Safety-modified episomal vectors for human gene therapy

• DOI: 10.1073/pnas.94.12.6450
• Publication Date: 2002-07-26T14:43:20Z
• Authors: Mark J. Cooper; Mara Lippa; Jennifer M. Payne; Georgia Hatzivass...; Erica Reifenberg; Behnaz Fayazi; Jose C. Perales; Laura J. Morrison; Dennis Templeton; Richard L. Piekarz; June Tan
• Keywords: DNA Replication; 0303 health sciences; Time Factors; Antigens, Polyomavirus Transforming; Genetic Vectors; Genetic Therapy; Simian virus 40; Polymerase Chain Reaction; Retinoblastoma Protein; Recombinant Proteins; Cell Line; Rats; 3. Good health; Kinetics; Mice; 03 medical and health sciences; Dogs; Cricetinae; Animals; Humans; Genes, Tumor Suppressor; Luciferases; DNA Primers

The effectiveness of ongoing gene therapy trials may be limited by the expression characteristics viral and plasmid-based vectors. To enhance levels heterologous expression, we have developed a safety-modified episomal vector that replicates extrachromosomally in human cells. This system employs simian virus 40 (SV40) large T antigen mutant (107/402-T) is deficient binding to tumor suppressor products, including p53, retinoblastoma, p107, yet retains replication competence. These SV40-based episomes replicate thousands copies 2-4 days after transfer multiple types cell lines, with lower activity hamster cells, no detectable dog, rat, murine lines. Importantly, 107/402-T has enhanced compared wild-type antigen; this finding due, part, inability p53 retinoblastoma inactivate function. We demonstrate level duration regulates observed copy number per cell. Compared standard plasmid constructs, encoding yield approximately 10- 100-fold unselected populations transient transfectants. determine if 107/402-T-based vivo, explants nude mice were directly injected liposome/DNA complexes. Using PCR-based assay, cells direct vivo transfer. data suggest will effective for cancer because high therapeutic genes transfectants should elimination.