PhD, Faculty of Health Sciences, University of the Witwatersrand / The vast potential of the RNA interference (RNAi) pathway as a new tool for the development of
therapeutic modalities has been quickly realised since its discovery in 1998. RNAi effector mimics have
been developed to successfully silence an array of disease-causing genetic elements. However,
because of the rapidly mutating genome of viruses such as the human immunodeficiency virus (HIV),
inhibition of replication cannot be sustained with single RNAi effector mimics. Instead, a combinatorial
approach is required, analogous to the cocktail of drugs necessary for successful highly active
antiretroviral therapy (HAART). Pioneering studies utilizing long hairpin RNAs (lhRNAs) showed that
the long double-stranded RNA stem region acts as a Dicer substrate and is processed into multiple
siRNA species. This intrinsic combinatorial property of lhRNAs was exploited in this thesis by
attempting to incorporate three non-contiguous potent siRNA sequences within a single lhRNA stem
expressed from an RNA Pol III promoter. Although significant knockdown of three independent HIV
target sequences was possible, the limitations of this approach became apparent when it was
observed that human Dicer does not function efficiently as a multiple turnover enzyme. The generation
of siRNA products therefore occurred in a gradient, with higher levels of siRNA produced from the base
of the hairpin stem and decreasing quantities generated towards the loop. Modifications to the
configuration of integrated siRNA sequences within the stem region enabled augmented RNAi activity
of siRNAs in the second position of the hairpin stem. This led to the notion that further manipulation of
the structural design of the stem duplex may improve efficacy of up to two siRNAs. Dual-targeting anti-HIV lhRNAs encoding only two highly effective siRNAs targeted against non-contiguous sites within the
tat, nef, LTR and int viral genes were therefore propagated. The spatial arrangement of two siRNA
sequences was extensively characterised within dual-targeting lhRNAs by inserting up to three random
base pairs at the junctions of siRNA encoding sequences and 5 bp preceding the terminal loop
sequence. A universally optimal hairpin design was identified which contained a single mismatched
base pair between two 19 bp + 2 nt siRNA sequences, as well as a terminal extension. Two powerful
dual-targeting lhRNA species, lhRNA-tat-nef +1 and lhRNA-LTR-int +1, each capable of producing two potent anti-HIV siRNA products in equal quantities were selected for incorporation into a combinatorial
RNAi system. These two effective dual-targeting lhRNAs were combined, adjacent to one another
within a single RNA Pol III-expressed transcript to create a novel lhRNA-based combinatorial RNAi
structure. This double lhRNA (dlhRNA) construct served as a precursor for four discrete highly
functional RNAi effector sequences which were capable of simultaneously silencing four unique HIV
target sites within the tat, nef, LTR and int genes. Furthermore, the ectopic expression of dlhRNAs did
not elicit activation of the interferon response, nor did it cause saturation of the endogenous miRNA
biogenesis pathway in vitro. In conclusion, the inherent combinatorial RNAi properties of long hairpin
RNAs were evaluated and the detailed analysis is presented in this thesis. Structurally optimised dualtargeting
lhRNAs subsequently formed the core components of a novel dlhRNA precursor which meets
all the requirements for an effective combinatorial RNAi strategy and therefore holds great promise for
mediating an effective and sustained gene therapy against HIV.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:wits/oai:wiredspace.wits.ac.za:10539/8758 |
| Date | 22 September 2010 |
| Creators | Saayman, Sheena Meg |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Format | application/pdf |
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