Safety-modified episomal vectors for human gene therapy

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