108. In Vitro Down-Regulation of Transgene Expression with Cre/loxP Mediated Recombination

Safe and durable transgene expression for the treatment of hepatic diseases has been reported utilizing several different viral vector systems. Following gene transfer, over-expression of some gene products may be undesirable. To this end, we prepared a recombinant AAV reporter vector that would be susceptible to molecular recombination that would terminate transgene expression. The AAV beta-galactosidase (LacZ) reporter vector (pAAV-LacZ) was modified to include loxP sites at two unique restriction enzyme sites flanking the LacZ expression cassette (pAAV-loxPLacZ). Sequential treatment of cultured human hepatoma cells (SKHep1) transfected with pAAV-loxPLacZ, followed by infection with a first generation adenovirus expressing the Cre recombinase (Ad/Cre) resulted in a 67% reduction of LacZ expression compared to control cells. Additional studies towards optimization of this molecular recombination as a form of reversible transgene expression are currently in progress. These in vitro studies represent an important step in the development of a safe, durable and reversible AAV-gene delivery system.