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

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dc.contributor.authorScholefield, Janineen_ZA
dc.contributor.authorGreenberg, L Jacquieen_ZA
dc.contributor.authorWeinberg, Marc Sen_ZA
dc.contributor.authorArbuthnot, Patrick Ben_ZA
dc.contributor.authorAbdelgany, Amren_ZA
dc.contributor.authorWood, Matthew J Aen_ZA
dc.date.accessioned2015-11-23T12:34:44Z
dc.date.available2015-11-23T12:34:44Z
dc.date.issued2009en_ZA
dc.description.abstractSpinocerebellar 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.en_ZA
dc.identifier.apacitationScholefield, J., Greenberg, L. J., Weinberg, M. S., Arbuthnot, P. B., Abdelgany, A., & Wood, M. J. A. (2009). Design of RNAi hairpins for mutation-specific silencing of ataxin-7 and correction of a SCA7 phenotype. <i>PLoS One</i>, http://hdl.handle.net/11427/15318en_ZA
dc.identifier.chicagocitationScholefield, Janine, L Jacquie Greenberg, Marc S Weinberg, Patrick B Arbuthnot, Amr Abdelgany, and Matthew J A Wood "Design of RNAi hairpins for mutation-specific silencing of ataxin-7 and correction of a SCA7 phenotype." <i>PLoS One</i> (2009) http://hdl.handle.net/11427/15318en_ZA
dc.identifier.citationScholefield, J., Greenberg, L. J., Weinberg, M. S., Arbuthnot, P. B., Abdelgany, A., & Wood, M. J. (2009). Design of RNAi hairpins for mutation-specific silencing of ataxin-7 and correction of a SCA7 phenotype. PloS one, 4(9), e7232. doi:10.1371/journal.pone.0007232en_ZA
dc.identifier.ris TY - Journal Article AU - Scholefield, Janine AU - Greenberg, L Jacquie AU - Weinberg, Marc S AU - Arbuthnot, Patrick B AU - Abdelgany, Amr AU - Wood, Matthew J A AB - 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. DA - 2009 DB - OpenUCT DO - 10.1371/journal.pone.0007232 DP - University of Cape Town J1 - PLoS One LK - https://open.uct.ac.za PB - University of Cape Town PY - 2009 T1 - Design of RNAi hairpins for mutation-specific silencing of ataxin-7 and correction of a SCA7 phenotype TI - Design of RNAi hairpins for mutation-specific silencing of ataxin-7 and correction of a SCA7 phenotype UR - http://hdl.handle.net/11427/15318 ER - en_ZA
dc.identifier.urihttp://hdl.handle.net/11427/15318
dc.identifier.urihttp://dx.doi.org/10.1371/journal.pone.0007232
dc.identifier.vancouvercitationScholefield J, Greenberg LJ, Weinberg MS, Arbuthnot PB, Abdelgany A, Wood MJA. Design of RNAi hairpins for mutation-specific silencing of ataxin-7 and correction of a SCA7 phenotype. PLoS One. 2009; http://hdl.handle.net/11427/15318.en_ZA
dc.language.isoengen_ZA
dc.publisherPublic Library of Scienceen_ZA
dc.publisher.departmentDivision of Human Geneticsen_ZA
dc.publisher.facultyFaculty of Health Sciencesen_ZA
dc.publisher.institutionUniversity of Cape Town
dc.rightsThis is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.en_ZA
dc.rights.holder© 2009 Scholefield et alen_ZA
dc.rights.urihttp://creativecommons.org/licenses/by/4.0en_ZA
dc.sourcePLoS Oneen_ZA
dc.source.urihttp://journals.plos.org/plosoneen_ZA
dc.subject.otherPlasmid constructionen_ZA
dc.subject.otherMicroRNAsen_ZA
dc.subject.otherRNA interferenceen_ZA
dc.subject.otherSmall interfering RNAsen_ZA
dc.subject.otherRNA stem-loop structureen_ZA
dc.subject.otherFluorescence imagingen_ZA
dc.subject.otherLuciferase assayen_ZA
dc.subject.otherSilent mutationen_ZA
dc.titleDesign of RNAi hairpins for mutation-specific silencing of ataxin-7 and correction of a SCA7 phenotypeen_ZA
dc.typeJournal Articleen_ZA
uct.type.filetypeText
uct.type.filetypeImage
uct.type.publicationResearchen_ZA
uct.type.resourceArticleen_ZA
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