3. Electronic Theses and Dissertations (ETDs) - All submissions
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Item Optimisation of expressed RNA interference effecters for the inhibition of hepatitis B virus ereplication(2010-02-23T12:53:51Z) Ely, AbdullahChronic 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.Item Inhibiting Hepatitus B virus replication with short hairpin RNA sequences that target the viral X open reading frame(2006-11-17T12:55:38Z) Ely, AbdullahChronic infection with the hepatitis B virus (HBV) is endemic to sub-Saharan Africa and south-east Asia where it is a major risk factor for the development of cirrhosis and hepatocellular carcinoma (HCC). Currently available therapy is only effective in a small subset of chronic carriers. The development of novel treatment modalities for the management of HBV therefore remains an important global medical objective. Sequence plasticity of the HBV genome is limited by its small size and the overlapping nature of its open reading frames (ORFs). These features make HBV an ideal target for therapy based on nucleic acid hybridization. The use of ribozymes (RNA enzymes) and antisense molecules to inhibit gene expression is well documented. The recent discovery of RNA interference (RNAi) has added to the arsenal of therapy based on nucleic acid hybridization. RNAi is the process whereby short RNA duplexes (called short interfering RNA or siRNA) mediate the sequence-specific post-transcriptional silencing of genes homologous in sequence to the siRNA. siRNA function by guiding a protein complex (RNA Induced Silencing Complex or RISC) to target mRNA for degradation or translational repression. The protein X ORF (HBx ORF) is a conserved region of the HBV genome and is common to all viral transcripts. HBx is required for infection by the virus and plays an important role in the establishment of chronic infections in vivo as well as in the development of HCC. RNAi targeted against the HBx ORF may therefore prove useful as treatment of chronic HBV infection. Plasmid based expression cassettes capable of endogenously generating short hairpin RNA (shRNA) targeted to the HBx ORF were constructed. The shRNA function as substrates for the RNAi machinery and are processed into siRNA. The ability of the expression cassettes to knockdown markers of HBV gene expression was tested in a human hepatoma cell line. A panel of 10 U6 promoter-driven shRNA expression vectors was generated. The U6 promoter (an RNA polymerase III promoter) is normally involved in the transcription of small nuclear RNA and as such is ideal for the generation of shRNA of precisely defined length. Three cytomegalovirus (CMV) promoter-driven shRNA expression cassettes incorporating ribozymes that produce defined hairpin sequences were also generated. The CMV promoter (an RNA polymerase II) promoter is involved in the transcription of large messenger RNA. Two hammerhead ribozymes lying 5’ and 3’ of the shRNA encoding sequence were incorporated into the cassette. Cis-cleavage by the ribozymes releases a shRNA of defined length thereby overcoming the limitations imposed by extraneous sequences from CMV promoter-driven transcription. U6 promoter-driven shRNA expression vectors efficiently knocked down markers of HBV replication in liver cells. The CMV promoter-driven expression vectors were incapable of inhibiting HBV gene expression; however shRNA generated in vitro from these vectors mediated efficient knockdown of HBV replication. shRNA-mediated inhibition of gene expression therefore holds promise as a novel treatment strategy for the management of HBV and other mobile genetic elements.