Browsing by Author "Scholefield, Janine"

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    Design of RNAi hairpins for mutation-specific silencing of ataxin-7 and correction of a SCA7 phenotype
    (Public Library of Science, 2009) Scholefield, Janine; Greenberg, L Jacquie; Weinberg, Marc S; Arbuthnot, Patrick B; Abdelgany, Amr; Wood, Matthew J A
    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.
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    Detailed investigation of the unstable (CAG) repeat and the immediate surrounding region of the IT15 gene in some South African families with Huntington disease
    (2005) Scholefield, Janine; Greenberg, Jacquie; Goliath, Rene
    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.
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    Development of a SCA7 patient-derived lymphoblast cell model for testing RNAi knock-down of the disease-causing gene
    (2011) Berkowitz, Danielle Claire; Greenberg, Jacquie; Scholefield, Janine; Weinberg, Marco
    Spinocerebellar ataxia type 7 (SCA7) is an inherited neurodegenerative disease caused by the expansion of a CAG repeat within the ataxin-7 gene. The South African SCA7 population has been shown to have arisen due to a founder effect, and a single nucleotide polymorphism (SNP) within ataxin-7 has been linked to the SCA7 mutation in all South African patients genotyped to date. Recently, this SNP has been exploited in a potential allele-specific RNA interference (RNAi) based therapy, in order to knock down the expression of the mutant transcript in heterozygous patients. Although this approach has been tested in an artificial cellbased model of SCA7, focus has shifted towards testing the therapy in SCA7 patient-derived transformed lymphoblast cell lines
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    RNAi based allele-specific silencing of the disease-causing gene in black South African patients with SCA7
    (2008) Scholefield, Janine; Greenberg, Jacquie
    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.