Hepatic Models in Precision Medicine: An African Perspective on Pharmacovigilance.

Pharmaceuticals are indispensable to healthcare as the burgeoning global population is challenged by diseases. The African continent harbors unparalleled genetic diversity, yet remains largely underrepresented in pharmaceutical research and development, which has serious implications for pharmaceuticals approved for use within the African population. Adverse drug reactions (ADRs) are often underpinned by unique variations in genes encoding the enzymes responsible for their uptake, metabolism, and clearance. As an example, individuals of African descent (14-34%) harbor an exclusive genetic variant in the gene encoding a liver metabolizing enzyme (CYP2D6) which reduces the efficacy of the breast cancer chemotherapeutic Tamoxifen. However, CYP2D6 genotyping is not required prior to dispensing Tamoxifen in sub-Saharan Africa. Pharmacogenomics is fundamental to precision medicine and the absence of its implementation suggests that Africa has, to date, been largely excluded from the global narrative around stratified healthcare. Models which could address this need, include primary human hepatocytes, immortalized hepatic cell lines, and induced pluripotent stem cell (iPSC) derived hepatocyte-like cells. Of these, iPSCs, are promising as a functional in vitro model for the empirical evaluation of drug metabolism. The scale with which pharmaceutically relevant African genetic variants can be stratified, the expediency with which these platforms can be established, and their subsequent sustainability suggest that they will have an important role to play in the democratization of stratified healthcare in Africa. Here we discuss the requirement for African hepatic models, and their implications for the future of pharmacovigilance on the African continent.

Viral Apoptosis Evasion via the MAPK Pathway by Use of a Host Long Noncoding RNA.

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.

Super-resolution microscopy reveals a preformed NEMO lattice structure that is collapsed in incontinentia pigmenti.

The NF-κB 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-κB activation. Such structures may permit proximity-based trans-autophosphorylation, leading to cooperative activation of the signalling cascade. We further show that NF-κB activation results in modification of these structures. Finally, we demonstrate that these structures are abrogated in cells derived from incontinentia pigmenti patients.

Polyglutamine disease: from pathogenesis to therapy.

Polyglutamine diseases are inherited neurodegenerative conditions arising from expanded trinucleotide CAG repeats in the disease-causing gene, which are translated into polyglutamine tracts in the resultant protein. Although these diseases share a common type of mutation, emerging evidence suggests that pathogenesis is complex, involving disruption of key cellular pathways, and varying with the disease context. An understanding of polyglutamine disease mechanisms is critical for development of novel therapeutics. Here we summarise theories of molecular pathogenesis, and examine ways in which this knowledge is being harnessed for therapy, with reference to work under way at the University of Cape Town. Despite a plethora of preclinical data, clinical trials of therapies for polyglutamine diseases have had only limited success. However, recently initiated trials, including those using gene silencing approaches, should provide valuable insights into the safety and efficacy of therapies directly targeting polyglutamine pathogenesis. This is particularly relevant in the South African context, where the frequencies of two polyglutamine diseases, spinocerebellar ataxia types 1 and 7, are among the highest globally.

Therapeutic gene silencing strategies for polyglutamine disorders.

Dominantly inherited polyglutamine disorders are chronic neurodegenerative diseases therapeutically amenable to gene-specific silencing strategies. Several compelling nucleic acid-based approaches have recently been developed to block the expression of mutant proteins and prevent toxic neurodegenerative sequelae. With such approaches, avoiding potential side effects caused by the concomitant ablation of the normal protein is an important objective. Therefore, allele-specific gene silencing is highly desirable; however, retaining wild type function is complex given that the common CAG mutation cannot be directly targeted, and might not be necessary or justifiable in all cases. Insights from polyglutamine gene function studies and the further development of allele-specific and other gene silencing methodologies will be important to determine the optimal therapeutic strategy for each polyglutamine disorder.

Design of RNAi hairpins for mutation-specific silencing of ataxin-7 and correction of a SCA7 phenotype.

Spinocerebellar ataxia type 7 is a polyglutamine disorder caused by an expanded CAG repeat mutation that results in neurodegeneration. Since no treatment exists for this chronic disease, novel therapies such post-transcriptional RNA interference-based gene silencing are under investigation, in particular those that might enable constitutive and tissue-specific silencing, such as expressed hairpins. Given that this method of silencing can be abolished by the presence of nucleotide mismatches against the target RNA, we sought to identify expressed RNA hairpins selective for silencing the mutant ataxin-7 transcript using a linked SNP. By targeting both short and full-length tagged ataxin-7 sequences, we show that mutation-specific selectivity can be obtained with single nucleotide mismatches to the wild-type RNA target incorporated 3' to the centre of the active strand of short hairpin RNAs. The activity of the most effective short hairpin RNA incorporating the nucleotide mismatch at position 16 was further studied in a heterozygous ataxin-7 disease model, demonstrating significantly reduced levels of toxic mutant ataxin-7 protein with decreased mutant protein aggregation and retention of normal wild-type protein in a non-aggregated diffuse cellular distribution. Allele-specific mutant ataxin7 silencing was also obtained with the use of primary microRNA mimics, the most highly effective construct also harbouring the single nucleotide mismatch at position 16, corroborating our earlier findings. Our data provide understanding of RNA interference guide strand anatomy optimised for the allele-specific silencing of a polyglutamine mutation linked SNP and give a basis for the use of allele-specific RNA interference as a viable therapeutic approach for spinocerebellar ataxia 7.