553. Silencing HBV Gene Expression with Pol II and Pol III Promoter-Derived Short Hairpin RNA

Abstract

Chronic infection with hepatitis B virus (HBV) occurs in approximately 6% of the world's population and is a major risk factor for cirrhosis and hepatocellular carcinoma (HCC). Current therapy for persistent HBV infection, including interferon alpha and nucleoside analogues, is often unsuccessful and developing effective HBV treatment remains an important global medical priority. The virus has a compact genome with limited sequence plasticity that makes it a good target for therapy that is based on nucleic acid hybridization. Thus, exploiting the RNA interference (RNAi) pathway to knock down HBV gene expression is a promising novel approach to treating HBV infection. To induce HBV gene silencing, synthetic siRNA duplexes or short hairpin RNA (shRNA) molecules derived from Pol III promoters have typically been employed to induce RNAi. Using these approaches, silencing is not targeted to the liver and potentially harmful non specific extrahepatic effects may occur. Pol II promoters, which are capable of precise transcription control, may be applied to effect more controlled silencing. The objective of this work was to assess the anti HBV silencing efficiency of transiently expressed short-hairpin RNAs (shRNA) regulated by a Pol II promoter and to compare their efficacy to equivalent Pol III-derived transcripts. A panel of 6 shRNAs regulated by U6 (Pol III) or CMV (Pol II) promoters were designed to target 3 regions of the conserved HBV X open reading frame. The HepG2.2.15 cell line, which constitutively produces HBV, and Huh7 human hepatoma cell line were transfected with plasmids encoding Pol II or Pol III shRNA expression cassettes. Huh7 cells were also cotransfected with pCH3091 or pCH3091-GFP HBV target vectors. pCH3091 is a plasmid that encodes all HBV sequences, and in pCH3091-GFP, the preS2-S region has been substituted with a sequence encoding the Enhanced Green Fluorescent Protein (EGFP). The effects of the shRNAs on HBV gene transcription were determined by measuring HBsAg, HBeAg, HBV transcripts and EGFP. shRNAs that are derived from both Pol II and Pol III promoters were capable of significant inhibition of HBV gene expression, although Pol III-derived shRNAs were stronger inducers of HBV gene silencing (95% vs 70% inhibition). To improve precisely controlled silencing, refinements in the design of Pol II cassettes that regulate strand bias and limit compromising effects of extra 5' and 3' sequences are being investigated.