PhD, Faculty of Health Sciences, University of the Witwatersrand, 2009 / Chronic infection with the hepatitis B virus (HBV) is a major risk factor for cirrhosis and hepatocellular carcinoma, which is the sixth most common cancer worldwide. Available treatment for chronic HBV infection has limited efficacy in preventing associated complications. The compact and multifunctional nature of the viral genome limits its mutability making HBV an ideal candidate for therapy based on nucleic acid hybridisation. The potent and specific gene silencing that can be achieved with RNA interference (RNAi) has fueled interest in exploiting this pathway as a therapeutic modality. Synthetic and expressed RNA sequences have been used to activate RNAi. These engineered sequences mimic natural substrates of the RNAi pathway, which allows them to enter and reprogramme the pathway to effect silencing of intended targets. Tradionally expressed RNAi activators have been transcribed as short hairpin RNA (shRNA) sequences from RNA polymerase III (Pol III) promoters. These shRNA mimic precursor microRNA (pre-miRNA) and consequently enter the RNAi pathway at a relatively late stage. Overexpression of shRNA sequences from Pol III promoters, specifically the U6 promoter, has been associated with toxic side effects and has raised concerns about the use of expressed RNAi activators. Another concern of developing therapeutic RNAi expression cassettes is the emergence of HBV mutants that are resistant to silencing by a single expressed RNAi effecter. These points have highlighted the need for the development expressed RNAi activators that are effective at low concentrations and capable of combinatorial silencing. To address these issues the aim of this study was to assess the feasibility of anti HBV effecter sequences that mimic an early substrate (viz. primary miRNA or pri-miRNA) of the RNAi pathway. Pri-miRNA expression is typically under the transcriptional control of Pol II promoters. Consequently RNAi activators that Abstract - xi - mimic pri-miRNA, so-called pri-miR shuttles, may be expressed from Pol II promoters. Initially a panel of shRNA expression cassettes driven by a Pol III promoter was constructed and silencing of HBV replication assessed. Pri-miR shuttles were then designed by incorporating guide sequences of the most effective anti HBV U6 shRNA into naturally occurring pri-miR-122 and pri-miR-31. Potent inhibition of viral replication was observed with both Pol III and Pol II-driven pri-miR shuttle expression cassettes in vitro and in vivo. Subsequently liver-specific pri-miR-122 and multimeric pri-miR-31 shuttle expression cassettes were created. Pri-miR-122 shuttle sequences expressed from the alpha-1 antitrypsin promoter and HBV basic core promoter exhibited the best liver-specific silencing. Polycistronic pri-miR-31 shuttle sequences were shown to produce multiple RNAi activators capable of silencing multiple target sequences. Silencing by the pri-miR shuttle sequences was independent of toxic effects that arise from induction of the interferon response or saturation of the endogenous miRNA pathway. Pri-miR shuttles clearly represent an improved option for the use of expressed shRNA and brings therapeutic RNAi technology a step closer to clinical application.