757. Enhancement of Gene Expression by a Recombinant Adeno-Associated Virus by Various Chemical Treatments

Abstract

A recombinant adeno-associated virus (rAAV) has drawn attention as a novel gene delivery system with the advantage of long-term and efficient gene expression, particularly in neuronal and skeletal muscle cells. However, transduction efficiency (TE) by rAAV is still low, which may explain its limited application, even in neuronal cells in vitro. To overcome this drawback of rAAV system, we attempted to enhance the transduction efficacy using many different kinds of chemicals, mostly modulating DNA synthesis, cell cycle or rAAV receptor expression. To investigate the effects of various chemical treatments on TE, we infected several human cancer cells lines (cervical Hela, colon HCT 116, breast MCF-7, hepatocellular carcinoma SK-Hep1, and glioma U 251 cancer cells) with rAAV-expressing b-galactosidase. We then examined the degree of x-gal staining 48 h after post-infection. Total 7 chemicals were employed to enhance TE by rAAV of Trichostatin A as an inducer of rAAV receptor expression, Aphidicolin and Hydroxyurea as a DNA damaging agent, Etopocide and Camptothecin as a DNA synthesis blocker, MG-132 as a proteosome inhibitor, and Tyrphostin-1 as a EGFR inhibitor. Hydorxyurea increased TE by 25- or 5-fold in Hela or HCT 116 cells, respectively. MG-132 increased TE by 8.5-fold in SK-Hep1 cells. Co-treatment of MG-132 and camptothecin enhanced TE by 12-fold in Hela cells. The combined treatment of hydroxyurea and tyrphostin-1 also enhanced the transduction efficiency by 12-fold in Hela cells. Finally, MTT assay consistently implied that the cytotoxicity was not observed in any concentrations of each chemical used in this study.