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949. Recombinant Simian Virus 40 Requires Lipid Rafts, but Not Caveolae, to Enter Cells
OTHER DNA VIRUSES 948. Use of Sv40 Recombinant Viruses as Vectors for Gene Therapy Maria Vera,1 Gloria G. Aseguinolaza,1 Carlos M. Rodriguez,1 Laura Martinez,1 Pedro Berraondo,1 Nerea Razquin,1 David S. Strayer,2 Jesus Prieto,1 Puri Fortes.1 1 Hepatology and Gene Therapy, University of Navarra, Pamplona, Navarra, Spain; 2Pathology Anatomy and Cell Biology, Thomas Jefferson University, Philadelphia, PA. Diabetes, hypoparathyroidism, cirrhosis and others are diseases caused by a defect in the expression of secreted proteins. Also, other secreted proteins such as cytokines can play an important role in the treatment of acquired diseases such as cancer and viral infections. Recombinant viruses expressing those proteins can be used as an alternative for the treatment of these diseases. SV40 (Simian Virus 40) viruses have several advantages for this purpose. They infect resting as well as cycling cells, they have a long term expression and they are not immunogenic. We have developed a new protocol that yields high titers of T-antigen deleted non-replicative recombinant SV40 viruses (rSV40). Using this technique, we have produced recombinant viruses expressing: rat insulin-like growth factor I (SVIGFI), murine interleukin 12 (SVmIL12), murine interleukin 15 (SVmIL15), woodchuck interleukin 12 (SVw12) and luciferase (rSVLUC) as a reporter gene. After high efficiency production, these viruses have been titered by in situ and quantitative PCR and assayed for recombinant gene expression. Different cell lines infected with the recombinant viruses efficiently expressed the exogenous proteins as detected by immunofluorescence. IGF-I, IL12 and IL15 were also detected in the supernatant of infected cultures by RIA and ELISA, indicating that SV40 infected cells maintain a regular secretory pathway. SV40-produced IL12 and IL15 were shown to be active by its ability to induce IFNg production in mouse splenocytes. All these results show that recombinant SV40 vectors are able to drive the expression of secreted functional proteins, so we decided to study the efficiency of rSV40 vectors “in vivo”. The effect of rSVIFG-I infection has been studied in the treatment of liver cirrhosis in rats. The immune-response against HIV gp120 protein has been assayed in mice after injection of dendritic cells infected with rSVmIL12 and rSVmIL15. Subcutaneous tumours in mice have been treated with rSVmIL15 and rSVmIL12 or dendritic cells transformed with these viruses. The results of these in vivo studies will be presented. In general, even if further studies still have to be done, we believe that rSV40 vectors will be an efficient system to treat diseases that need a long term expression of therapeutic proteins.
949. Recombinant Simian Virus 40 Requires Lipid Rafts, but Not Caveolae, to Enter Cells Jayanta Roy-Chowdhury,1 Siddhartha S. Ghosh,1 Alex W. Cohen,2 Huifang Huang,3 Namita Roy-Chowdhury,1 Chandan Guha,3 Michael Lisanti,2 David S. Strayer.4 1 Medicine and Molecular Genetics, Albert Einstein College of Medicine, New York, NY; 2Molecular Pharmacology, Albert Einstein College of Medicine, New York, NY, United States; 3 Radiation Oncology, Albert Einstein College of Medicine, New York, NY; 4Pathology, Thomas Jefferson University, Philadelphia, PA. Recombinant simian virus 40 (rSV40) vectors can be administered repeatedly without evoking host immune response. The immune transparency of rSV40 is thought to be related to the peculiarities of its intracellular trafficking. It has been reported that, in contrast to many other viruses, the wildtype SV40 (wtSV40), is internalized via cholesterol-rich lipid rafts from which it is sequentially translocated to caveolae, caveosomes, the endoplasmic reticulum and, finally, the nucleus. Since wtSV40 bypasses the lysosomes, Molecular Therapy Vol. 7, No. 5, May 2003, Part 2 of 2 Parts Copyright ® The American Society of Gene Therapy
capsid proteins of the input virus are not processed and presented to the cell surface. Immune response occurs only after de novo viral protein synthesis following T antigen (Tag) expression. As the Tag coding sequence is deleted in rSV40, viral proteins are not synthesized, which may explain its non-immunogenicity. In this study, to determine whether rSV40 follows the same route as the wildtype SV40, we generated an rSV40 that is deleted of all viral coding sequences and expresses E. coli β-galactosidase (β-gal), driven by an internal CMV promoter (rSV-LacZ). To determine the involvement of lipid rafts, we infected primary mouse skin fibroblasts with rSV-LacZ in the presence or absence of the sterol-binding agents, nystatin (5 mg/l, 6 hr) or filipin (1 μg/ml, 1hr). β-Gal staining after 48 hr of culture showed that both nystatin and filipin had inhibited transduction of the cells by 60-80%. To determine if rSV40 requires caveolae for internalization, we cultured skin fibroblasts from the caveolin-1 gene-disrupted mice (which do not form caveolae) and their congeneic wildtype counterparts. The cells were infected with 0 to 20 MOI of SV-LacZ for 16 hrs. β-gal staining after culturing the cells for 48 hr showed no significant difference in the percentage of transgene expressing cells between fibroblasts from the wildtype and caveolin 1-gene disrupted strains. CONCLUSION: rSV40 internalization involves lipid rafts, but caveolae are not required. The findings explain the transduction of a wide range of cells by rSV40, including those that do not contain caveolae.
950. Production of Replication-Defective HSV-1 Viral Vectors and Development of Precise Assay Systems To Analyze the Quality of Clinical-Grade HSV Vectors Ali Ozuer,1 James B. Wechuck,1 Steve K. Wendell,1 Darren P. Wolfe,1 William F. Goins,1 David M. Krisky,3 Mohammad M. Ataai,2 Joseph C. Glorioso.1 1 Molecular Genectics and Biochemistry, University of Pittsburgh, Pittsburgh, PA, United States; 2Chemical Engineering, University of Pittsburgh, Pittsburgh, PA, United States; 3Pathology, University of Pittsburgh, Pittsburgh, PA, United States. As the number of clinical and research applications employing herpes simplex virus (HSV) vectors increase, it becomes critical to develop methods for the large-scale manufacture of high titer vector stocks. Traditional methods to evaluate vector purity include a comparison of (i) viral titer assayed on the complementing cell line, (ii) the amount of protein in the preparation assayed by Bio-Rad total protein microassay, (iii) the number of RCV detected on noncomplementing cells and (iv) the integrity of the virion particle and the amount of cellular debris assessed by electron microscopy. Furthermore, development of an easy and inexpensive method to critically analyze vector production and quantify the production of infectious and defective particles will be required to meet FDA standards of vector lot release criteria. In order to maximize vector production, we have identified infection parameters that result in high-yield production of multiple immediate early (IE) gene deletion mutant HSV vectors in complementing cells. Virus yield was shown to correlate with various culture media conditions including: pH, glucose metabolism, lactate production, and serum level. We also found that complementing cell passage number, temperature and cell confluence affected the overall vector yield. We have also developed rapid, accurate and precise analysis systems to analyze the quality and the quantity of vector stocks. Our assay system contains two independent and complementary DNA quantification methods: a real-time quantitative PCR system using Taq polymerase and a micro-plate assay using PicoGreen dsDNA quantification reagent. This assay system has been optimized to quantify the amount of infectious versus defective HSV particles, host genomic DNA, and for the detection of replication competent virus (RCV). Our real-time quantitative PCR S365
949. Recombinant Simian Virus 40 Requires Lipid Rafts, but Not Caveolae, to Enter Cells Jayanta Roy-Chowdhury,1 Siddhartha S. Ghosh,1 Alex W. Cohen,2 Huifang Huang,3 Namita Roy-Chowdhury,1 Chandan Guha,3 Michael Lisanti,2 David S. Strayer.4 1 Medicine and Molecular Genetics, Albert Einstein College of Medicine, New York, NY; 2Molecular Pharmacology, Albert Einstein College of Medicine, New York, NY, United States; 3 Radiation Oncology, Albert Einstein College of Medicine, New York, NY; 4Pathology, Thomas Jefferson University, Philadelphia, PA. Recombinant simian virus 40 (rSV40) vectors can be administered repeatedly without evoking host immune response. The immune transparency of rSV40 is thought to be related to the peculiarities of its intracellular trafficking. It has been reported that, in contrast to many other viruses, the wildtype SV40 (wtSV40), is internalized via cholesterol-rich lipid rafts from which it is sequentially translocated to caveolae, caveosomes, the endoplasmic reticulum and, finally, the nucleus. Since wtSV40 bypasses the lysosomes, Molecular Therapy Vol. 7, No. 5, May 2003, Part 2 of 2 Parts Copyright ® The American Society of Gene Therapy
capsid proteins of the input virus are not processed and presented to the cell surface. Immune response occurs only after de novo viral protein synthesis following T antigen (Tag) expression. As the Tag coding sequence is deleted in rSV40, viral proteins are not synthesized, which may explain its non-immunogenicity. In this study, to determine whether rSV40 follows the same route as the wildtype SV40, we generated an rSV40 that is deleted of all viral coding sequences and expresses E. coli β-galactosidase (β-gal), driven by an internal CMV promoter (rSV-LacZ). To determine the involvement of lipid rafts, we infected primary mouse skin fibroblasts with rSV-LacZ in the presence or absence of the sterol-binding agents, nystatin (5 mg/l, 6 hr) or filipin (1 μg/ml, 1hr). β-Gal staining after 48 hr of culture showed that both nystatin and filipin had inhibited transduction of the cells by 60-80%. To determine if rSV40 requires caveolae for internalization, we cultured skin fibroblasts from the caveolin-1 gene-disrupted mice (which do not form caveolae) and their congeneic wildtype counterparts. The cells were infected with 0 to 20 MOI of SV-LacZ for 16 hrs. β-gal staining after culturing the cells for 48 hr showed no significant difference in the percentage of transgene expressing cells between fibroblasts from the wildtype and caveolin 1-gene disrupted strains. CONCLUSION: rSV40 internalization involves lipid rafts, but caveolae are not required. The findings explain the transduction of a wide range of cells by rSV40, including those that do not contain caveolae.
950. Production of Replication-Defective HSV-1 Viral Vectors and Development of Precise Assay Systems To Analyze the Quality of Clinical-Grade HSV Vectors Ali Ozuer,1 James B. Wechuck,1 Steve K. Wendell,1 Darren P. Wolfe,1 William F. Goins,1 David M. Krisky,3 Mohammad M. Ataai,2 Joseph C. Glorioso.1 1 Molecular Genectics and Biochemistry, University of Pittsburgh, Pittsburgh, PA, United States; 2Chemical Engineering, University of Pittsburgh, Pittsburgh, PA, United States; 3Pathology, University of Pittsburgh, Pittsburgh, PA, United States. As the number of clinical and research applications employing herpes simplex virus (HSV) vectors increase, it becomes critical to develop methods for the large-scale manufacture of high titer vector stocks. Traditional methods to evaluate vector purity include a comparison of (i) viral titer assayed on the complementing cell line, (ii) the amount of protein in the preparation assayed by Bio-Rad total protein microassay, (iii) the number of RCV detected on noncomplementing cells and (iv) the integrity of the virion particle and the amount of cellular debris assessed by electron microscopy. Furthermore, development of an easy and inexpensive method to critically analyze vector production and quantify the production of infectious and defective particles will be required to meet FDA standards of vector lot release criteria. In order to maximize vector production, we have identified infection parameters that result in high-yield production of multiple immediate early (IE) gene deletion mutant HSV vectors in complementing cells. Virus yield was shown to correlate with various culture media conditions including: pH, glucose metabolism, lactate production, and serum level. We also found that complementing cell passage number, temperature and cell confluence affected the overall vector yield. We have also developed rapid, accurate and precise analysis systems to analyze the quality and the quantity of vector stocks. Our assay system contains two independent and complementary DNA quantification methods: a real-time quantitative PCR system using Taq polymerase and a micro-plate assay using PicoGreen dsDNA quantification reagent. This assay system has been optimized to quantify the amount of infectious versus defective HSV particles, host genomic DNA, and for the detection of replication competent virus (RCV). Our real-time quantitative PCR S365