Inhibition of hepatitis B virus replication using synthetic antiviral RNA interference activators

A thesis submitted to the Faculty of Health Sciences, University of the Witwatersrand in fulfilment of the requirements for the degree of Doctor of Philosophy. Johannesburg 2016Chronic liver infection by hepatitis B virus (HBV) may lead to devastating clinical conditions that include hepatocellular carcinoma and cirrhosis. Approved antiHBV drugs do not completely eradicate the infection, leading to continued viral persistence in infected individuals. Inhibition of HBV replication using synthetic activators of RNA interference (RNAi) may provide a feasible strategy of developing superior antiviral drugs. The aim of this study was to evaluate the therapeutic utility of novel 2’-O-guanidinopropyl (GP) modified synthetic small interfering RNAs (siRNAs) to counter HBV replication in cultured mammalian cells and mice. Initially, single GP moieties were placed at different nucleotide positions of the guide strand of a potent antiHBV siRNA. Some GP-modified siRNAs enhanced antiHBV activity in vitro following transient transfection of Human hepatoma 7 (Huh7) cells with siRNAs and pCH-9/3091, a replication competent HBV target plasmid. These siRNAs inhibited the secretion of Hepatitis B surface antigen (HBsAg) by up to 95% in Huh7 cells. The level of knockdown exhibited by some modified siRNAs was statistically significant relative to that displayed by unmodified siRNA3 which achieved HBsAg silencing of 73%. Additionally, modified siRNAs were also capable of reducing RNA containing the X sequence in vitro by 88-93%. Impressively, some of these knockdown levels were statistically significant when compared to unmodified siRNA3, which achieved HBx knockdown of 83%. Quantitation of interferon (IFN) response genes by reverse transcription quantitative polymerase chain reaction (RTqPCR) and evaluation of cell viability by 3-(4,5-Dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) assay revealed no evidence of innate immune stimulation or cytotoxicity in cultured cells, respectively. Modified siRNAs also displayed moderate stability in 80% foetal calf serum (FCS). Target specificity was markedly improved by GP-modified siRNAs, especially those with seed modifications (comprising nucleotide position 2 to 8 from the 5’ end of the guide strand). The siRNA-mediated mRNA cleavage product was detected from transfected cells using 5’ Rapid Amplification of cDNA ends (5’ RACE). In the hydrodynamic mouse model, co-injection of GP-modified siRNAs and HBV plasmid vector led to HBsAg suppression of approximately 80-92% at day 3 and 77-96% at day 5 post-administration. The HBV knockdown levels observed at day 3 were statistically significant when compared to those displayed by unmodified siRNA3 which achieved HBsAg silencing of 58% during the same time frame. Furthermore, both sets of siRNAs also suppressed the number of circulating viral particle equivalents (VPEs) by 88- 90% at day 3 post-injection. HBV silencing efficacy of 70-75% and 65% was achieved by modified and unmodified siRNAs, respectively at day 5 post-administration. Finally, antiHBV efficacy of GP-modified siRNAs was tested in HBV transgenic mice following delivery of these RNAi effectors using cationic polyglutamate (PG) adjuvant liposomes. Both groups of antiHBV siRNAs effected HBsAg knockdown that ranged from 70-86% at day 3 to 7 post-administration as siRNA lipoplexes in HBV transgenic mice. In contrast to the unmodified siRNA3, GP-containing siRNAs achieved durable HBsAg silencing of 70% at day 14 post-administration, while the unmodified siRNA3 displayed a shorter duration of activity. As with HBsAg data, the GP-modified siRNAs also displayed silencing efficacy that was similar to the unmodified siRNA, reducing the number of circulating VPEs by 95% from day 3 to 7 post-injection. However, the unmodified siRNA3 lost efficacy by day 14 post-administration, while the GP-modified siRNAs displayed prolonged suppression by reducing the number of circulating VPEs by 75% during the same time interval. Intrahepatic RNA levels were also assessed in transgenic mice, in which GP3 siRNA3 significantly suppressed surface and core RNA levels by 40 and 42%, respectively at day 18 post-injection. The unmodified siRNA3 suppressed surface RNA levels by 20% and core RNA levels by 25% at day 21 post-administration. Furthermore, GP4 siRNA3 silenced both surface and core RNA levels by 42% during the same time period. Additionally, intrahepatic RNA quantitation revealed no induction of IFN response genes by either unmodified or GP-modified siRNAs. In contrast to mice that had received GP-modified siRNAs, significant induction of proinflammatory cytokine release was observed in mice treated with unmodified siRNAs. The siRNA-mediated mRNA cleavage product was also detected from liver samples following 5’ RACE analysis. Neither GPmodified nor unmodified siRNAs significantly induced toxicity in injected mice. Collectively, our data provide evidence that utilisation of GP-modified siRNAs and an efficient hepatotropic non-viral delivery system may be used as a strategy to counter chronic HBV infection.MT201

Cardiorespiratory fitness is associated with hard and light intensity physical activity but not time spent sedentary in 10–14 year old schoolchildren: the HAPPY study

Sedentary behaviour is a major risk factor for developing chronic diseases and is associated with low cardiorespiratory fitness in adults. It remains unclear how sedentary behaviour and different physical activity subcomponents are related to cardiorespiratory fitness in children. The purpose of this study was to assess how sedentary behaviour and different physical activity subcomponents are associated with 10–14 year-old schoolchildren's cardiorespiratory fitness

An international comparative study of blood pressure in populations of European vs. African descent

Background: The consistent finding of higher prevalence of hypertension in US blacks compared to whites has led to speculation that African-origin populations are particularly susceptible to this condition. Large surveys now provide new information on this issue. Methods: Using a standardized analysis strategy we examined prevalence estimates for 8 white and 3 black populations (N = 85,000 participants). Results: The range in hypertension prevalence was from 27 to 55% for whites and 14 to 44% for blacks. Conclusions: These data demonstrate that not only is there a wide variation in hypertension prevalence among both racial groups, the rates among blacks are not unusually high when viewed internationally. These data suggest that the impact of environmental factors among both populations may have been under-appreciated

The influence of the accessory genome on bacterial pathogen evolution

Bacterial pathogens exhibit significant variation in their genomic content of virulence factors. This reflects the abundance of strategies pathogens evolved to infect host organisms by suppressing host immunity. Molecular arms-races have been a strong driving force for the evolution of pathogenicity, with pathogens often encoding overlapping or redundant functions, such as type III protein secretion effectors and hosts encoding ever more sophisticated immune systems. The pathogens’ frequent exposure to other microbes, either in their host or in the environment, provides opportunities for the acquisition or interchange of mobile genetic elements. These DNA elements accessorise the core genome and can play major roles in shaping genome structure and altering the complement of virulence factors. Here, we review the different mobile genetic elements focusing on the more recent discoveries and highlighting their role in shaping bacterial pathogen evolution

Auditory Physiology

Contains reports on one research projects split into ten sections.National Institutes of Health (Grant 5 P01 NS13126)National Institutes of Health (Grant 5 RO1 NS18682)National Institutes of Health (Grant 5 RO1 NS20322)National Institutes of Health (Grant 5 RO1 NS20269)National Institutes of Health (Grant 5 PO1 NS23734)National Institutes of Health (Grant 5 T32 NS07047)Symbion, Inc

Signal Transmission in the Auditory System

Contains table of contents for Section 3, an introduction and reports on six research projects.National Institutes of Health Grant R01-DC-00194National Institutes of Health Contract P01-DC-00119National Institutes of Health Fellowship F32-DC00073National Institutes of Health Grant R01-DC00238National Institutes of Health Grant R01-DC00473National Institutes of Health Grant T32-DC00006National Institutes of Health Grant T32-DC00038National Institutes of Health Contract P01-DC00361National Institutes of Health Grant R01-DC00235National Institutes of Health Contract N01-DC2240

Signal Transmission in the Auditory System

Contains table of contents for Section 3, an introduction and reports on nine research projects.National Institutes of Health Grant 5 T32 NS07047National Institutes of Health Grant 5 P01 NS13126National Institutes of Health Grant 8 R01 DC00194National Institutes of Health Grant 5 R01 NS25995National Institutes of Health Grant 8 R01 DC00238National Institutes of Health Grant 5 R01 NS20322National Institutes of Health Grant 5 R01 DC00235National Institutes of Health Grant 5 R01 NS20269National Institutes of Health Grant 1 P01 NS23734Johnson and Johnson FoundationUnisys Corporation Doctoral Fellowshi

Genome-wide association study identifies a variant in HDAC9 associated with large vessel ischemic stroke

Genetic factors have been implicated in stroke risk but few replicated associations have been reported. We conducted a genome-wide association study (GWAS) in ischemic stroke and its subtypes in 3,548 cases and 5,972 controls, all of European ancestry. Replication of potential signals was performed in 5,859 cases and 6,281 controls. We replicated reported associations between variants close to PITX2 and ZFHX3 with cardioembolic stroke, and a 9p21 locus with large vessel stroke. We identified a novel association for a SNP within the histone deacetylase 9(HDAC9) gene on chromosome 7p21.1 which was associated with large vessel stroke including additional replication in a further 735 cases and 28583 controls (rs11984041, combined P = 1.87×10−11, OR=1.42 (95% CI) 1.28-1.57). All four loci exhibit evidence for heterogeneity of effect across the stroke subtypes, with some, and possibly all, affecting risk for only one subtype. This suggests differing genetic architectures for different stroke subtypes