Gene therapy using SV40-derived vectors: what does the future hold?

Effective genetic therapy requires both a fragment of genetic material to be used therapeutically and a means to deliver it. We began to study simian virus-40 (SV40) as a vector for gene transfer because available gene delivery vehicles did not provide for the full range of therapeutic uses. Other vectors are variably limited by immunogenicity, difficulties in production, restricted specificity, low titers, poor transduction efficiency, etc. In theory recombinant viral vectors based on SV40 (rSV40) should not, on the other hand, be similarly constrained. rSV40 vectors are easily manipulated and produced at very high titer, stable, lacking in immunogenicity, and capable of providing sustained high levels of transgene expression in both resting and dividing cells. The principle limitation of SV40-derived vectors is the size of the packageable insert (相似文献   

Protein blotting: principles and applications

Extensive studies on the DNA tumor virus Simian Virus 40 (SV40) have provided a wealth of information regarding the genome organization, regulation of viral gene expression, and the mechanism of DNA replication. SV40 can grow lytically in permissive monkey cells or the viral DNA can integrate into the host genome of nonpermissive rodent cells causing morphological transformation. The viral DNA exists as a minichromosome within the nuclei of lytically infected cells and, as a consequence of DNA replication, there is a significant amplification of the viral genome during infection. These properties suggested that SV40 could be developed as a transducing vector to introduce exogenous DNA into mammalian cells and to express this foreign DNA during the SV40 infectious cycle. In this article the properties of SV40 virus vectors and SV40 hybrid plasmid vectors are described and contrasted.     

A simian virus 5 (SV5) P/V mutant is less cytopathic than wild-type SV5 in human dendritic cells and is a more effective activator of dendritic cell maturation and function

Human epithelial cells infected with the parainfluenza virus simian virus 5 (SV5) show minimal activation of host cell interferon (IFN), cytokine, and cell death pathways. In contrast, a recombinant SV5 P/V gene mutant (rSV5-P/V-CPI-) overexpresses viral gene products and is a potent inducer of IFN, proinflammatory cytokines, and apoptosis in these cells. In this study, we have compared the outcomes of wild-type (WT) SV5 and rSV5-P/V-CPI- infections of primary human dendritic cells (DC), important antigen-presenting cells for initiating adaptive immune responses. We have tested the hypothesis that a P/V mutant which activates host antiviral responses will be a more potent inducer of DC maturation and function than WT rSV5, which suppresses host cell responses. Infection of peripheral blood mononuclear cell-derived immature DC with WT rSV5 resulted in high levels of viral protein and progeny virus but very little increase in cell surface costimulatory molecules or secretion of IFN and proinflammatory cytokines. In contrast, immature DC infected with the rSV5-P/V-CPI- mutant produced only low levels of viral protein and progeny virus, but these infected cells were induced to secrete IFN-alpha and other cytokines and showed elevated levels of maturation markers. Unexpectedly, DC infected with WT rSV5 showed extensive cytopathic effects and increased levels of active caspase-3, while infection of DC with the P/V mutant was largely noncytopathic. In mixed-culture assays, WT rSV5-infected DC were impaired in the ability to stimulate proliferation of autologous CD4+ T cells, whereas DC infected with the P/V mutant were very effective at activating T-cell proliferation. The addition of a pancaspase inhibitor to DC infected with WT rSV5 reduced cytopathic effects and resulted in higher surface expression levels of maturation markers. Our finding that the SV5 P/V mutant has both a reduced cytopathic effect in human DC compared to WT SV5 and an enhanced ability to induce DC function has implications for the rational design of novel recombinant paramyxovirus vectors based on engineered mutations in the viral P/V gene.     

Comparison of spontaneous and ultraviolet-induced mutagenesis on naked SV40 DNA and SV40 virus

Molecular aspects of mutagenesis in mammalian cells have been essentially analyzed using biological probes such as viruses and shuttle vector. Although the main data concerning the specificity of carcinogen-induced mutations are similar, the observed spontaneous mutation frequencies are significantly different when using one or the other model. This frequency is considerably higher with shuttle vectors than with viruses. We have performed an analysis of mutagenesis in order to determine if the obligatory transfection step associated with shuttle vector technology was responsible for the high mutation frequency found with these molecules. For this purpose simian virus 40 (SV40) genome used as virus or as naked DNA was introduced into permissive cells by viral infection or DNA transfection respectively. Our results show that transfection alone does not induce a higher mutation frequency on SV40 DNA the virus infection. Moreover, we have shown that the ultraviolet-light induced mutation spectrum was similar on the SV40 VP1 gene after viral infection or DNA transfection.     

Long-term gene expression in dividing and nondividing cells using SV40-derived vectors

Among the goals of gene therapy is long-term expression of delivered transgenes. Recombinant Tag-deleted SV40 vectors (rSV40s) are especially well suited for this purpose. rSV40s deliver transgene expression that endures for extended periods of time in tissue culture and in vivo, in both dividing and nondividing cells. These vectors are particularly effective in transducing some cell types that have been almost unapproachable using other gene delivery systems, such as quiescent hematopoietic progenitor cells and their differentiated derivatives. Other cellular targets include neurons, brain microglia, hepatocytes, dendritic cells, vascular endothelium, and others. Because rSV40s do not elicit neutralizing antibodies they are useful for in vivo gene delivery in settings where more than one administration may be desirable. The key characteristics of these vectors include their high production titers and therefore suitability for large cell pools, effectiveness in delivering intracellular proteins, and untranslated RNAs, maintenance of transgene expression at constant levels for extended times, suitability for constitutive or conditional promoters and for combinatorial gene delivery and ability to integrate into genomes of both dividing and nondividing cells.     

Function of small hydrophobic proteins of paramyxovirus

Mumps virus (MuV), a rubulavirus of the paramyxovirus family, causes acute infections in humans. MuV has seven genes including a small hydrophobic (SH) gene, which encodes a type I membrane protein of 57 amino acid residues. The function of the SH protein is not clear, although its expression is not necessary for growth of MuV in tissue culture cells. It is speculated that MuV SH plays a role in viral pathogenesis. Simian virus 5 (SV5), a closely related rubulavirus, encodes a 44-amino-acid-residue SH protein. Recombinant SV5 lacking the SH gene (rSV5DeltaSH) is viable and has no growth defect in tissue culture cells. However, rSV5DeltaSH induces apoptosis in tissue culture cells and is attenuated in vivo. Neutralizing antibodies against tumor necrosis factor alpha (TNF-alpha) and TNF-alpha receptor 1 block rSV5DeltaSH-induced apoptosis, suggesting that SV5 SH plays an essential role in blocking the TNF-alpha-mediated apoptosis pathway. Because MuV is closely related to SV5, we hypothesize that the SH protein of MuV has a function similar to that of SV5, even though there is no sequence homology between them. To test this hypothesis and to study the function of MuV SH, we have replaced the open reading frame (ORF) of SV5 SH with the ORF of MuV SH in a SV5 genome background. The recombinant SV5 (rSV5DeltaSH+MuV-SH) was analyzed in comparison with SV5. It was found that rSV5DeltaSH+MuV-SH was viable and behaved like wild-type SV5, suggesting that MuV SH has a function similar to that of SV5 SH. Furthermore, both ectopically expressed SV5 SH and MuV SH blocked activation of NF-kappaB by TNF-alpha in a reporter gene assay, suggesting that both SH proteins can inhibit TNF-alpha signaling.     

Single amino acid substitution in the V protein of simian virus 5 differentiates its ability to block interferon signaling in human and murine cells

Previous work has demonstrated that the V protein of simian virus 5 (SV5) targets STAT1 for proteasome-mediated degradation (thereby blocking interferon [IFN] signaling) in human but not in murine cells. In murine BF cells, SV5 establishes a low-grade persistent infection in which the virus fluxes between active and repressed states in response to local production of IFN. Upon passage of persistently infected BF cells, virus mutants were selected that were better able to replicate in murine cells than the parental W3 strain of SV5 (wild type [wt]). Viruses with mutations in the Pk region of the N-terminal domain of the V protein came to predominate the population of viruses carried in the persistently infected cell cultures. One of these mutant viruses, termed SV5 mci-2, was isolated. Sequence analysis of the V/P gene of SV5 mci-2 revealed two nucleotide differences compared to wt SV5, only one of which resulted in an amino acid substitution (asparagine [N], residue 100, to aspartic acid [D]) in V. Unlike the protein of wt SV5, the V protein of SV5 mci-2 blocked IFN signaling in murine cells. Since the SV5 mci-2 virus had additional mutations in genes other than the V/P gene, a recombinant virus (termed rSV5-V/P N(100)D) was constructed that contained this substitution alone within the wt SV5 backbone to evaluate what effect the asparagine-to-aspartic-acid substitution in V had on the virus phenotype. In contrast to wt SV5, rSV5-V/P N(100)D blocked IFN signaling in murine cells. Furthermore, rSV5-V/P N(100)D virus protein synthesis in BF cells continued for significantly longer periods than that for wt SV5. However, even in cells infected with rSV5-V/P N(100)D, there was a late, but significant, inhibition in virus protein synthesis. Nevertheless, there was an increase in virus yield from BF cells infected with rSV5-V/P N(100)D compared to wt SV5, demonstrating a clear selective advantage to SV5 in being able to block IFN signaling in these cells.     

Novel Therapeutic Approaches for Various Cancer Types Using a Modified Sleeping Beauty-Based Gene Delivery System

Successful gene therapy largely depends on the selective introduction of therapeutic genes into the appropriate target cancer cells. One of the most effective and promising approaches for targeting tumor tissue during gene delivery is the use of viral vectors, which allow for high efficiency gene delivery. However, the use of viral vectors is not without risks and safety concerns, such as toxicities, a host immune response towards the viral antigens or potential viral recombination into the host''s chromosome; these risks limit the clinical application of viral vectors. The Sleeping Beauty (SB) transposon-based system is an attractive, non-viral alternative to viral delivery systems. SB may be less immunogenic than the viral vector system due to its lack of viral sequences. The SB-based gene delivery system can stably integrate into the host cell genome to produce the therapeutic gene product over the lifetime of a cell. However, when compared to viral vectors, the non-viral SB-based gene delivery system still has limited therapeutic efficacy due to the lack of long-lasting gene expression potential and tumor cell specific gene transfer ability. These limitations could be overcome by modifying the SB system through the introduction of the hTERT promoter and the SV40 enhancer. In this study, a modified SB delivery system, under control of the hTERT promoter in conjunction with the SV40 enhancer, was able to successfully transfer the suicide gene (HSV-TK) into multiple types of cancer cells. The modified SB transfected cancer cells exhibited a significantly increased cancer cell specific death rate. These data suggest that our modified SB-based gene delivery system can be used as a safe and efficient tool for cancer cell specific therapeutic gene transfer and stable long-term expression.     

Naturally occurring substitutions in the P/V gene convert the noncytopathic paramyxovirus simian virus 5 into a virus that induces alpha/beta interferon synthesis and cell death

The V protein of the paramyxovirus simian virus 5 (SV5) is responsible for targeted degradation of STAT1 and the block in alpha/beta interferon (IFN-alpha/beta) signaling that occurs after SV5 infection of human cells. We have analyzed the growth properties of a recombinant SV5 that was engineered to be defective in targeting STAT1 degradation. A recombinant SV5 (rSV5-P/V-CPI-) was engineered to contain six naturally occurring P/V protein mutations, three of which have been shown in previous transfection experiments to disrupt the V-mediated block in IFN-alpha/beta signaling. In contrast to wild-type (WT) SV5, human cells infected with rSV5-P/V-CPI- had STAT1 levels similar to those in mock-infected cells. Cells infected with rSV5-P/V-CPI- were found to express higher-than-WT levels of viral proteins and mRNA, suggesting that the P/V mutations had disrupted the regulation of viral RNA synthesis. Despite the inability to target STAT1 for degradation, single-step growth assays showed that the rSV5-P/V-CPI- mutant virus grew better than WT SV5 in all cell lines tested. Unexpectedly, cells infected with rSV5-P/V-CPI- but not WT SV5 showed an activation of a reporter gene that was under control of the IFN-beta promoter. The secretion of IFN from cells infected with rSV5-P/V-CPI- but not WT SV5 was confirmed by a bioassay for IFN. The rSV5-P/V-CPI- mutant grew to higher titers than did WT rSV5 at early times in multistep growth assays. However, rSV5-P/V-CPI- growth quickly reached a final plateau while WT rSV5 continued to grow and produced a final titer higher than that of rSV5-P/V-CPI- by late times postinfection. In contrast to WT rSV5, infection of a variety of cell lines with rSV5-P/V-CPI- induced cell death pathways with characteristics of apoptosis. Our results confirm a role for the SV5 V protein in blocking IFN signaling but also suggest new roles for the P/V gene products in controlling viral gene expression, the induction of IFN-alpha/beta synthesis, and virus-induced apoptosis.     

Efficient CNS gene delivery by intravenous injection

We administered recombinant SV40-derived viral vectors (rSV40s) intravenously to mice with or without prior intraperitoneal injection of mannitol to deliver transgenes to the central nervous system (CNS). We detected transgene-expressing cells (mainly neurons) most prominently in the cortex and spinal cord; prior intraperitoneal mannitol injection increased CNS gene delivery tenfold. Intravenous injection of rSV40s, particularly with mannitol pretreatment, resulted in extensive expression of multiple transgenes throughout the CNS.     

The SH integral membrane protein of the paramyxovirus simian virus 5 is required to block apoptosis in MDBK cells

In some cell types the paramyxovirus simian virus 5 (SV5) causes little cytopathic effect (CPE) and infection continues productively for long periods of time; e.g., SV5 can be produced from MDBK cells for up to 40 days with little CPE. SV5 differs from most paramyxoviruses in that it encodes a small (44-amino-acid) hydrophobic integral membrane protein (SH). When MDBK cells were infected with a recombinant SV5 containing a deletion of the SH gene (rSV5DeltaSH), the MDBK cells exhibited an increase in CPE compared to cells infected with wild-type SV5 (recovered from cDNA; rSV5). The increased CPE correlated with an increase in apoptosis in rSV5DeltaSH-infected cells over mock-infected and rSV5-infected cells when assayed for annexin V binding, DNA content (propidium iodide staining), and DNA fragmentation (terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling assay). In rSV5DeltaSH-infected MDBK cells an increase in caspase-2 and caspase-3 activities was observed. By using peptide inhibitors of individual caspases it was found that caspase-2 and caspase-3 were activated separately in rSV5DeltaSH-infected cells. Expression of caspase-2 and -3 in rSV5DeltaSH-infected MDBK cells appeared not to require STAT1 protein, as STAT1 protein could not be detected in SV5-infected MDBK cells. When mutant mice homologous for a targeted disruption of STAT1 were used as a model animal system and infected with the viruses it was found that rSV5DeltaSH caused less mortality than wild-type rSV5, consistent with the notion of clearance of apoptotic cells in a host species.     

Efficient expression of small RNA polymerase III genes from a novel simian virus 40 vector and their effect on viral gene expression.

In the past, simian virus 40 (SV40) has been used as a cloning vehicle to clone foreign genes by substituting portions of the viral genome vital for viral replication. Propagation of these defective viruses required a helper virus and the recombinant viruses obtained could be grown only as a mixture. In this study, we describe a novel nondefective SV40 vector to clone small RNA polymerase III genes. Two small RNA polymerase III genes, an amber suppressor human serine tRNA gene and the adenovirus (Ad) VAI RNA gene, were cloned in the intron region of the large-T antigen gene of SV40 after deleting DNA sequences coding for the small-t polypeptide. The recombinant viruses grew to wild type levels and showed no growth defects. When CV-1p cells were infected with these viruses, the cloned RNA polymerase III genes were expressed at high levels at late times. Interestingly, large amounts VAI RNA in CV-1p cells infected with SV40-VA recombinant virus, did not enhance translation of viral mRNAs significantly but did lead to a 3 to 4 fold increase in the steady state levels of large-T mRNA suggesting a novel function for VAI RNA in SV40 infected monkey cells. Furthermore, VAI mutants which fail to function in Ad infected human cells also failed to enhance the levels of large-T mRNAs in monkey cells infected with SV40. The simple SV40 vector described here may be useful to study the structure and function of small RNA polymerase III genes in the context of a eucaryotic chromosome. In addition, the nondefective recombinant SV40 which expresses the suppressor tRNA gene at high levels may provide a useful helper system to propagate animal viruses with amber mutations in essential genes.     

Plant virus expression systems for transient production of recombinant allergens in Nicotiana benthamiana

In recent years, several studies have demonstrated the use of autonomously replicating plant viruses as vehicles to express a variety of therapeutic molecules of pharmaceutical interest. Plant virus vectors for expression of heterologous proteins in plants represent an attractive biotechnological tool to complement the conventional production of recombinant proteins in bacterial, fungal, or mammalian cells. Virus vectors are advantageous when high levels of gene expression are desired within a short time, although the instability of the foreign genes in the viral genome may present problems. Similar levels of foreign protein production in transgenic plants often are unattainable, in some cases because of the toxicity of the foreign protein. Now virus-based vectors are for the first time investigated as a means of producing recombinant allergens in plants. Several plant virus vectors have been developed for the expression of foreign proteins. Here, we describe the utilization of tobacco mosaic virus- and potato virus X-based vectors for the transient expression of plant allergens in Nicotiana benthamiana plants. One approach involves the inoculation of tobacco plants with infectious RNA transcribed in vitro from a cDNA copy of the recombinant viral genome. Another approach utilizes the transfection of whole plants from wounds inoculated with Agrobacterium tumefaciens containing cDNA copies of recombinant plus-sense RNA viruses.     

Fusion-mediated injection of SV40-DNA : Introduction of SV40-DNA into tissue culture cells by the use of DNA-loaded reconstituted Sendai virus envelopes

Sendai virus envelopes can be solubilized by non-ionic detergents such as Triton X-100. Removal of the detergent from a supernatant containing the solubilized viral envelope glycoproteins results in the formation of reconstituted fusogenic viral envelopes. When SV40-DNA is added to the reconstitution system, it is trapped within the viral envelope. Incubation of SV40-DNA-loaded Sendai virus envelopes with permissive cells (CV1 and TC7 cells) resulted in fusion-mediated injection of the trapped DNA, as was demonstrated by the ability of the injected cells to synthesize SV40-T-antigen. Quantitative estimation revealed that up to 20% of the injected cells were able to synthesize T-antigen. Loaded viral envelopes were able to inject SV40-DNA and to promote synthesis of T-antigen also in cells which are resistant to infection by intact SV40 viruses, such as F1' 1-4 cells. In addition, it is shown that reconstituted envelopes of Sendai virus are able to transfer membrane fragments from SV40 receptor-positive into SV40 receptor-negative cells, such as F1' 1-4 cells. After implantation of SV40 receptors, the F1' 1-4 cells became susceptible to infection by intact SV40 viruses.     

Autonomous parvovirus vectors

Parvoviruses are small, icosahedral viruses (approximately 25 nm) containing a single-strand DNA genome (approximately 5 kb) with hairpin termini. Autonomous parvoviruses (APVs) are found in many species; they do not require a helper virus for replication but they do require proliferating cells (S-phase functions) and, in some cases, tissue-specific factors. APVs can protect animals from spontaneous or experimental tumors, leading to consideration of these viruses, and vectors derived from them, as anticancer agents. Vector development has focused on three rodent APVs that can infect human cells, namely, LuIII, MVM, and H1. LuIII-based vectors with complete replacement of the viral coding sequences can direct transient or persistent expression of transgenes in cell culture. MVM-based and H1-based vectors with substitution of transgenes for the viral capsid sequences retain viral nonstructural (NS) coding sequences and express the NS1 protein. The latter serves to amplify the vector genome in target cells, potentially contributing to antitumor activity. APV vectors have packaging capacity for foreign DNA of approximately 4.8 kb, a limit that probably cannot be exceeded by more than a few percent. LuIII vectors can be pseudotyped with capsid proteins from related APVs, a promising strategy for controlling tissue tropism and circumventing immune responses to repeated administration. Initial success has been achieved in targeting such a pseudotyped vector by genetic modification of the capsid. Subject to advances in production and purification methods, APV vectors have potential as gene transfer agents for experimental and therapeutic use, particularly for cancer therapy.     

Viral Vectors for Gene Delivery and Expression in the CNS

Viral vectors have emerged as an important tool for manipulating gene expression in the adult mammalian brain. The adult brain is composed largely of nondividing cells, and therefore DNA viruses have become the vehicle of choice for neurobiologists interested in somatic gene transfer. Recombinant viral vectors based upon adenovirus or herpes simplex virus have been created in which a gene essential for viral replication is removed and a gene of interest is inserted in the viral genome. While this eliminates pathogenicity due to viral replication, retention of viral genes and continued expression of these genes may limit the potential of the current generation of vectors. Defective viral vectors represent a different approach, in which only viral recognition signals are used to allow packaging of foreign DNA into a viral coat while eliminating the possibility of viral gene expression within target cells. The defective HSV vector has been used to transfer genes into the adult rat brain. This vector has also been used for analysis of the preproenkephalin promoterin vivo,and important regions of this promoter have been identified using this technique. A modification ofin situPCR has been developed as an adjunctive tool for sensitively documenting the presence of vector DNA within target cells duringin vivopromoter studies. Finally, the adenoassociated virus vector has been used as the first fully defective DNA viral vector, which also eliminates any contamination by helper viruses. This vector can transfer genes into the mammalian brain and has shown significant behavioral recovery in a rodent model of Parkinson's disease. Future work will undoubtedly result in still more diverse and improved vectors; however, these studies have documented the importance of viral vectors to both basic neurobiology and the potential treatment of neurologic disease.     

Oncolytic adenoviruses in anticancer therapy: Current status and prospects

Lytic virus infection results in production of a virus progeny and lysis of the infected cell. Tumor cells are usually more sensitive to virus infection. Studies indicate that viral oncolysis provides a promising alternative approach to cancer therapy. The ability of viruses to selectively kill cancer cells is long known, but construction of virus variants with an improved therapeutic potential was impossible until recent advances in virus and cell molecular biology and the development of modern methods for directed modification of viruses. Adenoviruses are one of the best studied models of oncolytic viruses. These DNA viruses are convenient for genetic manipulation and show minimal pathogenicity. The review summarizes the data on the directions and approaches to generation of highly efficient variants of oncolytic adenoviruses. The approaches include introduction of directed genetic modifications into the virus genome, accelerated selection of oncolytic virus variants following treatment with mutagens, the use of adenoviruses as vectors to introduce therapeutic gene products, optimization of viral delivery systems, minimization of the negative effects from the host immune system, etc. The dynamic development of studies in the field holds promise that many variants of oncolytic adenoviruses will find clinical application in the nearest future.     

Activity of the simian virus 40 early promoter-enhancer in herpes simplex virus type 1 vectors is dependent on its position, the infected cell type, and the presence of Vmw175.

We have studied some of the parameters governing the expression of a foreign promoter-reporter gene construct incorporated into herpes simplex virus (HSV) type 1. These include the genetic background of the parental virus, the site of transgene insertion within the HSV genome, and the infected cell type. The genetic background of the vector constructs denoted delta 3 was an HSV type 1 mutant deleted for nearly the entire coding portion of Vmw175 (ICP4), the product of the essential immediate-early gene IE3. For vectors denoted +, the IE3 deletion had been repaired by marker rescue. We used as a reporter gene the bacterial chloramphenicol acetyltransferase (CAT) gene, driven by the simian virus 40 (SV40) early promoter and enhancer region. The SV40-cat hybrid gene was inserted either into the HSV thymidine kinase (TK) locus to create the vectors TKScat delta 3 and TKScat+ or into an intergenic site within the BamHI z fragment of the short unique portion of the viral genome to create the vectors GScat delta 3 and GScat+. In Vero and BHK cells infected with TKScat delta 3, CAT activity was first detected at 10 h postinfection and continued to accumulate until 36 h postinfection. In cells of primate origin infected with the replication-competent vector TKScat+, or in primate cells which complement the IE3 deficiency and which were infected with TKScat delta 3, CAT activity was significantly lower than in cells of rodent origin. However, levels of CAT were increased in the presence of cycloheximide, suggesting that the low production of CAT in primate cells was due to repression of SV40-cat hybrid gene expression. In contrast with results with TKScat delta 3 and TKScat+, CAT activity was not detectable in any of the tested cell types infected with GScat delta 3 or GScat+ except under conditions of cycloheximide reversal. These results show that while HSV gene products expressed in the presence of Vmw175 inhibited SV40-cat expression in the tk locus in a cell-type-specific manner, HSV gene products expressed in the presence or absence of Vmw175 inhibited SV40-cat expression in the BamHI z locus independently of cell type.