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    Targeted transcriptional silencing of hepatitis B virus using designer epigenome modifiers expressed from in vitro transcribed mRNA
    (University of the Witwatersrand, Johannesburg, 2025) Singh, Prashika; Ely, Abdullah; Arbuthnot, Patrick
    Approximately two-thirds of the world's population has been exposed to the hepatitis B virus (HBV), the causative agent of hepatitis B, with sub-Saharan Africa experiencing a high prevalence, where up to 8% of the population are chronic carriers. The persistence of HBV infection is largely due to the stable replicative intermediate, covalently closed circular DNA (cccDNA), which current treatments do not effectively target or eliminate. Consequently, a reliable cure for chronic hepatitis B (CHB) remains unavailable. Gene editing technologies, such as Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein (CRISPR/Cas), Transcription Activator-Like Effector Nucleases (TALENs), and Zinc Finger Nucleases (ZFNs), can directly target cccDNA and offer the potential to eliminate this reservoir. However, these methods are limited by the risk of off-target effects, including chromosomal deletions and translocations. In contrast, emerging epigenome editing technologies provide safer alternatives. Designer epigenome modifiers (DEMs) offer a less genotoxic option by modifying cccDNA epigenetically. DEMs, comprising DNA methyltransferase (DNMT) and Kruppel- associated box (KRAB) repressor domains along with a Transcription Activator-Like Effector- DNA binding domain (TALE-DBD) were designed to target the HBV surface (S), core (C), and polymerase (P) open reading frames (ORFs). Control DEMs lack a KRAB domain and contain catalytically inactive DNMT. Given the strategic positioning of CpG islands in the HBV genome, these regions make the viral genome an ideal target for DNA methylation. To improve the efficacy of already existing DEMs, this study evaluated the use of mRNA-based platforms (mRNA-based vectors, pmRNA-pA and pT7-(AG)) for expressing anti-HBV DEMs for the first time, allowing for targeted silencing of the S, C, and P ORFs of HBV in vitro. Sequences encoding the DEMs were cloned into an expression vector specifically designed for in vitro transcription of mRNA. In vitro transcribed anti-HBV DEM-encoding mRNA were transfected into liver-derived Huh7 and HEK 293T cells. Markers of viral replication, including HBV surface antigen (HBsAg) and viral surface and pregenomic RNA (pgRNA) and stimulation of interferon response genes were assessed. Bisulphite sequencing was used to confirm the methylation of HBV DNA, and 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays were conducted to assess the toxicity of the synthesized DEM RNA. The pT7- (AG) vector, featuring more stable regulatory sequences, a longer poly-A tract, and multiple termination codons, proved to be more effective than the original platform. DEMs expressed Abstract vi from this platform achieved a significant reduction in HBsAg levels, particularly with the complete substitution of uridine nucleotides with pseudouridine, incorporation of CleanCap® AG, and removal of dsRNA. HBsAg levels were reduced by 21-41 %, and up to 50-90 % reduction was observed with double transfection. Additionally, viral surface transcript levels decreased by 42-70 %, while pregenomic transcript levels decreased by 62-72 % correlating with increased trend in methylation at all three targeted CpG islands. There was no evidence of immune stimulation or metabolic toxicity, indicating that the observed transcriptional activity resulted from DEM activity and suggest that DEMs are safe. Furthermore, the SL and PL DEMs exhibited the greatest transcriptional silencing. This proof-of-concept study demonstrates that optimising mRNA stability, half-life, and immunogenicity can significantly enhance the efficacy of DEMs. However, further in vivo characterisation of these DEMs, along with an assessment of their duration of action at both on-target and off-target activities, is required. Nevertheless, targeted epigenome silencing of HBV through the use of epigenome editors expressed as in vitro transcribed mRNA represents a promising therapeutic approach for modulating viral replication and potentially treating CHB.
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    Sleep Characteristics Among In-School Adolescents In South-Western Nigeria: Pattern, Determinants And Association With Cardio-Metabolic Risk Factors
    (University of the Witwatersrand, Johannesburg, 2025) Olorunmoteni, Oluwatosin Eunice; Scheuermaier, Karine; Gómez-Olivé, Xavier
    Problematic sleep is a major threat to the well-being of adolescents globally. However, little is known about the sleep health of adolescents in Africa. Thus, this study aimed to map studies on adolescent sleep health in Africa, determine the sleep characteristics of Nigerian adolescents in rural versus urban communities and the association between their sleep health and cardio- metabolic risk factors. We conducted a scoping review by searching five databases from the inception (beginning) of each database to June 2023 using Joanna Briggs Institute (JBI) methodology. Furthermore, a quantitative, cross-sectional study was conducted among adolescents aged 13-19 in six rural and six urban schools in Osun State, Nigeria. We used structured questionnaires to measure sleep duration and quality, daytime sleepiness, and sleep apnea risk and used actigraphs on a subset. We measured their anthropometry, blood pressure and blood sugar. A continuous cardiometabolic (CMR) score was calculated using z-scores of mean arterial pressure (MAP), body mass index (BMI), waist circumference (WC), and random blood glucose. The data obtained was analysed using Stata 15 and SAS software. The 31 studies (77% cross-sectional) included in the scoping review were mainly focused on sleep quantity and quality. Inadequate sleep duration during weekdays was common among adolescents, often compensated by longer weekend sleep durations. Additionally, the prevalence of poor sleep quality and sleep disorders, such as insomnia and sleep-disordered breathing, was high across various regions. Younger age was reported as protective of good sleep health, while gender disparities in adolescent sleep health were noted across regions. The use of electronic devices at bedtime was a major risk factor for sleep disturbances among urban-dwelling adolescents while poor sleeping conditions impaired sleep, especially among adolescents in rural communities and for those in lower social class. Among the 900 participants in the quantitative study, (51% rural; 59% female; average age (SD) = 15.1(1.4) years), the adjusted odds of having poor sleep quality vii (p = .008) and daytime sleepiness (p = .007) were 2-fold higher among urban compared to rural adolescents. Poorer sleep quality was associated with a higher CMR score (β=0.02), BMI z-score (β=0.04) and WC z-score (β=0.03) (all p<0.05). Lower sleep duration was associated with a higher CMR score (β=0.04), BMI z-score (β=0.11), and WC z-score (β=0.07) (all p<0.05). Risk of sleep apnea was associated with higher CMR score (β=0.08), MAP z-score (β=0.17) and WC z-score (β=0.14) (all p<0.05). Using actigraphy for 170 adolescents, shorter total sleep time and earlier wake times were seen on weekdays than weekends. In conclusion, adolescents in rural communities had better sleep than their urban counterparts. Urban adolescents have later bedtimes, earlier wake times and shorter time in bed and total sleep time than rural adolescents. Furthermore, poor sleep health was associated with higher CMR. Interventions aiming to improve the sleep characteristics of adolescents are crucial, especially for urban-dwelling adolescents and may be important for preventing cardiometabolic disease
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    The Role of Copy Number Variants in the aetiology of developmental disorders in South Africa – a whole exome sequencing study
    (University of the Witwatersrand, Johannesburg, 2025) Louw, Nadja; Lombard, Zané; Carstens, Nadia
    Developmental disorders are rare conditions, causing a mental or physical impairment. Genetic heterogeneity and highly variable clinical manifestations poses great challenges in the diagnosis of developmental disorders. New sequencing technologies have become an important and cost-effective tool in the diagnosis of rare diseases and results can impact clinical practice significantly. Copy number variants (CNVs) play a major role in the pathogenesis of developmental disorders and thus it is important to investigate the presence of CNVs within these patient cohorts. The introduction of exome sequencing (ES) has allowed the detection of CNVs and single nucleotide variants (SNVs) exome wide with a single test. This is a valuable approach to implement especially in a limited resource setting like South Africa. Despite SNVs being well studied, the incorporation of CNV bioinformatics tools into variant calling pipelines has not been implemented routinely as there is no current gold standard for CNV detection from exome data. Very limited clinical CNV studies have been carried out on next generation sequencing (NGS) data in patients of African ancestry. This study thus aimed to identify the most appropriate bioinformatics approach to detect CNVs from exome data. Subsequently, it was implemented in a developmental disorder variant analysis pipeline for ES data generated by the Deciphering Developmental Disorders in Africa (DDD-Africa) study, to establish the role that CNVs play in this African cohort. The DDD-Africa study recruited and performed detailed clinical phenotyping and ES on 500 African patients with an undiagnosed developmental disorder. Four different bioinformatics tools (CANOES, CLAMMS, XHMM and InDelible) have been applied to a set of samples (N=100) with known CNVs which served as a truth set. Functional equivalence evaluation was carried out in order to identify the most effective CNV calling tool or combination of tools to implement on the DDD-Africa exome dataset. Implementing the chosen tools onto the first batch of ES data consisting of 287 participants, yielded a ~7% diagnostic yield. Integrating CNV detection tools into a standard variant analysis pipeline from ES data can improve diagnostic yield while also promoting an improved cost benefit for ES. These results could not only end the diagnostic odyssey, but may lead to better care and management for families with developmental disorders in Africa.
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    Exploring the HIV/AIDS infection, intimate partner violence experience and poor mental health syndemic and its association with parenting amongst women in Mpumalanga
    (University of the Witwatersrand, Johannesburg, 2024) Silima, Mpho
    Background: In South Africa, women disproportionately bear the burden of Intimate Partner Violence (IPV), HIV or AIDS, and poor mental health (MH), which significantly affects their parenting and children’s wellbeing. Harsh and inconsistent parenting, often resulting from these compounded stressors, negatively impacts the health and development of children. While there is extensive research on the impact of each epidemic on parenting, there is considerably less published research on the syndemic impact of IPV, HIV, and MH. A syndemic refers to the synergistic interaction of two or more co-existing epidemics where the combined impact is often greater than the sum of their individual effects, often leading to more severe health outcomes, particularly among vulnerable or marginalised populations. Aim: The overall aim of this PhD research was to explore the experiences and parenting practices among mothers and caregivers living with different combinations of the co-occurring IPV, MH, and HIV epidemics. Methods: This thesis draws upon three distinct studies, using an overall mixed-method study design. The first is a scoping review of 23 studies conducted between 2001-September 2023, following the JBI and Prisma-ScR guidelines. The second study is a nested qualitative study using in-depth interviews and arts-based methods with 20 women. Thematic data analysis using MAXQDA22 was used to analyse the data. The third study is a secondary data analysis of cross- sectional data of young adults who are primary caregivers of children under 18 from the third wave of a multigenerational longitudinal study. A total of 720 young women completed an interviewer administered questionnaire, and 547 were included in the final quantitative analysis. Quantitative data were analysed using Stata 17.0 using logistic regression and marginal effects models Results: This doctoral research contributes several new findings to the extant literature on parenting practices and the IPV, MH, and HIV syndemic. The scoping review identified primarily studies from the United States, that utilized quantitative, mainly longitudinal research designs. Findings from the review indicate living with the different combinations of IPV, MH and HIV led to increased likelihood of harsh and inconsistent parenting. Furthermore, the review viii revealed that while IPV was not directly associated with harsh parenting practices, the relationship was mediated by poor mental health. Anxiety and maternal depression were the most frequent disorders found in studies examining the co-occurrence of HIV-MH. Findings from the qualitative studies revealed that women living with the syndemic face numerous challenges due to these co-existing epidemics, with depression, suicidality, and post-traumatic stress disorder (PTSD) being most common. Participants adopted various coping strategies, including religion, acceptance, and family support, with harmful substance use most prevalent among those experiencing IPV. Regarding parenting, individual, interpersonal, and structural factors significantly influenced practices. The quantitative findings revealed that maternal (includes any woman who is a primary caregiver) use of emotional and physical violence was common, with women experiencing IPV and poor MH concurrently, having higher odds of using both physical and emotional violence towards their children, particularly emotional violence. The quantitative findings also revealed a synergistic relationship between IPV and MH, showing that the combined effect of these epidemics has a more severe impact on parenting than each independently However, combinations including HIV, such as HIV-MH and IPV-MH did not significantly increase the risk of maternal use of emotional violence Conclusion: This research highlights the severe effects of the IPV, MH syndemics on parenting among South African women. Multi-sectoral and multi-disciplinary interventions are needed to address the challenges experienced by mothers and caregivers living with the syndemic. To effectively reduce child maltreatment, interventions must tackle the IPV-MH syndemic in tandem. The overlap of these epidemics leads to detrimental parenting, strained relationships and inadequate provision of children’s needs. Additionally, mothers living with these syndemics often have lower levels of parenting satisfaction, which further compounds their challenges. Addressing these intersecting epidemics holistically is essential for improving the wellbeing of both parents and their childre