SAFETY-MODIFIED EPISOMAL VECTORS FOR HUMAN GENE-THERAPY

Citation
Mj. Cooper et al., SAFETY-MODIFIED EPISOMAL VECTORS FOR HUMAN GENE-THERAPY, Proceedings of the National Academy of Sciences of the United Statesof America, 94(12), 1997, pp. 6450-6455
Citations number
44
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Multidisciplinary Sciences
ISSN journal
0027-8424
Volume
94
Issue
12
Year of publication
1997
Pages
6450 - 6455
Database
ISI
SICI code
0027-8424(1997)94:12<6450:SEVFHG>2.0.ZU;2-S
Abstract
The effectiveness of ongoing gene therapy trials may be limited by the expression characteristics of viral and plasmid-based vectors, To enh ance levels of heterologous gene expression, we have developed a safet y-modified episomal expression vector that replicates extrachromosomal ly 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 hu man tumor suppressor gene products, including p53, retinoblastoma, and p107, yet retains replication competence, These SV40-based episomes r eplicate to thousands of copies by 2-4 days after gene transfer in mul tiple types of human cell lines, with lower activity in hamster cells, and no detectable activity in dog, rat, and murine cell lines, import antly, 107/402-T has enhanced replication activity compared with wild- type T antigen; this finding mag be due, in part, to the inability of p53 and retinoblastoma to inactivate 107/402-T function, We demonstrat e that the level and duration of 107/402-T expression regulates the ob served episomal copy number per cell, Compared with standard plasmid c onstructs, episomes encoding 107/402-T yield approximately 10- to 100- fold enhanced levels of gene expression in unselected populations of t ransient transfectants. To determine if 107/402-T-based episomes repli cate extrachromosomally in vivo, tumor explants in nude mice were dire ctly injected with Liposome/DNA complexes, Using a PCR-based assay, we demonstrate that SV40-based episomes replicate in human cells after d irect in vivo gene transfer, These data suggest that safety-modified S V40-based episomes will be effective for cancer gene therapy because h igh level expression of therapeutic genes in transient transfectants s hould yield enhanced tumor elimination.