Viral Vectors

Advances in genetic engineering and recombinant DNA technology have made it possible to use viruses as clinical tools to transfer therapeutic genes to treat human diseases. Viruses have developed effective mechanisms that promote their survival and replication in a host cell. These intrinsic mechanisms allows them to (a) evade host immune surveillance, (b) attach to the cell membrane, invade the cell, and enter the nucleus, and (c) direct the host cell replication machinery to allow viral multiplication. They are armed with features that meet gene therapy criteria. Research in the development of gene transfer vectors has, thus, focused on the use of these viral properties to facilitate the delivery of therapeutic genes to the nucleus of the cell where the genes can be amplified and expressed.

Hundreds of viruses have been identified and their genome and structure well characterized. This knowledge has allowed researchers to select those viruses with high tropism and low toxicity for the use in gene therapy. Many viruses, however, can only infect a limited number of cell types. Some viruses are pathogenic and will elicit a strong host immune response due to the production of viral proteins required for infectivity, major disadvantages to current gene therapy protocols. Other criteria essential to using a viralbased delivery system for therapeutic DNA include:

1. Stable gene transfer and expression

2. Long-term gene expression where a therapeutic product is needed to attenuate the disease

3. The use of replication-deficient viral vectors

4. Minimal intrinsic toxicity of the input vector and other associated pharmacological toxicity

5. High efficiency of transfection—a critical number of the transgene must enter a sufficient number of affected cells to elicit a significant biological response

6. Optimization of the delivery system to limit reinoculation

7. Use of vectors that do not result in insertional mutagenesis by activating oncogenes or by inactivating of other essential genes

Genetic engineering designs to improve the safety and efficiency of viral gene transfer aim to manipulate the host immune system to reduce immunological response to viral proteins, minimize infection by impairing the viral replication machinery to limit the viral particle to a single infectious cycle, and modify viral tropism and increase viral target specificity by alter ing viral envelope proteins. Designing second-generation viruses with decreased pathogenicity and high infectivity will increase the success of gene therapy in biomedical research.

A recombinant viral vector can be constructed by cutting the viral DNA and the gene of interest at specific sites with a restriction enzyme. The cut pieces are subsequently ligated with a DNA ligase to create a recombinant vector. Before the cutting and ligation procedure, the viral genome is first modified to remove key regulatory genes. This will render the virus replication deficient once it infects a host cell and create enough space for ligation of the new gene. Other accessory genes may be removed from the viral DNA to make the vector less immunogenic. Transfection of the recombinant viral genome into trans-complementing cells lines, stably transformed with the deleted regulatory coding cassettes from the viral genome, allows for replication and packaging of the recombinant viral DNA in vitro. Administration of a titer of recombinant virus obtained by this method should result in viral infection of the target tissue and expression of the inserted gene without viral replication or production of infectious progeny viruses in vivo (Fig. 5) (82,83).

a. Retrovirus. Retroviruses are RNA viruses that infect a wide variety of eukaryotic cells. The retroviral genome codes for three core genes—gag, pol, and env—which are flanked on either side by a long-term repeat (LTR) sequence (Fig. 6). The gag gene, located in the 5' region of the genome, encodes the viral core proteins. The pol gene is the second gene in the cassette and codes for three products: reverse transcriptase (RT), which allows the viral RNA to be transcribed into DNA after entry into a host cell; viral integrase (Int), which facilitates integration of viral DNA into the host DNA; and viral protease, which acts on the viral core proteins. The env gene is 3 ' of the pol gene and encodes the glycosylated viral envelope proteins, which determine the trop-ism of the virus.

The LTRs flank both ends of the genome and contain cis-acting sequences that regulate viral replication, transcription, and integration into the host genome. A packaging signal, which facilitates assembly of viral particles, is found immediately 3' of the 5' LTR. When a retrovirus infects a cell, its RNA is reversed-transcribed into DNA by RT. The DNA subsequently integrates into the host chromosome as a provirus and directs the synthesis of viral proteins by the host transcription and translation machinery. While most retroviruses are nonpathogenic, a few have been known to cause cancer. For example, the Rous sarcoma virus causes tumors of the connective tissue in chickens. The tumorigenic properties of these viruses have been associated with accessory genes called oncogenes.

Figure 5 Viral packaging: (1) transfection of the vital genome into the packaging cell to produce viral proteins; (2) transfection of recombinant viral DNA into the trans complementing packaging cell line and replication of the viral DNA; (3) release of infectious particles.

The most common retroviral vector used for gene transfer is the Moloney murine leukemia virus (MoMuLV). The viral genome is modified by deleting the three core genes—gag, pol, and env—before recombination with the target gene. The deleted segment of the viral genome is then replaced with the therapeutic gene of interest, and the recombinant vector can be grown to therapeutic titers by transfecting it into a suitable packaging cell line that has been stably transformed with the deleted viral core gene cassette. Replication and viral assembly proceed similar to infection with a wild-type virus (84-86). Recombinant viruses maintain their high targeting efficiency and stably integrate into the host genome. While long-

Figure 6 Schematic representation of a basic retrovirus genome showing the age, pol, and env genes, which are flanked by the long-terminal repeats (LTR). A packaging signal is located next to the 5' LTR.

Figure 6 Schematic representation of a basic retrovirus genome showing the age, pol, and env genes, which are flanked by the long-terminal repeats (LTR). A packaging signal is located next to the 5' LTR.

term gene expression is increased with stable integration, the potential risk of activating protooncogenes or inactivating essential host genes at the site of host genome integration are concerns when using this vector is gene therapy approaches.

New engineering technologies are being developed to modify the trop-ism of retroviruses by altering the envelope proteins. Such modifications are designed to facilitate the cell-specific targeting by the virus. Retroviruses, however, do not infect postmitotic cells. They require the active process of mitosis for entry into the nucleus, a feature that does not make them a suitable delivery system for post-mitotic tissues. The use of retroviral vectors in gene transfer therapies is also limited because of the size of the transgene insert that they can package and the low viral titers obtained in vitro. Currently, therapeutic genes larger than 9-10kb cannot be stably cloned into the retroviral genome (Table 1). In addition to these limitations, murine retroviruses are sensitive to inactivation by the human complement lysis-mediated pathway. Current research to minimize this sensitivity is focused on the use of human packaging cells lines or modification of the viral envelope proteins to produce complement-resistant retroviral pseudotypes. Despite these constraints, retroviruses are still the most widely used vectors in current gene delivery systems and account for approximately 40% of all gene therapy-related clinical trials. Stable expression of many therapeutic genes delivered by retroviruses has been observed for periods of over one year.

Several studies have shown a clinical potential of retroviruses as a gene delivery vehicle in the eye. In one report, modified retroviruses encoding either a urokinase-type plasminogen activator (u-PA) or a tissue-type plasminogen activator (t-PA) gene was successfully transfected into human RPE cell cultures. Ten weeks after infection, transfected cells were co-cultured with umbilical vein endothelial cells (HUVECs) and their effects on cell proliferation and wound healing assessed. Both transgenes were observed to express large amounts of biologically active products in the RPE cells. The expression of u-PA promoted HUVEC cell proliferation and wound healing, while t-PA or the control, noninfected RPE cells in nonconfluent cultures had no significant effect. Other retroviral constructs, designed to carry an internal opsin promoter fragment, were able to specifically direct

Table 1 Comparison of Vectors for Gene Therapy

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