et al., 1994), but a low activity CEA promoter was observed (Osaki et al., 1994). It is not clear which bases play a key role in the CEA promoter activity. It is possible that a more efficient CEA promoter can be constructed by

Gy, respectively, were required to reduce the surviving fraction to 0.01 (Figure 4A). When a combination of the same dose of 5-FC plus GCV was used, only 4.2 Gy were required to obtain the same survival fraction. The pCEAcdtk/5-FC+GCV system had a similar effect on GLC-82 cells (Figure 4B).

III. Discussion It is possible that the mutation in the CEA promoter can affect its activity and cell-specificity. In our experiments, the CEA promoter is obtained using a high fidelity DNA polymerase. The CEA promoter shows a higher activity in CEA-producing cells. In LoVo cells its activity was 3.14 times higher than that of the CMV promoter (using the E. coli CD as the indicator). The sequence of the CEA promoter used in our experiments is almost identical to that of the CEA promoter sequence published before (Richards, et al., 1993) except one base

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Xu et al: cd/tk fusion gene and radiation in cancer eradication changing some bases in the CEA promoter sequence, which may be much better suited for targeting expression of a suicide gene to CEA-producing tumor cells.

found that the bystander effect of the combined use is enhanced in vivo. Although HSV1-tk/GCV, E. coli cd/5-FC system could effectively kill tumor cells in vitro and in vivo, the efficiency between these two systems were different to some kinds of tumors. E. coli cd/5-FC therapy was more effective than HSV1-tk/GCV to pulmonary adenocarcinoma (Hoganson et al., 1996). In vivo, human colorectal carcinoma cells were more effectively eradicated by E. coli cd/GCV than HSV1-tk/GCV (Trinh et al., 1995). Most gastric-intestinal and lung carcinomas are CEA-positive. On the other hand, tumor microenvironment can determine the cell radiosensitivity, but the sensitivity of tumor cells to radiation also is dependent on intrinsic cellular factors. Both HSV1-tk/GCV and E. coli cd/5-FC could alter the cellular factors, and enhance the radiosensitivity (Kim et al., 1994, 1995; Khil et al., 1996; Rogulski et al., 1997). Most CEA-positive tumor cells, for example pulmonary adenocarcinoma cells, are not sensitive to radiation. Therefore, it is much more effective to use a combination of these two systems to kill these tumor cells.

Although the essential sequence for an active CEA promoter is known (Richards et al., 1995), the mechanism of activating CEA promoter is unclear. Our results indicate that the activities of CD and TK in different CEAproducing cell lines transfected with pCEAcd-tk are different. The enzymatic activity shows a positive relationship to the concentration of CEA in the cells. An active CEA promoter is determined by the interaction of a cis-acting sequence with trans-acting elements. We found that the nuclear proteins binding to the CEA promoter were different between LoVo and BEL-7402 using gel mobility shift assays (data not shown). The different enzymatic activities may reflect the different interactions involving these elements. Combined therapy to tumors can enhance the cytotoxicity and beneficial effect from each therapeutic regime. Using cotransfection of cells with HSV-tk and E. coli cd, Uckert, et al. (1998) found that the combination of the two genes was the most effective for killing tumor cells both in vitro and in vivo, and only this combination could cause complete eradication of tumors in vivo. Rogulski et al. (1997) revealed that the combined use of cd-tk/5-FC+GCV and radiation had a strong cytotoxic effect to 9L tumor cells. The best way to treat tumors is to kill only tumor cells without any severe damage to healthy cells. So it is important to limit the expression of a suicide gene only in tumor cells before using the prodrug. At present, two ways, targeting vectors and targeting transcription (see review by Miller & Whelan, 1997), can be used. We used the strategy of targeting transcription, and the pCEAcd-tk/5-FC+GCV system showed a strong cytotoxic effect to the CEA-producing cells. In addition, high concentration of GCV or 5-FC could cause remarkable nonspecific toxicity to nontransfected cells (Beck, et al., 1995, Cool, et al., 1996). Use of the combination of these two systems will reduce the dose of each prodrug, whereas the cytotoxic effect can be enhanced. In our experiments the doses of 5-FC and GCV are much lower than the â&#x20AC;&#x153;safeâ&#x20AC;? concentrations of these in human blood. If higher doses of the prodrugs are used, the pCEAcd-tk/5-FC+GCV might kill tumor cells even more efficiently reducing the possibility of converting tumor cells to become resistant.

There were some limitations for treating pulmonary adenocarcinoma cells by use retrovirus-mediated HSV-tk gene transfer (Zhang et al, 1997). Song et al. (1997) found that injection of a pcDNA3-liposome mixture could cause the highest expression of an exogenous gene in mouse lungs. In addition, the most common reason for mortality of patients with colon carcinoma is hepatic metastases. In normal lung and liver tissues, the CEA gene is not expressed. If pCEAcd-tk is non-specifically transfected into these normal cells, the suicide gene will not be expressed since the CEA promoter is in an inactive state. After use of prodrug, no toxic metabolite of the prodrug will be produced in the normal cells, thus reducing the side effects of suicide gene/prodrug therapy to normal cells. The therapeutic system, pCEAcd-tk/5-FC+GCV accompanied with low dose of radiation, may become a useful tool for the eradication of CEA-producing tumors.

IV. Materials and methods A. Vector construction Two primers were used to amplify the CEA promoter from the genomic DNA of peripheral blood cells from healthy blood donors, 5'-GTA TCG CGA ATC ATC CCA CCT TCC CAG AG-3' (sense), 5'-GGG AAG CTT TGT CTG CTC TGT CCT CCT C-3' (antisense). The high-fidelity Pwo polymerase (Boehringer Mannheim Co.) was used to amplify a 438-bp CEA promoter. The amplified fragment was cut with NruI and HindIII, and then the CMV promoter in pcDNA3 (Invitrogen) was replaced with this fragment, resulting in the vector pCEA, in which the CEA promoter fragment was ensured by direct

The mechanisms of the bystander effect of E. coli cd/5FC and HSV1-tk/GCV are not completely clear, but clear differences between these two systems have been observed (Denning & Pitts, 1997). The combined use of the two systems could promote the bystander effect (Rogulski, et al., 1997, Uckert, et al., 1998). In agreement with this we

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Xu et al: cd/tk fusion gene and radiation in cancer eradication

3

F i g u r e 4 . The radiative enhancing effect of pCEAcd-tk/5-FC+GCV. A: LoVo; B: GLC-82. 2 ¥10 cells/well were seeded on 96-well plates, and then 100 µM 5-FC and 0.5µM GCV were added. 72hrs later, the cells were irradiated with different doses of Xrays. After 6 days, the cell number in each well was estimated by MTT assay according to the standard calibration curves. SEMs (n=3) were omitted for clarity.

Military Medical University and Experimental Animal Center, Sun yat-sen University of Medical Sciences) were cultivated in RPMI 1640 (GIBCO-BRL) medium with 10% fetal calf serum, 100 units/ml penicillin and 100µg/ml streptomycin. No mycoplasma was detected by PCR. Cells were transfected with pCEAcd-tk by use of ESCORT transfection reagent (Sigma), and the positive clones were selected with G418(GIBCO-BRL) for fourteen days. These cells were used for measuring the enzymatic activities of E. coli CD and HSV1-TK, and for cytotoxicity assay.

dideoxynucleotide sequencing. The primers, (sense) 5'-GGG AAG CTT ACC ATG TCG AAT AAC GCTTTA C-3' (with a HindIII cut site in 5' end) and (antisense) 5'-CGC GGATCC TCC ACG TTT GTA ATC GAT GGC-3'(with a BamHI cut site in 5' end) were used to amplify the E. coli cd gene from chromosomal DNA of JM109 bacteria. In the sense primer, the initial context was changed into the Kozak sequence (Kozak, 1986). The stop codon (TGA) of E. coli cd was changed into GGA (encoding for glycine), leading to read through downstream HSV1-tk gene. The other two primers were used to amplify the HSV1-tk gene from the plasmid pHSV106 (GIBCO-BRL). The sense primer was 5'-CGC GGA TCC GGC GGG GGC GGT GGA GGA GGG GGT ATG GCT TCG TAC-3', in which there was a BamHI cut site and eight codons for glycine. The antisense primer was 5'-CGG GAA TTC CCT TCC GGT ATT GTC TCC TTC CGT-3'(with EcoRI cut site) (Rogulski et al., 1997). The ligation and identification of inserted fragments by using restriction enzyme analysis was carried out according to methods described (Sambrook et al., 1989). The amplified fragments were cut with relevant restriction enzymes, and then inserted into the MCS (multiple cloning site) of pCEA, resulting in the expression vector, pCEAcd-tk. Between the cd and tk, there was a linker which encoded ten glycines and one serine.

C. Enzymatic activities of CD, TK and cytotoxicity assay E. coli CD activity was measured according the method described (Austin & Huber, 1993). The buffer was 50 mM TrisHCl (pH7.3), 0.5 mM cytosine (Sigma). Specific activity was defined as nmol of cytosine deaminated/min/mg proteins. The 3 molar extinction coefficient was 1.038 ¥10 litre/mol/cm. TK activity was detected as follows: 25 µl of cell extract, 75 µl of reaction buffer contained 50 mM Tris-HCl (pH7.5), 10mM ATP, 10 mM MgCl2, 10 mM #-mercaptoethanol, 10 mM NaF, 3 50 µg/ml PMSF(Sigma) and 2µmol/L HdT (20ci/mmol). The mixture was incubated at 37 °C for 30 min, and then 100 µl of reaction mixture was dropped onto DE-81 filter paper (Whatman). The paper was washed with 95% ethanol three times, and then put in 5-ml scintillation liquid for measuring CPM. The relative activity of TK was defined as follows:

B. Cell culture and transfection The cell lines, LoVo, HT-29 (human colon carcinoma) and GLC-82 (human lung adenocarcinoma), SGC-7901 (human stomach carcinoma), BEL-7402 (human hepatoma) were used. LoVo, HT-29 (ATCC) and other cell lines (provided by the first

CPM/mg of proteins in the cells transfected with pCEAcd-tk ! 100% CPM/mg of proteins in the cells transfected with pcDNA3tk

114


Xu et al: cd/tk fusion gene and radiation in cancer eradication In pcDNA3tk, the CMV promoter drove the expression of HSV1-tk gene cut from pHSV106 with BglII and EcoRI.

cloning, sequencing, and expression of Escherichia coli cytosine deaminase. M o l . P h a r m a c o l . 43, 380-387.

The cytotoxicity assay was carried out by MTT (Sigma) 3 assay. In a 96-well culture plate, 2x10 cells/well were seeded, and the different concentrations of 5-FC (Sigma) and GCV (Roche) were added. After 72 hrs, 10 µl of MTT (5 mg/ml) was added into each well and incubated in 37 °C for 4hrs. The supernatant was discarded and 150 µl/well of DMSO was added. The absorbance (A) was measured at 570 nm. The survival rate=Atreated/Acontrol x100%. The experiment was performed three times.

Baranov, V., Yeung, M.M., and Hammarstrom, S. Y. (1 9 9 4 ). Expression of carcinoembryonic antigen and nonspecific cross-reacting 50-kDa antigen in human normal and cancerous colon mucosa: comparative ultrastructural study with monoclonal antibodies. Cancer R e s . 54(12), 3305-3314. Beck, C. Cayeux, S. Lupton, S.D. Dorken, B. and Blankenstein, T. (1 9 9 5 ). The thymidine kinase/ganciclovir-mediated "suicide" effect is variable in different tumor cells. Hum. Gene Ther. 6, 1525-1530.

The sensitivity of tumor cell expressing CD and TK was carried out according to the method described by Price & McMillan(1990). An X-ray instrument was used, and the dose rate was 106.82 cGR/min. The surviving fraction was calculated as follows:

Benedette, S., Dimeco, F., Pollo, B., Cirennei, N., Colombo B.M., Bruzzone, M.G., Cattaneo, E., Vescovi, A., Didonato, S., Colombo, M.P., and Finocchiaro, G. (1 9 9 7 ). Limited efficacy of the HSV-TK/GCV system or gene therapy of malignant gliomas and perspectives for the combined transduction of the interleukin-4 gene. Hum. Gene Ther. 8, 1345-1353.

(Cell number in the control well) divided by (Cell number in the radiated well or prodrug/radiation-treated well) x100%. The data and charts was processed and produced by the Department of Radiobiology, Tumor Hospital of China Academia.

Chen, C.J., Li, L.J, Maruya, A., and Shively, J.E. (1 9 9 5 ). In vitro and in vivo footprint analysis of the promoter of carcinoembryonic antigen in colon carcinoma cells: effects of interferon gamma treatment. C a n c e r R e s . 55, 3873-3882.

D. Radiosensitization

Cool V, Pirotte B, Gerard C, Dargent JL; Baudson N, Levivier M, Goldman S, Hildebrand J, Brotchi J and Velu T (1 9 9 6 ). Curative potential of herpes simplex virus thymidine kinase gene transfer in rats with 9L gliosarcoma. Hum. Gene Ther. 7, 627-635.

E . I n v i v o studies GLC-82 cells transfected with pCEAcd-tk were inoculated subcutaneously into 6-8 wk BALB/C-nu/nu mice, and tumors were allowed to grow for about one month. Afterwards, the tumor tissue was surgically removed and cut into 1-mm size fragments which were implanted under the renal capsules of BALB/C-nu/nu mice. 5-FC and GCV were delivered by intraperitoneal injection at days 2 and 3. 10 days later, the 2 3 animals were sucrificed. The tumor volume = (a xb ) /2 (mm ), where a is: the longest diameter of the tumor, and b: the shortest diameter. The tumor inhibition rate was calculated as follows: (Dn - Do in control) - (Dn - Do in subject )

Denning, C., and Pitts, J.D. (1 9 9 7 ). Bystander effects of different enzyme-prodrug systems for cancer gene therapy depend on different pathways for intercellular transfer of toxic metabolites, a factor that will govern clinical choice of appropriate regimes [see comments]. Hum. Gene Ther. 8, 1825-1835 Dimaio, J. M., Clary, B. M., Via, D.F., Coveney, E., Pappas, T.N., and Lyerly, H. K. (1 9 9 4 ). Directed enzyme pro-drug gene therapy for pancreatic cancer in vivo. Surgery. 116, 205-213.

x100%

Dn - Do in control

Egan, M. L., Pritchard, D. G., Todd, C.W., and Go,V.L. (1 9 7 7 ). Isolation andimmunochemical and chemical characterization of carcinoembryonic antigen-like substances in colon lavages of healthy individuals. Cancer Res. 37, 2638-2643.

Do: the tumor volume before translated into subrenal capsule; Dn: the tumor volume after the mouse was sacrificed.

Acknowledgements

Golumbek, P.T., Hamzek, F.M., Jaffee, E. M., Levitsky, H., Lietman, P.S., and Pardoll, D. M. (1 9 9 2 ). Herpes simplex-1 virus thymidine kinase gene is unable to completely eliminate live, nonimmunogenic tumor cell vaccines. J. Immunother. 12, 224-230.

We are indebted to Prof. Lin Lu and Drs. Yi-fang Chen for supplying vectors, and Department of Medicine of the First Military Medical University (Guangzhou, China) for supplying cell lines. This work is supported by the grants from China Fundation for Natural Sciences to X.Y. Wu, and from Research fundation of SUMS to D.S. Xu and X. Y. Wu.

Hoganson, D.K., Batra, R.K., Olsen, J.C., and Boucher, R.C. (1 9 9 6 ). Comparison of the effects of three different toxin genes and their level of experssion on cell growth and bystander effect in lung adenocarcinoma. C a n c e r R e s . 56, 1315-1323

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Gene Therapy and Molecular Biology Vol 1, page 123 Gene Ther Mol Biol Vol 3, 123-131. August 1999.

Efficacy of antiherpetic drugs in combined gene/chemotherapy of cancer is not affected by a specific nuclear or cytoplasmic compartmentation of herpes thymidine kinases Research Article

Bart Degrève1, Erik De Clercq1, Anna Karlsson2, and Jan Balzarini1 1

Rega Institute for Medical Research, Laboratory of Virology and Chemotherapy, B-3000 Leuven, Belgium

2

Karolinska Institute, Department of Immunology, Microbiology, Pathology and Infectious diseases, Division of Clinical Virology, S-141 86 Stockholm, Sweden __________________________________________________________________________________________________ C o r r e s p o n d i n g author: Jan Balzarini, Ph.D., Rega Institute for Medical Research, Katholieke Universiteit Leuven, Minderbroedersstraat 10, B-3000 Leuven, Belgium. Tel: +32-16-337352; Fax: +32-16-337340; E-mail, jan.balzarini@rega.kuleuven.ac.be Abbreviations AA, amino acid; ACV, 9-(2-hydroxyethoxymethyl)guanine (acyclovir); araT, 1-!-D-(arabinofuranosyl)thymine; BCV, (R)-9[(3,4-dihydroxybutyl)guanine] (buciclovir); BVDC, (E)-5-(2-bromovinyl)-2’-deoxycytidine; BVaraU, (E)-5-(2-bromovinyl)-1!-D-arabinofuranosyluracil; BVDU, (E)-5-(2-bromovinyl)-2’-deoxyuridine; FIAC, 1-(2’-fluoro-2’-deoxy-!-D-arabinofuranosyl)5-iodocytosine; FMAU, 1-(2’-fluoro-2’-deoxy-1-!-D-arabinofuranosyl)-5-methyluracil; GCV, 9-(1,3-dihydroxy-2propoxymethyl)guanine (ganciclovir); GFP, green fluorescent protein; HSV-1 and HSV-2, herpes simplex virus type 1 and herpes simplex virus type 2; LBV, (R)-9-[2,3-bis(hydroxymethyl)cyclobutyl]guanine (lobucavir, cyclobut-G, BMS180194); NLS, nuclear localization signal; PCV, 9-[4-hydroxy-3-(hydroxymethyl)but-1-yl]guanine (penciclovir); S-BVDU, (E)-5-(2bromovinyl)-2’-deoxy-4’-thiouridine; VZV , varicella-zoster virus. Received: 30 October 1998; accepted 10 November 1998

Summary Introduction of the herpes simplex virus type 1 (HSV-1) thymidine kinase (TK) gene in tumor c e l l s , f o l l o w e d b y t r e a t m e n t o f t h e t r a n s f e c t e d t u m o r c e l l s w i t h a n antiherpes drug has shown promise in the treatment of solid tumors. We have recently shown that the HSV-1 TK fused to green fluorescent protein (GFP) was localized almost exclusively in the nuclei of HSV-1 TK-GFP fusion gene-transfected human osteosarcoma cells, due to the presence of a nuclear localization signal (NLS) at the N-terminus of the HSV-1 TK. A deletion mutant, lacking the N-terminal 34 amino acids [ (AA1-34)HSV-1 TK-GFP], was distributed throughout the cytoplasm and nucleus of transfected tumor cells. In addition, varicella-zoster virus (VZV) TK-GFP, which lacks the NLS and which i s uniformly distributed i n the nucleus and cytoplasm o f the VZV TK-GFP genetransfected tumor cells, could be specifically targeted to the nucleus by ligating the HSV-1 TK nuclear localization signal to the VZV TK-GFP sequence. Two pairs of osteosarcoma cell lines stably expressing HSV-1 TK-GFP or VZV TK-GFP either in the nucleus or throughout the cell were established and compared for their sensitivity t o the cytostatic effects o f a variety of antiherpetic nucleoside analogues. In addition, the efficacy of nucleoside analogues in contributing to the bystander effect (i.e., the killing of non-transfected tumor cells by neighbouring TK genetransfected c e l l s after gap junctional transfer o f phosphorylated nucleoside metabolites), was evaluated using the HSV-1 TK-GFP and (AA1-34)HSV-1 TK-GFP gene constructs. From our e x p e r i m e n t s i t i s i n f e r r e d t h a t t h e r e i s no difference i n cytostatic activity o f the antiherpetic 123


Degrève et al: Antiherpetic drugs in combined gene/chemotherapy of cancer nucleoside analogues against TK gene-transfected cells, whether the TK activity is solely localized in the nucleus or spread over the nucleus and cytosol. Also, the bystander killing effect of the antiviral compounds was independent of the nature of the intracellular compartment in which the HSV-1 TK-GFP fusion protein was expressed.

I. Introduction The broad substrate specificity of the thymidine kinase (TK) of most herpes viruses, including herpes simplex virus type 1 (HSV-1), herpes simplex virus type 2 (HSV2) and varicella-zoster virus (VZV), can be exploited in the treatment of herpesvirus infections (De Clercq, 1993; 1995). Exquisitely potent antiherpetic nucleoside analogues including (E)-5-(2-bromovinyl)-2’-deoxyuridine (BVDU) and ganciclovir (GCV) have been developed, which owe their selective antiviral activity to their specific phosphorylation by the herpetic TK but not by the mammalian TK (Figure 1). This concept of the herpetic TK-dependent cytostatic effect of otherwise non-toxic nucleoside analogues was later introduced in the field of anticancer research. Balzarini and coworkers reported on the highly selective cytostatic activity of the antiherpetic drugs GCV and BVDU, and various structurally related derivatives thereof, against murine mammary carcinoma (FM3A) cells transfected with the HSV-1 or HSV-2 TK gene (Balzarini et al., 1985, 1987, 1993, 1994). Differences were found in the cytostatic potency of the drugs depending on the nature of the suicide gene (i.e. HSV-1 or HSV-2 TK gene). In 1992, Culver and coworkers showed complete regression of established brain tumors in rats after in situ transduction with the HSV-1 TK gene and subsequent treatment with GCV (Culver et al., 1992). Several clinical trials, all utilizing the HSV-1 TK/GCV system, are underway to assess the safety and efficacy of this combined gene/chemotherapy treatment for cancer (Oldfield et al., 1993; Culver et al., 1994; Freeman et al., 1995; Kun et al., 1995; Raffel et al., 1994). Recently, we demonstrated that VZV TK and a variety of pyrimidine nucleoside analogues represent appropriate alternatives for the HSV-1 TK/GCV combination therapy (Degrève et al., 1997). We also recently studied the intracellular localization of HSV-1, HSV-2 and VZV TK (Degrève et al., 1998). The herpetic TKs were expressed as fusion proteins with the green fluorescent protein (GFP) (Chalfie et al., 1994; Rizzuto et al., 1995; Youvan et al., 1996) in human OstTK- cells and their intracellular localization was examined using a fluorescence microscope. HSV-1 TK fused with GFP was almost exclusively localized in the nuclei of HSV-1 TK-GFP gene-transfected tumor cells. In contrast, introduction of the HSV-2 TK-GFP fusion gene gave rise to predominant cytosolic fluorescence. VZV TKGFP showed a uniformly distributed fluorescence pattern. When the N-terminal 34 amino acids (AAs) were deleted 124

from the HSV-1 TK-GFP construct, the resulting mutant fusion protein lost its specific nuclear localization. We proved that this 34 amino acid stretch was also capable of targeting VZV TK-GFP and GFP to the nucleus of genetransfected OstTK- cells, indicating that a nuclear localization signal (NLS) was present in this N-terminal part of HSV-1 TK. By site-directed mutagenesis of each of the positively charged amino acids at the N-terminus of HSV-1 TK, we were able to identify a nonapeptide, 25R-RT-A-L-R-P-R-R33, which is strictly required for specific nuclear localization of HSV-1 TK (Degrève et al., 1998).

F i g u r e 1 . Structural formulae of 4 representative test compounds.


Gene Therapy and Molecular Biology Vol 1, page 125 The expression of HSV-1, HSV-2 and VZV TK in different intracellular localizations prompted us to investigate whether the intracellular localization of a particular TK would influence the cytostatic effects of antiherpetic nucleoside analogues. Therefore, a series of antiherpetic pyrimidine and purine nucleoside analogues were evaluated for their inhibitory activity on the proliferation of OstTK- cells expressing either the nucleustargeted (wild-type) HSV-1 TK fused to GFP, the uniformly distributed "(AA1-34)HSV-1 TK-GFP (lacking the first 34 amino acids that contain the nuclear targeting signal nonapeptide), the wild-type uniformly distributed VZV TK-GFP and the nucleus-targeted NLS-VZV TKGFP (containing the HSV-1 TK AA1-34 NLS). We have also recently explored the ability of a variety of purine and pyrimidine nucleoside analogues to exert a bystander killing effect in mixed tumor cell populations (Degrève et al., 1999), i.e. the potency of the compounds to kill TK tumor cells that are neighbouring HSV-1 TK-GFP genetransfected cells upon gap junctional transfer of the phosphorylated compounds. We showed that purine nucleoside analogues (represented by GCV) have a far more pronounced bystander killer effect than pyrimidine nucleoside analogues (represented by BVDU), regardless of their potent inhibitory potential against the HSV-1 TKGFP gene-transfected tumor cells. We have now evaluated the impact of intracellular compartmentation (ie nucleus or cytoplasm) of HSV-1 TK-GFP on the bystander effect of purine and pyrimidine nucleoside analogues. Our experimental data revealed that the intracellular localization of HSV-1 TK-GFP or VZV TK-GFP expression has no significant influence on either the cytostatic effect or the bystander effect of antiviral nucleoside analogues. These findings argue against the compartmentation of nucleotide pools in mammalian cells and suggest that phosphorylated nucleoside anabolites can rapidly equilibrate between the nuclear and cytosolic compartments of the cell.

II. Results A. Intracellular targeting of HSV-1 TKGFP and VZV TK-GFP constructs The HSV-1 TK-GFP and "(AA1-34)HSV-1 TK-GFP gene constructs were stably introduced in OstTK - cells and the fluorescence pattern was subsequently visualized under a fluorescence microscope. The OstTK-/HSV-1 TK-GFP+ cell line, as described earlier by Degrève et al. (1998), expresses the wild-type HSV-1 TK-GFP fusion protein, which is targeted to the nucleus of the transfected cells (Figure 2, panel A). In contrast, OstTK-/"(AA134)HSV-1 TK-GFP + cells express an N-truncated form of HSV-1 TK-GFP in both the nucleus and cytosol (panel B). Transfection of the VZV TK-GFP gene construct (panel C) 125

gave rise to a uniformly distributed fluorescence pattern. Finally, ligation of the HSV-1 TK nuclear localization signal to the VZV TK-GFP construct resulted in a nuclear fluorescence pattern (panel D).

B. Effect of intracellular localization of HSV-1 TK-GFP and VZV TK-GFP on the cytostatic activity of antiviral compounds The cytostatic activity of a series of the antiherpetic pyrimidine and purine nucleoside analogues was evaluated against OstTK - cells stably expressing either HSV-1 TKGFP, "(AA1-34)HSV-1 TK-GFP, VZV TK-GFP or NLSVZV TK-GFP. Non-transfected OstTK- cells were included as a control. The selection of the compounds was based on previous studies on HSV TK and VZV TK gene-transfected tumor cells in our laboratory (i.e. the protoype antiherpetic pyrimidine nucleoside analogue (E)-5-(2-bromovinyl)-2’deoxyuridine (BVDU) and its closely related derivatives SBVDU, BVaraU and BVDC, the antiherpetic thymidine and cytidine analogues araT, FMAU and FIAC, the acyclic guanosine analogue 9-(1,3-dihydroxy-2-propoxymethyl) guanine (ganciclovir, GCV) and its derivatives ACV, BCV, LBV and PCV (Balzarini et al., 1985, 1987, 1993, 1994; Degrève et al., 1997). The structural formulae of 4 representative antiherpes nucleoside analogues are shown in Figure 1. Results are summarized in T a b l e 1. The pyrimidine nucleosides BVDU, S-BVDU and BVaraU inhibited non-transfected OstTK- cell proliferation only at concentrations that exceeded 850µM. BVDC and araT showed 50% inhibitory concentrations (IC50) still above 200 µM, while FIAC and FMAU were more inhibitory to the proliferation of OstTK- cells (IC50 values of 9 and 17 µM, respectively). In sharp contrast, the pyrimidine nucleoside analogues became exquisitely inhibitory after transfection of the osteosarcoma cells with the HSV-1 TKGFP, "(AA1-34)HSV-1 TK-GFP, VZV TK-GFP and NLS-VZV TK-GFP genes. The IC50 values for the individual compounds were essentially comparable for the two HSV-1 TK-GFP constructs and the two VZV TKGFP constructs, except for FIAC which displayed a sixfold lower inhibitory effect against OstTK-/"(AA134)HSV-1 TK-GFP+ cells than against OstTK -/HSV-1 TKGFP + cells. BVDU and BVDC (IC50 values for TK-GFP gene-transfected cells ranging from 0.035 to 0.36 µM) exhibited 50% inhibitory concentrations that were approximately 10-fold higher than those for the other pyrimidine nucleoside analogues, which were in the lower nanomolar concentration range. The highest selectivity indices (i.e. the ratio of the IC 50 value for non-transfected cells versus the IC 50 value for TK-GFP gene-transfected cells) were observed for BVaraU (up to 250,000), S-BVDU (up to 150,000) and AraT (up to 100,000). BVDU was intermediate (selectivity index of 20,000), while BVDC, FIAC and FMAU were 1,000 to 6,000-fold more


Degrève et al: Antiherpetic drugs in combined gene/chemotherapy of cancer cytostatic to the various HSV-1 TK-GFP fusion genetransfected cells than to non-transfected OstTK- cells. The purine nucleoside analogues that were included in our study exhibited 50% inhibitory concentrations for the growth of non-transfected OstTK- cells ranging from 18 µM (LBV) to 231 µM (PCV) ( Table 1). GCV, BCV and PCV showed IC50 values in the nanomolar concentration range for OstTK-/HSV-1 TK-GFP+ and OstTK-/"(AA134)HSV-1 TK-GFP+ cells, that is at concentrations that

were 15,000 to 47,000-fold lower than the concentrations required to inhibit the proliferation of the wild-type OstTKcells. ACV, which displayed the highest IC50 value among all antiherpetic nucleoside analogues (up to 0.14 µM) and LBV (due to its stronger inhibitory effect against nontransfected OstTK- cells) ranked among the compounds with the lowest selectivity index (1,000 and 2,000, respectively). As proved to be the case with the pyrimidine

F i g u r e 2 . The HSV-1 TK-GFP and VZV TK-GFP fusion constructs (shown on top of each picture) were transfected into OstTKcells. After selection of stable transfectants, the fluorescence pattern was evaluated using a FITC filter-equipped fluorescence microscope. (A) HSV-1 TK-GFP; (B) "(AA1-34)HSV-1 TK-GFP; (C) VZV TK-GFP; (D) NLS-VZV TK-GFP.

126


Gene Therapy and Molecular Biology Vol 1, page 127 T a b l e 1 . C y t o s t a t i c a c t i v i t y o f n u c l e o s i d e a n a l o g u e s a g a i n s t w i l d - t y p e ( O s t T K -) and TK-GFP gene-transfected OstTK - c e l l s IC50 (µM)a

BVDU S-BVDU

OstTK-

OstTK- /HSV-1 TK-GFP+

862 ± 192 b b

911 ± 105 b

BVaraU

942 ± 47

BVDC

209 ± 35 b

OstTK- /"(AA1-34) HSV-1 TK-GFP+

OstTK- /VZV TKGFP+

OstTK- /NLS-VZV TK-GFP+

0.035 ± 0.006 b

0.038 ± 0.022

0.091 ± 0.055

0.36 ± 0.13

0.008 ± 0.004

b

0.006 ± 0.001

0.007 ± 0.000

0.028 ± 0.026

0.004 ± 0.001

b

0.004 ± 0.002

0.009 ± 0.017

0.029 ± 0.024

0.10 ± 0.00

1.6 ± 0.7c,d

-

0.059 ± 0.019 b

araT

231 ± 27

b

0.004 ± 0.0006

FIAC

9.1 ± 6.7

FMAU

b

c,d

0.002 ± 0.000

0.78 ± 0.41

0.002 ± 0.0001

0.012 ± 0.001

-

-

17 ± 0.5

0.006 ± 0.0001

0.004 ± 0.002

-

-

GCV

44 ± 22 b

0.001 ± 0.0005 b

0.003 ± 0.002

6.3 ± 7.3

ACV

b

73 ± 29

0.059 ± 0.015

BCV

173 ± 67

LBV

18 ± 0.4 b

PCV

231 ± 13

b

b

b

0.006 ± 0.0000

0.008 ± 0.0008 b 0.013 ± 0.0022

b

14 ± 4

48 ± 12

d

-

0.004 ± 0.001

57 ± 10

d

-

0.008 ± 0.0002

4.4 ± 1.0 d

0.14 ± 0.04 b

-

0.009 ± 0.001

27 ± 4

d

-

a The IC50 was defined as the drug concentration required to inhibit cell proliferation by 50%. Data are the mean value (± SD) for at least 3 independent experiments. b Data taken from Degrève et al. (1999). c Data taken from Degrève et al. (1997), where non-fused VZV TK gene-transfected OstTK- cells were evaluated.

nucleoside analogues, the IC50 values of the purine nucleoside analogues did not depend on the intracellular compartment in which the TK was localized (T a b l e 1). The poor cytostatic effect of ganciclovir against OstTKcells expressing VZV TK-GFP and NLS-VZV TK-GFP was not unexpected, since this drug has poor, if any, affinity for VZV TK (Degrève et al., 1997).

C. Bystander effect The bystander effect of two pyrimidine (BVDU and S-BVDU) and two purine (GCV and LBV) nucleoside analogues was evaluated. We have recently demonstrated the superior bystander effect of purine versus pyrimidine nucleoside analogues in mixed OstTK- and OstTK-/HSV-1 TK-GFP+ tumor cell populations (Degrève et al., 1999). Mixed tumor cell populations were cultured in the presence of 5-fold dilutions of the test compounds, after which the viable cell number was assessed using a colorimetric assay, as described in Materials and methods (Figure 3). The thick line in each graph represents the theoretically predicted values, in case no bystander effect is active (for

127

example, 25% non-transfected cells in the mixed tumor cell culture should result in 25% cell viability at the end of the 3-day incubation period in the presence of a lethal concentration of the nucleoside analogue). As shown in Figure 3, the inefficient bystander effect exerted by BVDU and S-BVDU was not enhanced by changing the intracellular HSV-1 TK-GFP localization. The very weak bystander effect of BVDU in OstTK-/HSV-1 TK-GFP+ cells was even completely absent in OstTK-/"(AA134)HSV-1 TK-GFP+ cells. For S-BVDU, the cell viability curves, obtained using a colorimetric assay, exactly reflected the percentages of OstTK- and HSV-1 TK-GFP gene-transfected tumor cells (Figure 3). In sharp contrast with the pyrimidine nucleosides, the guanosine nucleoside analogues GCV and LBV exhibited a pronounced bystander effect which was dose-dependent. LBV was not tested at 50µM because of profound inhibition of OstTK- cell growth at this concentration (IC50 value, 18µM). Even at the lowest concentration tested (2 µM), bystander killing was still observed with GCV and LBV. At a concentration


Gene Therapy and Molecular Biology Vol 1, page 128

F i g u r e 3 . Bystander effect of nucleoside analogues in mixed cell cultures. The thick line in each graph represents the theoretically predicted values, in case no bystander effect was noted. Concentrations tested, 50 µM (squares), 10 µM (triangles), 2µM (circles). OstTK - were mixed with OstTK- /HSV-1 TK-GFP+ cells (black symbols, data taken from Degrève et al., 1999) or OstTK- /"(AA1-34)HSV-1 TK-GFP+ cells (open symbols).

fusion proteins were localized in the nucleus of HSV-1 TK-GFP gene-transfected tumor cells, in the cytosol of HSV-2 TK-GFP gene-transfected tumor cells and in both the nucleus and the cytosol of VZV TK-GFP genetransfected tumor cells. The N-terminal 34 amino acids of HSV-1 TK, the deletion of which resulted in the loss of specific nuclear localization of HSV-1 TK-GFP, were also sufficient to target the otherwise uniformly distributed VZV TK-GFP to the nucleus of gene-transfected cells. In the experiments described in this report, we evaluated whether the intracellular localization of HSV-1 TK-GFP or VZV TK-GFP would influence the cytostatic potential and bystander effect of the antiherpetic nucleoside analogues in TK-GFP gene-transfected osteosarcoma cells. As shown in Table 1, all evaluated nucleoside analogues showed exquisite cytostatic properties against HSV-1 TK-GFP and

of 10µM, the bystander effect of LBV was 2- to 3-fold more pronounced than that of the prototype compound GCV. Abolishing the specific nuclear localization of HSV-1 TK-GFP by deleting the N-terminal NLS, had no significant impact on the bystander effect of GCV and LBV, that was observed in the tumor cell cultures that expressed the HSV-1 TK-GFP solely in the nucleus (Figure 3).

III. Discussion We recently reported the differential intracellular localization of the TKs of three herpesviruses, i.e. HSV-1, HSV-2 and VZV in TK-GFP fusion gene-transfected osteosarcoma cells (Degrève et al., 1998). The TK-GFP 128


Gene Therapy and Molecular Biology Vol 1, page 129 VZV TK-GFP expressing tumor cells, the lowest 50% inhibitory concentrations being in the lower nanomolar range. The pronounced cytostatic effect of pyrimidine nucleosides like S-BVDU and BVaraU makes them promising candidate compounds for the combined gene/chemotherapy treatment of cancer, with selectivity indices markedly higher than that of GCV, the current drug of choice for HSV-1 TK gene-mediated tumor cell killing. Moreover, S-BVDU and BVaraU are resistant to glycosidic bond cleavage by mammalian dThd phosphorylases, a major advantage compared to the cleavage-susceptible parent compound BVDU. However, one should also take the bystander effect into account. The bystander effect was described as the ability of a drug to kill non-transfected tumor cells that were in close contact with HSV-1 TK gene-transfected cells in mixed tumor cell populations. Complete tumor eradication has been demonstrated with GCV even when as few as 10% of the tumor cell inoculum was transfected with the HSV-1 TK gene (Culver et al., 1992; Ram et al., 1993; Freeman et al., 1993). The succes of the combined herpesviral TK gene/chemotherapeutic approach seems to depend on the bystander effect, since current gene therapy vectors are not capable of introducing the viral thymidine kinase gene in 100% of the cells of a particular tumor. Instead, getting 1% of the tumor cells transfected is a more realistic goal. Moreover, the low fraction of herpesviral TK gene-transfected tumor cells should be uniformly distributed in the tumor to yield an optimal bystander effect, which is virtually impossible to achieve. We recently demonstrated that the in vitro bystander effect of purine nucleoside analogues was superior to that of pyrimidine nucleoside analogues in mixed OstTK- and OstTK-/HSV-1 TK-GFP+ cell cultures. The bystander effect exerted by pyrimidine nucleoside analogues (i.e., BVDU and derivatives) proved to be very ineffective or even absent in most cases. In contrast, most of the evaluated purine nucleoside analogues (in particular GCV and LBV) displayed potent bystander killing potencies. Therefore, the lower selectivity index of purine nucleoside analogues like GCV and LBV is well compensated by their superior bystander killing as compared to pyrimidine nucleoside analogues. It is clear from Table 1 that the cytostatic activities of the evaluated compounds were generally independent of the compartmentation of the HSV-1 TK-GFP in the cell. We could also conclude from our experiments that the Nterminal 34 amino acids of the HSV-1 TK are not important for enzyme activity, since the "(AA1-34)HSV-1 TK-GFP fusion protein was fully catalytically active in gene-transfected tumor cells. These findings corroborate the observation of Halpern and Smiley (1984) that the Nterminal 45 amino acids are not required for the catalytic activity of HSV-1 TK. The slightly higher IC50 values (at 129

most 4-fold) obtained for OstTK-/NLS-VZV TK-GFP+ cells compared with OstTK -/VZV TK-GFP+ cells could be attributed to differences in the expression level of the VZV TK-GFP and the NLS-VZV TK-GFP fusion proteins, rather than to the different localization of VZV TK-GFP in the tumor cells. Indeed, the lower expression of NLS-VZV TK-GFP is in agreement with a weaker fluorescence signal (Figure 2, panel C and D). Thus, the intracellular localization of the VZV TK-GFP fusion protein is not a determining factor in the cytostatic potency of the antiviral nucleoside analogues. These findings are in full agreement with the observations of Johansson et al. (1997) on the cytostatic activities of nucleoside analogues against 2’-deoxycytidine kinase (dCK) expressing tumor cell lines. Until recently, it had generally been assumed that enzymes required for nucleic acid synthesis (i.e., nucleoside kinases) are localized in the cytosol (dCK and TK1) or in the mitochondria [i.e., 2’-deoxyguanosine kinase (dGK) and TK2] (Arnér and Eriksson, 1995). However, dCK, that shows substrate specificity for 2’-deoxycytidine, 2’deoxyadenosine and several clinically important nucleoside analogues, has now been found to be predominantly localized in the nuclear compartment (Johansson et al., 1997). Moreover, Johansson and collaborators identified a nuclear targeting signal in the primary structure of human dCK and showed that this signal was required for nuclear import of the protein. Irrespective of the intracellular localization of dCK, no marked differences in the cytostatic activity of 1-!-D-arabinofuranosylcytosine (araC), 2’,3’dideoxy-2’,3’-difluorocytidine (dFdC, gemcitabine) and 2chloro-2’-deoxyadenosine (CdA) were noted. These data indicate that the nucleus and the cytosol do not have separate deoxynucleotide pools, and that phosphorylated nucleoside analogues are rapidly equilibrated between the nuclear and cytosolic compartments of the cell. Since the localization of nucleoside kinases in the cell does not seem to have any determining role as to their function, it is currently unclear why certain nucleoside kinases are localized in the nucleus (dCK, HSV-1 TK), others in the cytosol (HSV-2 TK, mammalian cytosolic TK1) and still others spread over the nucleus and cytosol (VZV TK). In conclusion, we found that the inhibitory effects of antiviral nucleoside analogues against herpes TK-GFP gene-transfected cells were not significantly altered by changing the intracellular localization (either nucleus or throughout the cell) of the HSV-1 TK-GFP or VZV TKGFP fusion protein. Also, the bystander effect of the antiviral nucleoside analogues was not affected by the intracellular targeting of HSV-1 TK-GFP. Our experimental data indicate that phosphorylated nucleoside analogues can rapidly equilibrate between the nuclear and cytosolic compartments of the cell before exerting their potent cytostatic effect.


Degrève et al: Antiherpetic drugs in combined gene/chemotherapy of cancer

IV. Materials and methods A. Compounds BVDU and BVDC were synthesized by P. Herdewijn and A. Van Aerschot at the Rega Institute for Medical Research (Katholieke Universiteit Leuven, Leuven, Belgium). S-BVDU was provided by the late R.T. Walker (University of Birmingham, Birmingham, U.K.). BVaraU was a kind gift of H. Machida (Yamasa Shoyu Co., Choshi, Japan). AraT was from Sigma Chemical Co. (St. Louis, MO), and also a kind gift from M. Sandvold and F. Myhren (Norsk Hydro, Porsgrunn, Norway). FIAC and FMAU were a kind gift of J.J. Fox (SloanKettering Institute, New York). GCV was from Syntex (Palo Alto, CA), ACV from the former Wellcome Research Laboratories (Research Triangle Park, NC), BCV from Astra Läkemedel (Sodertälje, Sweden) and LBV from Bristol-Myers Squibb (Princeton, NJ). PCV was obtained from I. Winkler (Hoechst, Frankfurt, Germany).

B. Cell Culture Adherent human osteosarcoma cells deficient in cytosol TK (OstTK- , ATCC CRL-8303) and all TK-GFP genetransfected OstTK- cells were maintained at 37°C in a humidified CO2 -controlled atmosphere, in MEM culture medium (Gibco, Paisley, U.K.), supplemented with 10% heatinactivated fetal calf serum (Biochrom KG, Berlin, Germany), 2mM L-glutamine (Gibco), 0.075% (w/v) NaHCO3 (Gibco), 0.5µl/ml geomycine (Gentamycin#, 40mg/ml, ScheringPlough, Madison, NJ) and 0.5µl/ml Amphotericin B (Fungizone#, 5mg/ml, Bristol-Myers Squibb).

C. Plasmid construction The construction of the HSV-1 TK-GFP, "(AA1-34)HSV-1 TK-GFP, VZV TK-GFP and NLS-VZV TK-GFP expression vectors has been described elsewhere (Degrève et al., 1998). Briefly, the coding sequence for the full-length and Ntruncated HSV-1 TK (lacking the first 34 amino acids) were amplified by PCR from the pMCTK plasmid kindly provided by Dr. D. Ayusawa (Yokohama City University, Japan), and cloned in the multiple cloning site of the pEGFP-N1 NTerminal Protein Fusion Vector (CLONTECH, Palo Alto, CA). The VZV TK coding sequence, PCR-amplified from the pRc/CMV/VZV TK plasmid (kindly provided by Dr. J. Piette, University of Liège, Belgium) was ligated with (NLS-VZV TKGFP) or without (VZV TK-GFP) the PCR-amplified sequence encoding for AA1-34 of HSV-1 TK in the pEGFP-N1 vector.

D. Stable transfection of tumor cells The construction of the OstTK - /HSV-1 TK-GFP+, OstTK/ "(AA1-34)HSV-1 TK-GFP+, OstTK- /VZV TK-GFP+ and OstTK- /NLS-VZV TK-GFP+ cell lines has been described elsewhere (Degrève et al., 1998). Briefly, the herpes virus TKGFP fusion constructs were introduced into OstTK - cells via membrane fusion-mediated transfer using plasmid/ liposome complexes (LipofectAMINE$ Reagent, Gibco), as described by the supplier. Stable fusion gene transfectants were isolated by maintaining the cell cultures in the presence of HAT

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medium (i.e. normal growth medium, supplemented with 100µM hypoxanthine, 0.4µM aminopterin and 16µM thymidine). Monoclonal transfected cell lines were obtained by plating the cells at clonal density in tissue culture plates (Corning, N.Y.), after which single colonies were isolated and expanded. A standard FITC filter-equipped fluorescence microscope was used to evaluate gene expression and GFP fusion protein localization.

E. Inhibition of tumor cell proliferation by antiherpetic compounds The cytostatic activity of antiviral nucleoside analogues against wild-type and herpes TK-GFP-expressing cells was evaluated as follows. 10 4 OstTK- , OstTK- /HSV-1 TK-GFP+, OstTK- /"(AA1-34)HSV-1 TK-GFP+, OstTK - /VZV TK-GFP+ or OstTK- /NLS-VZV TK-GFP+ cells/well were plated in 96-well microtiter plates (Falcon, Becton Dickinson, Franklin Lakes, NJ, USA) and subsequently incubated at 37°C, in a humidified CO2 -controlled atmosphere, in the presence of 5-fold dilutions (in normal growth medium) of the compounds. After 3 days, the number of cells was evaluated in a Coulter Counter (Coulter Electronics Ltd., Harpenden Hertz, U.K.). The IC50 was defined as the drug concentration required to inhibit tumor cell proliferation by 50%.

F. Bystander effect The procedure to evaluate the bystander effect of the compounds was as described elsewhere (Degrève et al., 1998). Briefly, OstTK- cells were mixed with HSV-1 TK-GFP genetransfected cells in percentages ranging from 0 to 100% (0, 0.2, 1, 5, 10, 25, 50, 75, 90 and 100%) transfected cells, and subsequently incubated in the presence of 5-fold dilutions (in 2% FCS-containing medium) of the compounds. After 3 days, i.e. the time needed by untreated cell cultures to reach confluency, cell viability was determined using the Cell Titer 96 Aqueous Non-radioactive MTT Cell Proliferation Assay (Promega, Madison, WI). Untreated cultures served as controls.

Acknowledgments We thank Christiane Callebaut for dedicated editorial help. This work was supported by Project 3-0180-95 from the Flemish “Fonds Voor Geneeskundig Wetenschappelijk Onderzoek”, Project 95/5 from the Belgian “Geconcerteerde Onderzoeksacties”, the Swedish Medical Research Council, the Medical Faculty of Karolinska Institute and the Harald and Greta Jeansson Foundation. Bart Degrève is recipient of an IWT fellowship from the “Vlaams Instituut voor de bevordering van het Wetenschappelijk-Technologisch onderzoek in de Industrie”.

References Arnér, E.S.J., and S. Eriksson. (1 9 9 5 ) Mammalian deoxyribonucleoside kinases. Pharmacol. Ther. 67,


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cancer. Gene Ther. 4, 1107-1114.

Balzarini, J., C. Bohman, and E. De Clercq. (1 9 9 3 ) Differential mechanism of cytostatic effect of (E)-5-(2bromovinyl)-2’-deoxyuridine, 9-(1,3-dihydroxy-2propoxymethyl)guanine, and other antiherpetic drugs on tumor cells transfected by the thymidine kinase gene of herpes simplex virus type 1 or type 2. J . B i o l . C h e m . 268, 6332-6337.

Degrève, B., M. Johansson, E. De Clercq, A. Karlsson, and J. Balzarini. (1 9 9 8 ) Differential intracellular compartmentalization of herpetic thymidine kinases (TKs) in TK gene-transfected tumor cells. Molecular characterization of the nuclear localization signal of herpes simplex virus type 1 TK. J . V i r o l . 72, 95359543.

Balzarini, J., C. Bohman, R.T. Walker, and E. De Clercq. (1 9 9 4 ) Comparative cytostatic activity of different antiherpetic drugs against herpes simplex virus thymidine kinase gene-transfected tumor cells. M o l . P h a r m a c o l . 45, 1253-1258.

Freeman S.M., C.N. Abboud, K.A. Whartenby, C.H. Packman, D.S. Koeplin, F.L. Moolten, and G.N. Abraham. (1 9 9 3 ) The “bystander effect”, tumor regression when a fraction of the tumor mass is genetically modified. C a n c e r R e s . 53, 5274-5283.

Balzarini, J., E. De Clercq, A. Verbruggen, D. Ayusawa, K. Shimizu, and T. Seno (1 9 8 7 ) Thymidylate synthase is the principal target enzyme for the cytostatic activity of (E)5-(2-bromovinyl)-2’-deoxyuridine against murine mammary carcinoma (FM3A) cells transformed with the herpes simplex virus type 1 and type 2 thymidine kinase gene. M o l . P h a r m a c o l . 32, 410-416.

Freeman, S.M., C. McCune, W. Robinson, C.N. Abboud, G.N. Abraham, C. Angel, and A. Marrogi. (1 9 9 5 ) The treatment of ovarian cancer with a gene modified cancer vaccine, A phase I study. H u m . G e n e T h e r . 6, 927939.

Balzarini, J., E. De Clercq, D. Ayusawa, and T. Seno. (1 9 8 5 ) Murine mammary FM3A carcinoma cells transformed with the herpes simplex virus type 1 thymidine kinase gene are highly sensitive to the growth-inhibitory properties of (E)-5-(2-bromovinyl)-2’-deoxyuridine and related compounds. FEBS Lett. 185, 95-100. Chalfie, M., Y. Tu, G. Euskirchen, W.W. Ward, and D.C. Prasher. (1 9 9 4 ) Green fluorescent protein as a marker for gene expression. S c i e n c e 263, 802-805. Culver, K.W., J. Van Gilder, C.J. Link, T. Carlstrom, T. Buroker, W. Yuh, K. Koch, K. Schabold, S. Doornbas, and B. Wetjen. (1 9 9 4 ) Gene therapy for the treatment of malignant brain tumors with in vivo tumor transduction with the herpes simplex thymidine kinase gene/ganciclovir system. Hum. Gene Ther. 5, 343379. Culver, K.W., Z. Ram, S. Wallbridge, H. Ishii, E.H. Oldfield, and R.M. Blaese. (1 9 9 2 ) In vivo gene transfer with retroviral vector-producer cells for treatment of experimental brain tumors. S c i e n c e 256, 1550-1552. De Clercq, E. (1 9 9 3 ) Antivirals for the treatment of herpesvirus infections. J . A n t i m i c r o b . C h e m o t h e r . 32, 121-132. De Clercq, E. (1 9 9 5 ) Trends in the development of new antiviral agents for the chemotherapy of infections caused by herpesviruses and retroviruses. R e v . M e d . V i r o l . 5, 149-164. Degrève, B., E. De Clercq, and J. Balzarini. (1 9 9 9 ) Bystander effect of purine nucleoside analogues in HSV-1 TK suicide gene therapy is superior to that of pyrimidine nucleoside analogues. Gene Ther. 6, in press. Degrève, B., G. Andrei, M. Izquierdo, J. Piette, K. Morin, E.E. Knaus, L.I. Wiebe, I. Basrah, R.T. Walker, E. De Clercq, and J. Balzarini. (1 9 9 7 ) Varicella-zoster virus thymidine kinase gene and antiherpetic pyrimidine nucleoside analogues in a combined gene/chemotherapy treatment for

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Halpern, M.E., and J.R. Smiley. (1 9 8 4 ) Effects of deletions on expression of the herpes simplex virus thymidine kinase gene from the intact viral genome, the amino terminus of the enzyme is dispensable for catalytic activity. J . V i r o l . 5 0 , 733-738. Johansson, M., S. Brismar, and A. Karlsson. (1 9 9 7 ) Human deoxycytidine kinase is located in the cell nucleus. P r o c . N a t l . A c a d . S c i . USA 94, 11941-11945. Kun, L.E., A. Gajjar, M. Muhlbauer, R.L. Heideman, R. Sanford, M. Brenner, A. Walter, J. Langston, J. Jenkins, and S. Facchini. (1 9 9 5 ) Stereotactic injection of herpes simplex thymidine kinase vector producer cells (PA317G1Tk1SvNa.7) and intravenous ganciclovir for the treatment of progressive or recurrent primary supratentorial pediatric malignant brain tumors. Hum. Gene Ther. 6, 1231-1255. Oldfield, E.H. 1993. Gene therapy for the treatment of brain tumors using intra-tumoral transduction with the thymidine kinase gene and intravenous ganciclovir. Clinical protocols. Hum. Gene Ther. 4, 36-69. Raffel, C., K. Culver, D. Kohn, M. Nelson, S. Siegel, F. Gillis, C.J. Link, and J.G. Villablanca. (1 9 9 4 ) Gene therapy for the treatment of recurrent pediatric malignant astrocytomas with in vivo tumor transduction with the herpes simplex thymidine kinase gene/ganciclovir system. Hum. Gene Ther. 5, 863-890. Ram Z., K.W. Culver, S. Walbridge, R.M. Blaese, E.H. Oldfield. (1 9 9 3 ) In situ retroviral-mediated gene transfer for the treatment of brain tumors in rats. C a n c e r R e s . 53, 83-88. Rizzuto, R., M. Brini, P. Pizzo, M. Murgia, and T. Pozzan. (1 9 9 5 ) Chimeric green fluorescent protein as a tool for visualizing subcellular organelles in living cells. Curr. B i o l . 5, 635-642. Youvan, D.C., and M.E. Michel-Beyerle. (1 9 9 6 ) Structure and fluorescence mechanism of GFP. Nature B i o t e c h n o l o g y 14, 1219-1220.


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Gene Therapy and Molecular Biology Vol 1, page 133 Gene Ther Mol Biol Vol 3, 133-148. August 1999.

Glioblastoma multiforme: molecular biology and new perspectives for therapy Review Article

Giorgio Pal첫, Luisa Barzon, and Roberta Bonaguro Institute of Microbiology, University of Padova Medical School, Padova, Italy __________________________________________________________________________________________________ Corresponding Author: Giorgio Pal첫, MD, Institute of Microbiology, Via A. Gabelli 63, 35121 Padova, Italy. Tel: +39-049-8272350; Fax: +39-049-8272355; E-mail: gpalu@microb.unipd.it K e y w o r d s : Therapy, gene therapy, brain tumors, gliomas, glioblastoma multiforme, molecular biology, pathogenesis, immunotherapy, neoangiogenesis, oncogenes A b b r e v i a t i o n : GBM, glioblastoma multiforme Received: 23 November 1998; accepted 30 November 1998

Summary Pathogenic features of glioblastoma multiforme and of other gliomas are reviewed in the present article. Emphasis is given to those genetic alterations which are involved in oncogenesis, to the p r o c e s s o f t u m o r n e o a n g i o g e n e s i s a n d t o t h e r o l e p l a y e d b y t h e i m m u n e s y s t e m i n controlling neoplastic growth. Aspects which are relevant to therapeutic interventions are also dissected, and gene therapy in particular. A new gene therapy approach that combines tumor suicide, via enzymedirected prodrug activation, and cytokine-promoted immune rejection i s reported, together with results from the first application of this approach in humans.

of patients for tumor recurrence within 6-12 months from treatment.

I. Introduction The outcome of malignant gliomas remains extremely poor, in spite of aggressive use of currently available therapies. Recent advances in elucidating the molecular biology of gliomas have led to the development of innovative therapeutic strategies. The more promising approaches involve gene therapy, aiming at increasing tumor cell chemosensitivity and/or immunogenicity, by transfer of genes expressing cytokines and prodrug activating enzymes.

Glioblastoma multiforme (grade IV astrocytoma) is usually located in the cerebral hemispheres, though it occasionally appears at other sites, such as the cerebellum, the brain stem and the spinal cord. Histology shows marked cytological diversity, ranging from tumors composed of small cells with scant cytoplasm to those composed of multinucleated giant cells. The World Health Organization (WHO) classification recognizes two distinct subvariants of the tumor: (i ) giant cell glioblastoma, characterized by a predominance of enormous, multinucleated giant cells and, on occasion, an abundant stromal reticulin network; and (i i ) gliosarcoma, in which hyperplastic vascular elements have undergone sarcomatous transformation.

Glioblastoma multiforme (GBM) represents 15-20% of all intracranial tumors and 50% of gliomas (Russel and Rubistein, 1989). It affects 5,000 Americans and 1,000 Italians every year, and typically occurs in adults, with a peak incidence in the fifth and sixth decades of life. It is a very aggressive tumor, with a uniform and profound morbidity. Because of its morbidity it contributes to the cost of cancer on a pro capite basis more than any other tumor. Despite surgery, radiotherapy and/or chemotherapy, the prognosis is extremely poor and has not substantially changed over the last two decades, death resulting in 80%

Current therapies for malignant gliomas include surgical removal of the tumor mass, which is mandatory for precise diagnosis, and irradiation. Although surgery improves the prognosis (Levin et al, 1993), the infiltrative behavior of malignant gliomas precludes their complete

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Pal첫 et al: Perspectives for therapy of glioblastoma multiforme resection, and 90% of GBM recur within 2 cm of the primary site. Postoperative radiotherapy is therefore commonly administered, with a significant improvement in survival (Hochberg and Pruitt, 1980; Walker et al, 1980). Despite surgery and irradiation, however, only a few patients are alive two years after diagnosis. Results of chemotherapy trials are disappointing (Hosli et al, 1998). This is due both to the tumor intrinsic chemoresistance (Petersdorf and Berger, 1996) and to the tumor location within the central nervous system, which limits the penetration of drugs (Janzer and Raff, 1987; Mak et al, 1995). Among malignant gliomas, GBM is the least responsive to medical treatment. Available protocols include both monochemotherapy and polychemotherapy regimens. Nitrosoureas are the leading drugs in glioma chemotherapy, with response rates as single agents varying from 10% to 40% (Young et al, 1973; Fewer et al, 1972; Hoogstraten et al, 1972). Other drugs, evaluated in monochemotherapy (Forsyth and Cairncross, 1996), occasionally showed clinically and radiologically objective responses. Among these are vincristine (Smart et al, 1968), procarbazine (Rodriguez et al, 1989), paclitaxel (Chamberlain and Kormanik, 1995; Prados et al, 1996), and temozolomide (Newlands et al, 1996). However, methodological bias present in most studies raise doubts about the validity of these results. The most commonly used polychemotherapy regimens for gliomas are PVC (i.e. a combination of CCNU, procarbazine, and vincristine) and MOP (i.e. a combination of procarbazine, vincristine, and mechlorethamine). Response rates (complete or partial) of 17-37% have been reported for glioblastomas (Levin et al, 1980; Coyle et al, 1990). More recently, interesting results have been obtained in GBM patients with the ICE regimen (ifosfamide, carboplatin and etoposide), although in association with severe hematological toxicity (Sanson et al, 1996). The role of PVC as adjuvant chemotherapy is controversial (Fine et al, 1993), and, overall, there is no clear-cut evidence that survival of glioblastoma patients is improved by chemotherapy (Hosli et al, 1998).

insurmountable task that gene replacement, or gene suppression, should simultaneously involve a number of different genes, and should be applied to all tumor cells to reverse the malignant phenotype. Hence, corrective gene therapy seems to be quite difficult to propose as a single therapeutic approach.

A. Genetic alterations 1. Oncogenes Several members of the protein-tyrosine kinase receptor family are over-expressed by gene amplification in malignant gliomas, including the epidermal growth factor receptor (EGFR), the platelet-derived growth factor receptor-! (PDGFR!) and the c-met genes (Furnari et al, 1995). A high percentage of glioblastomas also have EGFR gene rearrangements that may lead to the expression of a truncated, constitutively activated receptor. The transfer of a mutant human EGFR gene into glioblastoma cells caused constitutive self phosphorylation and a pronounced enhancement tumorigenicity in nude mice

II. Molecular biology of glioblastoma multiforme and corrective gene therapy As for most cancers, brain tumors derive from a multistep process of successive alterations, including loss of cell cycle control, neoangiogenesis and evasion of immune control. Figure 1 summarizes the genetic alterations associated with the malignant transformation of astrocytes. Most of these changes involve the loss of putative tumor suppressor genes or activation of proto-oncogenes.

o

Gene therapy of cancer, in its most direct form, should aim at replacing a mutated gene with its correct form, or at suppressing the abnormal oncogenic function. At present, however, such a corrective gene therapy, faces the

f

F i g u r e 1 . Simplified representation of oncogenes and tumor suppressor genes contributing to malignant progression of astrocytic tumors.

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Gene Therapy and Molecular Biology Vol 1, page 135 (Ekstrand et al, 1994; Nishikawa et al, 1994). Numerous strategies are currently being investigated to specifically inhibit EGFR using antibodies, immunoconjugates or antisense technology. The selective inhibition of EGFR in human GBM cells with kinase-deficient mutants inhibited cell proliferation and transforming efficiency in athymic mice (O'Rourke et al, 1997), and converted radioresistant human glioblastoma cells to a more sensitive phenotype (O'Rourke et al, 1998), providing a rationale for gene therapy applications.

cells with wild type p53 can significantly inhibit growth and neoangiogenesis, or can induce apoptosis in p53 mutant cells in several tumor models in vitro, including gliomas (Badie et al, 1995; Van Meir et al, 1995; GomezManzano et al, 1996). The presence of functional p53 has also been shown to modulate chemoresistance. Consequently, another possible advantage of the restoration of wild type p53 may be sensitization to chemotherapy and radiotherapy. Indeed, the combination of p53 gene transduction with radiation or chemotherapy (Lowe et al, 1994) has resulted in local tumor control superior to either therapy alone (Fujiwara T et al, 1994; Gjerset et al, 1995; Ngyuyen et al, 1996, Lang et al, 1998). This combined therapy is currently under investigation in clinical trials (Roth and Cristiano, 1997; Nielsen and Maneval, 1998).

Other dominant oncogenes, such as N-myc, fos, src, Hras or N-ras, and mdm2 are amplified and highly expressed in gliomas (Collins, 1993). GBM produce high levels of insulin-like growth factor I (IGF-I). When this alteration has been targeted by a vector expressing an antisense antiIGF-I gene, rejection of genetically altered rat C6 glioma cells was observed. Injection, even at a site distal to the tumor, caused regression of established brain GBM. Destruction of the tumor was mediated by a gliomaspecific T CD8+ (CTL) response (Trojan et al, 1993).

The cell cycle regulator genes provide an additional target for corrective gene therapy. The p105Rb product of the retinoblastoma tumor suppressor gene (Rb) is one of the most critical regulators of cellular proliferation. The Rb protein (pRb), when unphosphorylated, is responsible for arrest of cell cycle by inhibition of the activity of the E2F family of transcription factors. Normal cell cycle progression requires inactivation of Rb through phosphorylation by cyclin-dependent kinases (CDK). This process, in turn, is regulated by CDK inhibitors. Among these, p21 protein is induced directly by p53; p16 protein, and its homologue p15, specifically bind to and inhibit CDK4, and may therefore regulate Rb phosphorylation, and cell cycle progression. Dysregulation of cell cycle control is a frequent finding in malignant gliomas, like deletion or loss of expression of p16 and p15 tumor suppressor genes (Jen et al, 1994; Nishikawa et al, 1995), amplification of CDK4 (He et al, 1994), and deletion or mutation of the Rb tumor suppressor gene (Henson et al, 1994). Interestingly, both of the latter events take place when the p16 gene is intact and correctly expressed (He et al, 1995). Restoration of wild-type p16 gene in glioma cells through an adenoviral vector arrested cells in G0-G1 phases of the cell cycle (Fueyo et al, 1996) and suppressed glioma cells invasion in vitro (Chintala et al, 1997). Overexpression of p21 increases the susceptibility of glioblastoma cells to cisplatin-induced apoptosis (Kondo et al, 1996), whereas adenovirus-mediated transfer of exogenous E2F-1 protein induced massive apoptosis and suppressed glioma growth in vivo and in vitro (Fueyo et al, 1998). The possibility that E2F-responsive promoters may be more active in tumor cells relative to normal cells, because of loss of pRb function, has been exploited to design adenoviral vectors containing transgenes driven by the E2F-1 promoter for gene therapy of gliomas (Parr et al, 1997). These vectors showed tumor-selective gene expression in vivo and reduced toxicity of the normal tissue with respect to standard adenoviral vectors.

2. Genes associated to cell immortalization A role in cell immortalization has been proposed for telomerase, the RNA-protein complex that elongates telomeric DNA. Telomerase is expressed almost exclusively in cancer cells, but not in normal cells, suggesting the possibility that gene therapy may be applied to inhibit this function. A successful example of treatment via antisense oligonucleotides directed against human telomerase suppressed glioma cells growth and survival, both in vitro and in vivo, through the induction of apoptosis (Kondo et al, 1998). 3. Tumor suppressor genes Molecular and cytogenetic analyses of gliomas have shown frequent losses of genetic material, suggesting the inactivation of putative tumor suppressor genes. Loss of heterozygosity (LOH) has been described in chromosome 1p, 9p, 10p, 10q, 11p, 13q, 17p, 19q, and 22q, and in some cases the tumor suppressor gene involved in LOH has been identified. This is the case of the p53 tumor suppressor gene, which maps in 17p. Wild-type p53 protein is involved in G1 cell cycle arrest and apoptosis of DNA-damaged cells and is therefore crucial in preventing mutation or deletion of functional genes. Mutations of p53 seem to be an early event in glioma tumorigenesis, being frequently detected also in low grade astrocytomas. Along with p53 mutations, amplification of the mdm2 oncogene, whose product binds to and degrades p53, accounts for p53 inactivation in gliomas. Since p53 plays a key role in the pathogenesis of most cancers, it has raised great interest as a target for cancer gene therapy. Transduction of malignant

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Pal첫 et al: Perspectives for therapy of glioblastoma multiforme Inhibition or inactivation of genes/factors involved in DNA repair and/or cellular SOS response could represent a gene therapy approach that potentiates radiation therapy. In fact, inhibition of the RAD51 gene by antisense oligonucleotides enhanced the radiosensitivity of mouse malignant gliomas, both in vitro and in vivo, improving survival (Ohnishi et al, 1998). This gene, a homologue of the yeast RAD51 and E. coli RecA genes, is involved in repair of DNA double-strand breaks, in recombination repair, and in various SOS responses to DNA damage caused by gamma-irradiation and alkylating agents.

ribozymes against VEGF mRNA have been successfully employed to reduce VEGF expression in glioma cells (Ke et al 1998), once more suggesting a potential role for antiangiogenic gene therapy. Similarly, bFGF antisense cDNA decreased C6 glioma cells proliferation (Redekop and Naus, 1995). Besides inhibiting the production of angiogenic factors, a therapeutic intervention could also consist of providing tumors with antiangiogenic factors. Indeed, retroviral and adeno-associated viral vectors expressing a modified PF4 were reported to inhibit endothelial cells proliferation in vitro and the growth of intracerebrally implanted gliomas (Tanaka et al, 1997). Retroviral and adenoviral vectors transducing angiostatin gene increased apoptotic death of glioma tumor cells (Tanaka et al, 1998). Additionally, the intratumoral delivery of angiostatin gene by an adenoviral vector produced inhibition of tumor growth in vivo, suppression of neovascularization, and a marked increase of tumor cells apoptosis (Griscelli et al, 1998). Damage of tumor microvasculature was reported also in human malignant glioma xenografts, after gene therapy followed by radiotherapy. The treatment consisted of intratumoral injection of adenoviral vectors expressing tumor necrosis factor-! (TNF-!), under control of the Egr-1 promoter (Staba et al, 1998). The use of viral vectors containing radiation-inducible promoters, such as Egr-1, has the advantage of selectively, spatially, and temporally limiting the effects of the therapeutic gene in the radiation field. Recently, this strategy has yielded interesting results in rat 9L glioma cells (Manome et al, 1998).

Deletions of large regions or even of the entire copy of chromosome 10 are a genetic hallmark of GBM. At least two tumor suppressor genes located on chromosome 10 (one on each arm) have been demonstrated to participate to glial oncogenesis. A first candidate tumor suppressor gene, called PTEN (Phosphatase and tensin homologue deleted on chromosome TEN) was recently characterized (Li et al, 1997). The DNA region encoding PTEN is altered in glioblastoma multiforme, but not in lower grade astrocytic tumors (Tohma et al, 1998; Ichimura et al, 1998; Chiariello et al, 1998).

B. Neoangiogenesis Tumors may remain in a state of dormancy until they establish a blood supply for receiving oxygen and nutrients. The complex process of neoangiogenesis is regulated by numerous factors, some with angiogenic properties, i.e. vascular endothelial growth factor (VEGF), platelet derived growth factor (PDGF!), basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), interleukin-8, and by endogenous inhibitors of angiogenesis, i.e. thrombospondin-1, platelet factor 4 (PF4), angiostatin, endostatin. VEGF, which binds to two specific tyrosine-kinase receptors, called Flk-1 and Flt-1, has been demonstrated to play a key role in angiogenesis of gliomas. Indeed, VEGF and its receptors are downregulated in the normal adult brain, whereas, VEGF is highly produced by GBM cells. Since both flt-1 and flk-1 genes are expressed by proliferating endothelial cells of gliomas, this leads to the establishment of a paracrine loop. Moreover, VEGF expression is higher around necrotic areas and seems to be stimulated by hypoxia.

III. Suicide gene therapy Suicide gene therapy operates by tumor transduction of genes converting a prodrug into a toxic substance; independently, the gene product and the prodrug are nontoxic. The prototype of this approach exploits the selective intracellular phosphorylation of ganciclovir (GCV), driven by the herpes simplex virus thymidine kinase gene product (HSV-TK). This activation generates a toxic drug metabolite that inhibits DNA synthesis, inducing cell death. For in vivo gene transfer of the HSV-TK gene to malignant cells, packaging cells that produces retroviral vectors expressing HSV-TK, have been injected directly into the tumor to transduce replicating cells. An interesting feature of the HSV-TK/GCV system is the bystander killing of nontransduced cells.

Glioblastoma multiforme is one of the most highly vascularized solid neoplasms; therefore, treatments that target neoangiogenesis would be of great interest in clinical practice. Co-injection of rat C6 glioma cells, either subcutaneously or intracerebrally in nude mice, together with cells producing retroviral vectors encoding a dominant-negative mutant of the Flk-1 receptor showed inhibition of neoangiogenesis, reduction of tumor growth, and survival improvement (Millauer et al, 1994; 1996). Antisense VEGF oligonucleotides (Saleh et al, 1996) and

The mechanisms that are responsible for this effect have not been fully defined, but are likely to include: (i ) transfer of non-diffusible, phosphorylated GCV to neighboring cells through gap junctions; (i i ) endocytosis by nontransduced cells of cellular debris containing toxic GCV; and (i i i ) stimulation of host antitumor immune response. The therapeutic efficacy of the HSV-TK/GCV

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Gene Therapy and Molecular Biology Vol 1, page 137 system may be further increased by the use of adenoviral vectors, since these vectors can also transduce resting tumor cells. However, adenoviral vectors will infect also normal cells; hence, the inclusion of sequences able to restrict gene expression only in tumor cells can circumvent this problem. Selective tumor toxicity was obtained positioning the suicide gene under control of the E2Fresponsive promoter elements which are de-repressed in glioma cells (Parr et al, 1997).

have shown encouraging results both in vitro and in vivo (Khil et al, 1995; Kim et al, 1997). 9L glioma cells transduced with a retrovirus encoding a CD/HSV-TK fusion gene exhibited enhanced sensitivity to both GCV and 5-FC, as well as increased radiosensitivity (Rogulski et al, 1997). This experiment suggests the feasibility of a combined approach with two suicide genes associated with radiotherapy. Another prodrug activation system is represented by cytochrome P450 2B1 (the liver enzyme catalyzing cyclophosphamide and ifosfamide activation) gene transfer followed by cyclophosphamide or ifosfamide administration. Metabolites of these drugs produce interstrand DNA cross-linking in a cell cycle-independent fashion. C6 and 9L rat glioma cells, when stably transfected with the P450 2B1 gene, become highly sensitive to cyclophosphamide in in vitro and in vivo models (Wei et al, 1994; Manome et al, 1996). Rabbit cytochrome P450 isozyme CYP4B1, which converts the inert prodrugs 2-amonianthracene (2-AA) and 4-ipomeanol (4-IM) into highly toxic alkylating metabolites, also showed high antitumor effects, both in vitro and in vivo. The treatment had relatively low toxicity and was associated with a bystander effect, not requiring cell-to-cell contact (Rainov et al, 1998).

The effectiveness of suicide gene therapy has been explored for a variety of neoplasms, especially for refractory and localized diseases such as GBM. The HSVTK/GCV scheme has been demonstrated to be effective in animal models, by ex vivo and in vivo transduction with retroviral, adenoviral, or adenoviral-associated vectors expressing HSV-TK (Ezzeddine et al, 1991; Culver et al, 1992; Takamiya et al, 1992, 1993; Barba et al, 1993, 1994; Ram et al, 1993; Kim et al, 1994; Vincent et al, 1996; Maron et al, 1996; Mizuno et al, 1998). Novel gene delivery systems were also applied in a mouse model for gene therapy of meningeal gliomatosis. Liposomes coated with Sendai virus envelope protein were highly efficient in delivering the therapeutic gene in disseminated glioma cells (Mabuchi et al, 1997). A factor that may limit the effectiveness of HSVTK/GCV therapy is the GCV crossing through the bloodbrain barrier. This can be circumvented by the use of the bradykinin analogue and potent blood-brain barrier permeabilizer RMP-7, which, administered intravenously, increase the delivery of GCV into rat brain tumors, enhancing the cytotoxic and bystander effects of HSVTK/GCV (LeMay et al, 1998).

Another suicide gene system is based on E. coli purine nucleoside phosphorylase (PNP), which generates toxic purine nucleoside analogues intracellularly, either from 6methylpurine-2â&#x20AC;&#x2122;-deoxyriboside or arabinofuranosyl-2fluoroadenine monophosphate. Significant antitumor activity and low systemic toxicity were reported in nude mice bearing human malignant D54MG glioma tumors expressing PNP (Parker et al, 1997).

Several clinical trials exploiting the HSV-TK/GCV system have been initiated. It is too early to estimate the effectiveness of these therapeutic procedures. Notwithstanding the evidence for growth-suppressive activities of HSV-TK plus GCV, cure rates are low. Explanation for lack of complete response in humans may reside in the different biological behavior of GBM cells when injected into animals (Sturtz et al, 1997).

Phosphorylation of the prodrug cytosine arabinoside (ara-C) by deoxycyticine kinase (dCK) is a limiting step for activation. Thus, ara-C, a potent antitumor agent for hematological malignancies, has only minimal activity against most solid tumors. Transduction of the dCK cDNA by retroviral and adenoviral vectors also resulted in marked sensitization of glioma cell lines to ara-C in vitro, and in significant antitumor activity in vivo (Manome et al, 1996).

The antitumor effects elicited by HSV-TK/GCV prompted to explore other prodrug activating enzymes. A promising system exploits the ability of E. coli cytosine deaminase (CD) to convert the relatively non toxic 5fluorocytosine (5-FC) to the chemotherapeutic agent 5fluorouracil (5-FU). Significant antitumor effects of CD/5FC were observed in nude mice bearing tumors derived from C6 glioma cells and transduced with CD. A "bystander" effect could also be demonstrated (Ge et al, 1997), suggesting a potential role for gene therapy of glioblastoma.

Unlike other prodrug activating enzymes, E. coli gpt sensitizes cells to the prodrugs 6-thioxanthine (6TX) and 6thioguanine (6-TG), and confers resistance to different regimens (mycophenolic acid, xantine, and hypoxanthine), providing a means to select for gpt-positive cells. Rat C6 glioma cells transduced with a retroviral vector expressing the gpt gene exhibited significant 6TX and 6GT susceptibility and a "bystander" effect in vitro. An antiproliferative effect was demonstrated in vitro and in vivo (Tamiya et al, 1996; Ono et al, 1997).

As already reported for p53, both the CD and HSV-TK systems sensitize cancer cells to radiation. Animal models 137


Palù et al: Perspectives for therapy of glioblastoma multiforme Tumor suicide can also be achieved by direct infection of tumor cells with a conditionally replicative virus, i.e. an infectious agent able to replicate and to kill only dividing cells. In the case of tumors highly proliferating in the context of a completely post-mitotic tissue, such as the brain, gene transfer can ideally be obtained by using neurotropic herpes viral vectors, which are rendered conditionally replicative after deletion of non-essential genes (Lachmann and Efstathiou, 1997).

Despite the location in the central nervous system (CNS), a long-believed “immunologically privileged site”, glioblastoma cells may interact with immune cells. These interactions are mediated by receptor-ligand recognition during cell to cell contact and by a plethora of cytokines. An imbalance in the tumor-host relationship, resulting in deficit in some components of the response, may explain the aggressive growth of malignant gliomas. Before discussing the designed strategies to increase the immune response against glioblastoma cells, we review the more recent acquisitions in the “dialogue” between these neoplastic cells and the immune system (Dietrich et al, 1997).

Tumor specific cell death has already been provided in animal glioma models by HSV vectors, deleted in neurovirulence genes, such as " 34.5, thymidine kinase and ribonucleotide reductase (Chambers et al, 1995; Mineta et al, 1995; Boviatsis et al, 1994; Miyatake et al, 1997; McKie et al, 1996; Andreansky et al, 1996). Their direct injection into gliomas produced tumor regression with minimal bystander effects on surrounding normal tissue. Enhancement of replication of defective HSV vectors lacking " 34.5 gene and a significant reduction of tumor mass was observed combining ionizing radiation (Advani et al, 1998).

A. Antigenicity of glioblastoma cells The presence of specific antigens at the surface of tumor cells to be recognized by cells of the immune system is essential for the generation of a specific antitumor immune response. At present, no tumor antigen able to elicit an immune response has been identified in glioblastoma in vivo. MAGE-1 (melanoma antigen) was the first tumor-specific antigen to be identified (Van der Bruggen et al, 1991), and MAGE family members are expressed by some glioblastoma cell lines (Rimoldi et al, 1993), but not in uncultured tumors (De Smet et al, 1994). This observation could be explained with different levels of DNA methylation induced by culture, where MAGE expression was regulated by methylation (De Smet et al, 1995).

Another way to treat malignant gliomas emerged from the discovery that these tumors often express functional Fas (CD95) (Weller et al, 1994). Fas is a transmembrane glycoprotein belonging to the nerve growth factor/TNF receptor superfamily: when activated, Fas can transduce an apoptotic signal through its cytoplasmic domain. Apoptosis is triggered by the binding of Fas to its natural ligand (FasL) or by cross-linking with anti-Fas antibodies. A high proportion of human glioma cell lines are sensitive to apoptosis mediated by anti-Fas antibodies in vitro. Some other cell lines are resistant, but may be rendered sensitive after stable transfection with human Fas cDNA (Weller et al, 1995). These results offer new possibilities for treating gliomas with anti-Fas antibodies or soluble FasL. One possible drawback of such an approach is that other Fas-positive cells may be affected, like infiltrating leukocytes. Thus their activity may be reduced, restricting strategies relying upon simultaneous immune response enhancement.

Proteins that are structurally altered during malignant transformation, or that contribute to this process, are possible tumor antigen candidates. The frequent alterations of p53 in the early stages of carcinogenesis, for example, may provide new antigenic peptides that could trigger an immune response. Consistent with this possibility, specific cytotoxic T-cell clones were generated in vitro against mutated p53 protein (Houbiers et al, 1993). Additionally, in vivo immunization with mutated p53 peptide was shown to induce specific CTL clones able to lyse MHC-matched tumor cells expressing the mutated p53 gene (Noguchi et al, 1994).

IV. Immunotherapy of cancer

The future identification of glioblastoma-specific antigens would aid new treatment strategies.

Glioblastoma multiforme has the propensity to microscopically infiltrate normal structures since its early stage of development. This characteristic makes therapeutic success rather difficult by any approach. Moreover, it becomes virtually impossible to obtain specific targeting of all tumor cells and sparing of normal ones. Therefore, inducing an efficient immune response against malignant cells becomes an attractive and essential treatment strategy. In this perspective, the natural circulatory properties of cells of the immune system offer also an important support for the recognition of secondary lesions.

B. Tumor-induced immunosuppression A high proportion of glioblastomas have shown to be infiltrated by lymphocytes, mostly T lymphocytes, but also B and NK cells. Differently from what reported for other tumors, the level of lymphocyte infiltration does not relate with a better prognosis. In fact, tumor-infiltrating lymphocytes (TIL) of gliomas appear to be functionally defective. Abnormalities range from abnormal 138


Gene Therapy and Molecular Biology Vol 1, page 139 hypersensitivity responses, depressed response to mitogens, decreased humoral responses (CD4+ T helper cells deficit?), and impaired T cell-mediated cytotoxicity. These functional alterations may be explained, at least in part, by a defective high-affinity IL-2 receptor (IL-2 R) (Roszman et al, 1991). It has been demonstrated that glioblastoma cells produce and release soluble factors that are responsible for a depressed immune response. T lymphocytes from normal individuals exhibit immunologic abnormalities when grown in presence of supernatant obtained from glioblastoma cell line cultures (Roszman et al, 1991). The most important soluble suppressing factor seems to be TGF-#2. This cytokine acts as a growth-inhibitory factor (Sporn et al, 1986; Sporn et al, 1987), and has a defined variety of immunoregulatory properties, including: inhibition of: (i ) T cell proliferation; (i i ) IL-2R induction (Kehrl et al, 1986); (i i i ) cytokine production (Espevik et al, 1987; Espevik et al, 1988); (i v ) natural killer cell activity (Rook et al, 1986); (v) cytotoxic T cell development (Jin et al, 1989; Ranges et al, 1987); (v i ) LAK cell generation (Espevik et al, 1987; Jin et al, 1989); and (v i i ) production of tumor-infiltrating lymphocytes (Kuppner et al, 1989). Most cells secrete TGF-# in a latent form (Sporn et al, 1986), but glioblastoma cells have also the capacity to convert it to an active form, through proteolytic cleavage. This was demonstrated by an experimental work in which T-cell suppression mediated by TGF-#2 was inhibited when proteolytic enzymes were blocked by protease inhibitors (Huber et al, 1992). Other soluble factors, namely prostaglandin E2 (PGE2), IL-1 receptor (IL-1 R) antagonist, and interleukin-10 (IL10) may be implicated in immunosuppression, although in vivo intervention has not been fully defined. A potential down regulation of some immune functions was shown for IL-10 cytokine, which was produced both by GBM cells and normal brain tissue (Nitta et al, 1994; Merlo et al, 1993). Furthermore, in different animal models, human and murine IL-10 was demonstrated to stimulate the acquisition of a specific and efficient antitumor immune response (Berman et al, 1996).

C. Costimulatory molecules A complete T-cell effector function needs not only antigen presentation, but also the delivering of activation signals to the T cell, which is mediated by the so-called costimulatory molecules. Unresponsiveness of T cells (anergy) may be due to absence of the second signal, essentially given by the B7-CD28 interaction (June et al, 1994). Two other members of the B7 family have been cloned, B7.1 and B7.2; their counter-receptors on T cells are CD28 and CTLA-4, respectively. CD28 mediates stimulatory effects, while CTLA-4 appears to be a negative

regulator of T cell responses. Glioblastoma cells and monocytes that infiltrate the tumor are not expressing B7 costimulatory molecules, while monocytes in the normal tissue that surrounds the tumor are B7-positive (Tada et al, 1996). This suggests the possible intervention of local mechanisms able to down-regulate B7 expression in glioblastomas, impeding efficient T-cell priming and favoring T-cell anergy. Moreover, B7-CD28 interactions in the CNS have been shown to be essential to generate a valid CTL response towards viral antigens (Kuendig et al, 1996). Hence, restoring B7 expression by gene transfer might become an interesting task to elicit a proper immune recognition of glioblastoma cells, and an appropriate immune response.

V. Restoring a proper immune response Many approaches have been tented to restore a proper immune response towards malignant gliomas. As previously stated, T lymphocytes play a major role in the antitumor response, and priming of T lymphocytes requires antigen recognition, with or without help from APC. Since no specific antigens have yet been identified for glioblastomas, a vaccination approach has been proposed by administration of genetically modified tumor cells. Moreover, tumor cells transfected to produce various cytokines have been used to enhance lymphocyte responsiveness in animal models. The most interesting results were obtained with cells of murine glioma transfected with an expression vector containing the murine interleukin 7 cDNA (Aoki et al, 1992). IL-7 transfected glioma cells were vigorously rejected by a CD8+ T-cell-mediated immune response, that was proportional to the level of IL-7 production. Moreover, the response was tumor-specific, since no effect was observed against other syngeneic tumor cells (melanoma and fibrosarcoma cells). IL-7 is a very interesting cytokine being able to increase IL-2R ! chain expression on CD4+ T lymphocytes and to inhibit TGF-# mRNA expression and production by murine macrophages (Dubinett et al, 1993). IL-12 can also be considered a promising agent to enhance the antitumor response, since it augments T-cell and natural killer-cell activities, induces IFN-" production, and promotes the differentiation of uncommitted T cells to Th1 cells (Hendrzak and Brunda, 1995). Vector-mediated delivery of IL-12 into established tumors suppresses tumor growth (Caruso et al, 1996) and can induce immune responses against challenge tumors (Bramson et al, 1996). Moreover, IL-12 has other nonimmune properties such as anti-angiogenic effects (Voest et al, 1995). IL-4, another cytokine with pleiotropic functions, increases T cell proliferation and cytotoxicity, and enhances eosinophil and B cell proliferation and differentiation. It

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VI. Combined gene therapy approach in humans

also exerts direct anti-proliferative effects in vitro against many tumor cell lines (Tepper, 1993). Antitumor effects induced by IL-4-transfected cells were reported in nude mice, suggesting T cell-independent mechanisms (Tepper, 1993; Yu et al, 1993). A strong recruitment of eosinophils and subsequent inhibition of the tumor growth was noted. Eosinophil depletion was not performed as a control; hence a direct anti-proliferative effect mediated by IL-4 cannot be excluded. In any respect, a possible non-T-cell-dependent mechanism could be an advantage in the glioblastoma setting, considering the various abnormalities of T cell function.

The strict localization of glioblastomas in the CNS, with only exceptional metastases, makes these tumors candidates for approaches of direct intra-tumoral gene delivery. Retrovirus-mediated gene therapy of GBM is particularly attractive, since these viral vectors transduce only mitotically active cells, sparing the normal neuronal tissue composed of non-replicating cells. Gene therapy of brain tumors by intra-tumoral injection of retroviral vector producing cells (RVPC) in human patients was initiated by Oldfield and colleagues in 1993 (Oldfield et al, 1993). The gene being transferred was that expressing the herpes simplex thymidine kinase (HSV-TK), which conferred sensitivity to the anti-herpes drug ganciclovir. This treatment has proved free of toxicity and safe for there was no evidence of systemic spread of the retroviral vector (Long et al, 1998). However, a clinical benefit was limited to very small tumors (1.5 ml), probably because only malignant cells adjacent to the RVPC were transfected (Ram et al, 1997).

In an attempt to overcome the local immunosuppression mediated by TGF-#2 Fakhrai et al conducted an experimental work on rats by antisense gene therapy. 9L gliosarcoma transfected cells inoculated subcutaneously became highly immunogenic and were able to induce eradication of an established wild-type tumor (Fakhrai et al, 1996). The enhancement of antigen presentation and T cell costimulation has also been considered and may be achieved with genes coding for cytokines, like GM-CSF, costimulatory molecules, like B7, or CIITA, a transcription factor playing a critical role in the regulation of MHC class II molecules. Encouraging results have been reported in a murine melanoma model located in the CNS, whereby an efficient antitumor response was induced by subcutaneous vaccination with irradiated, GM-CSF-producing tumor cells (Sampson et al, 1996). Vaccination with cells co-transfected with B7 and IL-2 was able to mediate rejection of established tumors (Gaken et al, 1997), suggesting a possible application of such an intervention for the treatment of glioblastomas.

A new treatment strategy combining two different modalities, enzyme-directed prodrug activation (tumor suicide) along with cytokine-promoted tumor rejection, has been recently devised to amplify the antitumor response, and proved to be efficacious in animal models (Castleden et al, 1997) (Figure 2). A bicistronic retroviral vector coexpressing HSV-TK and human interleukin-2 genes has been designed to pursue this new approach of cancer gene therapy in humans (Pizzato et al, 1998).

F i g u r e 2 . Structure of a bicistronic retroviral vector for transduction of genes coding for a cytokine and a prodrugactivating enzyme, expressed via a cap-dependent and an internal ribosome entry site (IRES)-dependent mechanism, respectively. A selectable marker (neomycin phosphotransferase gene, neo) is expressed under the control of a SV40 promoter.

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F i g u r e 3 . Gene therapy approach for treatment of glioblastoma multiforme, via intratumoral stereotactic injection of cells producing a triple gene retroviral vector.

F i g u r e 4 . Contrast-enhanced MRI sagittal images of left parietal GBM lesion before (l e f t ), and one month after completion of GCV treatment (t o t h e r i g h t ).

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F i g u r e 5 . Histology of stereotactic biopsies from patients treated by HSV/TK-IL-2 combined gene therapy. A) Toluidine blue staining - Evidence of a large number of infiltrating inflammatory cells; Immunostaining with B ) monoclonal antibodies marking CD3+ cells; C) Mac 387 antibodies recognizing young/activated macrophages; D) monoclonal antibodies marking CD1+ infiltrating cells.

trials with thymidine kinase (Ram et al, 1997; Ostertag and Chiocca, personal communications).

After in vitro characterization of efficacy and safety (Pizzato et al, 1998), the vector was employed in a pilot study to treat four patients with recurrent glioblastoma multiforme (Colombo et al, 1997; Pal첫 et al, 1998) (Figure 3). A significant and sustained reduction (>50% of the initial volume) of the tumor mass (80 ml) was demonstrated by magnetic resonance imaging (MRI) and computerized tomography (CT) in one patient (Figure 4). In this case, the objective response was associated with a dramatic clinical improvement. The other three patients showed areas of tumor necrosis (2 ml) around the site of stereotactic RVPC injection and stabilized disease for a long period of time (11-12 months).

Interestingly, endothelial cells stained positive for TK by in situ hybridization, indicating that the vector had targeted the neo-vascular component, a highly replicative population in glioblastomas. This is consistent with an anti-angiogenic effect of this therapeutic approach, that, in addition to direct tumor suicide and immune activation, may be relevant to the bystander phenomenon and to the clinical response. It is noteworthy that IL-2 was measurable in the cerebral-spinal fluid, even after GCV treatment. This cytokine might have derived from an autocrine-paracrine secretion of recruited infiltrating immune-inflammatory cells, after primary expression in transduced cells.

In stereotactic biopsies taken before ganciclovir administration, large tumor infiltrates of immuneinflammatory cells (T lymphocytes, mostly CD4+ but also CD8+ granzyme B-positive cells, activated macrophages, NK cells, neutrophils) were present, notwithstanding the standard steroid therapy (Figure 5). The observed inflammatory response has never been reported in previous

Efforts to achieve more efficient gene transfer systems are being sought for. These include the development of new generation retroviral vectors, produced at higher titres and characterized by higher transduction efficiency. Strategies involving envelope pseudotyping, use of new

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Gene Therapy and Molecular Biology Vol 1, page 143 Bramson JL, Hitt M, Addison CL, Muller WJ, Gauldie J, Graham FL. (1 9 9 6 ) Direct intratumoral injection of an adenovirus expressing interleukin-12 induces regression and long-lasting immunity that is associated with highly localized expression of interleukin-12. Hum Gene Ther 7, 1995-2002

packaging cell lines of human origin, and substitution of promoter elements will contribute to the improvement of current available vectors. New therapeutic gene combinations should also be accomplished in order to promote a more generalized immune response. Genes for cytokines other than IL-2 (i.e., IL-4, IL-7, IL-12, GM-CSF) as well as genes targeting neoangiogenesis deserve further consideration for combined treatment approaches.

Caruso M, Pham-Nguyen K, Kwong Y-L, Xu B, Kosai K-I, Finegold M, Woo SLC, Chen SH. (1 9 9 6 ) Adenovirusmediated interleukin-12 gene therapy for metastatic colon cancer. Proc Natl Acad Sci USA 93, 11302-11306

The authors wish to acknowledge Fondazione Cassa di Risparmio di Padova e Rovigo and Regione Veneto for financial support.

Castleden SA, Chong H, Garcia-Ribas I, Melcher AA, Hutchinson G, Roberts B, Hart IR, Vile RG. (1 9 9 7 ) A family of bicistronic vectors to enhance both local and systemic antitumor effects of HSVTK or cytokine expression in a murine melanoma model. Hum Gene Ther 8, 2087-102.

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Gene Therapy and Molecular Biology Vol 1, page 133 Gene Ther Mol Biol Vol 3, 133-148. August 1999.

Glioblastoma multiforme: molecular biology and new perspectives for therapy Review Article

Giorgio Pal첫, Luisa Barzon, and Roberta Bonaguro Institute of Microbiology, University of Padova Medical School, Padova, Italy __________________________________________________________________________________________________ Corresponding Author: Giorgio Pal첫, MD, Institute of Microbiology, Via A. Gabelli 63, 35121 Padova, Italy. Tel: +39-049-8272350; Fax: +39-049-8272355; E-mail: gpalu@microb.unipd.it K e y w o r d s : Therapy, gene therapy, brain tumors, gliomas, glioblastoma multiforme, molecular biology, pathogenesis, immunotherapy, neoangiogenesis, oncogenes A b b r e v i a t i o n : GBM, glioblastoma multiforme Received: 23 November 1998; accepted 30 November 1998

Summary Pathogenic features of glioblastoma multiforme and of other gliomas are reviewed in the present article. Emphasis is given to those genetic alterations which are involved in oncogenesis, to the p r o c e s s o f t u m o r n e o a n g i o g e n e s i s a n d t o t h e r o l e p l a y e d b y t h e i m m u n e s y s t e m i n controlling neoplastic growth. Aspects which are relevant to therapeutic interventions are also dissected, and gene therapy in particular. A new gene therapy approach that combines tumor suicide, via enzymedirected prodrug activation, and cytokine-promoted immune rejection i s reported, together with results from the first application of this approach in humans.

of patients for tumor recurrence within 6-12 months from treatment.

I. Introduction The outcome of malignant gliomas remains extremely poor, in spite of aggressive use of currently available therapies. Recent advances in elucidating the molecular biology of gliomas have led to the development of innovative therapeutic strategies. The more promising approaches involve gene therapy, aiming at increasing tumor cell chemosensitivity and/or immunogenicity, by transfer of genes expressing cytokines and prodrug activating enzymes.

Glioblastoma multiforme (grade IV astrocytoma) is usually located in the cerebral hemispheres, though it occasionally appears at other sites, such as the cerebellum, the brain stem and the spinal cord. Histology shows marked cytological diversity, ranging from tumors composed of small cells with scant cytoplasm to those composed of multinucleated giant cells. The World Health Organization (WHO) classification recognizes two distinct subvariants of the tumor: (i ) giant cell glioblastoma, characterized by a predominance of enormous, multinucleated giant cells and, on occasion, an abundant stromal reticulin network; and (i i ) gliosarcoma, in which hyperplastic vascular elements have undergone sarcomatous transformation.

Glioblastoma multiforme (GBM) represents 15-20% of all intracranial tumors and 50% of gliomas (Russel and Rubistein, 1989). It affects 5,000 Americans and 1,000 Italians every year, and typically occurs in adults, with a peak incidence in the fifth and sixth decades of life. It is a very aggressive tumor, with a uniform and profound morbidity. Because of its morbidity it contributes to the cost of cancer on a pro capite basis more than any other tumor. Despite surgery, radiotherapy and/or chemotherapy, the prognosis is extremely poor and has not substantially changed over the last two decades, death resulting in 80%

Current therapies for malignant gliomas include surgical removal of the tumor mass, which is mandatory for precise diagnosis, and irradiation. Although surgery improves the prognosis (Levin et al, 1993), the infiltrative behavior of malignant gliomas precludes their complete

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Pal첫 et al: Perspectives for therapy of glioblastoma multiforme resection, and 90% of GBM recur within 2 cm of the primary site. Postoperative radiotherapy is therefore commonly administered, with a significant improvement in survival (Hochberg and Pruitt, 1980; Walker et al, 1980). Despite surgery and irradiation, however, only a few patients are alive two years after diagnosis. Results of chemotherapy trials are disappointing (Hosli et al, 1998). This is due both to the tumor intrinsic chemoresistance (Petersdorf and Berger, 1996) and to the tumor location within the central nervous system, which limits the penetration of drugs (Janzer and Raff, 1987; Mak et al, 1995). Among malignant gliomas, GBM is the least responsive to medical treatment. Available protocols include both monochemotherapy and polychemotherapy regimens. Nitrosoureas are the leading drugs in glioma chemotherapy, with response rates as single agents varying from 10% to 40% (Young et al, 1973; Fewer et al, 1972; Hoogstraten et al, 1972). Other drugs, evaluated in monochemotherapy (Forsyth and Cairncross, 1996), occasionally showed clinically and radiologically objective responses. Among these are vincristine (Smart et al, 1968), procarbazine (Rodriguez et al, 1989), paclitaxel (Chamberlain and Kormanik, 1995; Prados et al, 1996), and temozolomide (Newlands et al, 1996). However, methodological bias present in most studies raise doubts about the validity of these results. The most commonly used polychemotherapy regimens for gliomas are PVC (i.e. a combination of CCNU, procarbazine, and vincristine) and MOP (i.e. a combination of procarbazine, vincristine, and mechlorethamine). Response rates (complete or partial) of 17-37% have been reported for glioblastomas (Levin et al, 1980; Coyle et al, 1990). More recently, interesting results have been obtained in GBM patients with the ICE regimen (ifosfamide, carboplatin and etoposide), although in association with severe hematological toxicity (Sanson et al, 1996). The role of PVC as adjuvant chemotherapy is controversial (Fine et al, 1993), and, overall, there is no clear-cut evidence that survival of glioblastoma patients is improved by chemotherapy (Hosli et al, 1998).

insurmountable task that gene replacement, or gene suppression, should simultaneously involve a number of different genes, and should be applied to all tumor cells to reverse the malignant phenotype. Hence, corrective gene therapy seems to be quite difficult to propose as a single therapeutic approach.

A. Genetic alterations 1. Oncogenes Several members of the protein-tyrosine kinase receptor family are over-expressed by gene amplification in malignant gliomas, including the epidermal growth factor receptor (EGFR), the platelet-derived growth factor receptor-! (PDGFR!) and the c-met genes (Furnari et al, 1995). A high percentage of glioblastomas also have EGFR gene rearrangements that may lead to the expression of a truncated, constitutively activated receptor. The transfer of a mutant human EGFR gene into glioblastoma cells caused constitutive self phosphorylation and a pronounced enhancement tumorigenicity in nude mice

II. Molecular biology of glioblastoma multiforme and corrective gene therapy As for most cancers, brain tumors derive from a multistep process of successive alterations, including loss of cell cycle control, neoangiogenesis and evasion of immune control. Figure 1 summarizes the genetic alterations associated with the malignant transformation of astrocytes. Most of these changes involve the loss of putative tumor suppressor genes or activation of proto-oncogenes.

o

Gene therapy of cancer, in its most direct form, should aim at replacing a mutated gene with its correct form, or at suppressing the abnormal oncogenic function. At present, however, such a corrective gene therapy, faces the

f

F i g u r e 1 . Simplified representation of oncogenes and tumor suppressor genes contributing to malignant progression of astrocytic tumors.

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Gene Therapy and Molecular Biology Vol 1, page 135 (Ekstrand et al, 1994; Nishikawa et al, 1994). Numerous strategies are currently being investigated to specifically inhibit EGFR using antibodies, immunoconjugates or antisense technology. The selective inhibition of EGFR in human GBM cells with kinase-deficient mutants inhibited cell proliferation and transforming efficiency in athymic mice (O'Rourke et al, 1997), and converted radioresistant human glioblastoma cells to a more sensitive phenotype (O'Rourke et al, 1998), providing a rationale for gene therapy applications.

cells with wild type p53 can significantly inhibit growth and neoangiogenesis, or can induce apoptosis in p53 mutant cells in several tumor models in vitro, including gliomas (Badie et al, 1995; Van Meir et al, 1995; GomezManzano et al, 1996). The presence of functional p53 has also been shown to modulate chemoresistance. Consequently, another possible advantage of the restoration of wild type p53 may be sensitization to chemotherapy and radiotherapy. Indeed, the combination of p53 gene transduction with radiation or chemotherapy (Lowe et al, 1994) has resulted in local tumor control superior to either therapy alone (Fujiwara T et al, 1994; Gjerset et al, 1995; Ngyuyen et al, 1996, Lang et al, 1998). This combined therapy is currently under investigation in clinical trials (Roth and Cristiano, 1997; Nielsen and Maneval, 1998).

Other dominant oncogenes, such as N-myc, fos, src, Hras or N-ras, and mdm2 are amplified and highly expressed in gliomas (Collins, 1993). GBM produce high levels of insulin-like growth factor I (IGF-I). When this alteration has been targeted by a vector expressing an antisense antiIGF-I gene, rejection of genetically altered rat C6 glioma cells was observed. Injection, even at a site distal to the tumor, caused regression of established brain GBM. Destruction of the tumor was mediated by a gliomaspecific T CD8+ (CTL) response (Trojan et al, 1993).

The cell cycle regulator genes provide an additional target for corrective gene therapy. The p105Rb product of the retinoblastoma tumor suppressor gene (Rb) is one of the most critical regulators of cellular proliferation. The Rb protein (pRb), when unphosphorylated, is responsible for arrest of cell cycle by inhibition of the activity of the E2F family of transcription factors. Normal cell cycle progression requires inactivation of Rb through phosphorylation by cyclin-dependent kinases (CDK). This process, in turn, is regulated by CDK inhibitors. Among these, p21 protein is induced directly by p53; p16 protein, and its homologue p15, specifically bind to and inhibit CDK4, and may therefore regulate Rb phosphorylation, and cell cycle progression. Dysregulation of cell cycle control is a frequent finding in malignant gliomas, like deletion or loss of expression of p16 and p15 tumor suppressor genes (Jen et al, 1994; Nishikawa et al, 1995), amplification of CDK4 (He et al, 1994), and deletion or mutation of the Rb tumor suppressor gene (Henson et al, 1994). Interestingly, both of the latter events take place when the p16 gene is intact and correctly expressed (He et al, 1995). Restoration of wild-type p16 gene in glioma cells through an adenoviral vector arrested cells in G0-G1 phases of the cell cycle (Fueyo et al, 1996) and suppressed glioma cells invasion in vitro (Chintala et al, 1997). Overexpression of p21 increases the susceptibility of glioblastoma cells to cisplatin-induced apoptosis (Kondo et al, 1996), whereas adenovirus-mediated transfer of exogenous E2F-1 protein induced massive apoptosis and suppressed glioma growth in vivo and in vitro (Fueyo et al, 1998). The possibility that E2F-responsive promoters may be more active in tumor cells relative to normal cells, because of loss of pRb function, has been exploited to design adenoviral vectors containing transgenes driven by the E2F-1 promoter for gene therapy of gliomas (Parr et al, 1997). These vectors showed tumor-selective gene expression in vivo and reduced toxicity of the normal tissue with respect to standard adenoviral vectors.

2. Genes associated to cell immortalization A role in cell immortalization has been proposed for telomerase, the RNA-protein complex that elongates telomeric DNA. Telomerase is expressed almost exclusively in cancer cells, but not in normal cells, suggesting the possibility that gene therapy may be applied to inhibit this function. A successful example of treatment via antisense oligonucleotides directed against human telomerase suppressed glioma cells growth and survival, both in vitro and in vivo, through the induction of apoptosis (Kondo et al, 1998). 3. Tumor suppressor genes Molecular and cytogenetic analyses of gliomas have shown frequent losses of genetic material, suggesting the inactivation of putative tumor suppressor genes. Loss of heterozygosity (LOH) has been described in chromosome 1p, 9p, 10p, 10q, 11p, 13q, 17p, 19q, and 22q, and in some cases the tumor suppressor gene involved in LOH has been identified. This is the case of the p53 tumor suppressor gene, which maps in 17p. Wild-type p53 protein is involved in G1 cell cycle arrest and apoptosis of DNA-damaged cells and is therefore crucial in preventing mutation or deletion of functional genes. Mutations of p53 seem to be an early event in glioma tumorigenesis, being frequently detected also in low grade astrocytomas. Along with p53 mutations, amplification of the mdm2 oncogene, whose product binds to and degrades p53, accounts for p53 inactivation in gliomas. Since p53 plays a key role in the pathogenesis of most cancers, it has raised great interest as a target for cancer gene therapy. Transduction of malignant

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Pal첫 et al: Perspectives for therapy of glioblastoma multiforme Inhibition or inactivation of genes/factors involved in DNA repair and/or cellular SOS response could represent a gene therapy approach that potentiates radiation therapy. In fact, inhibition of the RAD51 gene by antisense oligonucleotides enhanced the radiosensitivity of mouse malignant gliomas, both in vitro and in vivo, improving survival (Ohnishi et al, 1998). This gene, a homologue of the yeast RAD51 and E. coli RecA genes, is involved in repair of DNA double-strand breaks, in recombination repair, and in various SOS responses to DNA damage caused by gamma-irradiation and alkylating agents.

ribozymes against VEGF mRNA have been successfully employed to reduce VEGF expression in glioma cells (Ke et al 1998), once more suggesting a potential role for antiangiogenic gene therapy. Similarly, bFGF antisense cDNA decreased C6 glioma cells proliferation (Redekop and Naus, 1995). Besides inhibiting the production of angiogenic factors, a therapeutic intervention could also consist of providing tumors with antiangiogenic factors. Indeed, retroviral and adeno-associated viral vectors expressing a modified PF4 were reported to inhibit endothelial cells proliferation in vitro and the growth of intracerebrally implanted gliomas (Tanaka et al, 1997). Retroviral and adenoviral vectors transducing angiostatin gene increased apoptotic death of glioma tumor cells (Tanaka et al, 1998). Additionally, the intratumoral delivery of angiostatin gene by an adenoviral vector produced inhibition of tumor growth in vivo, suppression of neovascularization, and a marked increase of tumor cells apoptosis (Griscelli et al, 1998). Damage of tumor microvasculature was reported also in human malignant glioma xenografts, after gene therapy followed by radiotherapy. The treatment consisted of intratumoral injection of adenoviral vectors expressing tumor necrosis factor-! (TNF-!), under control of the Egr-1 promoter (Staba et al, 1998). The use of viral vectors containing radiation-inducible promoters, such as Egr-1, has the advantage of selectively, spatially, and temporally limiting the effects of the therapeutic gene in the radiation field. Recently, this strategy has yielded interesting results in rat 9L glioma cells (Manome et al, 1998).

Deletions of large regions or even of the entire copy of chromosome 10 are a genetic hallmark of GBM. At least two tumor suppressor genes located on chromosome 10 (one on each arm) have been demonstrated to participate to glial oncogenesis. A first candidate tumor suppressor gene, called PTEN (Phosphatase and tensin homologue deleted on chromosome TEN) was recently characterized (Li et al, 1997). The DNA region encoding PTEN is altered in glioblastoma multiforme, but not in lower grade astrocytic tumors (Tohma et al, 1998; Ichimura et al, 1998; Chiariello et al, 1998).

B. Neoangiogenesis Tumors may remain in a state of dormancy until they establish a blood supply for receiving oxygen and nutrients. The complex process of neoangiogenesis is regulated by numerous factors, some with angiogenic properties, i.e. vascular endothelial growth factor (VEGF), platelet derived growth factor (PDGF!), basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), interleukin-8, and by endogenous inhibitors of angiogenesis, i.e. thrombospondin-1, platelet factor 4 (PF4), angiostatin, endostatin. VEGF, which binds to two specific tyrosine-kinase receptors, called Flk-1 and Flt-1, has been demonstrated to play a key role in angiogenesis of gliomas. Indeed, VEGF and its receptors are downregulated in the normal adult brain, whereas, VEGF is highly produced by GBM cells. Since both flt-1 and flk-1 genes are expressed by proliferating endothelial cells of gliomas, this leads to the establishment of a paracrine loop. Moreover, VEGF expression is higher around necrotic areas and seems to be stimulated by hypoxia.

III. Suicide gene therapy Suicide gene therapy operates by tumor transduction of genes converting a prodrug into a toxic substance; independently, the gene product and the prodrug are nontoxic. The prototype of this approach exploits the selective intracellular phosphorylation of ganciclovir (GCV), driven by the herpes simplex virus thymidine kinase gene product (HSV-TK). This activation generates a toxic drug metabolite that inhibits DNA synthesis, inducing cell death. For in vivo gene transfer of the HSV-TK gene to malignant cells, packaging cells that produces retroviral vectors expressing HSV-TK, have been injected directly into the tumor to transduce replicating cells. An interesting feature of the HSV-TK/GCV system is the bystander killing of nontransduced cells.

Glioblastoma multiforme is one of the most highly vascularized solid neoplasms; therefore, treatments that target neoangiogenesis would be of great interest in clinical practice. Co-injection of rat C6 glioma cells, either subcutaneously or intracerebrally in nude mice, together with cells producing retroviral vectors encoding a dominant-negative mutant of the Flk-1 receptor showed inhibition of neoangiogenesis, reduction of tumor growth, and survival improvement (Millauer et al, 1994; 1996). Antisense VEGF oligonucleotides (Saleh et al, 1996) and

The mechanisms that are responsible for this effect have not been fully defined, but are likely to include: (i ) transfer of non-diffusible, phosphorylated GCV to neighboring cells through gap junctions; (i i ) endocytosis by nontransduced cells of cellular debris containing toxic GCV; and (i i i ) stimulation of host antitumor immune response. The therapeutic efficacy of the HSV-TK/GCV

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Gene Therapy and Molecular Biology Vol 1, page 137 system may be further increased by the use of adenoviral vectors, since these vectors can also transduce resting tumor cells. However, adenoviral vectors will infect also normal cells; hence, the inclusion of sequences able to restrict gene expression only in tumor cells can circumvent this problem. Selective tumor toxicity was obtained positioning the suicide gene under control of the E2Fresponsive promoter elements which are de-repressed in glioma cells (Parr et al, 1997).

have shown encouraging results both in vitro and in vivo (Khil et al, 1995; Kim et al, 1997). 9L glioma cells transduced with a retrovirus encoding a CD/HSV-TK fusion gene exhibited enhanced sensitivity to both GCV and 5-FC, as well as increased radiosensitivity (Rogulski et al, 1997). This experiment suggests the feasibility of a combined approach with two suicide genes associated with radiotherapy. Another prodrug activation system is represented by cytochrome P450 2B1 (the liver enzyme catalyzing cyclophosphamide and ifosfamide activation) gene transfer followed by cyclophosphamide or ifosfamide administration. Metabolites of these drugs produce interstrand DNA cross-linking in a cell cycle-independent fashion. C6 and 9L rat glioma cells, when stably transfected with the P450 2B1 gene, become highly sensitive to cyclophosphamide in in vitro and in vivo models (Wei et al, 1994; Manome et al, 1996). Rabbit cytochrome P450 isozyme CYP4B1, which converts the inert prodrugs 2-amonianthracene (2-AA) and 4-ipomeanol (4-IM) into highly toxic alkylating metabolites, also showed high antitumor effects, both in vitro and in vivo. The treatment had relatively low toxicity and was associated with a bystander effect, not requiring cell-to-cell contact (Rainov et al, 1998).

The effectiveness of suicide gene therapy has been explored for a variety of neoplasms, especially for refractory and localized diseases such as GBM. The HSVTK/GCV scheme has been demonstrated to be effective in animal models, by ex vivo and in vivo transduction with retroviral, adenoviral, or adenoviral-associated vectors expressing HSV-TK (Ezzeddine et al, 1991; Culver et al, 1992; Takamiya et al, 1992, 1993; Barba et al, 1993, 1994; Ram et al, 1993; Kim et al, 1994; Vincent et al, 1996; Maron et al, 1996; Mizuno et al, 1998). Novel gene delivery systems were also applied in a mouse model for gene therapy of meningeal gliomatosis. Liposomes coated with Sendai virus envelope protein were highly efficient in delivering the therapeutic gene in disseminated glioma cells (Mabuchi et al, 1997). A factor that may limit the effectiveness of HSVTK/GCV therapy is the GCV crossing through the bloodbrain barrier. This can be circumvented by the use of the bradykinin analogue and potent blood-brain barrier permeabilizer RMP-7, which, administered intravenously, increase the delivery of GCV into rat brain tumors, enhancing the cytotoxic and bystander effects of HSVTK/GCV (LeMay et al, 1998).

Another suicide gene system is based on E. coli purine nucleoside phosphorylase (PNP), which generates toxic purine nucleoside analogues intracellularly, either from 6methylpurine-2â&#x20AC;&#x2122;-deoxyriboside or arabinofuranosyl-2fluoroadenine monophosphate. Significant antitumor activity and low systemic toxicity were reported in nude mice bearing human malignant D54MG glioma tumors expressing PNP (Parker et al, 1997).

Several clinical trials exploiting the HSV-TK/GCV system have been initiated. It is too early to estimate the effectiveness of these therapeutic procedures. Notwithstanding the evidence for growth-suppressive activities of HSV-TK plus GCV, cure rates are low. Explanation for lack of complete response in humans may reside in the different biological behavior of GBM cells when injected into animals (Sturtz et al, 1997).

Phosphorylation of the prodrug cytosine arabinoside (ara-C) by deoxycyticine kinase (dCK) is a limiting step for activation. Thus, ara-C, a potent antitumor agent for hematological malignancies, has only minimal activity against most solid tumors. Transduction of the dCK cDNA by retroviral and adenoviral vectors also resulted in marked sensitization of glioma cell lines to ara-C in vitro, and in significant antitumor activity in vivo (Manome et al, 1996).

The antitumor effects elicited by HSV-TK/GCV prompted to explore other prodrug activating enzymes. A promising system exploits the ability of E. coli cytosine deaminase (CD) to convert the relatively non toxic 5fluorocytosine (5-FC) to the chemotherapeutic agent 5fluorouracil (5-FU). Significant antitumor effects of CD/5FC were observed in nude mice bearing tumors derived from C6 glioma cells and transduced with CD. A "bystander" effect could also be demonstrated (Ge et al, 1997), suggesting a potential role for gene therapy of glioblastoma.

Unlike other prodrug activating enzymes, E. coli gpt sensitizes cells to the prodrugs 6-thioxanthine (6TX) and 6thioguanine (6-TG), and confers resistance to different regimens (mycophenolic acid, xantine, and hypoxanthine), providing a means to select for gpt-positive cells. Rat C6 glioma cells transduced with a retroviral vector expressing the gpt gene exhibited significant 6TX and 6GT susceptibility and a "bystander" effect in vitro. An antiproliferative effect was demonstrated in vitro and in vivo (Tamiya et al, 1996; Ono et al, 1997).

As already reported for p53, both the CD and HSV-TK systems sensitize cancer cells to radiation. Animal models 137


Palù et al: Perspectives for therapy of glioblastoma multiforme Tumor suicide can also be achieved by direct infection of tumor cells with a conditionally replicative virus, i.e. an infectious agent able to replicate and to kill only dividing cells. In the case of tumors highly proliferating in the context of a completely post-mitotic tissue, such as the brain, gene transfer can ideally be obtained by using neurotropic herpes viral vectors, which are rendered conditionally replicative after deletion of non-essential genes (Lachmann and Efstathiou, 1997).

Despite the location in the central nervous system (CNS), a long-believed “immunologically privileged site”, glioblastoma cells may interact with immune cells. These interactions are mediated by receptor-ligand recognition during cell to cell contact and by a plethora of cytokines. An imbalance in the tumor-host relationship, resulting in deficit in some components of the response, may explain the aggressive growth of malignant gliomas. Before discussing the designed strategies to increase the immune response against glioblastoma cells, we review the more recent acquisitions in the “dialogue” between these neoplastic cells and the immune system (Dietrich et al, 1997).

Tumor specific cell death has already been provided in animal glioma models by HSV vectors, deleted in neurovirulence genes, such as " 34.5, thymidine kinase and ribonucleotide reductase (Chambers et al, 1995; Mineta et al, 1995; Boviatsis et al, 1994; Miyatake et al, 1997; McKie et al, 1996; Andreansky et al, 1996). Their direct injection into gliomas produced tumor regression with minimal bystander effects on surrounding normal tissue. Enhancement of replication of defective HSV vectors lacking " 34.5 gene and a significant reduction of tumor mass was observed combining ionizing radiation (Advani et al, 1998).

A. Antigenicity of glioblastoma cells The presence of specific antigens at the surface of tumor cells to be recognized by cells of the immune system is essential for the generation of a specific antitumor immune response. At present, no tumor antigen able to elicit an immune response has been identified in glioblastoma in vivo. MAGE-1 (melanoma antigen) was the first tumor-specific antigen to be identified (Van der Bruggen et al, 1991), and MAGE family members are expressed by some glioblastoma cell lines (Rimoldi et al, 1993), but not in uncultured tumors (De Smet et al, 1994). This observation could be explained with different levels of DNA methylation induced by culture, where MAGE expression was regulated by methylation (De Smet et al, 1995).

Another way to treat malignant gliomas emerged from the discovery that these tumors often express functional Fas (CD95) (Weller et al, 1994). Fas is a transmembrane glycoprotein belonging to the nerve growth factor/TNF receptor superfamily: when activated, Fas can transduce an apoptotic signal through its cytoplasmic domain. Apoptosis is triggered by the binding of Fas to its natural ligand (FasL) or by cross-linking with anti-Fas antibodies. A high proportion of human glioma cell lines are sensitive to apoptosis mediated by anti-Fas antibodies in vitro. Some other cell lines are resistant, but may be rendered sensitive after stable transfection with human Fas cDNA (Weller et al, 1995). These results offer new possibilities for treating gliomas with anti-Fas antibodies or soluble FasL. One possible drawback of such an approach is that other Fas-positive cells may be affected, like infiltrating leukocytes. Thus their activity may be reduced, restricting strategies relying upon simultaneous immune response enhancement.

Proteins that are structurally altered during malignant transformation, or that contribute to this process, are possible tumor antigen candidates. The frequent alterations of p53 in the early stages of carcinogenesis, for example, may provide new antigenic peptides that could trigger an immune response. Consistent with this possibility, specific cytotoxic T-cell clones were generated in vitro against mutated p53 protein (Houbiers et al, 1993). Additionally, in vivo immunization with mutated p53 peptide was shown to induce specific CTL clones able to lyse MHC-matched tumor cells expressing the mutated p53 gene (Noguchi et al, 1994).

IV. Immunotherapy of cancer

The future identification of glioblastoma-specific antigens would aid new treatment strategies.

Glioblastoma multiforme has the propensity to microscopically infiltrate normal structures since its early stage of development. This characteristic makes therapeutic success rather difficult by any approach. Moreover, it becomes virtually impossible to obtain specific targeting of all tumor cells and sparing of normal ones. Therefore, inducing an efficient immune response against malignant cells becomes an attractive and essential treatment strategy. In this perspective, the natural circulatory properties of cells of the immune system offer also an important support for the recognition of secondary lesions.

B. Tumor-induced immunosuppression A high proportion of glioblastomas have shown to be infiltrated by lymphocytes, mostly T lymphocytes, but also B and NK cells. Differently from what reported for other tumors, the level of lymphocyte infiltration does not relate with a better prognosis. In fact, tumor-infiltrating lymphocytes (TIL) of gliomas appear to be functionally defective. Abnormalities range from abnormal 138


Gene Therapy and Molecular Biology Vol 1, page 139 hypersensitivity responses, depressed response to mitogens, decreased humoral responses (CD4+ T helper cells deficit?), and impaired T cell-mediated cytotoxicity. These functional alterations may be explained, at least in part, by a defective high-affinity IL-2 receptor (IL-2 R) (Roszman et al, 1991). It has been demonstrated that glioblastoma cells produce and release soluble factors that are responsible for a depressed immune response. T lymphocytes from normal individuals exhibit immunologic abnormalities when grown in presence of supernatant obtained from glioblastoma cell line cultures (Roszman et al, 1991). The most important soluble suppressing factor seems to be TGF-#2. This cytokine acts as a growth-inhibitory factor (Sporn et al, 1986; Sporn et al, 1987), and has a defined variety of immunoregulatory properties, including: inhibition of: (i ) T cell proliferation; (i i ) IL-2R induction (Kehrl et al, 1986); (i i i ) cytokine production (Espevik et al, 1987; Espevik et al, 1988); (i v ) natural killer cell activity (Rook et al, 1986); (v) cytotoxic T cell development (Jin et al, 1989; Ranges et al, 1987); (v i ) LAK cell generation (Espevik et al, 1987; Jin et al, 1989); and (v i i ) production of tumor-infiltrating lymphocytes (Kuppner et al, 1989). Most cells secrete TGF-# in a latent form (Sporn et al, 1986), but glioblastoma cells have also the capacity to convert it to an active form, through proteolytic cleavage. This was demonstrated by an experimental work in which T-cell suppression mediated by TGF-#2 was inhibited when proteolytic enzymes were blocked by protease inhibitors (Huber et al, 1992). Other soluble factors, namely prostaglandin E2 (PGE2), IL-1 receptor (IL-1 R) antagonist, and interleukin-10 (IL10) may be implicated in immunosuppression, although in vivo intervention has not been fully defined. A potential down regulation of some immune functions was shown for IL-10 cytokine, which was produced both by GBM cells and normal brain tissue (Nitta et al, 1994; Merlo et al, 1993). Furthermore, in different animal models, human and murine IL-10 was demonstrated to stimulate the acquisition of a specific and efficient antitumor immune response (Berman et al, 1996).

C. Costimulatory molecules A complete T-cell effector function needs not only antigen presentation, but also the delivering of activation signals to the T cell, which is mediated by the so-called costimulatory molecules. Unresponsiveness of T cells (anergy) may be due to absence of the second signal, essentially given by the B7-CD28 interaction (June et al, 1994). Two other members of the B7 family have been cloned, B7.1 and B7.2; their counter-receptors on T cells are CD28 and CTLA-4, respectively. CD28 mediates stimulatory effects, while CTLA-4 appears to be a negative

regulator of T cell responses. Glioblastoma cells and monocytes that infiltrate the tumor are not expressing B7 costimulatory molecules, while monocytes in the normal tissue that surrounds the tumor are B7-positive (Tada et al, 1996). This suggests the possible intervention of local mechanisms able to down-regulate B7 expression in glioblastomas, impeding efficient T-cell priming and favoring T-cell anergy. Moreover, B7-CD28 interactions in the CNS have been shown to be essential to generate a valid CTL response towards viral antigens (Kuendig et al, 1996). Hence, restoring B7 expression by gene transfer might become an interesting task to elicit a proper immune recognition of glioblastoma cells, and an appropriate immune response.

V. Restoring a proper immune response Many approaches have been tented to restore a proper immune response towards malignant gliomas. As previously stated, T lymphocytes play a major role in the antitumor response, and priming of T lymphocytes requires antigen recognition, with or without help from APC. Since no specific antigens have yet been identified for glioblastomas, a vaccination approach has been proposed by administration of genetically modified tumor cells. Moreover, tumor cells transfected to produce various cytokines have been used to enhance lymphocyte responsiveness in animal models. The most interesting results were obtained with cells of murine glioma transfected with an expression vector containing the murine interleukin 7 cDNA (Aoki et al, 1992). IL-7 transfected glioma cells were vigorously rejected by a CD8+ T-cell-mediated immune response, that was proportional to the level of IL-7 production. Moreover, the response was tumor-specific, since no effect was observed against other syngeneic tumor cells (melanoma and fibrosarcoma cells). IL-7 is a very interesting cytokine being able to increase IL-2R ! chain expression on CD4+ T lymphocytes and to inhibit TGF-# mRNA expression and production by murine macrophages (Dubinett et al, 1993). IL-12 can also be considered a promising agent to enhance the antitumor response, since it augments T-cell and natural killer-cell activities, induces IFN-" production, and promotes the differentiation of uncommitted T cells to Th1 cells (Hendrzak and Brunda, 1995). Vector-mediated delivery of IL-12 into established tumors suppresses tumor growth (Caruso et al, 1996) and can induce immune responses against challenge tumors (Bramson et al, 1996). Moreover, IL-12 has other nonimmune properties such as anti-angiogenic effects (Voest et al, 1995). IL-4, another cytokine with pleiotropic functions, increases T cell proliferation and cytotoxicity, and enhances eosinophil and B cell proliferation and differentiation. It

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VI. Combined gene therapy approach in humans

also exerts direct anti-proliferative effects in vitro against many tumor cell lines (Tepper, 1993). Antitumor effects induced by IL-4-transfected cells were reported in nude mice, suggesting T cell-independent mechanisms (Tepper, 1993; Yu et al, 1993). A strong recruitment of eosinophils and subsequent inhibition of the tumor growth was noted. Eosinophil depletion was not performed as a control; hence a direct anti-proliferative effect mediated by IL-4 cannot be excluded. In any respect, a possible non-T-cell-dependent mechanism could be an advantage in the glioblastoma setting, considering the various abnormalities of T cell function.

The strict localization of glioblastomas in the CNS, with only exceptional metastases, makes these tumors candidates for approaches of direct intra-tumoral gene delivery. Retrovirus-mediated gene therapy of GBM is particularly attractive, since these viral vectors transduce only mitotically active cells, sparing the normal neuronal tissue composed of non-replicating cells. Gene therapy of brain tumors by intra-tumoral injection of retroviral vector producing cells (RVPC) in human patients was initiated by Oldfield and colleagues in 1993 (Oldfield et al, 1993). The gene being transferred was that expressing the herpes simplex thymidine kinase (HSV-TK), which conferred sensitivity to the anti-herpes drug ganciclovir. This treatment has proved free of toxicity and safe for there was no evidence of systemic spread of the retroviral vector (Long et al, 1998). However, a clinical benefit was limited to very small tumors (1.5 ml), probably because only malignant cells adjacent to the RVPC were transfected (Ram et al, 1997).

In an attempt to overcome the local immunosuppression mediated by TGF-#2 Fakhrai et al conducted an experimental work on rats by antisense gene therapy. 9L gliosarcoma transfected cells inoculated subcutaneously became highly immunogenic and were able to induce eradication of an established wild-type tumor (Fakhrai et al, 1996). The enhancement of antigen presentation and T cell costimulation has also been considered and may be achieved with genes coding for cytokines, like GM-CSF, costimulatory molecules, like B7, or CIITA, a transcription factor playing a critical role in the regulation of MHC class II molecules. Encouraging results have been reported in a murine melanoma model located in the CNS, whereby an efficient antitumor response was induced by subcutaneous vaccination with irradiated, GM-CSF-producing tumor cells (Sampson et al, 1996). Vaccination with cells co-transfected with B7 and IL-2 was able to mediate rejection of established tumors (Gaken et al, 1997), suggesting a possible application of such an intervention for the treatment of glioblastomas.

A new treatment strategy combining two different modalities, enzyme-directed prodrug activation (tumor suicide) along with cytokine-promoted tumor rejection, has been recently devised to amplify the antitumor response, and proved to be efficacious in animal models (Castleden et al, 1997) (Figure 2). A bicistronic retroviral vector coexpressing HSV-TK and human interleukin-2 genes has been designed to pursue this new approach of cancer gene therapy in humans (Pizzato et al, 1998).

F i g u r e 2 . Structure of a bicistronic retroviral vector for transduction of genes coding for a cytokine and a prodrugactivating enzyme, expressed via a cap-dependent and an internal ribosome entry site (IRES)-dependent mechanism, respectively. A selectable marker (neomycin phosphotransferase gene, neo) is expressed under the control of a SV40 promoter.

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F i g u r e 3 . Gene therapy approach for treatment of glioblastoma multiforme, via intratumoral stereotactic injection of cells producing a triple gene retroviral vector.

F i g u r e 4 . Contrast-enhanced MRI sagittal images of left parietal GBM lesion before (l e f t ), and one month after completion of GCV treatment (t o t h e r i g h t ).

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F i g u r e 5 . Histology of stereotactic biopsies from patients treated by HSV/TK-IL-2 combined gene therapy. A) Toluidine blue staining - Evidence of a large number of infiltrating inflammatory cells; Immunostaining with B ) monoclonal antibodies marking CD3+ cells; C) Mac 387 antibodies recognizing young/activated macrophages; D) monoclonal antibodies marking CD1+ infiltrating cells.

trials with thymidine kinase (Ram et al, 1997; Ostertag and Chiocca, personal communications).

After in vitro characterization of efficacy and safety (Pizzato et al, 1998), the vector was employed in a pilot study to treat four patients with recurrent glioblastoma multiforme (Colombo et al, 1997; Pal첫 et al, 1998) (Figure 3). A significant and sustained reduction (>50% of the initial volume) of the tumor mass (80 ml) was demonstrated by magnetic resonance imaging (MRI) and computerized tomography (CT) in one patient (Figure 4). In this case, the objective response was associated with a dramatic clinical improvement. The other three patients showed areas of tumor necrosis (2 ml) around the site of stereotactic RVPC injection and stabilized disease for a long period of time (11-12 months).

Interestingly, endothelial cells stained positive for TK by in situ hybridization, indicating that the vector had targeted the neo-vascular component, a highly replicative population in glioblastomas. This is consistent with an anti-angiogenic effect of this therapeutic approach, that, in addition to direct tumor suicide and immune activation, may be relevant to the bystander phenomenon and to the clinical response. It is noteworthy that IL-2 was measurable in the cerebral-spinal fluid, even after GCV treatment. This cytokine might have derived from an autocrine-paracrine secretion of recruited infiltrating immune-inflammatory cells, after primary expression in transduced cells.

In stereotactic biopsies taken before ganciclovir administration, large tumor infiltrates of immuneinflammatory cells (T lymphocytes, mostly CD4+ but also CD8+ granzyme B-positive cells, activated macrophages, NK cells, neutrophils) were present, notwithstanding the standard steroid therapy (Figure 5). The observed inflammatory response has never been reported in previous

Efforts to achieve more efficient gene transfer systems are being sought for. These include the development of new generation retroviral vectors, produced at higher titres and characterized by higher transduction efficiency. Strategies involving envelope pseudotyping, use of new

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Gene Therapy and Molecular Biology Vol 1, page 143 Bramson JL, Hitt M, Addison CL, Muller WJ, Gauldie J, Graham FL. (1 9 9 6 ) Direct intratumoral injection of an adenovirus expressing interleukin-12 induces regression and long-lasting immunity that is associated with highly localized expression of interleukin-12. Hum Gene Ther 7, 1995-2002

packaging cell lines of human origin, and substitution of promoter elements will contribute to the improvement of current available vectors. New therapeutic gene combinations should also be accomplished in order to promote a more generalized immune response. Genes for cytokines other than IL-2 (i.e., IL-4, IL-7, IL-12, GM-CSF) as well as genes targeting neoangiogenesis deserve further consideration for combined treatment approaches.

Caruso M, Pham-Nguyen K, Kwong Y-L, Xu B, Kosai K-I, Finegold M, Woo SLC, Chen SH. (1 9 9 6 ) Adenovirusmediated interleukin-12 gene therapy for metastatic colon cancer. Proc Natl Acad Sci USA 93, 11302-11306

The authors wish to acknowledge Fondazione Cassa di Risparmio di Padova e Rovigo and Regione Veneto for financial support.

Castleden SA, Chong H, Garcia-Ribas I, Melcher AA, Hutchinson G, Roberts B, Hart IR, Vile RG. (1 9 9 7 ) A family of bicistronic vectors to enhance both local and systemic antitumor effects of HSVTK or cytokine expression in a murine melanoma model. Hum Gene Ther 8, 2087-102.

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Gene Therapy and Molecular Biology Vol 3, page 149 Gene Ther Mol Biol Vol 3, 149-155. August 1999.

Gene-based vaccine strategies against cancer Review Article

Daniel Lee1, Ken Wang 1, Liesl K. Nottingham2, Jim Oh1, David B. Weiner1, and Jong J. Kim1 1

Department of Pathology and Laboratory Medicine; 2Department of Otolaryngology/Head and Neck Surgery

University of Pennsylvania, Philadelphia, PA 19104 __________________________________________________________________________________________________ Corresponding Author: Jong J. Kim, Ph.D., Department of Pathology and Laboratory Medicine, University of Pennsylvania, 505 Stellar-Chance, 422 Curie Blvd., Philadelphia, PA 19104. Tel: (215) 662-2352; Fax: (215) 573-9436; E-mail: jonger@seas.upenn.edu Received: 30 September 1998; accepted: 7 October 1998

Summary In recent years, the characterization of gene-based cancer vaccines has been an important step in the development of different treatment options for human carcinoma. These particular vaccines m a k e u s e o f p r o t e i n s t h a t a r e s p e c i f i c a l l y p r o d u c e d a t v e r y h i g h l e v e l s by tumor c e l l s . These tumor-associated antigens (TAAs) are not o n l y used i n diagnostic situations, but also i n the development of cancer vaccines. In this review we will focus on two well characterized TAAs, carcinoembryonic antigen (CEA) and prostate specific antigen (PSA). The two methods of i n v i v o delivery we will examine are recombinant vaccinia virus and nucleic acid immunization. The TAA g e n e c a n b e c l o n e d i n t o v a c c i n i a v i r u s a n d the viral infection stimulates an adequate immune response in the host. In the case of nucleic acid immunization, DNA constructs encoding for TAAs are directly injected into the host and are taken up by its cells. The cells express the specific encoded antigen upon which the immune system acts. The effects o f CEA recombinant vaccinia virus (rV-CEA) have been characterized i n rodents, macaques, and humans. It was shown that the vaccine induced both humoral and cellular immune responses in mice and monkey models. In a phase I clinical trial, a CEA-specific cytotoxic Tlymphocyte response was observed. The effects of a CEA DNA vaccine were investigated in both mice and dogs and both humoral and cellular immune responses were found as well. A recombinant vaccinia virus expressing PSA was tested in rhesus monkeys and induced a PSA-specific long term cellular immune response. Experiments were also performed injecting a PSA DNA construct into both mice and rhesus monkeys. PSA-specific humoral and cellular immune responses were observed in both cases. All these experimental approaches demonstrate the efficacy and advantages of genebased cancer vaccine strategies and support further clinical investigations.

Thus, researchers are continually investigating novel and more effective treatment strategies for various forms of cancer. Research, in recent years, has turned toward the use of vaccines to treat cancer. To this end, several proteins produced by tumor cells became a target for vaccine development. These tumor-associated antigens are predominantly expressed in a tissuespecific manner and are expressed at greatly increased levels in affected cells. Besides being important

I. Introduction Although advances in science have led to countless theories and methods designed to combat human carcinoma, the battle is far from being over. Surgical excision of tumors, drug therapies, and chemotherapy have been effective in certain cases but in other situations, particularly when the tumor has begun to metastasize, effective treatment is far more difficult and far less potent.

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Lee et al: Gene-based vaccine strategies against cancer diagnostic aids, these antigens represent appropriate targets for the development of cancer vaccines (Sogn et al, 1993).

(Kaufman et al, 1991). Among its many advantages is that it greatly enhances the immune response when coupled with a weak immunogen such as a TAA. Through recombinant DNA technology, TAA genes can be cloned into the vaccinia viral vector and this recombinant vaccinia virus can be used to stimulate an effective immune response. Another advantage is that it can infect professional antigen presenting cells (APCs), such as dendritic cells or macrophages, and express the antigen along with MHC class I and/or class II complexes (Tsang et al, 1995). Finally, the stability and efficiency of vaccinia allows it to successfully incorporate fairly large inserts, which is advantageous in the context of cloning the genes for different TAAs (Kaufman et al, 1991). Potential disadvantages are toxicity effects, immunogenecity to the virus, and risk of viral reversion. Moreover, recombinant vaccinia viruses cannot be used to target specific cells.

Tumor-associated antigens (TAA) are proteins produced by tumor cells which can be presented on the cell surface in the context of major histocompatibility complexes (Kelley and Cole, 1998). Recently, these antigens have been the focus of study as a viable option for immunotherapy of various types of cancer. In this review we will examine the progress in the investigation of the immunological effects of two such TAAs, carcinoembryonic antigen (CEA) and prostate specific antigen (PSA).

II. Background The use of therapeutic cancer vaccines has several distinct advantages. The immune response can be directed against carcinomas with a high degree of specificity. They can also generate immunological memory, for continued protection. The immune response induced by the vaccine can be modified or enhanced with other forms of immunotherapy such as using cytokines and other cellular therapies (Jones and Mitchell, 1996). Gene-based cancer vaccine strategies have yielded promising results, and several different methods of in vivo delivery are currently being explored (Roth and Cristiano, 1997). Two such approaches are recombinant vaccinia virus and nucleic acid, or DNA immunization (Table 1).

DNA vaccination is a relatively new approach towards disease prophylaxis and/or treatment. DNA expression cassettes introduced in vivo can be taken up and expressed by host cells, leading to the production of specific foreign proteins. The presence of these foreign proteins can then elicit specific humoral and cellular immune responses against the foreign antigens (Wolff et al, 1990; Tang et al, 1992; Wang et al, 1993; Ulmer et al, 1993). This technique can be applied more widely than delivery through a recombinant vaccinia virus because there is no limitation on the size and type of nucleic acid used (Roth and Cristiano 1997). DNA vaccines are non-replicating, thereby minimizing the risk of any primary infections. It is also possible to alter or delete undesirable genes, such as those which may inhibit the immune response. More recently, the

Vaccinia virus is one of the most heavily investigated viral delivery vehicles; it is a type of pox virus which was used in the successful eradication of smallpox (Kantor et al, 1992a). It is extremely immunogenic and is capable of stimulating both humoral and cellular immune responses

Vaccinia

DNA

Advantages

Disadvantages

-highly immunogenic -infects APCs -induces both humoral and cellular responses -large insert size

-toxicity -risk of viral reversion -no targeting -induces vaccinia specific immune response

-possible to specifically target cells -low immunogenicity -no limit on size and type of nucleic acid -difficult to incorporate into cells in vivo -induces both humoral and cellular responses -non-replicating -able to genetically alter and enhance -use of molecular adjuvants to modulate response -repeated use without decrease in effect

T a b l e 1 . Comparison of recombinant vaccinia virus and nucleic acid immunization as in vivo delivery vehicles for gene-based cancer vaccine therapy.

use of molecular adjuvants such as cytokines and costimulatory molecules has proven to be effective in modulating and directing the desired immune responses 150


Gene Therapy and Molecular Biology Vol 3, page 151 (Kim et al, 1998). Nucleic acid immunization is promising in the development of vaccinations for a wide array of pathogens, including cancer (Kim et al, In Press). Using DNA expression cassettes, DNA sequences that encode certain cancer proteins, such as those found in colon cancer or prostate cancer, are introduced into host cells. These cells then synthesize the antigenic cancer proteins which can then elicit an immune response against those proteins. The first clinical studies for DNA vaccines tested the effects of the HIV-1 env/rev DNA vaccine in HIV-infected patients (MacGregor et al, 1998). Each patient in the trial received three injections each separated by ten weeks with increasing dosage (3 dosage groups of 5 subjects) of envelope vaccine. The clinical results reveal no significant clinical or laboratory adverse effects measured in all three dosage groups (30, 100, 300 Âľg). The immunized individuals developed increased antibody responses to envelope proteins and peptides after receiving the 100 Âľg dose of env/rev. Some increased cellular responses were also observed. These preliminary results demonstrate that the injection of even relatively low doses of a single immunogen DNA vaccine can augment both existing humoral and cellular immune responses in humans in a safe and tolerant manner.

III. Gene-based cancer vaccine strategies using CEA Human CEA is a 180-kDa glycoprotein expressed in elevated levels in 90% of gastrointestinal malignancies, including colon, rectal, stomach, and pancreatic tumors, 70% of lung cancers, and 50% of breast cancers (Zaremba et al, 1997, Kelley and Cole, 1998). CEA is also found in human fetal digestive organ tissue, hence the name carcinoembryonic antigen (Foon et al, 1995). It has been discovered that CEA is expressed in normal adult colon epithelium as well, albeit at far lower levels (Conry et al, 1996a). Sequencing of CEA shows that it is associated with the human immunoglobulin gene superfamily and that it may be involved in the metastasizing of tumor cells (Foon et al, 1995).

immunization were predominantly mediated by CEAspecific CD8+ T-cell response (Abrams et al, 1997). Splenocytes from rV-CEA immunized C57BL/6 mice were adoptively transferred to syngeneic immune deficient, tumor-bearing mice. They exhibited strong anti-tumor activity compared to splenocytes transferred from nonimmunized mice. Adoptive transfer of CD4+, but not CD8 + T cells did not show anti-tumor activity. However, transfer of CD8+, but not CD4 + T cells still showed some antitumor response, although this response was less compared to when both CD8+ and CD4+ cell populations are present. CD4+ cells therefore may play an important helper or regulatory role in anti-tumor responses. Immunization of mice with rV-CEA induced anti-tumor activity that was mediated mainly by CD8+ cells, but both CD8+ and CD4+ cells were necessary to acheive optimal anti-tumor responses (Abrams et al, 1997). The effects of rV-CEA vaccination were further characterized in experimental trials with non-human primates. After injection, the rhesus macaques of the experimental group showed both humoral and cellular immune responses to CEA. The immunization also resulted in toxic effects such as mild fever, irritation of the skin near the injection point, and lymphadenopathy (Kantor et al, 1992b). The results of this experiment along with the results from various rodent experiments demonstrated potential utility and limitations of the rVCEA vaccine. Additional information in this regard has been provided in the clinical setting. Tsang, et al. in conjunction with the National Cancer Institute, recently conducted a phase I clinical trial testing the effects of rV-CEA in 26 patients with advanced metastatic carcinoma (Tsang et al, 1995). Peripheral blood lymphocytes (PBLs) were taken from patients both before and after vaccination and analyzed for their response to specific CEA peptides with human leukocyte antigen (HLA) class I-A2 motifs. It was observed that CEA-specific MHC class I restricted cytotoxic T-lymphocyte response could be elicited (Tsang et al, 1995). However, following the first vaccination, there was an anti-vaccinia immune response which suppressed the effects of subsequent vaccinations (Kelley and Cole, 1998).

A. CEA recombinant vaccinia virus vaccine Recombinant vaccinia virus expressing the human CEA gene (rV-CEA) has been investigated as a potential therapy for colon and other gastrointestinal carcinomas. A number of groups have shown that immunization of these constructs into rodents induced both cellular and humoral responses. More importantly, immunization with rV-CEA led to antigen-specific inhibition of tumor growth in mice. Using an adaptive transfer experiment, Abrams, et. al. found that anti-tumor responses after rV-CEA

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B. CEA DNA vaccine The immune response to nucleic acid vaccination using a CEA DNA construct was characterized in a murine model. The CEA insert was cloned into a vector containing the cytomegalovirus (CMV) early promoter/enhancer and injected intramuscularly. CEA spe-


Gene Therapy and Molecular Biology Vol 3, page 152

Humoral response

Cellular response

rV-PSA

+

+

PSA DNA

+

+

T a b l e 2 . Induction of PSA-specific immune responses in rhesus macaques.

human PSA (rV-PSA) were studied in rodent as well as in non-human primate models (Hodge et al, 1995). Hodge, et al. investigated the immunological effects of a recombinant vaccinia virus expressing human PSA (rVPSA) in rhesus monkeys. Because of the high degree of similarity between the rhesus and human prostate gland and PSA (>90%), this animal model was well suited to accurately assess the effects of rV-PSA. Murine and other models did not share this homology. A control group receiving high-dose V-Wyeth, a group receiving low-does rV-PSA and a group receiving high-dose rV-PSA were all given 3 injections at four week intervals. Before the initial injection, one monkey in each group was given a prostatectomy in order to mimic the situation of human patients who have undergone the same procedure. Following injection, the rhesus monkeys exhibited the expected low-grade fever and other symptoms of vaccinia infection. It was found that the monkeys receiving the high dose rV-PSA vaccination expressed long term cellular immune responses specific to PSA (Table 2). Also, there was no difference in the immune response of the monkeys who had their prostates removed (Hodge et al, 1995). Much like the experiments with rV-CEA, this experiment showed the effectiveness of rV-PSA in inducing an immune response in macaques.

cific humoral and cellular responses were detected in the immunized mice. These responses were comparable to the immune response generated by rV-CEA (Conry et al, 1994). The CEA DNA vaccine was also characterized in a canine model, where sera obtained from dogs injected intramuscularly with the construct demonstrated an increase in antibody levels (Smith et al, 1998). Cellular immune responses quantified using the lymphoblast transformation (LBT) assay also revealed proliferation of CEA-specific lymphocytes. Therefore a CEA nucleic acid vaccine was able to induce both arms of the immune responses (Smith et al, 1998). CEA DNA vaccines are currently being investigated in humans.

IV. Gene-based cancer vaccine strategies using PSA Prostate cancer is the most common form of cancer and the second most common cause of cancer related death in American men (Boring et al, 1994). The appearance of prostate cancer is much more common in men over the age of fifty (Gilliland and Keys, 1995). Three of the most widely used treatments are surgical excision of the prostate and seminal vesicles, external bean irradiation, and androgen deprivation. However, conventional therapies lose their efficacy once the tumor has metastasized, which is the case in more than half of initial diagnoses (Wei et al, 1997, Ko et al, 1996).

B. PSA DNA Vaccine

PSA is a serine protease and a human glandular kallikrein gene product of 240 amino acids which is secreted by both normal and transformed epithelial cells of the prostate gland (Wang et al, 1982; Watt et al, 1986). Because cancer cells secrete much higher levels of the antigen, PSA level is a particularly reliable and effective diagnostic indicator of the presence of prostate cancer (Labrie et al, 1992). PSA is also found in normal prostate epithelial tissue and its expression is highly specific (Wei et al, 1997).

The immune responses induced by a DNA vaccine encoding for human PSA has been investigated in a murine model. The vaccine construct was constructed by cloning a gene for PSA into expression vectors under control of a CMV promoter (Figure 1). The expression of 30 kD PSA protein was determined in vitro using immunoprecipitation following a transfection with the PSA construct (F i g u r e 1 ). In vivo expression of PSA was determined by intramuscularly injecting BALB/C mice with the DNA vaccine and performing an immunohistochemistry analysis on their quadriceps muscles (Figure 2).

A. PSA recombinant vaccinia virus vaccine

Following the injection of the PSA DNA construct (pCPSA), various assays were performed to measure both

Recombinant vaccinia virus

vaccines expressing

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Figure 1. Construction and in vitro expression of PSA DNA vaccine. The complete coding sequence of PSA was cloned into pCDNA3 vector. Expression of PSA was assayed by immunoprecipitation with !-PSA antibodies. The immunoprecipitated sample was analyzed by SDS-PAGE (12%).

Figure 2. Immunohistochemical assay for expression of PSA on muscle cells. Frozen muscle sections were prepared from DNA injected animals and stained with !-PSA antibody. Positive antigen expression is illustrated by PSAspecific staining and representative examples of in vivo expression are highlighted with black arrows. A) A slide from a leg immunized with PSA vaccine and stained with !-PSA antibody. B ) A slide from control plasmid immunized leg stained with !-PSA antibody.

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Gene Therapy and Molecular Biology Vol 3, page 154 Improvement of hepatitis B virus DNA vaccines by plasmids coexpressing hepatitis B surface antigen and interleukin-2. J Virol 71, 169-178.

the humoral and cellular immune responses of the mice (Kim et al, In Press). PSA-specific immune responses induced in vivo by immunization were characterized by enzyme-linked immunosorbent assay (ELISA), T helper proliferation cytotoxic T lymphocyte (CTL), and flow cytometry assays. Strong and persistent antibody responses were observed against PSA for at least 180 days following immunization. In addition, a significant T helper cell proliferation was observed against PSA protein. Immunization with pCPSA also induced MHC Class I CD8+ T cell-restricted cytotoxic T lymphocyte response against tumor cell targets expressing PSA. The induction of PSA-specific humoral and cellular immune responses following injection with pCPSA was also observed in rhesus macaques (Table 2).

Conry RM, LoBuglio AF, Kantor J, Schlom J, Loechel F, Moore SE, Sumerel LA, Barlow DL, Abrams S, Curiel DT. (1 9 9 4 ) Immune response to a carcinoembryonic antigen polynucleotide vaccine. Cancer Res 54, 1164-1168. Conry RM, LoBuglio AF, Curiel DT. ( 1 9 9 6 a ) Polynucleotide-mediated immunization therapy of cancer. S e m i n O n c o l 23, 135-147. Conry RM, Widera G, LoBuglio AF, Fuller JT, Moore SE, Barlow DL, Turner J, Yang NS, Curiel DT. ( 1 9 9 6 b ) Selected strategies to augment polynucleotide immunization. Gene Ther 3, 67-74. Foon KA, Chakraborty M, John WJ, Sherratt A, Kohler H, Bhattacharya-Chatterjee M. ( 1 9 9 7 ) Immune response to the carcinoembryonic antigen in patients treated with an anti-idiotype antibody vaccine. J C l i n I n v e s t 96, 334342.

V. Conclusion Research involving different gene-based vaccines demonstrate that they can induce effective immune responses in a variety of animal models, including rodents and macaques as well as in humans. This effect was found in both methods of in vivo delivery, though differences remain between the two. Although recombinant vaccinia virus may produce more potent immune responses than DNA, it has many side effects such as eliciting an immune response against the virus itself. This immune response reduces the effectiveness of subsequent innoculations. DNA, while less immunogenic, can be used repeatedly with less adverse side effects. Furthermore, coadministration of molecular adjuvants with DNA vaccine constructs enhance the level of antigen-specific immune responses (Kim et al, 1997a,b; Conry et al, 1996b; Kim and Weiner, 1997; Chow et al, 1997; Sin et al, 1998).

Gilliland FD, Keys CR. ( 1 9 9 5 ) Male genital cancers. Cancer 75, 295-315. Hodge JW, Schlom J, Donohue SJ, Tomaszewski JE, Wheeler CW, Levine BS, Gritz L, Panicali D, Kantor JA. ( 1 9 9 5 ) A recombinant vaccinia virus expressing human prostatespecific antigen (PSA): safety and immunogenicity in a non-human primate. Int J Cancer 63, 231-237. Jones VE, Mitchell, MS. ( 1 9 9 6 ) Therapeutic vaccines for melanoma: progress and problems. T r e n d s B i o t e c h 14, 349-355. Kantor J, Irvine K, Abrams S, Kaufman H, DiPietro J, Schlom J. ( 1 9 9 2 a ) Antitumor activity and immune responses induced by a recombinant carcinoembryonic antigenvaccinia virus vaccine. J N a t l C a n c e r I n s t 84, 10841091. Kantor J, Irvine K, Abrams S, Snoy P, Olsen R, Greiner J, Kaufman H, Eggensperger D, Schlom J. ( 1 9 9 2 b ) Immunogenicity and safety of a recombinant vaccinia virus vaccine expressing the carcinoembryonic antigen gene in a nonhuman primate. Cancer R e s 52, 69176925.

Additional studies are warranted to optimize these strategies. Areas of future study could focus on controlling the immune responses induced by these therapies and further explore their effects on humans. It would be advantageous to modulate and refine the effects of these vaccines in order to gain optimal response. There are a number of ongoing clinical studies that will help ascertain how to best use gene-based therapies.

Kaufman H, Schlom J, Kantor J. ( 1 9 9 1 ) A recombinant vaccinia virus expressing human carcinoembryonic antigen (CEA). Int J Cancer 48, 900-907. Kelley JR, Cole DJ. ( 1 9 9 8 ) Gene therapy strategies utilizing carcinoembryonic antigen as a tumor associated antigen for vaccination against solid malignancies. Gene Ther M o l B i o l 2, 14-30.

References Abrams SI, Hodge JW, McLaughlin JP, Steinberg SM, Kantor JA, Schlom J. (1 9 9 7 ) Adoptive immunotherapy as an in vivo model to explore antitumor mechanisms induced by a recombinant anticancer vaccine. J Immunother 20, 4859.

Kim JJ and Weiner DB ( 1 9 9 7 ) DNA/genetic vaccination for HIV. Springer Sem Immunopathol 19, 174-195. Kim JJ, Bagarazzi ML, Trivedi N, Hu Y, Chattergoon MA, Dang K, Mahalingam S, Agadjanyan MG, Boyer JD, Wang B, Weiner DB ( 1 9 9 7 a ) Engineering of in vivo immune responses to DNA immunization via co-delivery of costimulatory molecule genes. N a t B i o t e c h 15, 641645.

Boring CC, Squires TS, Tong T. ( 1 9 9 4 ) Cancer statistics, 1994. Ca: A Cancer Journal for Clinicians 44, 726. Chow YH, Huang WL, Chi WK, Chu YD, Tao MH. ( 1 9 9 7 )

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Gene Therapy and Molecular Biology Vol 3, page 155 Kim JJ, Ayyvoo V, Bagarazzi ML, Chattergoon MA, Dang K, Wang B, Boyer JD, Weiner DB. ( 1 9 9 7 b ) In vivo engineering of a cellular immune response by coadministration of IL-12 expression vector with a DNA immunogen. J . I m m u n o l . 158, 816-826. Kim JJ, Trivedi NN, Nottingham L, Morrison L, Tsai A, Hu Y, Mahalingam S, Dang K, Ahn L, Doyle NK, Wilson DM, Chattergoon MA, Chalian AA, Boyer JD, Agadjanyan MG, Weiner DB. ( 1 9 9 8 ) Modulation of amplitude and direction of in vivo immune responses by coadministration of cytokine gene expression cassettes with DNA immunogens. Eur J Immunol 28, 1089-1103. Kim JJ, Trivedi NN, Mahalingam S, Morrison L, Tsai A, Chattergoon MA, Dang K, Patel M, Ahn L, Chalian AA, Boyer JD, Kieber-Emmons T, Agadjanyan MG, Weiner DB. Molecular and immunological analysis of genetic prostate specific antigen (PSA) vaccine. O n c o g e n e In Press. Ko SC, Gotoh A, Thalmann GN, Zhau HE, Johnston DA, Zhang WW, Kao C, Chung LWK. ( 1 9 9 6 ) Molecular therapy with recombinant p53 adenovirus in an androgenindependent, metastatic human prostate cancer model. Hum Gene Ther 7, 1683-1691. Labrie F, Dupont A, Suburu R, Cusan L, Tremblay M, Gomez JL, Edmond J. ( 1 9 9 2 ) Serum prostate specific antigen as pre-screening test for prostate cancer. J U r o l 147, 84652. MacGregor RR, Boyer JD, Ugen KE, Lacy KE, Bagarazzi ML, Chattergoon MA, Baine Y, Higgins TJ, Ciccarelli RB, Coney LR, Ginsberg RS, Weiner DB ( 1 9 9 8 ) First human trial of a DNA-based vaccine for treatment of HIV-1 infection: safety and host response. J I n f D i s 178, 92100. Roth JA and Cristiano RJ. (1 9 9 7 ) Gene therapy for cancer: what have we done and where are we going? J Natl Cancer Inst 89, 21-39. Sin JI, Kim JJ, Boyer JD, Huggins C, Higgins T, Weiner DB. ( 1 9 9 8 ) In vivo modulation of immune responses and protective immunity against herpes simplex virus-2 infection using cDNAs expressing Th1 and Th2 Type Cytokines in gD DNA Vaccination. J Virol In Press. Smith BF, Baker HJ, Curiel DT, Jiang W, Conry RM ( 1 9 9 8 ) Humoral and cellular immune responses of dogs immunized with a nucleic acid vaccine encoding human carcinoembryonic antigen. Gene Ther 5, 865-868. Sogn JA, Finerty JF, Heath AK, Shen GLC, Austin FC. ( 1 9 9 3 ) Cancer vaccines: the perspective of the Cancer Immunology Branch, NCI. A n n a l s N Y A c a d S c i 690, 322-330. Tang D, DeVit M, Johnston S. ( 1 9 9 2 ) Genetic immunization is a simple method for eliciting an immune response. Nature 356, 152-154. Tsang KY, Zaremba S, Nieroda CA, Zhu MZ, Hamilton JM, Schlom J. (1 9 9 5 ) Generation of human cytotoxic T-cells specific for human carcinoembryonic antigen epitopes from patients immunized with recombinant vaccinia-CEA

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vaccine. J Natl Cancer Inst 87, 982-990. Ulmer JB, Donnelly J, Parker SE, Rhodes GH, Felgner PL, Dwarki VL, Gromkowski SH, Deck R, DeVitt CM, Friedman A, Hawe LA, Leander KR, Marinez D, Perry H, Shiver JW, Montgomery D, Liu MA. ( 1 9 9 3 ) Heterologous protection against influenza by injection of DNA encoding a viral protein. S c i e n c e 259, 1745-1749. Wang B, Ugen KE, Srikantan V, Agadjanyan MG, Dang K, Refaeli Y, Sato A, Boyer J, Williams WV, Weiner DB. ( 1 9 9 3 ) Gene inoculation generates immune responses against human immunodeficiency virus type 1. P r o c . N a t l . A c a d . S c i . U S A 90, 4156-4160. Wang MC, Kuriyama M, Papsidero LD, Loor RM, Valenzyela LA, Murphy GP, Chu TM. ( 1 9 8 2 ) Prostate antigen of human cancer patients. Meth Cancer Res 19, 179-197. Watt KWK, Lee PJ, Timkulu TM, Chan WP, Loor R. ( 1 9 9 6 ) Human prostate-specific antigen: structural and functional similarity with serine proteases. P r o c N a t l A c a d S c i USA 83, 3166-3170. Wei C, Willis RA, Tilton BR, Looney RJ, Lord EM, Barth RK, Frelinger JG. ( 1 9 9 7 ) Tissue-specific expression of the human prostate-specific antigen gene in transgenic mice: Implications for tolerance and immunotherapy. Proc Natl Acad Sci USA 94, 6369-6374. Wolff JA, Malone RW, Williams P, Chong W, Acsadi G, Jani A, Felgner PL. ( 1 9 9 0 ) Direct gene transfer into mouse muscle in vivo. S c i e n c e 247, 1465-1468. Zaremba S, Barzaga E, Zhu M, Soares N, Tsang KY, Schlom J. ( 1 9 9 7 ) Identification of an enhancer agonist cytotoxic T lymphocyte peptide from human carcinoembryonic antigen. Cancer Res 57, 4570-4577.


Gene Therapy and Molecular Biology Vol 3, page 157 Gene Ther Mol Biol Vol 3, 157-165. August 1999.

Rational vaccine design through the use of molecular adjuvants Review Article

Jong J. Kim1, Liesl K. Nottingham2, Jim Oh1, Daniel Lee1, Ken Wang 1, Mera Choi3, Tzvete Dentchev 1, Darren Wilson, Devin M. Cunning2, Ara A. Chalian 2, Jean Boyer, Jeong I. Sin1, and David B. Weiner 1 1

Department of Pathology and Laboratory Medicine; 2Department of Otolaryngology/Head and Neck Surgery, University of Pennsylvania, Philadelphia, PA 19104, 3Bryn Mawr College _________________________________________________________________________________________________ Corresponding Author: Jong J. Kim, Ph.D., Department of Pathology and Laboratory Medicine, University of Pennsylvania, 505 Stellar-Chance, 422 Curie Blvd., Philadelphia, PA 19104. Tel: (215) 662-2352; Fax: (215) 573-9436; Email: jonger@seas.upenn.edu Received: 18 September 1998; accepted: 25 September 1998

Summary Nucleic acid immunization is an important vaccination strategy which delivers DNA constructs encoding for a specific immunogen into the host. These expression cassettes transfect the host cells, which become the i n v i v o protein source for the production of antigen. This antigen then is the focus of the resulting immune response. This vaccination technique is being explored as an immunization strategy against a variety o f infectious diseases as w e l l as cancer. The first generation DNA immunization experiments have shown that the DNA vaccines’ ability to elicit humoral and cellular responses i n v i v o i n a s a f e a n d well-tolerated manner i n various model systems, including humans. As we explore the next generation of DNA vaccines, our goal is to refine the current strategy t o elicit more clinically efficacious immune responses. A more c l i n i ca l l y ef fec t i ve v a cc i ne m a y need t o elicit a more specific immune response against the targeted pathogen. It would be a distinct advantage to design immunization strategies which can be “focused” according to the correlates of protection known for the particular pathogen. In order to focus the immune responses induced from DNA immunization, we have investigated the co-delivery o f genes for immunologically important molecules, such as costimulatory molecules and cytokines which play critical regulatory and signaling roles in immunity. We and others have shown that the use of these molecular adjuvants could enhance and modulate immune responses induced by DNA immunogens. Co-administration of costimulatory molecules (CD80 and CD86), proinflammatory cytokines (IL-1 , T N F - , and TNF- ), Th1 cytokines (IL-2, IL-12, IL-15, and IL-18), Th2 cytokine (IL-4, IL-5 and IL-10), and GM-CSF with DNA vaccine constructs led to modulation of the magnitude and direction (humoral or cellular) of the immune responses. These studies demonstrate the potential utility of molecular adjuvant strategy as an important tool for the development of more rationally designed vaccines.

with natural infection (Stasney et al, 1955; Paschkis et al, 1955; Ito, 1960). Nucleic acid or DNA inoculation is an important vaccination technique which delivers DNA constructs encoding specific immunogens directly into the host (Wolff et al, 1990; Tang et al, 1992; Wang et al, 1993; Ulmer et al 1993; Kim et al, 1997a; Agadjanyan et

I. Introduction Although the injection of DNA into tissues was originally reported in the 1950s, the technology has gained more attention in recent years as a safe means of mimicking in vivo protein production normally associated 157


Kim et al: Vaccine design using molecular adjuvants al, 1997, Tascon et al, 1996, Conry et al, 1996). This injection results in the subsequent expression of the foreign gene in that host and the presentation of the specific encoded proteins to the immune system. DNA vaccine constructs are produced as small circular vehicles or plasmids. These plasmids are constructed with a promoter site which starts the transcription process, an antigenic DNA sequence and a messenger RNA stop site containing the poly A tract necessary for conversion of the messenger RNA sequence into the antigen protein by the ribosomal protein manufacturing machinery (F i g u r e 1 ). This antigen then is the focus of the resulting immune response. This vaccination technique is being explored as an immunization strategy against cancer as well as a variety of infectious diseases including AIDS.

1997; Chattergoon et al, 1997). Since DNA vaccines are non replicating and the vaccine components are produced within the host cells, they can be constructed to function safely with the specificity of a subunit vaccine. However, DNA vaccine cassettes produce immunological responses that are more similar to live vaccine preparations. By directly introducing DNA into the host cell, the host cell is essentially directed to produce the antigenic protein, mimicking viral replication or tumor cell marker presentation in the host. This process has been reported to generate both antibody and cell mediated, particularly cytotoxic T cell-mediated, immunity (Figure 2). Unlike a live attenuated vaccine, conceptually there is little risk from reversion to a disease-causing pathogen from the injected DNA, and there is no risk for secondary infection as the material injected is not-living and not-infectious. In addition, genes which lead to undesired immunologic inhibition or cross-reactivity (autoimmunity) may be either altered or deleted altogether. Finally, DNA vaccines can be manipulated to present a particular genome of the pathogen or display specific tumor antigens in nonreplicating vectors (Figure 1).

II. Potential advantages of DNA vaccines Nucleic acid immunization may afford several potential advantages over traditional vaccination strategies such as whole killed or live attenuated virus and recombinant protein-based vaccines (Kim and Weiner,

Figure 1. Potential immunologic targets for DNA vaccination against HIV-1. These targets include env, gag, and pol genes as well as the four accessory genes.

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Figure 2. Induction of antigen-specific humoral and cellular immune responses.

III. Molecular adjuvants as a immune modulation strategy The overall objective of any immunization strategy is to induce specific immune responses which protect the immunized individual from a given pathogen over his or her lifetime. One major challenge in meeting this goal is that the correlates of protection from an individual pathogen vary from one infectious agent to the next. The first generation DNA immunization experiments have shown that the DNA vaccinesâ&#x20AC;&#x2122; ability to elicit humoral and cellular responses in vivo in a safe and well-tolerated manner in various model systems, including humans. As we explore the next generation of DNA vaccines, our goal is to refine the current strategy to elicit more clinically efficacious immune responses. A more clinically effective vaccine may need to elicit a more specific immune response against the targeted pathogen. It would be a distinct advantage to design immunization strategies which can be targeted according to the correlates of protection 159

known for the particular pathogen (Figure 3). Such refinement could be accomplished by co-delivering genes for immunologically important molecules, such as costimulatory molecules and cytokines which play critical regulatory and signaling roles in immunity (Kim and Weiner, 1997). These molecular adjuvant constructs could be co-administered along with immunogen constructs to modulate the magnitude and direction (humoral or cellular) of the immune responses induced (Figure 4). There has been several reports of immune modulation by protein delivered cytokines. However, the results in general appeared marginal. More recently, we and others have focused on analyzing immune responses induced to such gene delivery. Raz et al. observed that intramuscular injections of plasmids encoding IL-2, IL-4, or TGF-!1 modestly modulated immune responses to transferrin protein delivered at a separate site (Raz et al, 1993). IL-2 immunization resulted in an enhancement of antibody and T helper proliferative responses while TGF-!1 immunization reduced anti-transferrin responses.


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Figure 3. The potential utility of the molecular adjuvant network. Tailoring the induction of specific immune responses by vaccination programs against viral, bacterial, or parasitic diseases could be beneficial.

Figure 4. Cytokines as immune response regulators. Cytokines play critical roles in the immune and inflammatory responses. Based upon their specific function in the immune system these cytokines could be further grouped as proinflammatory, Th1, and Th2 cytokines. Along with costimulatory molecules, these cytokines also play important roles in the activation and proliferation of T and B cells.

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Gene Therapy and Molecular Biology Vol 3, page 161 gD protein with the gene plasmids encoding for Th1-type (IL-2, 12, 15, 18) and Th2-type (IL-4, IL-10) cytokines in an effort to drive immunity induced by vaccination. We then analyzed the vaccine modulatory effects on resulting immune phenotype and on the mortality and the morbidity of the immunized animals following HSV lethal challenge. We observed Th1 cytokine gene coadministration not only enhanced survival rate, but also reduced the frequency and severity of herpetic lesions following intravaginal HSV challenge (Figure 6). On the other hand, co-injection with Th2 cytokine genes increased the rate of mortality and morbidity of the challenged mice. Again, among the Th1 type cytokine genes tested IL-12 was particularly a potent adjuvant for the gD DNA vaccination.

IV. Modulation of immune responses using cytokine molecular adjuvants In order to focus the immune responses induced from DNA immunization, we have investigated the co-delivery of molecular adjuvants. We first reported that coimmunization of GM-CSF genes with DNA vaccine constructs increases antigen-specific antibody and T helper cell proliferation responses while co-immunization with IL-12 genes results in weaker antibody responses and enhanced T helper cell proliferation (Kim et al, 1997b,c). In addition, IL-12 co-immunization resulted in a significant enhancement of CTL responses. Importantly, we observed a significant enhancement of CTL response in vivo with the co-administration of murine IL-12 genes with four different HIV-1 DNA immunogens (gag/pol, envelope, vif, and nef) which were CD8+ T cell- and MHC class I-restricted. In contrast, almost no effect on CTL induction was observed with the genes for GM-CSF in these studies. Moreover, Iwasaki et al. (1997) reported that GM-CSF and IL-12 co-delivery with DNA immunogen encoding for influenza NP resulted in enhanced cellular immune responses. Moreover, Agadjanyan et al. (1997) reported that co-administration of IL-12 genes with HIV-2 DNA immunogen resulted in a dramatic enhancement of both Th and CTL responses. Furthermore, coadministration of IL-12 genes with DNA immunogens strongly directed the antigen specific immune response towards a Th1 type immunity and induced delayed type hypersensitivity (DTH) to contact allergens as an in vivo model of the Th1 response (Kim et al, 1998a). In addition to these reports, Chow et al. reported that either injection of plasmid co-expressing hepatitis B surface antigen (HBsAg) and IL-2 or co-injection of IL-2 genes with plasmid expressing HBsAg resulted in the enhancement of both antibody and T helper cell responses (Chow et al, 1997).

V. Modulation of immune responses using costimulatory molecule adjuvants The generation of the T cell immune response is a complex process that requires the engagement of T cells with professional APCs such as dendritic cells, macrophages, and B cells. These professional APCs possess large surface areas for interaction with T cells. They also express high levels of MHC class I and II molecules, adhesion molecules, and costimulatory molecules which are critical for efficient antigen presentation and T cell activation. Professional APCs initiate T cell activation by binding antigenic peptideMHC complexes to T cell receptor molecules. In addition, the APCs provide secondary signals through the ligation of costimulatory molecules with their receptors (CD28/CTLA-4) present on T cells. These costimulatory signals are required for the clonal expansion and differentiation of T cells. The blocking of this additional costimulatory signal leads to T cell anergy (Schwartz et al, 1992). Among different costimulatory molecules, CD80 and CD86 have been observed to provide potent immune signals ( Lanier et al, 1995, Linsley et al, 1990).

More recently, we investigated the induction and regulation of immune responses from the co-delivery of proinflammatory cytokines (IL-1", TNF-", and TNF-!), Th1 cytokines (IL-2, IL-15, and IL-18), and Th2 cytokines (IL-4, IL-5 and IL-10) (Figure 5) (Kim et al, 1998b). We observed enhancement of antigen-specific humoral response with the co-delivery of Th2 cytokines IL-4, IL-5, and IL-10 as well as that of IL-2 and IL-18. A dramatic increase in antigen-specific T helper cell proliferation was seen with IL-2 and TNF-" co-injections. In addition, we observed a significant enhancement of the cytotoxic response with the co-administration of TNF-" and IL-15 genes with HIV-1 DNA immunogens. These increases in CTL response were both MHC class I-restricted and CD8+ T cell-dependent. We also investigated whether the Th1 or Th2-type immune responses are more important for protection from HSV-2 infection (Sin et al, 1998). We codelivered DNA expression construct encoding for HSV-2

The CD80 and CD86 molecules are surface glycoproteins and members of immunoglobulin superfamily which are expressed only on professional APCs (Lanier et al, 1995, Linsley et al, 1990, June et al,1994). Although both CD80 and CD86 molecules interact with either CD28 or CTLA-4 molecules on T cells, CD80 and CD86 expression seem to be differentially regulated. CD86 is constitutively expressed by the APCs whereas CD80 is expressed only after activation of these cells (Freeman et al, 1989; Azuma et al, 1993; Freedman et al, 1991). Thus, CD86 may be important in the early interactions between APCs and T cells during the induction phase of the immune response.

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Figure 5. Each cytokine gene was cloned into expression plasmids under the control of a CMV promoter.

Figure 6. Protection from lethal HSV-2 challenge. Each group of mice (n=10) was immunized with gD DNA vaccines (60 Âľg), and/or cytokine genes (40 Âľg) at 0 and 2 weeks. Three weeks after the second immunization, mice (n=8) were challenged i.v. with 200 x LD 50 of HSV2 strain 186 (7 x 10 5 pfu).

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Gene Therapy and Molecular Biology Vol 3, page 163 candidates, such as chemokines, should be further developed and tested. Cumulatively, these studies demonstrate the potential utility of molecular adjuvant strategy as an important tool for the development of more rationally designed vaccines.

We recently reported that CD86 molecules play a prominent role in the antigen-specific induction of CD8+ cytotoxic T lymphocytes when delivered as vaccine adjuvants (Figure 7) (Kim et al, 1997a). Coadministration of CD86 cDNA along with DNA encoding HIV-1 antigens intramuscularly dramatically increased antigen-specific T-cell responses without a significant change to the level of the humoral response. This enhancement of cytotoxic T lymphocyte (CTL) response was both major histocompatibility complex (MHC) class I-restricted and CD8+ T cell-dependent. Similar results have been obtained by other investigators who also found that CD86, not CD80 co-expression results in the enhancement of T cell-mediated immune responses (Tsuji et al, 1997; Iwasaki et al, 1997). Accordingly, we speculate that engineering of non-professional APCs such as muscle cells to express CD86 costimulatory molecules could empower them to prime CTL precursors. On the other hand, the enhancement effect of CD86 co-delivery could also have been mediated through the direct transfection of a small number of professional APCs residing within the muscle tissue. Subsequently, these cells could have greater expression of costimulatory molecules and could in theory become more potent.

References Stasney, J., Cantarow, A., and Paschkis, K.E. (1 9 5 5 ). Production of Neoplams by Injection of Fractions of Mammalian Neoplasms. Cancer Res. 11, 775-782. Paschkis, K. E., Cantarow, A., Stasney, J. (1 9 5 5 ). Induction of Neoplasms by Injection of Tumor Chromatin. J . N a t l . Cancer Inst. 15:1525-1532. Ito, Y. (1 9 6 0 ). A Tumor-producing factor extracted by phenol from papillomatous tissue of cottontail rabbits. V i r o l o g y 12, 596-601. Wolff, J. A., R. W. Malone, P. Williams, W. Chong, G. Acsadi, A. Jani, P. L. Felgner. (1 9 9 0 ). Direct gene transfer into mouse muscle in vivo. S c i e n c e . 247, 14651468. Tang, D., M. DeVit, S. Johnston. (1 9 9 2 ). Genetic immunization is a simple method for eliciting an immune response. Nature. 356, 152-154. Wang, B., K. E. Ugen, V. Srikantan, M. G. Agadjanyan, K. Dang, Y. Refaeli, A. Sato, J. Boyer, W. V. Williams, D. B. Weiner. (1 9 9 3 ). Gene inoculation generates immune responses against human immunodeficiency virus type 1. P r o c . N a t l . A c a d . S c i . U S A 90, 4156-4160.

VI. Future directions As summarized in Figure 8, we observed that significant modulation was possible using molecular adjuvants. This cytokine gene adjuvant network underscores an important level of control in the induction of specific immune responses to tailor vaccination programs more closely to the correlates of protection which vary from disease to disease. This type of fine control of vaccine and immune therapies was previously very difficult to obtain. Controlling the magnitude and direction of the immune response could be advantageous in a wide variety of vaccine strategies. For instance, in a case where T cell mediated response is paramount, but the humoral response may not be needed or even be harmful, IL-12 genes could be chosen as the immune modulator to be co-delivered with a specific DNA immunogen. On the other hand, for building vaccines to target extracellular bacteria, for example, IL-4, IL-5 or IL-10 genes could be co-injected. Furthermore, in cases where both CD4+ T helper cells and antibodies play more important roles in protection, GM-CSF as well as IL-2 could be co-delivered. Lastly, in cases where all three arms of immune responses are critical, TNF-" could be co-injected to give a combined enhancement of antibody, T helper cell, and CTL responses. In this regard it will be important to examine combination delivery in the presence or the absence of costimulatory genes to further control the immune responses. Furthermore, additional molecular adjuvant

Ulmer, J. B., J. Donnelly, S. E. Parker, G. H. Rhodes, P. L. Felgner, V. L. Dwarki, S. H. Gromkowski, R. Deck, C. M. DeVitt, A. Friedman, L. A. Hawe, K. R. Leander, D. Marinez, H. Perry, J. W. Shiver, D. Montgomery, M. A. Liu. (1 9 9 3 ). Heterologous protection against influenza by injection of DNA encoding a viral protein. S c i e n c e 259, 1745-1749. Kim, J. J., M.L. Bagarazzi, N. Trivedi, Y. Hu, M.A. Chattergoon, K. Dang , S. Mahalingam, M.G. Agadjanyan, J.D. Boyer, B. Wang, D.B. Weiner. (1 9 9 7 a ). Engineering of In Vivo Immune Responses to DNA Immunization Via Co-Delivery of Costimulatory Molecule Genes. Nature Biotech. 15, 641-645. Agadjanyan, M. G., N.N.Trivedi, S. Kudchodkar, M. Bennett, W. Levine, A. Lin, J. Boyer, D. Levy, K. Ugen, J.J. Kim, D.B. Weiner. (1 9 9 7 ). An HIV-2 DNA vaccine induces cross reactive immune responses against HIV-2 and SIV. AIDS Human Retrov 13, 1561-1572. Tascon, R. E., M.J. Colston, S. Ragno, E. Stavropoulos, D. Gregory,, and D. B. Lowrie. (1 9 9 6 ). Vaccination against tuberculosis by DNA injection. Nature Med. 2, 888-92. Conry, R. M., G. Widera, A.F. LoBuglio, J.T. Fuller, S.E. Moore, D.L. Barlow, J. Turner, N.-S. Yang, D.T. Curiel. (1 9 9 6 ). Selected strategies to augment polynucleotide immunization. Gene Ther 3, 67-74.

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Figure 7. Co-expression of HIV-1 envelope gp120 protein with CD86 on muscle cells. Frozen muscle sections were prepared from DNA injected animals and stained with FITC-labeled (green) "-CD86 antibodies and Texas Red-labeled (red) "-gp120 antibodies. (A) A slide from a leg immunized with pCDNA3 (control vector) was stained with "-CD86 and "-gp120. (B ) A slide from a leg immunized with pCEnv+pCD86 was stained with "-CD86 and "-gp120 antibodies.

Figure 8. A summary of the each cytokine co-administration effects on antibody (y-axis), T helper (x-axis), and cytotoxic T lymphocyte responses (z-axis). Each cytokine is plotted on the 3-D axis according to its effects on the three modes of immune response.

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Gene Therapy and Molecular Biology Vol 3, page 165 Kim, J. J. and D.B. Weiner. (1 9 9 7 ). DNA/genetic vaccination for HIV. S p r i n g e r S e m I m m u n o p a t h o l 19, 174-195.

June, C., J. A. Bluestone, L. M. Nadler, C. B. Thompson. (1 9 9 4 ). The B7 and CD28 receptor families. I m m u n o l . Today 15, 321-333.

Chattergoon, M., J. Boyer, , and D. B. Weiner. (1 9 9 7 ). Genetic immunization: a new era in vaccines and immune therapies. FASEB J. 11:753-763.

Freeman, G. J., A. S. Freedman, J. M. Segil, G. Lee, J. F. Whitman, L. M. Nadler. (1 9 8 9 ). B7 a new member of the Ig superfamily with unique expression on activated and neoplastic B cells. J . I m m u n o l. 143, 2714-2722.

Raz, E., A. Watanabe, S. M. Baird, R. A. Eisenberg, T. B. Parr, M. Lotz, T. J. Kipps, D. A. Carson. (1 9 9 3 ). Systemic immunological effects of cytokine genes injected into skeletal muscle. P r o c . N a t l . A c a d . S c i . USA 90, 4523-4527.

Azuma, M., D. Ito, H. Yagita, K. Okumura, J. H. Phillips, L. L. Lanier, C. Somoza. (1 9 9 3 ). B70 antigen is a second ligand for CTLA-4 and CD28. Nature 366, 76-79. Freedman, A. S., G. J. Freeman, K. Rhynhart, L. M. Nadler. (1 9 9 1 ). Selective induction of B7/BB-1 on interferon-#stimulated monocytes: a potential mechanism for amplification of T cell activation through the CD28 pathway. C e l l . I m m u n o l . 137, 429-437.

Kim, J. J., V. Ayyvoo, M. L. Bagarazzi, M. A. Chattergoon, K. Dang, B. Wang , J. D. Boyer, D. B. Weiner. (1 9 9 7 b ). In vivo Engineering of a Cellular Immune Response by Co-administration of IL-12 Expression Vector with a DNA Immunogen. J. Immunol. 158, 816-826.

Tsuji, T., K. Hamajima, N. Ishii, I. Aoki, J. Fukushima, K.Q. Xin, S. Kawamoto, S. Sasaki, K. Matsunaga, Y. Ishigatsubo, K. Tani, T. Okubo, K. Okuda. (1 9 9 7 ). Immunomodulatory effects of a plasmid expressing B7-2 on humanimmunodeficiency virus-1-specific cell-mediated immunity induced by a plasmid encoding the viral antigen. Euro. J. Immuno. 27, 782-787.

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Iwasaki, A., B.J. Stiernholm, A.K. Chan, N.L. Berstein, B.H. Barber. (1 9 9 7 ). Enhanced CTL responses mediated by plasmid DNA immunogens encoding costimulatory molecules and cytokines. J . Immunol. 158, 45914601.

Chow, Y.-H., W. -L. Huang, W. -K Chi, Y. -D Chu, M. -H Tao. (1 9 9 7 ). Improvement of hepatitis B virus DNA vaccines by plasmids coexpressing hepatitis B surface antigen and interleukin-2. J . V i r o l . 71, 169-178. Kim, J. J., N. N. Trivedi, L. Nottingham, L. Morrison,A. Tsai, Y. Hu, S. Mahalingam, K. Dang, L. Ahn, N. K. Doyle, D. M. Wilson, M. A. Chattergoon, A. A. Chalian, J. D. Boyer, M. G. Agadjanyan, D. B. Weiner. (1 9 9 8 b ). Modulation of Amplitude and Direction of In Vivo Immune Responses By Co-Administration Of Cytokine Gene Expression Cassettes With DNA Immunogens. Eur. J. Immunol. 28, 1089-1103. Sin, J. I., J. J. Kim, J. D. Boyer, C. Huggins, T. Higgins, D. B. Weiner. (1 9 9 8 ). In Vivo Modulation of Immune Responses and Protective Immunity against Herpes Simplex Virus-2 Infection using cDNAs expressing Th1 and Th2 Type Cytokines in gD DNA Vaccination. J . V i r o l . In Press. Schwartz, R. H. (1 9 9 2 ). Costimulation of T lymphocytes: the role of CD29, CTLA-4, and B7/BB1 in interleukin-2 production and immunotherapy. C e l l 71, 1065-1068. Lanier, L. L., S. Oâ&#x20AC;&#x2122;Fallon, C. Somoza, J. H. Phillips, P. S. Linsley, K. Okumura, D. Ito, M. Azuma. (1 9 9 5 ). CD80 (B7) and CD86 (B70) provide similar costimulatory signals for T cell proliferation, cytokine production, and generation of CTL. J . I m m u n o l. 154, 97-105. Linsley, P. S., E. A. Clark, J .A. Ledbetter. (1 9 9 0 ). The T cell antigen, CD28, mediates adhesion with B cells by interacting with activation antigen, B7/BB-1. P r o c . Natl. Acad. Sci. USA 87, 5031-5035.

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Gene Therapy and Molecular Biology Vol 3, page 167 Gene Ther Mol Biol Vol 3, 167-177. August 1999.

In vivo production of therapeutic antibodies by engineered cells for immunotherapy of cancer and viral diseases Review Article

Mireia Pelegrin, Danièle Noël, Mariana Marin, Estanislao Bachrach, Robert M. Saller*, Brian Salmons*, and Marc Piechaczyk Institut de Génétique Moléculaire de Montpellier, UMR 5535, CNRS, 1919 route de Mende, 34293 Montpellier Cédex 05, France * Bavarian Nordic Research Institute, Fraunhoferstr. 18B, 82152 Martinsried, Germany __________________________________________________________________________________________________ Correspondence: Marc Piechaczyk, Ph.D. Tel: + (33) 4.67.61.36.68; Fax + (33) 4.67.04.02.45; E-mail: piechaczyk@jones.igm.cnrs-mop.fr Received: 18 November 1998; accepted: 25 November 1998

Summary Our recently developed ability to produce human monoclonal antibodies, together with that of reshaping antibody molecules, offers new tools for treating a number of human diseases. Direct injection of purified antibodies, or of antibody-related molecules, to patients would, however, not always be possible or desirable. This is especially true in the case of long-term therapies for at least two reasons. One is the high cost of antibodies certified for human use. The other is the possibility of neutralizing anti-idiotypic immune responses as a result of repeated injection of m a s s i v e d o s e s o f a n t i b o d y . I n v i v o production of therapeutic antibodies through either genetic modification of patients' cells or implantation of antibody-producing cells might overcome both of t h e s e h u r d l e s . S e v e r a l c e l l t y p e s s u i t a b l e f o r u s e i n c e l l / g e n e therapy protocols, such as skin fibroblasts, keratinocytes, myogenic cells and hepatocytes, are capable of producing monoclonal antibodies i n v i t r o upon gene transfer. Furthermore, the grafting of engineered myogenic cells permits the long-term systemic delivery o f recombinant antibodies i n immunocompetent mice. Importantly, antibodies produced both i n v i t r o and i n v i v o , retain the specificity and the affinity of the parental antibody and no anti-idiotypic response i s detected i n mice producing ectopic antibodies. Long-term systemic delivery of such antibodies into mice can also be achieved through the implantation o f antibody-producing c e l l s encapsulated into a new biocompatible material, cellulose sulphate. Importantly, no inflammation occurs at capsule implantation sites over periods as long as 10 months. Moreover, no anti-idiotypic response develops against antibodies released by encapsulated cells. Encapsulation of antibody-producing cells in immunoprotective devices should offer multiple advantages over genetic modification of patients' cells. These include protection against immune cells of treated individuals, the possibility of easy removal of implanted cells as w e l l as that o f implantation o f non-autologous c e l l s . Taken together, these observations demonstrate that long-term i n v i v o production and systemic delivery of monoclonal antibodies is technically feasible. Application o f this technology t o the treatment o f various viral and autoimmune diseases as well as that of cancer is currently underway.

I. Introduction Specific antibodies can be generated against virtually any type of molecule since antigens can be proteins, nucleic acids, lipids or glucids. They can also be self or

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foreign. The potential of clinical applications for antibodies is thus enormous and concerns a wide range of diseases including cancer, viral infections, transplant rejection, autoimmunity, toxic shock, rheumatoid arthritis, and restenosis (Chester and Hawkins, 1995).


Pelegrin et al: Engineering cells to produce therapeutic antibodies for immunotherapy of cancer Since the discovery of monoclonal antibodies in 1975, various antibody-based therapies have been tested, mostly for treating patients suffering from cancer. However, the poor efficiency of the first monoclonal antibodies used in clinical trials, the development of neutralizing immune responses by patients against antibodies of animal origin and the long periods of time necessary for forming a proper view of the efficacy of treatments have momentarily tempered the initial enthusiasm raised by this technology. Nevertheless, the therapeutic successes obtained during the past years (Scott and Welt, 1997) and the rapid developments of antibody engineering have brought therapeutic monoclonal antibodies back to the fore. Among the therapeutic successes, one can mention a variety of anti-idiotypic antibodies for treating B lymphoma (White et al., 1996) and the now commercially available chimeric antibody ICED-C2B8, which is more efficient than conventional chemotherapy for treating nonHodgkinâ&#x20AC;&#x2122;s lymphomas (Maloney et al., 1997; Marwick, 1997). The main initial drawback met when administering monoclonal antibodies in human patients, namely the immunogenicity of murine antibodies, can now be overcome following several approaches (Figure 1). These include : (i ) the humanization of animal antibodies using site-directed mutagenesis possibly assisted by computerized molecular modeling (Wawrzynczak, 1995); (i i ) the generation of hybridomas from transgenic mice harboring the human immunoglobulins loci substituted for the mouse loci (Bruggemann and Taussig, 1997; Mendez et al., 1997); (i i i ) the construction of hybridomas from activated human B lymphocytes (Wawrzynczak, 1995); and

(i v ) the screening of bacteriophage libraries expressing human immunoglobulins at their surface (Marks and Marks, 1996; Rader and Barbas, 1997). In addition, gene engineering now allows both the improvement of intrinsic properties of antibodies, such as affinity and avidity, the grafting of new effector or enzymatic functions as well as the construction of new antibody-based molecules such as single chain Fv, bispecific antibodies (Chester and Hawkins, 1995; Wawrzynczak, 1995). In conclusion, molecular engineering of antibodies, together with the possibility of generating human monoclonal antibodies, provide us with new antibodies and antibody-related molecules which will, undoubtedly, find clinical therapeutical applications, especially in the field of gene therapy (Pelegrin et al., 1998).

II. A gene/cell therapy approach for the systemic delivery of therapeutic antibodies. In theory, the simplest mode of administration of therapeutic antibodies consists of repeated intraveinous injection. However, the high cost of antibodies produced under gmp (good manufacturing practice) conditions makes most monoclonal antibodies uneconomic for long-term treatments (several months to several years) on a large scale since numerous antibody-based therapies would involve several tens to several hundreds of mg of antibody per month and per patient. Therefore, clinical application of therapeutic antibodies in the long-term is limited by the necessity of finding financially acceptable delivery systems.

Generation of hybridomas from transgenic mice expressing human immunoglobulins genes

Humanisation of animal monoclonal antibodies

Production of monoclonal antibodies for long-term use in humans.

Screening of libraries of bacteriophages expressing human immunoglobulins

Generation of human hybridomas from human B lymphocytes

F i g u r e 1 . Generation of monoclonal antibodies suitable for long-term use in humans.

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To solve this issue, a new gene/cell therapy based on the in vivo production of ectopic antibodies through either the genetic modification of patients' cells or the implantation of antibody-producing cells encapsulated within immunoprotective devices are currently being developed in the laboratory. These delivery systems should not only render long-term therapeutic antibody treatments cost-effective but should also provide an additional therapeutic benefit. Continuous and sustained delivery of antibodies at a low, but therapeutic, level should permit the suppression, or at least the delay, of neutralizing antiidiotypic immune responses which often develop when massive doses of purified immunoglobulins are repeatedly injected (see below).

III. Potential applications of the in vivo production of ectopic antibodies. A first and obvious clinical target for therapeutic antibodies produced in vivo is cancer. Long-term production of ectopic antibodies could, thus, be used in the context of surveillance treatments for preventing relapse after a primary treatment consisting of surgery, chemo- or radiotherapy. Providing the basis for future protocols, several antibodies, cytostatic or cytocidic for tumor cells, have already been characterised (Old, 1995; Riethm端ller et al., 1993; Scott and Welt, 1997; Vitetta and Uhr, 1994). Some of them have even been used with success in various clinical trials based on passive administration of purified immunoglobulins (Table 1) (Scott and Welt, 1997). A second target is life-threatening viral diseases, such as AIDS, for which no satisfactory treatment is available to date. The therapeutic antibodies could be virusneutralizing antibodies, antibodies toxic for virusproducing cells or antibodies specific for cell surface molecules required for viral infection. Supporting the notion that such treatments can be efficiently applied to the curing of viral diseases, transgenic mice expressing a neutralizing antibody are protected from lethal infection by the lymphocytic choriomeningitis virus (Seiler et al., 1998). Also supporting the view of the potential utility of such treatments, it was recently shown that blocking the entry of HIV into target cells by administration of a short peptide (T20) can provide potent inhibition of HIV replication in patients suffering from AIDS (Kilby et al., 1998). In addition, several monoclonal antibodies with a neutralizing effect on HIV, including primary virus isolates, are already available and might be used for passive immunoprophylaxy of AIDS in the future (Table 2) (Burton, 1997; Burton and Montefiori, 1997). These antibodies are directed against the envelope glycoprotein subunits, gp120 and gp41, and have originally been characterised in in vitro inhibition assays. Some of them can even synergise for inhibiting HIV (Mascola et al., 1997) and SHIV (Li et al., 1998) replication. Finally, some of these antibodies are able to inhibit HIV replication in SCID mice grafted with human peripheral

blood lymphocytes (Burton et al., 1994; Gauduin et al., 1997). A third therapeutic application would be the treatment of certain chronic inflammatory diseases such as rheumatoid arthritis. In vitro experiments and recent clinical data have shown that TNF-! is a critical inflammatory mediator of this autoimmune disease and might therefore represent a molecular target for specific immunotherapy (Maini et al., 1995). Indeed, it has been shown that administration of anti-TNF-! monoclonal antibodies causes an improvement in the health of rheumatoid arthritis-suffering patients, thus providing evidence that such antibodies might represent efficacious drugs for long-term treatments of this disease (Elliott et al., 1994; Maini et al., 1995). However, the elevated doses necessary for obtaining therapeutic effects as well as their high cost still restrict the use of these antibodies on a large scale. Besides therapy, in vivo production of monoclonal antibodies may also have applications in the laboratory. Although the construction of transgenic mice could, most often, be envisaged to reach the same goal, genetic modification of somatic cells of animals or implantation of antibody-producing cells are expected to represent more versatile and less time-consuming techniques, especially when production of combinations of antibodies is desired. A first example of this application would be the development of new animal models of human autoimmune diseases in which the humoral immune response is responsible for, or contributes to, the development of the pathology (Rose and Bona, 1993). Another interesting application would be continuous cell type-specific ablation for studying the biological role of certain cell lineages in living animals. According to this approach, cytotoxic antibodies recognizing specific cell surface markers would be delivered continuously into the bloodstream of living animals where they could kill cells immediately upon appearance (for example, after a differentiation step) of the cognate antigen at their surface. A third application, called "phenotypic knock-out", could be the systemic delivery of antibodies neutralizing the activity of circulating antigens. Demonstrating the relevance of this approach, expression in the central nervous system of transgenic mice of a monoclonal antibody directed against substance P was able to inhibit the activity of this neuropeptide and was shown to be useful for studying the mechanisms of action of the latter (Piccioli et al., 1995).

IV. In vivo production of antibodies by genetically modified cells. Plasmocytes are the terminally differentiated cells of the B lineage which are responsible for the production and the release of antibodies into the bloodstream (Piccioli et al., 1995). Because of their short life-span (several days to few weeks), they cannot be used for long-term gene/cell therapy. Moreover, they already produce an immunoglobu-


T a b l e 1 . A n t i b o d y and a n t i b o d y - b a s e d m o l e c u l e s used for immunotherapy o f cancer. This list is not exhaustive. (°) corresponds to radiolabelled antibodies and (*) corresponds to immunotoxins. Details of clinical trials are to be found in the indicated references. Agent

Antigen

Disease

Reference

° [131 I ]-anti-B1 (mouse Mab)

CD20

B-cell lymphoma

Kaminsky et al., 1996; Press et al., 1995

° [90 Y]-anti-CD20 (mouse Mab)

CD20

B-cell lymphoma

Knox, 1996

° [90 Y]-anti-idiotype (mouse Mab)

Idiotype

B-cell lymphoma

White et al., 1996

* IgG HD37-dgA [deglycosylated ricin A] (mouse Mab)

CD19

B-cell lymphoma

Stone et al., 1996

* RF84-dgA [deglycosylated ricin A] (mouse Mab)

CD22

B-cell lymphoma

Amlot et al, 1993

IDEC-C2B8 (human-mouse chimeric Mab)

CD20

B-cell lymphoma

Maloney et al. 1997

M195 (mouse humanised Mab)

CD33

Acute Myeloid Leukemia

Caron et al., 1994; Jurcic et al. 1995

° [131 I]-M195 (mouse humanised Mab)

CD33

Acute Myeloid Leukemia

Jurcic et al., 1995

CAMPATH-1H (humanised Mab)

CDw52

Chronic lymphocytic leukemia

Osterborg et al., 1997

17-1A (mouse Mab)

Epithelial membrane antigen (EMA)

Colorectal carcinoma

Riethmuller et al., 1994

° [125 I]-A33 (murine Mab)

A33

Colorectal carcinoma

Welt et al., 1996; Daghighian et al., 1996

Anti-Le y B3-liked to Pseudomonas exotoxin (murine Mab)

Ley -Antigen

Colorectal carcinoma

Pai, 1996

MFE-23 (scFv antibody)

carcinoembryonic antigen (CEA)

Colorectal carcinoma

Begent et al., 1996

rhuMabHER (humanised Mab)

p185 HER2

Breast cancer

Baselga et al., 1996

° [131 I]-cG250 (human-mouse chimeric Mab)

G250

Renal carcinoma

Surfus et al., 1996; Steffens et al., 1997

lin, the expression of which might interfere with the production of the therapeutic antibody. It has long been known that several eukaryotic cell types such as yeast and certain insect cells in addition to certain mammalian cell lines can produce antibodies upon appropriate genetic modification (for references, see Noël et al., 1997). Interestingly, this observation raised the possibility that a variety of non-plasmocytic cells could be used for production of immunoglobulins. Indeed, we have recently shown that a number of cell types amenable to genetic modification and suitable for graft to humans can secrete antibodies (Noël et al., 1997). These cells include myogenic cells, hepatocytes, keratinocytes and skin

fibroblasts. It is, however, likely that their number will increase in the near future. Furthermore, geneticallymodified myogenic cells (Noël et al., 1997) and fibroblasts (unpublished results) grafted to mice were shown to be capable to sustain systemic delivery of cloned antibodies for several months (Figure 2). Importantly, the antibodies expressed ectopically in vitro and in vivo retained the specificity and the kinetic and thermodynamic characteristics of the parental antibody secreted by lymphocytic cells, as assayed using the BIAcore technology (Noël et al., 1997). These data indicate that several (and possibly all) non-B cell types possess the machinery requi-


Table 2. HIV-neutralizing human monoclonal antibodies. Details can be found in the references indicated. Agent

Antigen

In vitro neutralisation

In vivo neutralisation

R e fe r e nces

2F5

gp41 (linear amino acid sequence ELDKWA)

potent neutralisation of a broad range of primary isolates of HIV

delayed seroconversion and decrease in the viral load of chimpanzees infected with primay isolates

Muster et al, 1994 D’Souza et al, 1997 Conley et al, 1996

IgGb12

gp120 (epitope overlapping the CD4 binding domain and the V2 loop)

potent neutralisation of a broad range of primary isolates of HIV

inhibition of primary isolates of HIV in hu-PBL/SCID mice

Burton et al., 1994 Gaudin et al., 1997 Kessler et al., 1997

2G12

gp120 (epitope overlapping the V3 loop and the V4 region)

potent neutralisation of a broad range of primary isolates of HIV

Trkola, 1996 D’Souza et al, 1997

694/98D

gp120 (V3 loop)

neutralisation of several laboratory strains of HIV, activation of complement

Gorny et al., 1993 Spears et al., 1993

F105

gp120 (CD4 binding domain)

neutralisation of several laboratory strains and primary isolates of HIV

Posner et al., 1993

red for both production and correct folding of antibodies. So far, the production of antibodies by engineered cells has proved weak as compared to the production by cells of the B lineage. However, it is very likely that poor production results, not from the inability of the various cell types to make and secrete antibodies, but rather from poor expression of the retroviral vectors used for gene transfer. Improvement of the latter will thus constitute a major step towards efficient antibody-based gene therapy.

Figure 2. Systemic production of cloned antibodies through grafting of genetically m o d i f i e d m y o g e n i c c e l l s . Primary myoblasts are isolated from mouse muscle biopsies and expanded ex vivo. Following retroviral gene transfer of the cloned monoclonal antibody, stably transduced cells producing the antibody are selected and amplified for implantation into recipient mice. Myogenic cells are grafted by simple injection into the tibialis anterior muscle of mice treated with cardiotoxin. The antibody produced is released into the bloodstream (For more details, see Noël et al., 1997; Pelegrin et al., 1998).


Pelegrin et al: Engineering cells to produce therapeutic antibodies for immunotherapy of cancer

V. In vivo production of antibodies by encapsulated cells. Systemic production of antibodies in mice implanted with cells encapsulated into various biocompatible materials has been achieved by several groups (Okada et al., 1997; Pelegrin et al., 1998; Savelkoul et al., 1994). In the context of gene therapy, capsules are interesting for at least two reasons. First, they constitute immunoprotective devices since the size of their pores can be adjusted in order to allow the diffusion of small molecules (such as nutrients and antibodies) through them but can prevent the passage of cells. In other words, encapsulated cells, which are efficiently retained within capsules, are protected from immune cells of the host which cannot enter the matrix. This property is important with regard to the versatility of the capsule approach since non-autologous, or even xenogenic, cells can potentially be implanted into individuals (Figure 3 ) . Second, capsules offer an advantage with respect to safety since, in contrast to grafted cells, they can easily be removed by simple surgery if, for any reason, the treatment needs to be terminated.

Several types of polymers have been used to encapsulate antibody-producing cells for implantation into mice. These include cellulose sulphate (Dautzenberg et al., in press; Pelegrin et al., 1998), alginate (Savelkoul et al., 1994) and alginate-poly(L)lysine-alginate (Okada et al., 1997). The various matrices used differ in their physical and mechanical properties with cellulose sulphate (Dautzenberg et al., in press) offering advantages over the other two which either rapidly deteriorate or induce an inflammatory response, respectively. Interestingly, cellulose sulphate capsules (Figure 4) implanted subcutaneously form neoorgans which are extensively vascularized within days and are stable for at least 10 months (Figure 5) (Pelegrin et al., 1998). This is certainly beneficial for two reasons. Antibody uptake by the blood is favored and a better supply of nutrients is achieved, thus favoring cell survival in the capsules. Alternatively, other biocompatible immunoprotective devices, such as polyethersulfone fibers, might be used to replace capsules for implantation of antibody-producing cells in vivo (DĂŠglon et al., 1996). So far, only encapsulation of cells with short life-span within capsules, such as hybridoma cells, has been tested for transient antibody production in vivo. It will thus be crucial to test whether long-lived primary cells or cell lines can also be used for long-term production. This seems possible since primary skin fibroblasts have already been shown to survive longer than one year in vivo when encapsulated in alginate-poly-L-lysine alginate (Tai and Sun, 1993). Work is currently underway to address this issue.

VI. Overcoming some of the possible hurdles. The possible development of an immune response against the ectopic antibody and/or the antibody-producing cells is a major threat for this therapeutic strategy. This response can thus potentially be cellular and/or humoral. In case of grafting engineered autologous cells, a cytotoxic response against antibody-producing cells is very unlikely to occur; this is because secreted antibodies are not foreign molecules, provided they are of human origin or of the species in which the experiments are being conducted. However, it cannot yet be ruled out that ectopic

Figure 3. Systemic production of antibodies by implantation of encapsulated antibody-producing c e l l s . Established cell lines validated for human use can be genetically modified to produce therapeutic antibodies upon gene transfer. Selected antibody-producing cells can be amplified and encapsulated in immunoprotective devices (see text and Dautzenberg et al., in press). Capsules are implanted subcutaneously by simple surgical treatment for systemic delivery of therapeutic antibodies.

172


F i g u r e 4 . C e l l u l o s e s u l p h a t e c a p s u l e s . A . Production of cellulose sulphate capsules. Cells are resuspended in a cellulose sulphate solution. Droplets of the suspension are generated and dropped into a solution of polymerization catalyst (PDADMAC). Capsules form within 90 second. After washing with the appropriate medium, they can be implanted immediately or kept in culture for several days to several weeks before use (Dautzenberg et al., in press). B . Cellulose sulphate capsules containing hybridoma cells. These capsules have an average diameter of 0.6 mm. The dark zones correspond to encapsulated cells

Figure 5. Neo-organ formation following implantation of e n c a p s u l a t e d c e l l s . Cellulose sulphate capsules containing antibody-producing cells are vascularised within a few days when implanted subcutaneously (Pelegrin et al., 1998). In this experiment, a group of 10 capsules (C) was implanted. Within 3 days they were wrapped in a pouch of loose connective tissue (CT) which rapidly underwent peripheral vascularization (PV). Later, blood vessels extended into the inner part (IV ) of this pouch for irrigation of the neo-organ.


antibodies can be degraded producing antigenic peptides presentable by MHC class I molecules when expressed in non-B cells. If this occurs a cytotoxic T cell response against such cells could be triggered. This issue merits thorough analysis. The situation is quite different in the case of capsule implantation : even if encapsulated cells are non-MHCmatched or xenogenic, they cannot be destroyed by host cytotoxic T cells because no physical contact is allowed between the two types of cells. However, xenogenic cells are sometimes killed by a mechanism involving complement-mediated lysis. An appropriate choice of xenogenic cells and/or adapted strategies for protecting cells from complement will, thus, be necessary. It is likely that cellular debris released from the capsules could trigger a cytotoxic T cell response directed against encapsulated cells. However, more than being a drawback, this response should present an advantage with respect to the safety: in case of accidental escape from capsules (after breakage, for example), antibody-producing cells released into the bloodstream would immediately be destroyed by circulating T cells. A more serious threat is the possible generation of humoral anti-idiotypic responses against therapeutic antibodies. Such immune responses were observed in patients treated with repeated injections of high doses of purified antibodies. Sometimes, but not always, they could even neutralize the treatment (Isaacs, 1990). It must, however, be emphasized, that in no clinical trial performed so far, were the antibodies of human origin : at best, they were humanized murine antibodies. Moreover, it is not clear whether the observed anti-idiotypic responses were primary responses against the idiotypes of the injected antibodies or just parts of responses directed against whole non-self proteins. In contrast with these observations, no detectable antiidiotypic response was observed in mice producing a model anti-human thyroglobulin monoclonal antibody upon grafting engineered myogenic cells (unpublished data) or upon implantation of cellulose sulphate capsules containing hybridoma cells (Pelegrin et al., 1998). Even though these first data are encouraging, such studies need to be extended to a number of other immunoglobulins to establish whether ectopic monoclonal antibodies produced in vivo are immunogenic or not. It is also possible that the concentration of antibody released systemically is crucial in triggering anti-idiotypic responses. In this case, determining the threshold levels of antibody required for the mounting of the immune response will be crucial for developing efficient long-term antibody-based gene therapies. Inducible expression systems, such as the tetracycline system of Bujard and co-workers, might reveal invaluable tools for adjusting the concentration of antibody delivered into the bloodstream of patients.

VII. Conclusions Using model systems, we have demonstrated the feasibility of the in vivo production and systemic delivery of antibodies by engineered cells. Our work thus sets up the technical basis for a new gene/cell therapy approach aimed at the long-term treatment of patients suffering from a variety of severe diseases such as cancer, viral diseases and various autoimmune diseases. The two major issues which must now be solved before application of this novel therapeutical strategy to humans are, (i ) the optimization of the in vivo production of antibodies and, (i i ), the validation of its therapeutical value in several animal models of human diseases. For optimization in antibody production, several approaches have already been considered. The use of cell lines certified for human use and amenable to encapsulation certainly constitutes a promising approach for various reasons including efficiency, cost-effectiveness and safety. Nevertheless, using in vivo injectable vectors, such as adenoviruses and AAV, for long-term production of monoclonal antibodies in vivo is also a promising approach. Finally, we have recently been able to protect mice from developing a lethal retroviral disease using systemic delivery of antibodies by antibody-producing cells, thus providing the first demonstration of the therapeutical potential of the approach. Extension of this study to other animal diseases is currently under investigation and should pave the way to human applications.

Acknowledgments. This work was supported by grants from the Centre National de la Recherche Scientifique, the Ligue Nationale contre le Cancer, the Association de Recherche contre le Cancer (ARC), the Agence Nationale de Recherche contre le Sida (ANRS) and the EC Biotech Program. We are grateful to Anna Oates for careful correction of the manuscript.

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