RNAi based allele-specific silencing of the disease-causing gene in black South African patients with SCA7

Includes abstract.Includes bibliographical references (leaves 118-133).The polyglutamine disorders are a subgroup of inherited neurodegenerative disorders with a common mutation which confers toxicity via a polyglutamine tract in the protein leading ultimately to various forms of neurodegeneration. One of these disorders, spinocerebellar ataxia 7 (SCA7) exists at a higher frequency in South Africa, than elsewhere in the world, and a founder effect has been demonstrated in South Africa, such that every patient tested thus far is linked to a common ancestor. The manipulation of RNA interference (RNAi) has been used with increasing success to selectively knockdown the expression of disease-causing genes at the RNA level. Thus, the possibility of applying this method to SCA7 in South Africa was considered. However, the wild-type allele of ataxin-7 is likely to be necessary for cellular function therefore a form of allele-specific silencing is required, such as a SNP linked to the mutation

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

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

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

Detailed investigation of the unstable (CAG) repeat and the immediate surrounding region of the IT15 gene in some South African families with Huntington disease

Includes bibliographical references (leaves 113-124).The primary aim of this study was to investigate the origins of the HD mutation in South Africa (SA) by constructing a single nucleotide polymorphism (SNP) haplotype around the IT15 gene and to determine how many haplotypes there are in SA. Haplotypes were created by genotyping 6 SNPs in a total of 15 HD families. These families were comprised of seven Caucasian, 6 Mixed Ancestry and two Black African families

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

The data that support the findings of this study are available from the corresponding authors upon request.International audienceThe NF-kB 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-kB activation. Such structures may permit proximity-based trans-autophosphorylation, leading to cooperative activation of the signalling cascade. We further show that NF-kB activation results in modification of these structures. Finally, we demonstrate that these structures are abrogated in cells derived from incontinentia pigmenti patients

Image_1_Viral Apoptosis Evasion via the MAPK Pathway by Use of a Host Long Noncoding RNA.TIFF

<p>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.</p

Image_2_Viral Apoptosis Evasion via the MAPK Pathway by Use of a Host Long Noncoding RNA.TIFF

<p>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.</p

The translational gap for gene therapies in low- and middle-income countries

Gene therapy is at the forefront of modern medicine. Operating at the genomic level, these sophisticated technologies are designed to address the root cause of disease. This class of medicines is made possible by advances in genetic engineering and cellular delivery technologies. As scientific understanding behind disease prevention, diagnosis, and treatment improves in tandem with technological innovation, gene therapies will hopefully become safe and effective treatment options for a wide range of genetic and non-genetic diseases. However, as the medicinal scope of gene therapies expands, consideration must be given to who will benefit and what proactive steps must be taken to widen development and access potential, particularly in areas carrying high burden diseases likely to benefit from their introduction