title: Safety-modified episomal vectors for human gene therapy.
abstract: 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.
authors.affiliation: Department of Medicine, Case Western Reserve University School of Medicine, 10900 Euclid Avenue, Cleveland, OH 44106, USA.
authors.forename: M J
authors.initials: MJ
authors.lastname: Cooper
authors.forename: M
authors.initials: M
authors.lastname: Lippa
authors.forename: J M
authors.initials: JM
authors.lastname: Payne
authors.forename: G
authors.initials: G
authors.lastname: Hatzivassiliou
authors.forename: E
authors.initials: E
authors.lastname: Reifenberg
authors.forename: B
authors.initials: B
authors.lastname: Fayazi
authors.forename: J C
authors.initials: JC
authors.lastname: Perales
authors.forename: L J
authors.initials: LJ
authors.lastname: Morrison
authors.forename: D
authors.initials: D
authors.lastname: Templeton
authors.forename: R L
authors.initials: RL
authors.lastname: Piekarz
authors.forename: J
authors.initials: J
authors.lastname: Tan
mesh_terms: D000818:Animals; D000952:Antigens, Polyomavirus Transforming / Q000096:biosynthesis / Q000235:genetics; D002460:Cell Line; D006224:Cricetinae; D017931:DNA Primers; D004261:DNA Replication; D004285:Dogs; D016147:Genes, Tumor Suppressor; D015316:Genetic Therapy / Q000379:methods* / Q000592:standards; D005822:Genetic Vectors; D006801:Humans; D007700:Kinetics; D008156:Luciferases / Q000096:biosynthesis; D051379:Mice; D016133:Polymerase Chain Reaction; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis; D016160:Retinoblastoma Protein / Q000378:metabolism; D013539:Simian virus 40 / Q000235:genetics; D013997:Time Factors; D014162:Transfection; D014407:Tumor Cells, Cultured; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism; D001616:beta-Galactosidase / Q000096:biosynthesis
chemical_list: D000952:Antigens, Polyomavirus Transforming; D017931:DNA Primers; D011994:Recombinant Proteins; D016160:Retinoblastoma Protein; D016159:Tumor Suppressor Protein p53; D008156:Luciferases; D001616:beta-Galactosidase