579. Combinatorial RNA Interference Therapy Prevents Selection of Pre-Existing HBV Variants in Human Liver Chimeric Mice

Abstract

BACKGROUND & AIMS: Selection of drug-resistant viral variants is a major cause of treatment failure in chronic hepatitis B virus (HBV) infection. We previously identified a highly potent shRNA, S1, which, when delivered by an adeno-associated viral vector, effectively inhibits HBV replication in HBV transgenic mice. Here, we investigated the impact of a pre-existing shRNA-resistant HBV variant on the efficacy of shRNA therapy and propose the use of combination shRNA therapy to prevent viral escape. METHODS: We applied the “PICKY” software to systemically screen the HBV genome, then used hydrodynamic transfection and HBV transgenic mice to identify additional highly potent shRNAs for use, together with S1, as combination therapy. Human liver chimeric mice were infected with a mixture of wild-type and T472C HBV, a S1-resistant HBV variant, then viral dynamics were monitored during treatment with a single or combined shRNAs. RESULTS: We identified five highly potent shRNAs that reduced serum HBV DNA levels by 900- to 8,500-fold in HBV transgenic mice. In HBV-infected human liver chimeric mice, the presence of T472C mutant compromised the therapeutic efficacy of S1 and resulted in replacement of wild-type HBV by T472C HBV. In contrast, combinatorial therapy using S1 and P28, one of five potent shRNAs, markedly reduced titers for both wild-type and T472C HBV Interestingly, treatment with P28 alone led to the emergence of escape mutants with mutations in the P28 target region. CONCLUSIONS: The presence of viral quasispecies in HBV patients might compromise efficacy of RNAi therapy by selecting shRNA-resistant HBVs. Combinatorial RNAi therapy can minimize the escape of resistant HBV mutants.