Mice were sacrificed 2 days later, and liver lysates were analyzed for dual luciferase activity. As shown in Fig. 6, 73% (P INK 128 ic50 < 0.01) inhibition of the RLuc-HCV UTR1 reporter was observed; similarly, 67% (P < 0.01), 93% (P < 0.01), and 80% (P < 0.01) inhibition of the RLuc-HCV-UTR3, Core, and NS5B reporters was observed, respectively. Consistent with the in vitro and in vivo

data using plasmids to express the cluster, no inhibition of the RLuc-HCV UTR2 reporter was observed. These data demonstrate that AAV vectors can efficiently deliver miRNAs to the liver, and four of the five miRNAs expressed from Cluster 1 are effective inhibitors of HCV. To evaluate the scAAV8-HCV-miR-Cluster 1 for hepatocellular toxicity, cohorts of mice were injected with one of four doses of the vector (5 × 108, 5 × 109, 5 × 1010, 5 × 1011 vg/mouse), and ALT levels were measured at multiple time

points over the course of 10 weeks. No elevations in ALT were observed at any time, even at the highest dose of vector, which is approximately five-fold higher than the dose of scAAV-shRNA vectors that resulted in hepatic toxicity.11 Thus, the use of a polycistronic miRNA scaffold to express anti-HCV RNAi effectors appears to be safer than using shRNAs to mediate PARP inhibitor RNAi.13 In this study, we exploited the endogenous RNAi mechanism to design a novel treatment for HCV infection, because the current therapy is not equally effective against all HCV genotypes and has numerous side effects.1 In designing this alternative strategy, we took advantage of the results gleaned from previous attempts to inhibit HCV using RNAi. In particular, we relied on the literature to identify HCV target sequences, and incorporated validated siRNA and shRNA sequences6 into the endogenous miR-17-92 cluster. The use of a polycistronic miRNA to express five RNAi effectors that target different regions of HCV increases the likelihood of inhibiting the virus. In addition, four of the five RNAi effectors

target conserved regions of all six HCV genotypes, providing 上海皓元 broader applicability to this approach than drugs currently in use and those in development. We used miRNAs, rather than shRNAs, to mediate RNAi to avoid interference with the endogenous miRNA pathway.12-14 The mature miRNAs were designed to mimic the secondary structure of their endogenous counterparts and to have low internal stability at their 5′ ends, because these characteristics have been associated with preferential incorporation of the guide strand into the RNA-induced silencing complex,24, 25 a feature that will minimize off-target effects. The use of a liver-specific promoter to express the miRNAs ensured expression in hepatocytes, which will also minimize potential off-target effects.