3. Electronic Theses and Dissertations (ETDs) - All submissions
Permanent URI for this communityhttps://wiredspace.wits.ac.za/handle/10539/45
Browse
2 results
Search Results
Item Post-transcriptional inhibition of human immunodeficiency virus type 1 (HIV-1) using combinatorial RNA interference (RNAi) expression vectors(2010-07-09T11:00:09Z) Barichievy, SamanthaRecent estimates indicate that globally there are over 33 million people infected with Human Immunodeficiency Virus type 1 (HIV-1). The epidemic is particularly severe in sub-Saharan Africa which accounts for 67% of all infected individuals and 72% of AIDS deaths in 2007. While current therapies, particularly in combination as a cocktail of highly active antiretroviral therapy (HAART), have had an important positive impact on the morbidity and mortality of HIV-related illness, there remain significant limitations. These include toxicities, resistance and the inability to eradicate a latent infection. In addition, most therapeutic agents have been developed to target HIV-1 subtype B, which affects individuals predominantly in Western Europe and North America. These concerns, along with ensuring patient compliance with treatment and the high cost of improved treatment regimens, have prompted the search for innovative and globally-effective therapies to eradicate HIV infection. More recently, gene therapy strategies based on the naturally-occurring RNA interference (RNAi) pathway has provided an exciting new mechanism to inhibit rogue gene expression. RNAi represents a set of highly conserved cellular pathways whereby double-stranded RNA (dsRNA) precursors are processed into shorter dsRNAs by the successive action of ribonucleases Drosha and Dicer. Processed, 21-23 nucleotide, short interfering RNAs (siRNAs) or antisense RNAs (asRNAs), associate with members of the Argonaute family of proteins to regulate gene expression at the transcriptional and post-transcriptional level. By exploiting the biogenesis of the endogenous mammalian RNAi pathway, several exogenous RNAi pathway mimics have been developed to inhibit unique sequences, including viral targets such as HIV. In the context of HIV, a combinatorial system that allows for the simultaneous suppression of multiple targets is important in preventing viral mutational escape of this rapidly evolving pathogen. The studies presented in this thesis add significantly to the newly emerging body of research on combinatorial RNAi strategies by focusing on the two novel technological approaches using mammalian expression systems. Both RNA Pol III-generated long-hairpin RNAs (lhRNAs) and RNA Pol II-generated polycistronic primary microRNAs (pri-miRNAs) were developed as systems for generating combinatorial RNAi precursors from single transcriptional units that induce post-transcriptional silencing of several highly conserved sequences of HIV-1. These included established therapeutic sites targeted to coding and non-coding regions of the HIV-1 long terminal repeat (LTR), Polymerase, Tat and Integrase. Expressed lhRNAs with ~63 bp duplex dsRNA regions and defined 5' and 3' termini were targeted to the transcribed region of the HIV 5' LTR and effectively suppressed two distinct sites within this region across both subtypes B and C HIV sequences. In addition, to assess whether lhRNAs could inhibit basal levels of HIV transcription, the lhRNAs were shown to suppress Tat-mediated (processive) and Tat-independent (non-processive) transcription when targeting episomal and integrated LTR-driven sequences. Portions of the lhRNAs that produced the most active siRNAs were dissected by using tiled LTR targets cloned into a luciferase reporter gene and by using northern blot analyses. Dicer-processing of expressed lhRNAs was shown to be most effective from the base of the duplex and decreased in efficiency towards the loop, suggesting that a gradient of siRNAs production is possible from a single lhRNA but with decreasing efficacy. This work laid the foundation for improved expressed lhRNAs whereby multiple unique anti-HIV siRNAs were produced from a single lhRNA. The second combinatorial RNAi strategy made use of RNA Pol II-expressed pri-miRNA mimics, where each mimic was derived from a different endogenous scaffold. Polycistronic transcripts consisting of four different pri-miRNA scaffolds and targeting four separate sites in HIV were tested in several combinatorial systems. The pri-miRNA backbone chosen was shown to dramatically affect the concentration and inhibitory efficacy of each generated effector strand, and this was largely independent of the sequence used. A strategy to combine four of the most effective pri-miRNA scaffolds into one expression cassette was developed and significant inhibition of an HIV infectious molecular clone as well as a wild type HIV isolate was demonstrated. Finally, in an attempt to uncover additional asRNAs capable of inducing inhibition via transcriptional gene silencing of the HIV LTR promoter, indiscriminate cell-wide gene activation was shown to occur as result of an unintended off-target effect. These observations demonstrated that caution should be exercised when interpreting RNA-induced gene activation results. Overall, this thesis provides a detailed description of the efficacy of two novel combinatorial RNAi approaches based on single promoter expression systems that are aimed at generating multiple RNAi effector sequences targeted to HIV. These approaches pave the way towards a better understanding of the efficacy of combinatorial RNAi and an effective and sustained gene therapy of HIV.Item Mutation rates in mycobacterial hosts with altered Dna metabolic activity(2006-02-08) Barichievy, SamanthaThe completion of the genome sequence of Mycobacterium tuberculosis strain H37Rv revealed that 10% of the coding capacity is devoted to two, large multigene families that are characterised by repeat sequences. These are the PE and PPE families that code for acidic, glycine rich proteins. A subgroup of the PE family is the polymorphic GC rich sequence (PGRS) gene subfamily. Genome comparisons of clinical isolates of M. tuberculosis have confirmed the polymorphic character of some of these genes suggesting they may be analogous to the contingency loci found in other pathogenic bacteria. Certain PE-PGRS proteins play a direct role in virulence in M. marinum, other PE-PGRS genes are cell surface associated, and some PE-PGRS proteins are variable surface antigens, supporting a potential role in host pathogen interactions. A reporter assay designed to investigate mutations in a PE-PGRS repeat-containing sequence was used to assess mutation rates in various M. smegmatis host strains by fluctuation analysis. A wide spectrum of mutations was observed and the evidence suggests that slipped-strand mispairing between proximal and distal PGRS sequences located in cis is the predominant type of mutational event at such loci. Moreover, slipped-strand mispairing at such loci occurs at a moderately higher rate than base substitution mutagenesis and is mediated by the normal replicative polymerase.