Inhibition of hepatitis C virus replication using adeno-associated virus vector delivery of an exogenous anti-hepatitis C virus microrna cluster

Abstract

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 cluster 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 cluster. 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 3 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 H epatitis C virus (HCV) infection remains a major worldwide health care problem, because approximately 3% of the world population is chronically infected with this virus, which causes viral hepatitis and can lead to cirrhosis and hepatocellular carcinoma.1 HCV replicates in the cytoplasm by a virally encoded RNA-dependent RNA polymerase (nonstructural protein 5B [NS5B]), and like most RNA polymerases, NS5B has low fidelity and incorporates mutations into its genome at a rate of $10 À4 base substitutions/nucleotide, 2 generating $one mutation per round of replication. Thus, HCV shows extraordinary genetic diversity with six major genotypes, at least 50 subtypes, and millions of quasispecies. This feature of HCV has made vaccine and drug development extremely challenging. Although HCV infections are currently managed with a combination of pegylated interferon-a and ribavirin, this regimen is successful in achieving a sustained virological response in only approximately 50% of patients infected with

Short Hairpin RNA-Mediated Gene Silencing

“…The human mir-17-92 cluster on chromosome 13, which encodes a 1 kb pri-miRNA polycistronic transcript that produces seven mature miRNAs, was modi fi ed to encode up to four embedded shRNA sequences, leading to effective HIV inhibition ( 96 ) . Similar studies have used the chicken miR-126 cluster to express shRNAs targeting in fl uenza ( 97 ) , the human mir106b-mir93-mir2548 cluster to express anti-HIV molecules ( 149 ) , and fi ve of the miRNAs from the miRNA-17-92 cluster were modi fi ed to target different regions of the HCV genome ( 150 ) .…”

Expression of Duplex shRNAs through a Lentiviral Vector against Cellular and Viral Genes Inflicts Sustained Inhibition of Hepatitis C Virus Replication

“…To address the issue HCV-infected cells were transduced with a lentiviral vector and we observed effective viral inhibition for both single and dual shRNA (Fig.7a-d, 8a, b). Few contemporary studies have also shown similar synergistic effects on HCV inhibition upon transduction with multiple shRNA expressing adenoviral or adeno-associated viral vectors [14,15,27,28].…”

“…HCV often causes chronic infections and, therefore, a consistent presence of effective doses of siRNA would be DOI: 10.1159/000492220 obligatory for a sustained virological response (SVR), which is feasible either by frequent deliveries of exogenous siRNA or in vivo expression of shRNA [13,14]. However, repetitive administration of exogenous siRNA may not be feasible in the clinical setting; therefore, approaches for in vivo expression of shRNAs have been evaluated [15,16]. Lentiviral vectors derived from retroviruses are an attractive delivery option for stable expression of shRNA due to their ability to transduce many cell types, including dividing and nondividing cells [17].…”

RNA Interference-Based Therapeutics: Molecular Platforms for Infectious Diseases

“…Further, the authors suggested the potential of their system as a promising alternative to the existing anti-HCV treatments. 147 RNAi based anti-viral HBV therapeutic approach for viral and cellular targeting shown in Figure 7.…”