Browsing by Author "Scholefield, Janine"

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    An African perspective on genetically diverse human induced pluripotent stem cell lines
    (2024-10) Hurrell, Tracey; Naidoo, Jerolen; Ntlhafu, Tiro; Scholefield, Janine
    Human induced pluripotent stem cell-derived models are a well-established preclinical tool, with the ability to retain the genetics of the individual from which they are derived. Here we comment on the global representation and accessibility of such cellular tools from African population groups.
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    Assessing potential drug-drug interactions between clofazimine and other frequently used agents to treat drug-resistant tuberculosis
    (2024-04) Kengo, A; Nabeemeeah, F; Denti, P; Sabet, R; Okyere-Manu, G; Abraham, P; Weisner, L; Mosala, MH; Tshabalala, Sibongile; Scholefield, Janine
    Clofazimine is included in drug regimens to treat rifampicin/drug-resistant tuberculosis (DR-TB), but there is little information about its interaction with other drugs in DR-TB regimens. We evaluated the pharmacokinetic interaction between clofazimine and isoniazid, linezolid, levofloxacin, and cycloserine, dosed as terizidone. Newly diagnosed adults with DR-TB at Klerksdorp/Tshepong Hospital, South Africa, were started on the then-standard treatment with clofazimine temporarily excluded for the initial 2 weeks. Pharmacokinetic sampling was done immediately before and 3 weeks after starting clofazimine, and drug concentrations were determined using validated liquid chromatography-tandem mass spectrometry assays. The data were interpreted with population pharmacokinetics in NONMEM v7.5.1 to explore the impact of clofazimine co-administration and other relevant covariates on the pharmacokinetics of isoniazid, linezolid, levofloxacin, and cycloserine. Clofazimine, isoniazid, linezolid, levofloxacin, and cycloserine data were available for 16, 27, 21, 21, and 6 participants, respectively. The median age and weight for the full cohort were 39 years and 52 kg, respectively. Clofazimine exposures were in the expected range, and its addition to the regimen did not significantly affect the pharmacokinetics of the other drugs except levofloxacin, for which it caused a 15% reduction in clearance. A posteriori power size calculations predicted that our sample sizes had 97%, 90%, and 87% power at P < 0.05 to detect a 30% change in clearance of isoniazid, linezolid, and cycloserine, respectively. Although clofazimine increased the area under the curve of levofloxacin by 19%, this is unlikely to be of great clinical significance, and the lack of interaction with other drugs tested is reassuring.
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    CRISPR-Cas9 mediated knockout of NDUFS4 in human iPSCs: A model for mitochondrial complex I deficiency
    (2024-11) Goolab, Shivani; Terburgh, T; Du Plessis, C; Scholefield, Janine
    Mitochondrial diseases, often caused by defects in complex I (CI) of the oxidative phosphorylation system, currently lack curative treatments. Human-relevant, high-throughput drug screening platforms are crucial for the discovery of effective therapeutics, with induced pluripotent stem cells (iPSCs) emerging as a valuable technology for this purpose. Here, we present a novel iPSC model of NDUFS4-related CI deficiency that displays a strong metabolic phenotype in the pluripotent state. Human iPSCs were edited using CRISPR-Cas9 to target the NDUFS4 gene, generating isogenic NDUFS4 knockout (KO) cell lines. Sanger sequencing detected heterozygous biallelic deletions, whereas no indel mutations were found in isogenic control cells. Western blotting confirmed the absence of NDUFS4 protein in KO iPSCs and CI enzyme kinetics showed a ~56 % reduction in activity compared to isogenic controls. Comprehensive metabolomic profiling revealed a distinct metabolic phenotype in NDUFS4 KO iPSCs, predominantly associated with an elevated NADH/NAD+ ratio, consistent with alterations observed in other models of mitochondrial dysfunction. Additionally, β-lapachone, a recognized NAD+ modulator, alleviated reductive stress in KO iPSCs by modifying the redox state in both the cytosol and mitochondria. Although undifferentiated iPSCs cannot fully replicate the complex cellular dynamics of the disease seen in vivo, these findings highlight the utility of iPSCs in providing a relevant metabolic milieu that can facilitate early-stage, high-throughput exploration of therapeutic strategies for mitochondrial dysfunction.
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    The emerging molecular biology toolbox for the study of long noncoding RNA biology
    (Future Medicine Ltd, 2017-10) Fok, Ezio T; Scholefield, Janine; Fanucchi, Stephanie; Mhlanga, Musa
    Long noncoding RNAs (lncRNAs) have been implicated in many biological processes. However, due to the unique nature of lncRNAs and the consequential difficulties associated with their characterization, there is a growing disparity between the rate at which lncRNAs are being discovered and the assignment of biological function to these transcripts. Here we present a molecular biology toolbox equipped to help dissect aspects of lncRNA biology and reveal functionality. We outline an approach that begins with a broad survey of genome-wide, high-throughput datasets to identify potential lncRNA candidates and then narrow the focus on specific methods that are well suited to interrogate the transcripts of interest more closely. This involves the use of imaging-based strategies to validate these candidates and observe the behaviors of these transcripts at single molecule resolution in individual cells. We also describe the use of gene editing tools and interactome capture techniques to interrogate functionality and infer mechanism, respectively. With the emergence of lncRNAs as important molecules in healthy and diseased cellular function, it remains crucial to deepen our understanding of their biology.
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    Impact of donor CYP3A5 genotype on pharmacokinetics of tacrolimus in South African paediatric liver transplant patients
    (2024-04) Wheeler, Caitlin A; Masimirembwa, C; Mthembu, B; Botha, J; Scholefield, Janine; Fabian, J
    In the paediatric liver transplant programme in Johannesburg, South Africa (SA), tacrolimus is the calcineurin inhibitor of choice, comprising an essential component of the immunosuppression regimen. It is characterised by a narrow therapeutic index and wide interpatient variability, necessitating therapeutic drug monitoring of whole-blood concentrations. Pharmacogenetic research, although not representative of SA population groups, suggests that single-nucleotide polymorphisms within the cytochrome P450 3A5 (CYP3A5) gene contribute to the variability in tacrolimus dosing requirements. The rs776746 polymorphism, CYP3A5*3, results in a splice defect and a non-functional enzyme. Clinically, to reach the same tacrolimus concentration-to-dose ratio (CDR), expressors (CYP3A5*1/*1 and *1/*3) require a higher tacrolimus dose than non-expressors (*3/*3).
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    Making gene editing accessible in resource limited environments: Recommendations to guide a first-time user
    (2024) Goolab, Shivani; Scholefield, Janine
    The designer nuclease, CRISPR-Cas9 system has advanced the field of genome engineering owing to its programmability and ease of use. The application of these molecular scissors for genome engineering earned the developing researchers the Nobel prize in Chemistry in the year 2020. At present, the potential of this technology to improve global challenges continues to grow exponentially. CRISPR-Cas9 shows promise in the recent advances made in the Global North such as the FDA-approved gene therapy for the treatment of sickle cell anaemia and β-thalassemia and the gene editing of porcine kidney for xenotransplantation into humans affected by end-stage kidney failure. Limited resources, low government investment with an allocation of 1% of gross domestic production to research and development including a shortage of skilled professionals and lack of knowledge may preclude the use of this revolutionary technology in the Global South where the countries involved have reduced science and technology budgets. Focusing on the practical application of genome engineering, successful genetic manipulation is not easily accomplishable and is influenced by the chromatin landscape of the target locus, guide RNA selection, the experimental design including the profiling of the gene edited cells, which impacts the overall outcome achieved. Our assessment primarily delves into economical approaches of performing efficient genome engineering to support the first-time user restricted by limited resources with the aim of democratizing the use of the technology across low- and middle-income countries. Here we provide a comprehensive overview on existing experimental techniques, the significance for target locus analysis and current pitfalls such as the underrepresentation of global genetic diversity. Several perspectives of genome engineering approaches are outlined, which can be adopted in a resource limited setting to enable a higher success rate of genome editing-based innovations in low- and middle-income countries.
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    Prime editing: Therapeutic advances and mechanistic insights
    (2024-11) Murray, JB; Harrison, PT; Scholefield, Janine
    We are often confronted with a simple question, “which gene editing technique is the best?”; the simple answer is “there isn’t one”. In 2021, a year after prime editing first made its mark, we evaluated the landscape of this potentially transformative advance in genome engineering towards getting treatments to the clinic [1]. Nearly 20% of the papers we cited were still in pre-print at the time which serves to indicate how early-stage the knowledge base was at that time. Now, three years later, we take a look at the landscape and ask what has been learnt to ensure this tech is broadly accessible, highlighting some key advances, especially those that push this towards the clinic. A big part of the appeal of prime editing is its ability to precisely edit DNA without double stranded breaks, and to install any of the 12 possible single-nucleotide conversion events as well as small insertions and/or deletions, or essentially any combination thereof. Over the last few decades, other transformative and Nobel prize-winning technologies that rely on Watson-Crick base-pairing such as PCR, site-directed mutagenesis, RNA interference, and one might say, “classic” CRISPR, were swiftly adopted across labs around the world because of the speed with which mechanistic rules governing their efficiency were determined. Whilst this perspective focuses on the context of gene therapy applications of prime editing, we also further look at the recent studies which have increased our understanding of the mechanism of PEs and simultaneously improved the efficiency and diversity of the PE toolbox.
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    Super-resolution microscopy reveals a preformed NEMO lattice structure that is collapsed in incontinentia pigmenti
    (Nature, 2016-09) Scholefield, Janine; Henriques, R; Savulescu, AF; Fontan, E; Boucharlat, A; Laplantine, E; Smahi, A; Israel, A; Agou, F; Mhlanga, Musa
    The NF- B pathway has critical roles in cancer, immunity and inflammatory responses. Understanding the mechanism(s) by which mutations in genes involved in the pathway cause disease has provided valuable insight into its regulation, yet many aspects remain unexplained. Several lines of evidence have led to the hypothesis that the regulatory/sensor protein NEMO acts as a biological binary switch. This hypothesis depends on the formation of a higher-order structure, which has yet to be identified using traditional molecular techniques. Here we use super-resolution microscopy to reveal the existence of higher-order NEMO lattice structures dependent on the presence of polyubiquitin chains before NF- B activation. Such structures may permit proximity-based trans-autophosphorylation, leading to cooperative activation of the signalling cascade. We further show that NF- B activation results in modification of these structures. Finally, we demonstrate that these structures are abrogated in cells derived from incontinentia pigmenti patients.
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    The generation of human induced pluripotent stem cell lines from individuals of Black African ancestry in South Africa
    (2024-12) Naidoo, Jerolen; Hurrell, Tracey; Scholefield, Janine
    The lack of equitable representation of African diversity in scientific resources, such as genome-wide association studies and human induced pluripotent stem cell (hiPSC) repositories, has perpetuated inequalities in the advancement of health research. HiPSCs could be transformative in regenerative and precision medicine, therefore, the generation of diverse lines is critical in the establishment of African-relevant preclinical cellular models. HiPSC lines were derived from two healthy donors of Black African ancestry using Sendai virus reprogramming of dermal fibroblasts, and characterised to confirm stemness markers, trilineage differentiation, and genetic integrity. These hiPSCs represent a valuable resource for modelling African relevant disease biology.
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    Viral Apoptosis Evasion via the MAPK Pathway by Use of a Host Long Noncoding RNA
    (Frontiers Media SA, 2018-08) Barichievy, Samantha; Naidoo, Jerolen; Boullé, M; Scholefield, Janine; Parihar, SP; Coussens, AK; Brombacher, F; Sigal, A; Mhlanga, Musa M
    An emerging realization of infectious disease is that pathogens can cause a high incidence of genetic instability within the host as a result of infection-induced DNA lesions. These often lead to classical hallmarks of cancer, one of which is the ability to evade apoptosis despite the presence of numerous genetic mutations that should be otherwise lethal. The Human Immunodeficiency Virus type 1 (HIV-1) is one such pathogen as it induces apoptosis in CD4+ T cells but is largely non-cytopathic in macrophages. As a consequence there is long-term dissemination of the pathogen specifically by these infected yet surviving host cells. Apoptosis is triggered by double-strand breaks (DSBs), such as those induced by integrating retroviruses like HIV-1, and is coordinated by the p53-regulated long noncoding RNA lincRNA-p21. As is typical for a long noncoding RNA, lincRNA-p21 mediates its activities in a complex with one of its two protein binding partners, namely HuR and hnRNP-K. In this work, we monitor the cellular response to infection to determine how HIV-1 induces DSBs in macrophages yet evades apoptosis in these cells. We show that the virus does so by securing the pro-survival MAP2K1/ERK2 cascade early upon entry, in a gp120-dependent manner, to orchestrate a complex dysregulation of lincRNA-p21. By sequestering the lincRNA-p21 partner HuR in the nucleus, HIV-1 enables lincRNA-p21 degradation. Simultaneously, the virus permits transcription of pro-survival genes by sequestering lincRNA-p21's other protein partner hnRNP-K in the cytoplasm via the MAP2K1/ERK2 pathway. Of particular note, this MAP2K1/ERK2 pro-survival cascade is switched off during T cell maturation and is thus unavailable for similar viral manipulation in mature CD4+ T cells. We show that the introduction of MAP2K1, ERK2, or HDM2 inhibitors in HIV-infected macrophages results in apoptosis, providing strong evidence that the viral-mediated apoptotic block can be released, specifically by restoring the nuclear interaction of lincRNA-p21 and its apoptosis protein partner hnRNP-K. Together, these results reveal a unique example of pathogenic control over mammalian apoptosis and DNA damage via a host long noncoding RNA, and present MAP2K1/ERK2 inhibitors as a novel therapeutic intervention strategy for HIV-1 infection in macrophages.