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A Packaging System for SV40 Vectors without Viral Coding Sequences
ANALYTICAL BIOCHEMISTRY ARTICLE NO.
254, 139–143 (1997)
AB972417
A Packaging System for SV40 Vectors without Viral Coding Sequences Bingliang Fang,*,1 Patricia Koch,* Michael Bouvet,† Lin Ji,* and Jack A. Roth* *Section of Thoracic Molecular Oncology, Department of Thoracic and Cardiovascular Surgery, and †Department of Surgical Oncology, University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030
Received May 16, 1997
SV40 vectors have been used as expression vectors for mammalian cells since the early 1980s. More recently, they have been used as gene transfer vectors in mice and in human peripheral blood cells. Here we described a system for packaging SV40 vectors without viral coding sequences. Recombinant adenovirusexpressing SV40 capsids can effectively package plasmids that contain the SV40 replication origin. The final yield of infectious SV40 vector is about 3 1 105, with a SV40 to adenoviral vector ratio of about 1000:1. Helper adenoviruses can be effectively heat-inactivated with no effect on the infectivity of SV40 vectors. q 1997 Academic Press
SV40 virus is one of the most thoroughly studied mammalian viruses. Since the 1980s it has been a popular viral vector for expression of foreign genes in mammalian cells (reviewed in 1, 2). More recently, the SV40 vector has been reevaluated for its use in in vivo gene transfer and been found able to mediate persistent transgene expression in mice (3, 4). SV40 vectors were conventionally generated by replacing either later or early regions with a foreign gene, after which recombinant viral vectors were propagated with a wild-type SV40 virus or a temperature mutant as a helper. The resulting viral preparation contained a mixture of recombinant and helper at a ratio of about 3:7 (2). With the advent of COS-7, a cell line transformed with an origin-defective mutant of SV40 and capable of supporting the lytic cycle of SV40 viruses with deletions in their early regions (5), helper-virus-free generation of recombinant vectors whose early regions are re1 To whom correspondence should be addressed at Department of Thoracic and Cardiovascular Surgery, University of Texas M. D. Anderson Cancer Center, Box 109, 1515 Holcombe Blvd., Houston, TX 77030. Fax: 713/794-4901.
placed became possible. However, this system was limited by the size of transgenes that could be accommodated (õ2.5 kb) and the possible expression of the retained late region, which might result in an immune response to transduced cells expressing viral genes. Replication-defective adenoviral vectors as a means of gene delivery have lately been rigorously studied in vitro and in vivo because of their high transduction efficiency in a variety of tissues and cell types from various species. Moreover, since deletion of the E1 region renders adenoviruses defective in the lytic cycle, we have tested the hypothesis that SV40 vectors containing only the replication origin from the viral genome can be generated in COS-7 cells when supplemented with capsid proteins by adenoviral vectors expressing SV40 late genes. Since the adenoviral vector itself is believed to be replication defective, only the recombinant SV40 vector would be produced and packaged. Removal of all SV40 coding sequences would also theoretically increase the packaging capacity up to 5 kb, comparable to the capacity of E1-adenoviral vectors. Furthermore, host cellular immune responses to the viral antigens in the transduced cells would be effectively eliminated because no viral gene would exist. As a first step to proving our hypothesis, we therefore asked whether an adenoviral vector containing an SV40 late-gene-expressing cassette is capable of packaging a plasmid that contains the SV40 replication origin. MATERIALS AND METHODS
Cell culture. 293 and COS-7 cells were obtained from American Type Culture Collection (Rockville, MD) and maintained in Dulbecco’s modified Eagle’s medium (DMEM)2 containing 4.5 g/l glucose, 10% FBS, 100 U/ml penicillin, and 100 mg/ml streptomycin. 2
Abbreviations used: DMEM, Dulbecco’s modified Eagle’s medium; GFP, green fluorescent protein; CMV, cytomegalovirus; AAV, adenoassociated viral vector. 139
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Construction of recombinant adenovirus. SV40 DNA was purchased from Sigma (St. Louis, MO). pAD1/CMV was constructed by replacing the RSV-LTR in pADL.1/RSV (6) with the CMV early promoter. pAD1/CMV-CAP, which contains SV40 late genes driven by the CMV promoter, was constructed by inserting a 2.5-kb fragment from the SV40 viral genome (bp 273–2771) at the EcoRV site of pAD1/CMV. pAD1/ SV-CAP was constructed by inserting a 3.27-kb TaqIand BclI-digested fragment from SV40 at the ClaI/ BamHI site of pXCJL.1 (gift of Dr. F. Graham, McMaster University, Canada). Recombinant adenoviruses Ad/CMV-CAP and Ad/SV-CAP were constructed by cotransfecting 293 cells with a 35-kb ClaI fragment from dl324 and pAD1/CMV-CAP (for Ad/CMV-CAP) or pAD1/SV-CAP (for Ad/SV-CAP). The recombinants were identified by restriction digestion of viral genomes with BamHI. Packaging of recombinant SV40 vector. For use in preparing recombinant SV40 vectors, plasmid pEGFPN1, which contains the SV40 replication origin and a green fluorescent protein (GFP)-expressing cassette driven by CMV, was obtained from Clontech (Palo Alto, CA). In brief, COS-7 cells were seeded at 1 1 106/ 10-cm dish and then infected with adenoviral vector at an m.o.i. of 500 1 h prior to transfection. Plasmid DNA was transfected into cultured cells by calcium phosphate methods (7). Cells were then trypsinized and suspended in 1 ml medium. Cell suspensions were frozen and thawed three times to release the virus. Then, cell debris was removed after centrifugation at 13,000 rpm for 5 min. Titration of recombinant SV40 virus. Titers of infectious particles were determined by an end-point titer assay [median tissue culture-infective dose (TCID50)]. In brief, COS-7 cells were plated onto a 96-well microtiter plate at 104 cells/100 ml/well. Viral stocks were serially diluted with DMEM containing 10% bovine calf serum and then transferred in quadruplicate to COS7-seeded plates at 100 ml/well. After culture for 2 days, the plates were examined under a fluorescent microscope and scored for the presence of GFP. Titers were determined using the Titerprint Analysis program (8). DNA assay. Aliquots of viral stock (0.37 ml each) were digested with 100 mg DNase I for 20 min to remove cellular DNA. Each mixture was then digested with 100 mg proteinase K in 1% SDS, 20 mM EDTA at 557C for 2 h. After extraction with phenol, viral DNA was precipitated by ethanol. The final product was dissolved in 30 ml H2O. The DNA was then subjected to polymerase chain reaction (PCR) as described previously (6) using the following PCR primers: (i) 5*-acgcaaatgggcggtag-3* and 5*-cgctgaacttgtggccg-3* for CMV and GFP; (ii) 5*-gacactctatgcctgtg-3* and 5*-gagcagtggtggaatgc-3* for SV40 large T antigen gene; (iii)
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5*-ccttgtaccggaggtgatc-3* and 5*-cacactctatcacccactg-3* for the adenovirus E1A region; and (iv) 5*-ggaaatatgactacgtc-3* and 5*-aagtccacgcctacatg-3* for the adenovirus E4 region. RESULTS
Recombinant adenovirus-expressing SV40 capsid. Two recombinant adenoviruses, Ad/CMV-CAP and Ad/ SV-CAP, each containing an SV40-capsid-expressing cassette, were constructed (Fig. 1). Ad/CMV-CAP contains SV40 late genes driven by the human CMV immediate-early (CMV-IE) gene promoter, one of the strongest promoters in a variety of cells (9). The SV40 late genes in Ad/SV-CAP are driven by a SV40 late promoter, the same as in the SV40 virus. Thus, the splicing of the late mRNAs and the ratio of the late proteins in Ad/SV-CAP-infected cells remained the same as in SV40-infected cells. The recombinants were identified by DNA assays. A single plaque from each construct was expanded and titrated on 293 cells. Packaging of SV40 vector. Plasmid pEGFP-N1 was used to test whether the recombinant adenoviruses containing an SV40 late-gene-expressing cassette were able to package plasmids having an SV40 replication origin. PEGFP-N1 is about nine-tenths the size of the SV40 genome and contains a GFP-expressing cassette driven by the CMV promoter. It also contains a 356-bp DNA fragment from the SV40 genome (SV40 nucleotides 5177 to 290) consisting of an SV40 replication origin and a cis-acting DNA signal for encapsidation (10). There are no SV40 coding sequences in the plasmid. To package pEGFP-N1 in SV40 capsids, COS-7 cells were infected with recombinant adenovirus at an m.o.i. of 500 1 h prior to transfection. Preliminary experiments showed that over 70% of COS-7 cells were transduced by adenovirus at an m.o.i. of 500 (data not shown). The cells were then transfected with pEGFPN1, and the medium was changed 5 h after transfection. Four days after transfection, the cells were harvested and cell lysates were titrated on COS-7 cells for the presence of GFP-expressing vector (Table 1). No detectable GFP-expressing vector was found when Ad/ SV-CAP or control virus was used to package pEGFPN1. In contrast, GFP-expressing vectors were typically 105/106 cells when packaged with Ad/CMV-CAP. To verify that transduction of GFP is SV40-mediated, 2 ml of rabbit anti-SV40 polyclonal antibody (a gift of Dr. J. S. Butel, Baylor College of Medicine, Houston, TX) was added to 100 ml of vector aliquot, and the mixture was incubated at 377C for 1 h. A rabbit anti-human adenovirus antiserum was used as a control. The antiserum–vector mixtures were then titrated on COS-7 cells. While expression of GFP was not changed after incubation with anti-human adenovirus antiserum, it was completely abrogated by adding anti-SV40 antise-
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FIG. 1. Schematics of two recombinant adenoviruses, each of which contains an SV40 capsid expression cassette. The expression cassette is inserted in the adenoviral E1 region, from left to right in Ad/CMV-CAP and from right to left in Ad/SV-CAP. The nucleotide numbers above each construct correspond to the SV40 genome. The numbers below each construct indicate the positions of the expression cassette relative to adenoviral genome map units.
rum to the vector preparation. Thus, the transduction of GFP is SV40 mediated. To determine the time course of the packaging, COS7 cells were harvested over time after infection with Ad/CMV-CAP and transfection with pEGFP-N1. Cell lysates were then titrated using TCID50 as the end point. GFP-expressing vector was detected 1 day after transfection, and its level peaked at day 4 (Fig. 2). In subsequent experiments, the cells were harvested at 4 days after infection and transfection. Detection of helper virus in vector preparations by PCR and plaque assay. Since the SV40 vector described here contains only the SV40 replication origin, the chances of generating wild-type SV40 virus by recombination should be very low. Furthermore, E1-deleted recombinant adenovirus is believed to be replication defective and so should not be packaged. To test for the presence of SV40-GFP vector, wild-type SV40, and recombinant adenovirus in vector preparations, viral DNA was isolated after digestion of cell lysates with DNase I. Viral DNA was then subjected to 30 cycles of PCR with primers specific for CMV-GFP, adenoviral E1, adenoviral E4, and SV40 large T genes. The presence of CMV-GFP and adenoviral E4 was readily detected by PCR; however, adenoviral E1 and SV40 large T were not detected at all (Figs. 3A and 3B). To determine the amount of recombinant adenovirus in the vec-
tor preparations, cell lysates were titrated by plaque assay on 293 and COS-7 cells. While no plaques were titrated on COS-7 cells, about 1.9 1 102 plaque-forming units was titrated on 293 cells. Thus, no contamination by wild-type SV40 was detected by either PCR or plaque assay; however, the helper recombinant adenovirus was present at low titers in the vector preparations. This result is consistent with our previous observation that the E1-deleted recombinant adenovirus still replicates at low levels when cells were infected at a high m.o.i. (11). Heat inactivation of helper virus. SV40 virus is known to be relatively resistant to heat inactivation, while adenovirus is known to be heat labile. To test whether the helper recombinant adenovirus can be heat inactivated without affecting the titer of the SV40 vector, vector preparations were incubated at 567C for
TABLE 1
Titration of SV40 Vectors Packaged with Recombinant Adenoviruses Adenovirus
Titer of SV40 vectors
Ad/CMV-CAP Ad/SV-CAP Ad/CMV-LacZ Ad/RSV-Luc
3.1 1 105 ND ND ND
Note. ND, not detectable.
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FIG. 2. Time course of production the SV40-GFP vector. Titers were determined by TCID50 assay in COS-7 cells. Values represent means ({standard error) of two duplicated experiments.
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FIG. 3. DNA analysis of vector preparations. (A) PCR analysis of CMV-GFP expression in COS-7 cells. Cells were transfected with pEGFPN1 and followed by mock infection (lane 2) or infection with Ad/CMV-CAP (lane 3), Ad/SV-CAP (lane 4), and Ad/CMV-LacZ (lane 5). Cell lysates were treated with DNase I, and the DNAs isolated were subsequently subjected to PCR to detect the presence of CMV-GFP. pEGFPNl was used as positive control (lane 1). Lane M, 100-bp ladder. (B) PCR analysis of DNAs isolated from COS-7 cells transfected with pEGFP-N1 and infected with Ad/CMV-CAP. PCR analyses were performed to detect (1) CMV-GFP, (2) SV40 large T, (3) adenoviral E4, and (4) adenoviral E1. Lane M, 100-bp ladder; /, positive control; v, testing DNA.
30 min and subsequently titrated on 293 cells by plaque assay and on COS-7 cells by TCID50 assay. A non-heatinactivated vector preparation was used as a positive control and a mock-infected preparation as a negative control. While the titer for GFP-expressing vector remained unchanged after heat inactivation, the level of plaque-forming units for adenovirus dropped from 2 1 102 to undetectable (data not shown). DISCUSSION
Here we have demonstrated that a plasmid containing the SV40 replication origin can be packaged into SV40 vector. Moreover, several pieces of our data strongly imply that transduction of pEGFP-N1 after packaging with Ad/CMV-CAP is SV40 mediated. First, packaging with control virus did not produce vectors that could transduce pEGFP-N1. Second, PCR assays showed no pEGFP-N1 DNA in cell lysates containing plasmid packaged with control virus, whereas pEGFPN1 DNA was readily detected in cell lysates containing plasmid packaged with Ad/CMV-CAP. Third, heat inactivation diminished the titer of infectious adenovirus to undetectable levels but had no effect on the titer of GFP-expressing vector. Finally, incubation with antiSV40 antiserum completely eliminated GFP transduction. Together, these results ruled out protein- or adenovirus-mediated transduction of GFP and pointed to Ad/CMV-CAP as the only one of the two adenoviruses containing an SV40 late-protein-expressing cassette that could possibly package pEGFP-N1. SV40 virus has long been considered nonpathogenic in humans. Indeed, the contamination of early preparations of polio vaccine by wild-type SV40 virus produced
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no significant side effects (12, 13). However, the recent finding of the SV40 genome in some tumor samples has raised the alarm of possible SV40 pathogenesis in humans (14–16). Nevertheless, the complete removal of viral coding sequences from SV40 will generate a vector similar to and presumably just as safe as retroviral or adeno-associated viral vectors (AAV). Moreover, unlike retroviral vector, SV40 vector can easily be concentrated to high titer, and unlike AAV, SV40 is double stranded and will not require helpers for transgene expression. More recently, it was reported that SV40 capsid proteins synthesized in insect cells are capable of packaging plasmid into SV40 pseudovirions in vitro. Thus, replication-competent virus can be effectively eliminated (17). Yet, despite being one of the first vectors used for gene expression in mammalian cells, SV40 is not a common vector in the field of gene therapy. Therefore, further research must be done before it can be generally used in human gene therapy. Its current limitations include low titer and low transduction efficiencies in human cells. The problem of low titer may not be difficult to solve. Theoretically, SV40 vector can be purified to titers as high as those for adenoviral vectors. Moreover, though the procedure described here results in a vector lacking viral coding sequences, it also produces a titer of about 105 before concentration and purification, a level comparable to that for retrovirus vectors. The vector titer reported here is higher than that obtained by in vitro packaging but lower than that produced with a helper SV40 virus (17,18). It may therefore reflect different ways of vector preparation and titration. Transduction efficiency may be a more diffi-
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cult problem. Though SV40 can reportedly transduce human hemopoietic and peripheral blood cells (18, 19), we have found that the transduction efficiency of SV40 vector in human cells is low and that no detectable gene transduction occurs in human lung carcinoma cell lines H1299 and A549 after infection of both lines with the SV40-GFP vector at m.o.i. of 1 and 5. We have seen, however, few GFP-transduced cells among K562 cells infected with the SV40-GFP vector at an m.o.i. of 1 (data not shown). Thus, changes in the SV40 capsid may have to be made before SV40 vectors can be used efficiently for human gene therapy. ACKNOWLEDGMENTS We thank Dr. J. S. Butel for providing us the anti-SV40 antiserum used in this study, Jude Richard for editorial review, and Monica Contreras for assistance in preparing the manuscript. This study was funded by a grant from the University of Texas Physicians Referral Service, a Specialized Program of Research Excellence (SPORE) in Lung Cancer grant (P50-CA70907), and a sponsored research agreement with Introgen Therapeutics, Inc. (Austin, TX).
REFERENCES 1. Elder, J. T., Spritz, R. A., and Weissman, S. M. (1981) Annu. Rev. Genet. 15, 295–340. 2. Hamer, D. H.(1980) in Genetic Engineering (Setlow, J. K., and Holaender, A., Eds.), pp. 83–101, Plenum Press, New York. 3. Strayer, D. S., and Milano, J. (1996) Gene Ther. 3, 581–587.
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4. Strayer, D. S. (1996) J. Biol. Chem. 271, 24741–24746. 5. Gluzman, Y. (1981) Cell 23, 175–182. 6. Fang, B., Eisensmith, R. C., Li, X. H., Finegold, M. J., Shedlovsky, A., Dove, W., and Woo, S. L. (1994) Gene Ther. 1, 247–254. 7. Graham, F. L., and Van Der Eb, A. J. (1973) Virology 52, 456– 467. 8. Lynn, D. (1992) Biotechniques 12, 880–881. 9. Guo, Z. S., Wang, L. H., Eisensmith, R. C., and Woo, S. L. C. (1996) Gene Ther. 3, 802–810. 10. Oppenheim, A., Sandalon, Z., Peleg, A., Shaul, O., Nicolis, S., and Ottolenghi, S. (1992) J. Virol. 66, 5320–5328. 11. Fang, B., Koch, P., and Roth, J. A. (1997) J Virol. 71, 4798– 4803. 12. Fraumeni, J. F., Jr. (1963) J. Am. Med. Assoc. 185, 713–718. 13. Lewis, A. M., Jr. (1973) in Biohazards in Biological Research (Hellman, A., Oxman, M. N., and Pollack, R., Eds.), pp. 96–113, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 14. Lednicky, J. A., Garcea, R. L., Bergsagel, D. J., and Butel, J. S. (1995) Virology 212, 710–717. 15. Strickler, H. D., Goedert, J. J., Fleming, M., Travis, W. D., Williams, A. E., Rabkin, C. S., Daniel, R. W., and Shah, K. V. (1996) Cancer Epidemiol. Biomarkers Prev. 5, 473–475. 16. Carbone, M., Rizzo, P., Procopio, A., Giuliano, M., Pass, H. I., Gebhardt, M. C., Mangham, C., Hansen, M., Malkin, D. F., Bushart, G., Pompetti, F., Picci, P., Levine, A. S., Bergsagel, J. D., and Garcea, R. L. (1996) Oncogene 13, 527–535. 17. Sandalon, Z., Dalyot-Herman, N., Oppenheim, A. B., and Oppenheim, A. (1997) Human Gene Ther. 8, 843–849. 18. Oppenheim, A., Peleg, A., Fibach, E., and Rachmilewits, E. A. (1986) Proc. Natl. Acad. Sci. USA 83, 6925–6929. 19. Strayer, D. S., Kondo, R., Milano, J., and Duan, L. X. (1997) Gene Ther. 4, 219–225.
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AB972417
A Packaging System for SV40 Vectors without Viral Coding Sequences Bingliang Fang,*,1 Patricia Koch,* Michael Bouvet,† Lin Ji,* and Jack A. Roth* *Section of Thoracic Molecular Oncology, Department of Thoracic and Cardiovascular Surgery, and †Department of Surgical Oncology, University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030
Received May 16, 1997
SV40 vectors have been used as expression vectors for mammalian cells since the early 1980s. More recently, they have been used as gene transfer vectors in mice and in human peripheral blood cells. Here we described a system for packaging SV40 vectors without viral coding sequences. Recombinant adenovirusexpressing SV40 capsids can effectively package plasmids that contain the SV40 replication origin. The final yield of infectious SV40 vector is about 3 1 105, with a SV40 to adenoviral vector ratio of about 1000:1. Helper adenoviruses can be effectively heat-inactivated with no effect on the infectivity of SV40 vectors. q 1997 Academic Press
SV40 virus is one of the most thoroughly studied mammalian viruses. Since the 1980s it has been a popular viral vector for expression of foreign genes in mammalian cells (reviewed in 1, 2). More recently, the SV40 vector has been reevaluated for its use in in vivo gene transfer and been found able to mediate persistent transgene expression in mice (3, 4). SV40 vectors were conventionally generated by replacing either later or early regions with a foreign gene, after which recombinant viral vectors were propagated with a wild-type SV40 virus or a temperature mutant as a helper. The resulting viral preparation contained a mixture of recombinant and helper at a ratio of about 3:7 (2). With the advent of COS-7, a cell line transformed with an origin-defective mutant of SV40 and capable of supporting the lytic cycle of SV40 viruses with deletions in their early regions (5), helper-virus-free generation of recombinant vectors whose early regions are re1 To whom correspondence should be addressed at Department of Thoracic and Cardiovascular Surgery, University of Texas M. D. Anderson Cancer Center, Box 109, 1515 Holcombe Blvd., Houston, TX 77030. Fax: 713/794-4901.
placed became possible. However, this system was limited by the size of transgenes that could be accommodated (õ2.5 kb) and the possible expression of the retained late region, which might result in an immune response to transduced cells expressing viral genes. Replication-defective adenoviral vectors as a means of gene delivery have lately been rigorously studied in vitro and in vivo because of their high transduction efficiency in a variety of tissues and cell types from various species. Moreover, since deletion of the E1 region renders adenoviruses defective in the lytic cycle, we have tested the hypothesis that SV40 vectors containing only the replication origin from the viral genome can be generated in COS-7 cells when supplemented with capsid proteins by adenoviral vectors expressing SV40 late genes. Since the adenoviral vector itself is believed to be replication defective, only the recombinant SV40 vector would be produced and packaged. Removal of all SV40 coding sequences would also theoretically increase the packaging capacity up to 5 kb, comparable to the capacity of E1-adenoviral vectors. Furthermore, host cellular immune responses to the viral antigens in the transduced cells would be effectively eliminated because no viral gene would exist. As a first step to proving our hypothesis, we therefore asked whether an adenoviral vector containing an SV40 late-gene-expressing cassette is capable of packaging a plasmid that contains the SV40 replication origin. MATERIALS AND METHODS
Cell culture. 293 and COS-7 cells were obtained from American Type Culture Collection (Rockville, MD) and maintained in Dulbecco’s modified Eagle’s medium (DMEM)2 containing 4.5 g/l glucose, 10% FBS, 100 U/ml penicillin, and 100 mg/ml streptomycin. 2
Abbreviations used: DMEM, Dulbecco’s modified Eagle’s medium; GFP, green fluorescent protein; CMV, cytomegalovirus; AAV, adenoassociated viral vector. 139
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Construction of recombinant adenovirus. SV40 DNA was purchased from Sigma (St. Louis, MO). pAD1/CMV was constructed by replacing the RSV-LTR in pADL.1/RSV (6) with the CMV early promoter. pAD1/CMV-CAP, which contains SV40 late genes driven by the CMV promoter, was constructed by inserting a 2.5-kb fragment from the SV40 viral genome (bp 273–2771) at the EcoRV site of pAD1/CMV. pAD1/ SV-CAP was constructed by inserting a 3.27-kb TaqIand BclI-digested fragment from SV40 at the ClaI/ BamHI site of pXCJL.1 (gift of Dr. F. Graham, McMaster University, Canada). Recombinant adenoviruses Ad/CMV-CAP and Ad/SV-CAP were constructed by cotransfecting 293 cells with a 35-kb ClaI fragment from dl324 and pAD1/CMV-CAP (for Ad/CMV-CAP) or pAD1/SV-CAP (for Ad/SV-CAP). The recombinants were identified by restriction digestion of viral genomes with BamHI. Packaging of recombinant SV40 vector. For use in preparing recombinant SV40 vectors, plasmid pEGFPN1, which contains the SV40 replication origin and a green fluorescent protein (GFP)-expressing cassette driven by CMV, was obtained from Clontech (Palo Alto, CA). In brief, COS-7 cells were seeded at 1 1 106/ 10-cm dish and then infected with adenoviral vector at an m.o.i. of 500 1 h prior to transfection. Plasmid DNA was transfected into cultured cells by calcium phosphate methods (7). Cells were then trypsinized and suspended in 1 ml medium. Cell suspensions were frozen and thawed three times to release the virus. Then, cell debris was removed after centrifugation at 13,000 rpm for 5 min. Titration of recombinant SV40 virus. Titers of infectious particles were determined by an end-point titer assay [median tissue culture-infective dose (TCID50)]. In brief, COS-7 cells were plated onto a 96-well microtiter plate at 104 cells/100 ml/well. Viral stocks were serially diluted with DMEM containing 10% bovine calf serum and then transferred in quadruplicate to COS7-seeded plates at 100 ml/well. After culture for 2 days, the plates were examined under a fluorescent microscope and scored for the presence of GFP. Titers were determined using the Titerprint Analysis program (8). DNA assay. Aliquots of viral stock (0.37 ml each) were digested with 100 mg DNase I for 20 min to remove cellular DNA. Each mixture was then digested with 100 mg proteinase K in 1% SDS, 20 mM EDTA at 557C for 2 h. After extraction with phenol, viral DNA was precipitated by ethanol. The final product was dissolved in 30 ml H2O. The DNA was then subjected to polymerase chain reaction (PCR) as described previously (6) using the following PCR primers: (i) 5*-acgcaaatgggcggtag-3* and 5*-cgctgaacttgtggccg-3* for CMV and GFP; (ii) 5*-gacactctatgcctgtg-3* and 5*-gagcagtggtggaatgc-3* for SV40 large T antigen gene; (iii)
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5*-ccttgtaccggaggtgatc-3* and 5*-cacactctatcacccactg-3* for the adenovirus E1A region; and (iv) 5*-ggaaatatgactacgtc-3* and 5*-aagtccacgcctacatg-3* for the adenovirus E4 region. RESULTS
Recombinant adenovirus-expressing SV40 capsid. Two recombinant adenoviruses, Ad/CMV-CAP and Ad/ SV-CAP, each containing an SV40-capsid-expressing cassette, were constructed (Fig. 1). Ad/CMV-CAP contains SV40 late genes driven by the human CMV immediate-early (CMV-IE) gene promoter, one of the strongest promoters in a variety of cells (9). The SV40 late genes in Ad/SV-CAP are driven by a SV40 late promoter, the same as in the SV40 virus. Thus, the splicing of the late mRNAs and the ratio of the late proteins in Ad/SV-CAP-infected cells remained the same as in SV40-infected cells. The recombinants were identified by DNA assays. A single plaque from each construct was expanded and titrated on 293 cells. Packaging of SV40 vector. Plasmid pEGFP-N1 was used to test whether the recombinant adenoviruses containing an SV40 late-gene-expressing cassette were able to package plasmids having an SV40 replication origin. PEGFP-N1 is about nine-tenths the size of the SV40 genome and contains a GFP-expressing cassette driven by the CMV promoter. It also contains a 356-bp DNA fragment from the SV40 genome (SV40 nucleotides 5177 to 290) consisting of an SV40 replication origin and a cis-acting DNA signal for encapsidation (10). There are no SV40 coding sequences in the plasmid. To package pEGFP-N1 in SV40 capsids, COS-7 cells were infected with recombinant adenovirus at an m.o.i. of 500 1 h prior to transfection. Preliminary experiments showed that over 70% of COS-7 cells were transduced by adenovirus at an m.o.i. of 500 (data not shown). The cells were then transfected with pEGFPN1, and the medium was changed 5 h after transfection. Four days after transfection, the cells were harvested and cell lysates were titrated on COS-7 cells for the presence of GFP-expressing vector (Table 1). No detectable GFP-expressing vector was found when Ad/ SV-CAP or control virus was used to package pEGFPN1. In contrast, GFP-expressing vectors were typically 105/106 cells when packaged with Ad/CMV-CAP. To verify that transduction of GFP is SV40-mediated, 2 ml of rabbit anti-SV40 polyclonal antibody (a gift of Dr. J. S. Butel, Baylor College of Medicine, Houston, TX) was added to 100 ml of vector aliquot, and the mixture was incubated at 377C for 1 h. A rabbit anti-human adenovirus antiserum was used as a control. The antiserum–vector mixtures were then titrated on COS-7 cells. While expression of GFP was not changed after incubation with anti-human adenovirus antiserum, it was completely abrogated by adding anti-SV40 antise-
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FIG. 1. Schematics of two recombinant adenoviruses, each of which contains an SV40 capsid expression cassette. The expression cassette is inserted in the adenoviral E1 region, from left to right in Ad/CMV-CAP and from right to left in Ad/SV-CAP. The nucleotide numbers above each construct correspond to the SV40 genome. The numbers below each construct indicate the positions of the expression cassette relative to adenoviral genome map units.
rum to the vector preparation. Thus, the transduction of GFP is SV40 mediated. To determine the time course of the packaging, COS7 cells were harvested over time after infection with Ad/CMV-CAP and transfection with pEGFP-N1. Cell lysates were then titrated using TCID50 as the end point. GFP-expressing vector was detected 1 day after transfection, and its level peaked at day 4 (Fig. 2). In subsequent experiments, the cells were harvested at 4 days after infection and transfection. Detection of helper virus in vector preparations by PCR and plaque assay. Since the SV40 vector described here contains only the SV40 replication origin, the chances of generating wild-type SV40 virus by recombination should be very low. Furthermore, E1-deleted recombinant adenovirus is believed to be replication defective and so should not be packaged. To test for the presence of SV40-GFP vector, wild-type SV40, and recombinant adenovirus in vector preparations, viral DNA was isolated after digestion of cell lysates with DNase I. Viral DNA was then subjected to 30 cycles of PCR with primers specific for CMV-GFP, adenoviral E1, adenoviral E4, and SV40 large T genes. The presence of CMV-GFP and adenoviral E4 was readily detected by PCR; however, adenoviral E1 and SV40 large T were not detected at all (Figs. 3A and 3B). To determine the amount of recombinant adenovirus in the vec-
tor preparations, cell lysates were titrated by plaque assay on 293 and COS-7 cells. While no plaques were titrated on COS-7 cells, about 1.9 1 102 plaque-forming units was titrated on 293 cells. Thus, no contamination by wild-type SV40 was detected by either PCR or plaque assay; however, the helper recombinant adenovirus was present at low titers in the vector preparations. This result is consistent with our previous observation that the E1-deleted recombinant adenovirus still replicates at low levels when cells were infected at a high m.o.i. (11). Heat inactivation of helper virus. SV40 virus is known to be relatively resistant to heat inactivation, while adenovirus is known to be heat labile. To test whether the helper recombinant adenovirus can be heat inactivated without affecting the titer of the SV40 vector, vector preparations were incubated at 567C for
TABLE 1
Titration of SV40 Vectors Packaged with Recombinant Adenoviruses Adenovirus
Titer of SV40 vectors
Ad/CMV-CAP Ad/SV-CAP Ad/CMV-LacZ Ad/RSV-Luc
3.1 1 105 ND ND ND
Note. ND, not detectable.
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FIG. 2. Time course of production the SV40-GFP vector. Titers were determined by TCID50 assay in COS-7 cells. Values represent means ({standard error) of two duplicated experiments.
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FIG. 3. DNA analysis of vector preparations. (A) PCR analysis of CMV-GFP expression in COS-7 cells. Cells were transfected with pEGFPN1 and followed by mock infection (lane 2) or infection with Ad/CMV-CAP (lane 3), Ad/SV-CAP (lane 4), and Ad/CMV-LacZ (lane 5). Cell lysates were treated with DNase I, and the DNAs isolated were subsequently subjected to PCR to detect the presence of CMV-GFP. pEGFPNl was used as positive control (lane 1). Lane M, 100-bp ladder. (B) PCR analysis of DNAs isolated from COS-7 cells transfected with pEGFP-N1 and infected with Ad/CMV-CAP. PCR analyses were performed to detect (1) CMV-GFP, (2) SV40 large T, (3) adenoviral E4, and (4) adenoviral E1. Lane M, 100-bp ladder; /, positive control; v, testing DNA.
30 min and subsequently titrated on 293 cells by plaque assay and on COS-7 cells by TCID50 assay. A non-heatinactivated vector preparation was used as a positive control and a mock-infected preparation as a negative control. While the titer for GFP-expressing vector remained unchanged after heat inactivation, the level of plaque-forming units for adenovirus dropped from 2 1 102 to undetectable (data not shown). DISCUSSION
Here we have demonstrated that a plasmid containing the SV40 replication origin can be packaged into SV40 vector. Moreover, several pieces of our data strongly imply that transduction of pEGFP-N1 after packaging with Ad/CMV-CAP is SV40 mediated. First, packaging with control virus did not produce vectors that could transduce pEGFP-N1. Second, PCR assays showed no pEGFP-N1 DNA in cell lysates containing plasmid packaged with control virus, whereas pEGFPN1 DNA was readily detected in cell lysates containing plasmid packaged with Ad/CMV-CAP. Third, heat inactivation diminished the titer of infectious adenovirus to undetectable levels but had no effect on the titer of GFP-expressing vector. Finally, incubation with antiSV40 antiserum completely eliminated GFP transduction. Together, these results ruled out protein- or adenovirus-mediated transduction of GFP and pointed to Ad/CMV-CAP as the only one of the two adenoviruses containing an SV40 late-protein-expressing cassette that could possibly package pEGFP-N1. SV40 virus has long been considered nonpathogenic in humans. Indeed, the contamination of early preparations of polio vaccine by wild-type SV40 virus produced
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no significant side effects (12, 13). However, the recent finding of the SV40 genome in some tumor samples has raised the alarm of possible SV40 pathogenesis in humans (14–16). Nevertheless, the complete removal of viral coding sequences from SV40 will generate a vector similar to and presumably just as safe as retroviral or adeno-associated viral vectors (AAV). Moreover, unlike retroviral vector, SV40 vector can easily be concentrated to high titer, and unlike AAV, SV40 is double stranded and will not require helpers for transgene expression. More recently, it was reported that SV40 capsid proteins synthesized in insect cells are capable of packaging plasmid into SV40 pseudovirions in vitro. Thus, replication-competent virus can be effectively eliminated (17). Yet, despite being one of the first vectors used for gene expression in mammalian cells, SV40 is not a common vector in the field of gene therapy. Therefore, further research must be done before it can be generally used in human gene therapy. Its current limitations include low titer and low transduction efficiencies in human cells. The problem of low titer may not be difficult to solve. Theoretically, SV40 vector can be purified to titers as high as those for adenoviral vectors. Moreover, though the procedure described here results in a vector lacking viral coding sequences, it also produces a titer of about 105 before concentration and purification, a level comparable to that for retrovirus vectors. The vector titer reported here is higher than that obtained by in vitro packaging but lower than that produced with a helper SV40 virus (17,18). It may therefore reflect different ways of vector preparation and titration. Transduction efficiency may be a more diffi-
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cult problem. Though SV40 can reportedly transduce human hemopoietic and peripheral blood cells (18, 19), we have found that the transduction efficiency of SV40 vector in human cells is low and that no detectable gene transduction occurs in human lung carcinoma cell lines H1299 and A549 after infection of both lines with the SV40-GFP vector at m.o.i. of 1 and 5. We have seen, however, few GFP-transduced cells among K562 cells infected with the SV40-GFP vector at an m.o.i. of 1 (data not shown). Thus, changes in the SV40 capsid may have to be made before SV40 vectors can be used efficiently for human gene therapy. ACKNOWLEDGMENTS We thank Dr. J. S. Butel for providing us the anti-SV40 antiserum used in this study, Jude Richard for editorial review, and Monica Contreras for assistance in preparing the manuscript. This study was funded by a grant from the University of Texas Physicians Referral Service, a Specialized Program of Research Excellence (SPORE) in Lung Cancer grant (P50-CA70907), and a sponsored research agreement with Introgen Therapeutics, Inc. (Austin, TX).
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