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

Scholefield J, Greenberg LJ, Weinberg MS, Arbuthnot PB, Abdelgany A, Wood MJ - PLoS ONE (2009)

Bottom Line: Spinocerebellar ataxia type 7 is a polyglutamine disorder caused by an expanded CAG repeat mutation that results in neurodegeneration.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.

View Article: PubMed Central - PubMed

Affiliation: Division of Human Genetics/MRC/UCT Human Genetics Research Unit, Institute of Infectious Disease and Molecular Medicine, Faculty of Health Sciences, University of Cape Town, Cape Town, South Africa.

ABSTRACT
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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Related in: MedlinePlus

RNAi-based shRNAs and target vectors designed for atxn7 knockdown.(A) Convention for the diagram follows Schwarz and colleagues (16). Sequences of expected single and double mismatch guide strands (underlined) processed from an shRNA format indicating the site of the primary mismatch to the G allele of the wild-type atxn7 target. Sequence context of both the wild-type and mutant targets are shown. Functional asymmetry was induced by creating G:U mis-pairings in the 3′ end of the anti-guide strand; these are highlighted in bold. The shRNAs were labelled according to the position of the primary mismatch relative to the 5′ end of the guide strand; followed by any additional mismatches. The nomenclature is based on a 21bp guide strand after cleavage of the anti-sense strand by Dicer. (B) Schematic representations of the fused target-reporter cassettes. atx7-G-luc and atx7-A-luc are the wild-type and mutant reporter siCHECK plasmids used in the luciferase assay, and included a short region of 60 bp of atxn7 target sequence spanning the SNP fused to the Renilla luciferase reporter gene. atx7-10-G-eGFP, atx7-100-A-eGFP, atx7-100-A-DsRED are the 3 reporter plasmids used in the full-length hemi- and heterozygous assays and consist of the full-length atxn7 cDNA. The wild-type construct (atx7-10-G-eGFP) has a CAG repeat length of 10 incorporating the G allele of the SNP, fused to eGFP. The two mutant constructs (atx7-100-A-eGFP, atx7-100-A-DsRED) have CAG repeat lengths of 100, as well as the A allele of the SNP, and are fused to eGFP and DsRED respectively.
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pone-0007232-g001: RNAi-based shRNAs and target vectors designed for atxn7 knockdown.(A) Convention for the diagram follows Schwarz and colleagues (16). Sequences of expected single and double mismatch guide strands (underlined) processed from an shRNA format indicating the site of the primary mismatch to the G allele of the wild-type atxn7 target. Sequence context of both the wild-type and mutant targets are shown. Functional asymmetry was induced by creating G:U mis-pairings in the 3′ end of the anti-guide strand; these are highlighted in bold. The shRNAs were labelled according to the position of the primary mismatch relative to the 5′ end of the guide strand; followed by any additional mismatches. The nomenclature is based on a 21bp guide strand after cleavage of the anti-sense strand by Dicer. (B) Schematic representations of the fused target-reporter cassettes. atx7-G-luc and atx7-A-luc are the wild-type and mutant reporter siCHECK plasmids used in the luciferase assay, and included a short region of 60 bp of atxn7 target sequence spanning the SNP fused to the Renilla luciferase reporter gene. atx7-10-G-eGFP, atx7-100-A-eGFP, atx7-100-A-DsRED are the 3 reporter plasmids used in the full-length hemi- and heterozygous assays and consist of the full-length atxn7 cDNA. The wild-type construct (atx7-10-G-eGFP) has a CAG repeat length of 10 incorporating the G allele of the SNP, fused to eGFP. The two mutant constructs (atx7-100-A-eGFP, atx7-100-A-DsRED) have CAG repeat lengths of 100, as well as the A allele of the SNP, and are fused to eGFP and DsRED respectively.

Mentions: In order to identify effective post-transcriptional RNAi sequences targeting the atxn7 linked SNP, multiple short hairpin RNAs (shRNAs) were screened, each incorporating the weak G:U mismatch created by the targeted SNP with respect to the wild-type transcript successively at positions 10 to 16 from the 5′ end of the active shRNA guide sequence (Figure 1A). These shRNAs were screened in an assay in which a short 60 bp target of either the mutant or the wild-type ataxin7 gene sequence was inserted in the 3′UTR of Renilla luciferase and normalised to background Firefly luciferase (Figure 1B). Nucleotide mismatches incorporated at shRNA positions 11-16 all showed effective discrimination between the wild-type and mutant targets (Figure 2A), shR-P15 demonstrating the greatest discrimination with the wild-type target minimally affected retaining 90% expression and the mutant knocked down to nearly 50% relative to a non-specific shRNA control (p<0.05). However, overall target knockdown was less efficient than for shR-P12 (wild-type and mutant targets knocked down to 40% and 20% respectively) and shR-P14 (wild-type and mutant targets knocked down to 45% and 20% respectively). Interestingly, the shR-P16 construct showed little mutant-specific discrimination, knocking down both wild-type and mutant targets with high efficiency (20% and 10% remaining of the wild-type and mutant targets), and indicating high tolerance of the single nucleotide mismatch incorporated at this predicted shRNA guide strand position. Given the weak nature of the atxn7 G:U nucleotide mismatch, we investigated whether enhanced wild-type:mutant discrimination could be achieved by incorporating a range of secondary mismatches in addition to the primary nucleotide mismatch (Figure 1A), however improved mutant selectivity was not obtained (Figure S1A). It was noted that the inclusion of secondary mismatches resulted in decreased levels of knockdown at almost all positions tested indicating that the secondary mismatch ablated efficient RNAi against both targets.


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

Scholefield J, Greenberg LJ, Weinberg MS, Arbuthnot PB, Abdelgany A, Wood MJ - PLoS ONE (2009)

RNAi-based shRNAs and target vectors designed for atxn7 knockdown.(A) Convention for the diagram follows Schwarz and colleagues (16). Sequences of expected single and double mismatch guide strands (underlined) processed from an shRNA format indicating the site of the primary mismatch to the G allele of the wild-type atxn7 target. Sequence context of both the wild-type and mutant targets are shown. Functional asymmetry was induced by creating G:U mis-pairings in the 3′ end of the anti-guide strand; these are highlighted in bold. The shRNAs were labelled according to the position of the primary mismatch relative to the 5′ end of the guide strand; followed by any additional mismatches. The nomenclature is based on a 21bp guide strand after cleavage of the anti-sense strand by Dicer. (B) Schematic representations of the fused target-reporter cassettes. atx7-G-luc and atx7-A-luc are the wild-type and mutant reporter siCHECK plasmids used in the luciferase assay, and included a short region of 60 bp of atxn7 target sequence spanning the SNP fused to the Renilla luciferase reporter gene. atx7-10-G-eGFP, atx7-100-A-eGFP, atx7-100-A-DsRED are the 3 reporter plasmids used in the full-length hemi- and heterozygous assays and consist of the full-length atxn7 cDNA. The wild-type construct (atx7-10-G-eGFP) has a CAG repeat length of 10 incorporating the G allele of the SNP, fused to eGFP. The two mutant constructs (atx7-100-A-eGFP, atx7-100-A-DsRED) have CAG repeat lengths of 100, as well as the A allele of the SNP, and are fused to eGFP and DsRED respectively.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2747278&req=5

pone-0007232-g001: RNAi-based shRNAs and target vectors designed for atxn7 knockdown.(A) Convention for the diagram follows Schwarz and colleagues (16). Sequences of expected single and double mismatch guide strands (underlined) processed from an shRNA format indicating the site of the primary mismatch to the G allele of the wild-type atxn7 target. Sequence context of both the wild-type and mutant targets are shown. Functional asymmetry was induced by creating G:U mis-pairings in the 3′ end of the anti-guide strand; these are highlighted in bold. The shRNAs were labelled according to the position of the primary mismatch relative to the 5′ end of the guide strand; followed by any additional mismatches. The nomenclature is based on a 21bp guide strand after cleavage of the anti-sense strand by Dicer. (B) Schematic representations of the fused target-reporter cassettes. atx7-G-luc and atx7-A-luc are the wild-type and mutant reporter siCHECK plasmids used in the luciferase assay, and included a short region of 60 bp of atxn7 target sequence spanning the SNP fused to the Renilla luciferase reporter gene. atx7-10-G-eGFP, atx7-100-A-eGFP, atx7-100-A-DsRED are the 3 reporter plasmids used in the full-length hemi- and heterozygous assays and consist of the full-length atxn7 cDNA. The wild-type construct (atx7-10-G-eGFP) has a CAG repeat length of 10 incorporating the G allele of the SNP, fused to eGFP. The two mutant constructs (atx7-100-A-eGFP, atx7-100-A-DsRED) have CAG repeat lengths of 100, as well as the A allele of the SNP, and are fused to eGFP and DsRED respectively.
Mentions: In order to identify effective post-transcriptional RNAi sequences targeting the atxn7 linked SNP, multiple short hairpin RNAs (shRNAs) were screened, each incorporating the weak G:U mismatch created by the targeted SNP with respect to the wild-type transcript successively at positions 10 to 16 from the 5′ end of the active shRNA guide sequence (Figure 1A). These shRNAs were screened in an assay in which a short 60 bp target of either the mutant or the wild-type ataxin7 gene sequence was inserted in the 3′UTR of Renilla luciferase and normalised to background Firefly luciferase (Figure 1B). Nucleotide mismatches incorporated at shRNA positions 11-16 all showed effective discrimination between the wild-type and mutant targets (Figure 2A), shR-P15 demonstrating the greatest discrimination with the wild-type target minimally affected retaining 90% expression and the mutant knocked down to nearly 50% relative to a non-specific shRNA control (p<0.05). However, overall target knockdown was less efficient than for shR-P12 (wild-type and mutant targets knocked down to 40% and 20% respectively) and shR-P14 (wild-type and mutant targets knocked down to 45% and 20% respectively). Interestingly, the shR-P16 construct showed little mutant-specific discrimination, knocking down both wild-type and mutant targets with high efficiency (20% and 10% remaining of the wild-type and mutant targets), and indicating high tolerance of the single nucleotide mismatch incorporated at this predicted shRNA guide strand position. Given the weak nature of the atxn7 G:U nucleotide mismatch, we investigated whether enhanced wild-type:mutant discrimination could be achieved by incorporating a range of secondary mismatches in addition to the primary nucleotide mismatch (Figure 1A), however improved mutant selectivity was not obtained (Figure S1A). It was noted that the inclusion of secondary mismatches resulted in decreased levels of knockdown at almost all positions tested indicating that the secondary mismatch ablated efficient RNAi against both targets.

Bottom Line: Spinocerebellar ataxia type 7 is a polyglutamine disorder caused by an expanded CAG repeat mutation that results in neurodegeneration.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.

View Article: PubMed Central - PubMed

Affiliation: Division of Human Genetics/MRC/UCT Human Genetics Research Unit, Institute of Infectious Disease and Molecular Medicine, Faculty of Health Sciences, University of Cape Town, Cape Town, South Africa.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus