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.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:wits/oai:wiredspace.wits.ac.za:10539/7544 |
| Date | 23 February 2010 |
| Creators | Ely, Abdullah |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Format | application/pdf |
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