393. Site-Directed Integration in Human Genome by Lentiviral Vectors Containing HIV-1 Integrase/Polydactyl Zinc Finger Fusion Proteins

Abstract

Studies on site directed integration using fusion proteins may lead to a new approach for inserting exogenous genes at specific chromosomal sites and improve the therapeutic application of current retroviral and lentiviral vectors. Using an in trans method (Holmes-Son, et al, J Virol., 2000;74:11548), we incorporated the synthetic polydactyl zinc finger E2C fused to the N- (E2C/IN) or C- (IN/E2C) terminus of IN into HIV-1 virions. E2C contains six zinc finger domains and recognizes a unique contiguous 18-bp sequence (e2c) located within the 5'untranslated region of erbB2 gene on human chromosome 17. These viruses were infectious and capable of performing integration at a level ranging from 6 to 12% of viruses containing wild-type (WT) IN. Human Hela cells, an ovary epithelial cell line, were infected by viruses containing WT-IN or fusion proteins. Chromosomal DNA from infected cells was isolated three days post-infection and the number of proviruses was determined using a quantitative real-time PCR assay. The specificity of directed integration was quantified by comparing the number of proviruses near the E2C recognition site (between 100 bp upstream and downstream of e2c) to that of the whole genome. The number of proviruses around the e2c site was six to eleven times higher with viruses containing the E2C fusion proteins (IN/E2C or E2C/IN) than those containing the WT-IN. The results indicated that the IN-E2C fusion protein strategy is capable of directing the integration of lentiviral DNA into a predetermined site in human genome.