3. Electronic Theses and Dissertations (ETDs) - All submissions
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Item Expression of the hepatitis b virus genome in chronic hepatitis b carriers(1986-12-15) Bowyer., Sheila. Mary.The methylation status of CCGG sites in hepatitis B virus (HBV) DNA was examined to determine whether methylation could be responsible for the selective expression of the HBV surface gene in the progression of chronic hepatitis B infection and hepatocellular carcinoma. Total cellular DNA, determined to have sufficient HBV DNA, was digested with the restriction endonucleases Mspl and Hpall, to determine whether the HBV DNA was methylated, or HindiII, to determine whether the HBV DNA was integrated. The cleavage fragments were analysed by Southern blotting and hybridisation to •32p- labelled HBV DNA.Item Liver-targeted transcription activator-like effector repressors that inactivate HBV cccDNA(2017) Kaldine, HaajiraThe hepatitis B virus (HBV) continues to be a global health threat as chronic infection may lead to cirrhosis and hepatocellular carcinoma (HCC). Current treatments are limited in efficacy and do not target the stable HBV covalently closed circular DNA (cccDNA) minichromosome which forms the template for viral replication and is responsible for persistence of the infection. Using gene editing technologies to disable cccDNA presents a potential approach for treating HBV infection. Transcription activator-like effector (TALE) proteins provide specific and adaptable DNA binding modules, which can be used to generate engineered proteins capable of modifying DNA. Transcription activator-like effector nuclease (TALEN) mediated cleavage of cccDNA has been shown to effectively inhibit HBV replication. However, the approach to transcriptionally silence cccDNA, instead of cleaving it, may overcome the risk of unwanted host DNA cleavage. Repressor transcription activator-like effectors (rTALEs), which target and transcriptionally silence genes, have shown potential as antiviral agents. Here we generated Krüppel-associated box (KRAB)-based rTALEs targeted to the surface open reading frame (ORF) and HBx promoter region of HBV cccDNA to inhibit transcription. The rTALEs were placed under the transcriptional control of the liver-specific modified murine transthyretin (mTTR) promoter, to restrict activity to hepatocytes thereby reducing the potential for off-target activity. In vitro the mTTR-driven rTALEs were shown to tissue specifically decrease secreted HBV surface antigen (HBsAg) levels by 63 - 92 %. Additionally, the mTTR-driven rTALEs were shown to tissue specifically decrease surface mRNA levels by 65 – 81 % and pregenomic RNA levels by 60 - 76 %. These results indicate that the KRAB domain was able to effectively suppress transcription from the basic core, Pre-S1 and/or vi Pre-S2 promoters which otherwise regulates HBV transcription. Furthermore, the observed inhibition was not associated with cytotoxicity or off-target effects. The work presented here is a proof-of-concept study demonstrating that highly specific transcriptional repressors designed to target and inhibit HBV viral replication without altering the genetic sequence or causing mutations in the host genome may be a promising antiviral approach. The capabilities of this technology to directly target cccDNA and inhibit its transcription, could contribute to addressing a global health problem.Item Development of an mRNA vaccination strategy for the prevention and treatment of HBV infection(2017) Lamb, CamillaPersistent HBV infection carries an elevated risk of developing cirrhosis and hepatocellular carcinoma (HCC). Infection is preventable by immunisation with a recombinant protein vaccine encompassing the major surface antigen (HBsAg) of Hepatitis B virus (HBV). The vaccine is globally administered resulting in a notable decrease in global carrier rates. However, some recipients (5-10%) remain as hypo- or non-responders associated with minimal to no protective antibody levels. HBV-specific DNA-vaccines have demonstrated potential for induction of potent antibody responses and target-specific activation of cellular immunity with purported application in chronic HBV infection (CHB) treatment. Furthermore, in situ production of HBsAg allows for inclusion of neutralising and cross-specific preS epitopes absent from the current vaccine. Immunisation with mRNA accompanies a superior safety profile and offers several other advantages over DNA, but no such vaccine targeting HBV exists. To develop an anti-HBV mRNA-based vaccine, vectors engineered for mRNA production were constructed. From the production vectors, LHBs (large HBsAg) and SHBs (small or major HBsAg) mRNA was synthesised by in vitro transcription using phage T7 polymerase, and translated in transfected cells to produce detectable LHBs and SHBs. SHBs was readily secreted while LHBs was retained intracellularly. This investigation constitutes a preliminary step towards the preclinical development of an anti-HBV mRNA-based prophylactic and therapeutic vaccine formulation and has provided proof-of-principle that LHBs encompassing preS epitopes is expressible in situ from synthetic mRNA transcripts. Further developments will include modification of the mRNA synthesis protocol to comply with GMP regulations and functional testing of the vaccine candidate in appropriate animal models. Keywords: HBV, HCC, HBsAg, preS, DNA-vaccines, CHB, mRNA-based vaccineItem A molecular analysis of genes involved in the cell cycle in southern African blacks with hepatocellular carcinoma(2014-05-22) Martins, Carla Suzana PintoHepatocellular carcinoma (HCC) is a leading cause of death in both Africa and Asia. It is multifactorial in aetiology and complex in its pathogenesis. Genes that might affect tumour progression, invasion, and metastasis are good candidates to investigate in attempting to understand the transformation process. The p53, RBI, BRCA1, BRCA2, WT1 and Ecadherin genes were analysed for allelic imbalance/loss of heterozygosity (LOH), polymorphisms, and mutations. Tumour and non-tumorous liver tissue from 25 southern African blacks were examined, using polymerase chain reaction restriction fragment length polymorphisms (PCR-RFLP) and PCR-single stranded conformational polymorphisms (SSCP), sequencing, and Southern blotting techniques. Allele frequencies for polymorphisms at the WEI, D13S137, D13S120, D13S127, D17S855, D16S301, and D16S260 loci were determined in 20 random African blacks using microsatellite analysis to determine allele frequencies, polymorphism information content (PIC) and diversity (H) values. To our knowledge this has not been done previously for these loci in this population. The chromosomal region l i p 13, containing the VV77 gene, and the gene itself has been reported to be deleted in 4.5% of HCCs. LOH was detected at the WT1 locus for 1/13 HCCs (8%) in this study. The RBI gene has been described to be mutated in 32.4% (China), 33.3% (Korea), 29% and 50% tJapan), and 27% (Australia), of advanced stage HCCs. In our study LOH at this locus was found in 3/19 HCCs (16%). Our finding of LOH at the BRCA2 locus in 2/20 HCCs (10%) supports the previously proposed notion that BRCA2 may function as a tumour suppressor gene in a hormone-related pathway in the liver, and that it may in some way be involved in HCC. No conclusive findings were made for any o f the other loci. Microsatellite instability was detected in 3/22 (14%) individuals. We propose that microsatellite/genomic instability may play a role in a subset of HCCs only. O f this population, 27 % had the specific p53 codon 249 AGG-AGT mutation in some tumour and non-tumorous liver. This was expected as the great majority of the individuals were from Mozambique, a country where heavy aflatoxin exposure is prevalent. All the loci examined in the allele frequency studies proved to be highly informative, showing high PIC and ED values, and should therefore be useful in population studies.