1000. In Vitro and In Vivo Evaluation of Optimized Short-Hairpin RNAs for the Treatment of the Hepatitis C Virus Using Expression Cassettes Producing One, Two or Three Anti HCV shRNAs

Abstract

Infection with the Hepatitis C virus (HCV) represents a major world-wide health problem. Current therapies consisting of small molecule pharmaceuticals with or without interferon are most often inadequate due to the generation of viral escape mutants. RNA interference (RNAi) based treatments using both short-interfering RNAs (siRNAs) and short-hairpin RNAs (shRNAs) have been proposed as new therapeutic modalities. As HCV is a chronic disease, we are interested in sustained expression of anti-HCV RNAs, and thus have focused on the expression of short-hairpin RNAs (shRNAs) that can elicit an RNAi response against the HCV genome. We aligned multiple HCV sequences and, and identified 30 potential RNAi targets. As a first step, the siRNAs corresponding to these targets were synthesized and tested in vitro against HCV-luciferase fusion reporter constructs, as well as subgenomic and full length HCV replicons. We identified 8 siRNAs that were capable of efficiently silencing the expression of luciferase-HCV fusions and of the HCV replicon. The corresponding shRNAs were cloned in expression cassettes downstream of ubiquitous promoters. We generated expression cassettes capable of expressing either 1, 2 or 3 shRNAs simultaneously. The shRNAs were evaluated in vitro for silencing of an HCV-luciferase fusion reporter, as well as subgenomic and/or full length HCV replicons in the primary hepatocyte cell line Huh7. The shRNAs that inhibited the luciferase reporter by greater than 80% in vitro, were selected for in vivo analysis. Tail vein injection of mice with single shRNA expression cassettes and the HCV-luciferase reporter resulted in up to 70% inhibition of luciferase. Analysis of shRNA expression cassettes that generate two or three shRNAs targeted to different conserved regions of the HCV genome is ongoing and will be reported on.