Successful disabling of the 5' UTR of HCV using adeno-associated viral vectors to deliver artificial primary microrna mimics

Abstract

Chronic hepatitis C virus (HCV) infection is a major health concern and is strongly associated with cirrhosis, hepatocellular carcinoma and liver related mortality. The current standard treatment for HCV is a combination of interferon-based therapies and ribavirin, which only produces sustained viral suppression in 40-50% of patients. Thus, the development of new treatments for HCV infection is critical. The HCV genome is the template for both protein translation and viral replication and, being RNA, is amenable to direct genetic silencing by RNA interference (RNAi). HCV is a highly mutable virus with error prone RNA replication and it has been previously reported that the virus can escape RNAi-mediated treatments through various point mutations. This has highlighted the importance of developing RNAi-based therapy that simultaneously targets multiple regions of the HCV genome. Thus, five artificial primary miRNA (pri-miRNA) were designed to mimic the naturally occurring monomeric pri-miRNA-31. The natural guide sequence on the 5’ arm of the pri-miRNA-31 was replaced with sequences complementary to different regions of the 5’ UTR of HCV. Potent knockdown of an HCV reporter was seen with four of the five constructs, and these were used to generate polycistronic cassettes, which showed impressive silencing of an HCV target. To further their application as a gene therapy recombinant adeno-associated viral (rAAV) vectors that express the polycistronic pri-miRNA mimics were generated. Two different promoter sequences were used to direct the expression of the polycistronic constructs. Ubiquitously expressed CMV and liver-specific mTTR promoters were used to generate rAAVs. All of the vectors enter liver- derived cells efficiently and significantly knock down the expression of an HCV target and showed dramatic inhibition of HCV replicon replication. The expressed polycistronic pri-miRNA mimics did not induce any off-target effects, such as stimulation of the immune response and saturation of the RNAi pathway. All the pri-miRNA mimics within the polycistronic cassettes were processed according to their intended design. The anti-HCV rAAVs developed have the potential to be an effective therapy that may contribute to the eradication of HCV.