Street Broad Scientific
Review
Engineering a Robust Gene Network in Pseudotyped Packaging Cells for in vivo Production of Ecotropic Protein Bound Retroviral Vectors: Prospectives and future directions from a comprehensive review of existing literature Param Sidhu ABSTRACT: An emerging question in the fields of both synthetic biology and gene therapy is the potential for use of a retroviral vector in cell targeted protein and drug delivery. The vector should be able to hold DNA for transduction, produce proteins or substances for delivery and bind those substances to its surface membrane. The goal of this review was, therefore, to determine the feasibility of introducing a robust, multifaceted gene network into pseudotyped HEK-293 packaging cells in order to produce retroviral vectors. The vectors would be assembled using packaging cell enzymatic machinery in vivo. The applications of the work include selective tumor necrosis and targeted drug delivery. I. Overview of Retroviral Vectors Introduction to Gene Therapy Gene therapy refers to disease treatment involving the modification, insertion, or deletion of genes. Alterations to the genetic library of an organism results in modified protein production, which ultimately has physiological implications for the organism. There are four major ways in which gene therapy seeks to address diseases: insertion of a functional gene, replacement of a dysfunctional gene with a normal gene, repair of abnormal genes through selective reverse mutation, and regulation of protein production by modifying DNA (Anson). In gene therapy, the modification or insertion of genetic information is conducted using carrier molecules known as vectors. This transport agent is typically a benign virus or bacteria that has been genetically altered to carry normal human DNA to the organism’s cells. The vector will infect or enter the cell, introducing its genetic plasmid into the genome of the cell to induce perceptible changes in protein production. There are currently four major vectors that are used in gene therapy: herpes simplex viruses, which are cold sore causing viruses that very specifically target neurons; adeno-associated viruses, which attach to a specific binding site on chromosome 19; adenoviruses, which are a class of viruses with double stranded DNA that cause a number of respiratory and eye infections; and retroviruses, which are a group of viruses that can create double-stranded DNA copies of their RNA genomes for integration into host cells. The scope of this article will focus on retroviral vectors. Overview and Structur of Retroviral Vectors Retroviruses were first discovered as cell free oncogenic factors in chickens, but have subsequently been determined to be present in a large variety of animal species. Retroviral virions are typically spherical particles 80-100 nanometers in length. At the heart of the virion are two identical cop-
ies of RNA genome molecules, which are tRNA primers responsible for reverse transcription. The capsid contains a variety of proteins. The gag gene codes for proteins responsible for virion maturation, proteins generated by the pol gene are responsible for synthesis of viral DNA, and proteins created by the env gene are responsible for entry of the virion into the cell. The plasma membrane itself is derived from the host cell’s lipid bilayer membrane and contains the envelope proteins. Retroviruses also elicit a wide range of pathologic conditions in their host; retroviruses range in action from completely vestigial, such as the spumaviruses, to being somewhat aggressive in their progression, such as Human Immunodeficiency Virus. The large number of benign types of retroviruses makes it an excellent candidate for use in gene therapy as there is typically not as significant an immune response (Templeton). Retroviral vectors themselves can be developed using a variety of packaging methods, can have all DNA removed, and can store up to eight kilobase of exogenous DNA. These viruses also have pre-coded genetic sequences responsible for regulation and replication of viral DNA. These predefined functions give retroviruses a pertinent use in gene therapy. Components of Retroviral Vectors A simple backbone of a vector system is composed of three components: the lipid encapsulated retroviral protein capsid, the viral proteins, and the vector genome. There are currently two types of vector construction: MMLV vectors and retroviral DNA vectors. The Moloney murine leukemia virus (MMLV) vector is the most commonly used kind of vector for gene therapy. This kind of vector is separated into two components: the vector and the packaging cell line. The vector is composed of a lipid encapsulated retroviral protein layer, which contains the vector genome with the viral DNA “knockeddown.” This means that only proteins coding for reverse transcription, integration, and packaging are left in Volume 1 | 2011-2012 | 29
Street Broad Scientific
Review
Figure 1. “A diagram depicting the current mechanism of action in a Moloney murine leukemia virus (MMLV) vector.” Source: Verma, et al
the viral genome. The desired genes replace the proteins ordinarily coding for viral replication and virion maturation (Verma et al).The genes to be delivered by the vector are inserted or cloned into the genome construct and its expression is promoted by the 5’ long terminal repeat. The second component of the vector is a packaging cell line that delivers all the viral proteins coded for by the gag, pol, and env genes to the vector in trans. Proteins responsible for insertion of the vector genome into a host cell (transduction) are contained within the packaging construct and become active upon binding with the cell surface (Templeton). The second type of retroviral vector, which is significantly less common, is the retroviral DNA vector. These are typically composed of vector plasmids inserted between the long terminal repeat promoters of the original virus genome. This allows for simple plasmid manipulation. The use of this vector however, is beyond the scope of this article. Efficacy and mechanisms of action The most attractive features of the retroviral vector are its unique life cycle and expedient design of the retroviral genome. A retrovirus binds to a host cell through interaction of the Env glycoprotein with receptors on the cell surface. This results in fusion of the cell lipid bilayer with the viral sheath. Upon binding of the retrovirus to 30 | 2011-2012 | Volume 1
host cell, the virus converts its RNA genome into double stranded DNA, which is then efficiently integrated into the cell genome. This robust process occurs as the reverse transcriptase enzyme converts viral RNA into double stranded DNA, which is then randomly inserted into the host cell genome by the enzyme integrase. This enables efficient protein synthesis as the provirus (the integrated DNA) is transcribed as normal cellular genetic material. Thus, the viral proteins and the viral RNA genome can be transcribed without the need for de novo protein synthesis – the virus itself does not need to produce infectious proteins or particles, which minimizes an immune response to the vector (Anson). The genetic structure of the retrovirus also makes it an attractive vector. The DNA based proviral form allows for simple molecular manipulation. We can easily create a replication defective (non-reproducing) form by manipulating cis and trans elements of the genome. The cis elements of the retroviral DNA (biologically active nucleic acids) and the trans elements (proteins coding sequences) are non-overlapping. Thus, the two can easily be separated in the manufacture of replication defective vectors: the nucleic acid elements can be introduced on a transfer vector construct and protein elements can be expressed using standard recombinant plasmid expression systems. By constructing helper cell lines that produce the trans-acting viral gene products, we can propagate the cis components in manufacture of a fully replication defective cell line. In
Review this way, the resulting provirus will be free of viral DNA responsible for viral replication and cell lysis. The resulting vector is replication dead and therefore nonmalignant and suitable for protein introduction (Markwoitz et al). The diagram below explains in detail the mechanism for production of retroviral vectors and the action of said vectors on target cells. suitable for protein introduction (Markwoitz et al). The diagram below explains in detail the mechanism for production of retroviral vectors and the action of said vectors on target cells. II. Advantages of Retroviral Vectors Defective Vector System A replication defective vector system refers to a retroviral vector without genes responsible for viral reproduction. This defect occurs naturally in certain mouse retroviruses because part of the normal viral genome has been replaced with a cDNA copy of a cellular oncogene. This type of vector is beneficial in gene therapy because its viral proteins do not have to be introduced to the host cell; rather, proteins can simply be provided in trans by the producer cell. No de novo protein synthesis is required in maintenance of the provirus, thus, immune response commonly associated with viral protein transfer and production is minimized. The repression of replication and reduced immune response associated with replication defective retroviruses makes them an attractive agent for protein and drug transfer (Hindmarsh et al). Well Documented Integration One of the primary advantages of retroviral vectors over others is that they are able to integrate their genetic information into the host cell efficiently, and the mechanism for this has been well documented. The process begins when the virus RNA genome is reverse transcribed into linear DNA before being converted into double stranded DNA. The ends of the long terminal repeats found at the termini of the linear viral DNA are recognized by integrase. The next step is formation of preintegration complexes, large protein structures composed of linear viral DNA; several viral proteins including matrix reverse transcriptase, nucleocapsid, and viral integrase; and at least two cellular proteins, high-mobility-group [HMG-I(Y)] and barrier to autointegration factor (BAF). This complex enters the nucleus via the nuclear pores or after the disintegration of the nuclear membrane in cell division. Once the complex enters the nucleus and associates with the chromosomes, viral integrase is released. This enzyme catalyzes the insertion of viral DNA into the host genome by bringing the linear viral DNA together with the host DNA. Lastly, a two base pair sequence is lost from each end of the viral DNA, four to seven base pairs are duplicated on the ends of the host DNA, and integrase binds the host and viral DNA. Cellular proteins mediate
Street Broad Scientific repair of damage to the newly generated provirus from the binding process. The excellent documentation and efficient insertion of desired genes into target cells make retroviruses ideal vectors for DNA transduction (Anson). Flexible Component Organization and Gene Expression Although in a rudimentary retroviral vector system the 5’ long terminating repeats acts as a promoter for the ordinarily coding for viral replication and virion maturation (Verma et al).The genes to be delivered by the vector are inserted or cloned into the genome construct and its expression is promoted by the 5’ long terminal repeat. The second component of the vector is a packaging cell line that delivers all the viral proteins coded for by the gag, pol, and env genes to the vector in trans. Proteins responsible for insertion of the vector genome into a host cell (transduction) are contained within the packaging construct and become active upon binding with the cell surface (Templeton).
III. Goals in Developing Retroviral Vectors Target Specificity One of the foremost goals in vector design is creating a vector that can target specific cells. Creating feature of a vector would grant it a significantly greater clinical relevance because it would transduce only in targeted cells. Development of this technology would allow in vivo delivery of a vector to an afflicted cell and would allow for treatment of specific cells using gene therapy. Proteins on the viral membrane surface mediate the mechanism by which a virus is guided towards its target cell in nature. The interaction of viral surface proteins with receptors on the cell surface detery mines entry of the virus into the cell. Thus, one step toward creating a target specific vector involves tagging the vector surface with the appropriate proteins to interact with the surface of the target cell. Upon binding of a vector to a cell surface receptor, the receptor will either cause a change in the physiological protein structure of the virus to grant it entry or it will cause acidification of the viral sheath to induce structural changes. This type of targeting is known as vector pseudotyping. Regulated Gene Expression Maintaining an appropriate level of gene expression in cells with transduced vector DNA has been problematic in retroviral vectors in the past. Integration of a vector genome into the provirus in the host cell is an advantage of current retroviral vectors, however, regulation of the gene expression of the vector DNA has not been effectively documented. This problem is true of all types of vectors; it is not limited to retroviruses in particular. There are two main barriers to effective gene regulation: interaction of the cell with vector promoter sequences and interaction of the host immune system with vector generated proteins. Although a promoter in the vector genome may drive gene Volume 1 | 2011-2012 | 31
Street Broad Scientific expression in vitro, the in vivo action of the promoter sequence is unpredictable – interaction of the sequence with existing promoters or override of the promoter by cellular promoters minimizes control over the vector gene expression. The immune system also has the ability to recognize foreign promoters that have been integrated into the host genome and inactivate them. Suppression of the long terminal repeat promoter has been studied and will be further reviewed in Section V. The second factor that limits gene regulation is interaction of the immune system with vector produced proteins. Immune identification of endogenous foreign proteins, even if produced by the host cell’s machinery, causes mediated cell death in vectortransduced cells. Untreated, the loss of vector viability is inevitable even if promoter sequences operate correctly (Verma et al). One way that scientists have circumvented this problem is by using proteins that limit T-Cell response. This was exemplified in the study regarding Y-interferon transfer into tumor cells, conducted by Dr. Gansbacher at Sloan-Kettering Cancer Center. In the work, roviral vectors were used to introduce the γ-interferon (IFN-γ) gene into CMS-5 cells.” T-Cell Activation was repressed and this allowed for a long term, stable expression of the endogenous proteins. Future study should seek to minimize immune response to vector proteins and allow for the efficient regulation of gene expression. Effective Integration The inherent risk of mutagenesis following an ineffective integration has been a point of study since 2002, when a group of ten monkeys exposed to myeloblatic irradiation and subsequently transplanted with hematopoetic stem cells treated with a viral vector (Templeton). The introduction of the replication competent retroviral vector resulted in mutagenesis of the target cells. It is therefore
Figure 1. “Components and functionality of a gene regulatory network.” Source: U.S. Department of Energy Genome Programs 32 | 2011-2012 | Volume 1
Review pertinent for us to ensure comprehensive error checks after integration of vector genome. IV. Overview of Regulatory Gene Networks Introduction to Gene Regulatory Networks A gene regulatory network is a collection of DNA, which has its expression regulated by the interaction of secondary products of gene expression (including proteins and mRNA). A regulatory network typically involves translation of modular DNA into mRNA sequences that code for specific proteins, which can include structural proteins, enzymatic proteins, and transcription factors. Every module of DNA evaluates and responds to a number of inputs, using activators and repressors to gain transcriptional control over epigenetic expression. Networks are capable of completing a variety of tasks – they are quite versatile and robust. For example, depending on the protein coded for by genes in the network, endogenous protein concentration can be upregulated, biologically catalyzed reactions can be induced, and transcription factor binding to promoter sequences can activate genes. A regulatory gene network can essentially be thought of as a network of computational units in the sense that the network functions on logic connectivity and ordinary stochastic processes. This means that most of the functionality of the network is provided by using simple logic operators like “or” functions, “and” functions” and “switch” functions”. Although the basis of the network is simple, complex arrangement of the nodes (DNA elements) of the network can cause stochastic behavior. The caustic nature of these networks is also significant to study – they arrangement of DNA sequences is intentional and caustic – it provides temporal regulation of intergene connectivity. The potential for engineering synthetic genetic regulatory networks is an emerging point of study in the field of synthetic biology that may have lucrative applications in retroviral vector gene therapy (Lu et al). Types of Gene Circuity The most significant types of genetic circuitry involved with the creation of a robust gene network are the gene oscillator, cell-cell communicator, and genetic toggle switch controller. The gene oscillator is capable of manipulating a network architecture based in fluid positive feedback loops to cause an oscillation in the expression of a gene. The most famous cell-cell communicator developed thus far was that developed by Thomas Bulter. His circuit used “a threshold concentration of acetate to induce gene expression by acetate kinase and part of the nitrogenregulation two-component system.” His communicator essentially made an artificial quorum sensor in E. Coli that allowed the bacteria to respond to changes in its environment. J.J. Collitns characterized the genetic toggle switch controller effectively at the turn of the century in his
Review
Street Broad Scientific
renowned study “Construction of a genetic toggle switch in Escherichia coli.” The controller is composed of two repressible promoter sequences and is flipped between two stable states to switch between expressions of two genes or to turn the expression of a single gene on and off. Other genetic circuits that have been developed include digital logic evaluators, filters, and sensors, but those topics are out of the scope of this review. The diagram in Figure 2 represents the typical functionality of a regulatory gene network. Challenges of Engineering Regulatory Networks Although significant strides have been made in the field of synthetic biology since the pioneering of the inaugural devices by J.J. Collins and associates, current gene networks lack robustness that would allow them to demonstrate predictable behaviors. An insufficient library of modular component parts prevents effective computational modeling in silico – it is therefore our lack of characterized interoperable parts that prevents the construction rather than a flaw in the construction method itself. Therefore, a significant time in any synthetic biology project will error checks after integration of vector genome. Retroviral vectors were used to introduce the γ-interferon (IFN-γ) gene into CMS-5 cells.”T-Cell Activation was repressed and this allowed for a long term, stable expression of the endogenous proteins. Future study should seek to minimize immune response to vector proteins and allow for the efficient regulation of gene expression.
Volume 1 | 2011-2012 | 33
Street Broad Scientific References
Anson, Donald S. “Retroviral Vectors.” Genetic Vaccines and Therapy. Biomed Central, 13 Aug. 2004. Web. 17 Feb. 2012. <http:// www.gvt-journal.com/content/2/1/9>.
Anson, Donald S. “The Use of Retroviral Vectors for Gene Therapy-what Are the Risks? A Review of Retroviral Pathogenesis and Its Relevance to Retroviral Vector-mediated Gene Delivery.” Pub Med Central - NCBI. National Institutes of Health. Web. 17 Feb. 2012. <http://www.ncbi.nlm.nih.gov/pmc/articles/PMC515179/>.
Butler, Thomas, Sun-Gu Lee, Wilson W. Wong, Eileen Fung, Michael R. Connon, and James C. Liao. “Design of Artificial Cell–cell Communication Using Gene and Metabolic Networks.” PNAS. org. Proceedings of the National Academies of Sciences of the US. Web.
Cole, Caroline, Jian Qiao, Timothy Kottke, Rosa M. Diaz, Atique Ahmed, Luis Sanchez-perez, Gregory Brunn, Jill Thompson, John Chester, and Richard G. Vile. “Tumor-Targeted, Systemic Delivery of Therapeutic Viral Vectors.” MedScape Today Scientific News. Web. 17 Feb. 2012. <http://www.medscape.com/viewarticle/514625_4>.
Cronin, Michelle, Ali R. Akin, Sara A. Collins, Jeff Meganck, JaeBeom Kim, Chwanrow Baban, Susan A. Joyce, Gooitzen M. Van Dam, Ning Zhang, Douwe Van Sinderen, Gerald C. O’Sullivan, Noriyuki Kasahara, Cormac C. Gahan, Kevin P. Francis, and Mark Tangney. “PLoS ONE: High Resolution In Vivo Bioluminescent Imaging for the Study of Bacterial Tumour Targeting.” PLoS ONE : Accelerating the Publication of Peer-reviewed Science. Web. 17 Feb. 2012. <http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030940>. “Engineered Bacteria Effectively Target Tumors, Enabling Tumor Imaging Potential in Mice.” Science Daily: News & Articles in Science, Health, Environment & Technology. Web. 17 Feb. 2012. <http://www.sciencedaily.com/releases/2012/01/120125172319. htm>.
Gansbacher, Bernd, Rajat Bannerji, Brian Daniels, Karen Zier, Kathy Cronin, and Eli Gilboa. “Retroviral Vector-mediated γ-Interferon Gene Transfer into Tumor Cells Generates Potent and Long Lasting Antitumor Immunity.” Cancer Research. AACR Journals, 15 Dec. 1990. Web. 17 Feb. 2012. <http://cancerres. aacrjournals.org/content/50/24/7820.short>. Ghani, Karim, Sylvine Cottin, Pedro Otavio De Campos-Lima, Marie-Christine Caron, and Manuel Caruso. “Characterization of an Alternative Packaging System Derived from the Cat RD114 Retrovirus for Gene Delivery.” The Journal of Gene Medicine. Wiley Online Library, 8 June 2009. Web. 17 Feb. 2012. <http://onlinelibrary.wiley.com/doi/10.1002/jgm.1351/full>.
Hindmarsh, Patrick, and Jonathan Leis. “Retroviral DNA Integration.” Pubmed Central - NCBi. National Institutes of Health, 1999. Web. 17 Feb. 2012. <http://www.ncbi.nlm.nih.gov/pmc/articles/PMC98978/>. Hu, Wei-Shau, and Vinay K. Pathak. “Design of Retroviral Vectors and Helper Cells for Gene Therapy.” Pharmocological Reviews. Aspet Journals, 1 Dec. 2000. Web. 17 Feb. 2012. <http://pharmrev. aspetjournals.org/content/52/4/493.full#title12>. Hughes, S. H., and H. E. Varmus. “Principles of Retroviral Vector
34 | 2011-2012 | Volume 1
Review Design.” NCBI Bookshelf. Ed. J. M. Coffin. National Institutes of Health / Cold Spring Harbor Laboratory Press, 1997. Web. 17 Feb. 2012. <http://www.ncbi.nlm.nih.gov/books/NBK19423/>.
Janes, Sam. “Mesenchymal Stem Cell Delivery of TRAIL Can Eliminate Metastatic Cancer.” Cancer Research. AACR Journals. Web. 17 Feb. 2012. <http://cancerres.aacrjournals.org/content/69/10/4134. full>.
Markowitz, D., S. Goff, and A. Bank. “A Safe Packaging Line for Gene Transfer: Separating Viral Genes on Two Different Plasmids.” Journal of Virology. American Society for Microbiology. Web. 17 Feb. 2012. <http://jvi.asm.org/content/62/4/1120.abstract>. Sorge, Joe, Dowain Wright, Valerie D. Erdman, and Ann E. Cutting. “Amphotropic Retrovirus Vector System for Human Cell Gene Transfer.” Pubmed Central - NCBI. National Institutes of Health. Web. 17 Feb. 2012. <http://www.ncbi.nlm.nih.gov/pmc/articles/ PMC368980/pdf/molcellb00151-0068.pdf>.
Stricker, Jessee, Scott Cookson, Matthew R. Bennett, William H. Mather, Lev A. Tsimring, and Jeff Hasty. “A Fast, Robust and Tunable Synthetic Gene Oscillator.” Access : Nature. Nature: International Weekly Journal of Science, 29 Oct. 2008. Web. 17 Feb. 2012. <http://www.nature.com/nature/journal/v456/n7221/full/nature07389.html>. Sugimoto, Y., A. Aksentijevich, M. M. Gottesman, and I. Patsman. “Efficient Expression of Drug-selectable Genes in Retroviral Vectors under Control of an Internal Ribosome Entry Site.” PubMed.org NCBI. National Institutes of Health, July 1994. Web. 17 Feb. 2012. <http://www.ncbi.nlm.nih.gov/pubmed/7764914>. Templeton, Nancy Smyth. Gene and Cell Therapy: Therapeutic Mechanisms and Strategies. New York: Marcel Dekker, 2004. Print.
“The V-ras Oncogene Inhibits the Expression of Differentiation Markers and Facilitates Expression of Cytokeratins 8 and 18 in Mouse Keratinocytes.” Wiley Online Library. Web. 17 Feb. 2012. <http:// onlinelibrary.wiley.com/doi/10.1002/mc.2940030608/abstract>. Verma, Inder M., and Nikunj Somia. “Gene Therapy – Promises, Problems and Prospects.” Western Washington University. Web. 17 Feb. 2012. <http://fire.biol.wwu.edu/trent/trent/genetherapy2.pdf>.
Yu, S. F., T. Von Ruden, P. W. Kantoff, C. Garber, M. Seiberg, U. Ruther, W. F. Anderson, E. F. Wagner, and E. Gilboa. “Self-inactivating Retroviral Vectors Designed for Transfer of Whole Genes into Mammalian Cells.” Abstract - PNAS.org. Proceedings of the National Academies of Sciences of the US, 1 May 1986. Web. 17 Feb. 2012. <http://www.pnas.org/content/83/10/3194.short>.
Acknowledgements Dr. Myra Halpin The North Carolina School of Science and Mathematics Research in Chemistry Program Duke University Pratt School of Engineering 2012 Duke iGEM Mentors Dr. Jingdong Tian Dr. Nicolas Buchler Dr. Charles Gersbach Mr. Aakash Indurkhya