Inhibition of Hepatitis C Virus Replication Using Adeno-Associated Virus Vector Delivery of an Exogenous Anti-Hepatitis C Virus MicroRNA Cluster

RNA interference (RNAi) is being evaluated as an alternative therapeutic strategy for hepatitis C virus (HCV) infection The use of viral vectors encoding short hairpin RNAs (shRNAs) has been the most common strategy employed to provide sustained expression of RNAi effectors However, overexpression and incomplete processing of shRNAs has led to saturation of the endogenous miRNA pathway, resulting in toxicity The use of endogenous microRNAs (miRNAs) as scaffolds for short interfering (siRNAs) may avoid these problems, and miRNA clusters can be engineered to express multiple RNAi effectors, a feature that may prevent RNAi-resistant HCV mutant generation We exploited the endogenous miRNA-17-92 duster to generate a polycistronic primary miRNA that is processed into five mature miRNAs that target different regions of the HCV genome All five anti-HCV miRNAs were active, achieving up to 97% inhibition of Renilla luciferase (RLuc) HCV reporter plasmids Self-complementary recombinant adeno-associated virus (scAAV) vectors were chosen for therapeutic delivery of the miRNA duster Expression of the miRNAs from scAAV inhibited the replication of cell culture propagated HCV (HCVcc) by 98%, and resulted in up to 93% gene silencing of RLuc-HCV reporter plasmids in mouse liver No hepatocellular toxicity was observed at scAAV doses as high as 5 x 10(11) vector genomes per mouse, a dose that is approximately five-fold higher than doses of scAAV-shRNA vectors that others have shown previously to be toxic in mouse liver Conclusion. We have demonstrated that exogenous anti-HCV miRNAs induce gene silencing, and when expressed from scAAV vectors inhibit the replication of HCVcc without inducing toxicity The combination of an AAV vector delivery system and exploitation of the endogenous RNAI pathway is a potentially viable alternative to current HCV treatment regimens (HEPATOLOGY 2010,52 1877-1887)