Limits...
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.

Show MeSH

Related in: MedlinePlus

Investigation of aggregate formation.(A) Representative confocal images of cells transfected with (i, ii) mutant (atx7-100-A-DsRED) alone; (iii, iv) wild-type (atx7-10-G-eGFP) alone. (i) Image visualized under red fluorescence, (ii) red fluorescence merged with the bright field, (iii) green fluorescence, and (iv) green fluorescence merged with the bright field. (B) Representative confocal images of cells co-transfected with wild-type and mutant expression plasmids in addition to (i–iii) shR-NS or (iv–vi) shR-P16. (i) and (iv) show images under green fluorescence to reveal wild-type protein, (ii) and (v) show images under red fluorescence to reveal mutant protein, and (iii) and (vi) show images then merged under green and red fluorescence and bright field. Scale bar represents 10 µm. C. Cells expressing eGFP and/or DsRED were counted according to whether they contained aggregates or a dispersed pattern of expression of mutant and wild-type ataxin-7. Cells were transfected with wild-type target (atx-10-G-eGFP) alone; mutant target (atx7-100-A-DsRED) alone; mutant (atx7-100-A-DsRED), wild-type (atx-10-G-eGFP) and shR-NS (non-specific shRNA); mutant, wild-type and shR-P16. Cells were counted separately in the red and the green filter by collecting 3 representative images from each well and combining the total number. This was performed for each indicated combination in biological triplicate, yielding standard deviations. The bars comprise the total number of cells counted in each transfection; separated according to whether they contained aggregates (blue) or a dispersed pattern of expression (grey). Note that the decrease in expression from target vectors transfected alone to co-transfected cells is due to a promoter occlusion effect of co-expression of targets and not due to the addition of shR-NS which has no effect upon the target vectors (data not shown). Statistically significant differences in % of aggregate containing cells are indicated by corresponding p values.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2747278&req=5

pone-0007232-g005: Investigation of aggregate formation.(A) Representative confocal images of cells transfected with (i, ii) mutant (atx7-100-A-DsRED) alone; (iii, iv) wild-type (atx7-10-G-eGFP) alone. (i) Image visualized under red fluorescence, (ii) red fluorescence merged with the bright field, (iii) green fluorescence, and (iv) green fluorescence merged with the bright field. (B) Representative confocal images of cells co-transfected with wild-type and mutant expression plasmids in addition to (i–iii) shR-NS or (iv–vi) shR-P16. (i) and (iv) show images under green fluorescence to reveal wild-type protein, (ii) and (v) show images under red fluorescence to reveal mutant protein, and (iii) and (vi) show images then merged under green and red fluorescence and bright field. Scale bar represents 10 µm. C. Cells expressing eGFP and/or DsRED were counted according to whether they contained aggregates or a dispersed pattern of expression of mutant and wild-type ataxin-7. Cells were transfected with wild-type target (atx-10-G-eGFP) alone; mutant target (atx7-100-A-DsRED) alone; mutant (atx7-100-A-DsRED), wild-type (atx-10-G-eGFP) and shR-NS (non-specific shRNA); mutant, wild-type and shR-P16. Cells were counted separately in the red and the green filter by collecting 3 representative images from each well and combining the total number. This was performed for each indicated combination in biological triplicate, yielding standard deviations. The bars comprise the total number of cells counted in each transfection; separated according to whether they contained aggregates (blue) or a dispersed pattern of expression (grey). Note that the decrease in expression from target vectors transfected alone to co-transfected cells is due to a promoter occlusion effect of co-expression of targets and not due to the addition of shR-NS which has no effect upon the target vectors (data not shown). Statistically significant differences in % of aggregate containing cells are indicated by corresponding p values.

Mentions: It is well documented that mutant polyQ proteins form intracellular aggregates which are associated with, if not directly causative of, disease pathogenesis [27]. In addition, it has been shown that mutant ATXN7 protein recruits wild-type ATXN7 into intracellular inclusion-like aggregates [28]. In our studies, confocal microscopy showed that while the mutant protein (CAG100) forms large distinct aggregates, expression of the wild-type protein (CAG10) alone resulted in minimal aggregate formation (Figure 5A). However, heterozygous co-expression of both wild-type and mutant atxn7 genes reproduces previous reports [28] in which the wild-type ATXN7 protein is found to co-localize with aggregates of mutant protein (Figure 5Bi–iii). Transfection of mutation-specific shR-P16 into these cells resulted not only in decreased numbers of cells with mutant protein aggregates, but also in wild-type protein normalised to a non-aggregated, more diffuse pattern of cellular localisation seen when the wild-type is transfected alone and typical of the non-disease state (Figure 5Biv–vi). To quantify these findings, the number of cells containing detectable aggregates or showing a dispersed pattern of wild-type protein localisation was determined following shR-P16 treatment and compared to that seen with the non-specific control (Figure 5C). This data shows a highly significant decrease in the number of cells expressing mutant protein aggregates in the presence of shR-P16, corroborating data from the fluorescent heterozygous assay. More importantly, these results further support the original observation that, not only does expression of both mutant and wild-type protein result in a significantly increased percentage of aggregates containing wild-type protein (p<0.05), but that treatment with shR-P16 promotes restoration of the wild-type protein expression pattern.


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)

Investigation of aggregate formation.(A) Representative confocal images of cells transfected with (i, ii) mutant (atx7-100-A-DsRED) alone; (iii, iv) wild-type (atx7-10-G-eGFP) alone. (i) Image visualized under red fluorescence, (ii) red fluorescence merged with the bright field, (iii) green fluorescence, and (iv) green fluorescence merged with the bright field. (B) Representative confocal images of cells co-transfected with wild-type and mutant expression plasmids in addition to (i–iii) shR-NS or (iv–vi) shR-P16. (i) and (iv) show images under green fluorescence to reveal wild-type protein, (ii) and (v) show images under red fluorescence to reveal mutant protein, and (iii) and (vi) show images then merged under green and red fluorescence and bright field. Scale bar represents 10 µm. C. Cells expressing eGFP and/or DsRED were counted according to whether they contained aggregates or a dispersed pattern of expression of mutant and wild-type ataxin-7. Cells were transfected with wild-type target (atx-10-G-eGFP) alone; mutant target (atx7-100-A-DsRED) alone; mutant (atx7-100-A-DsRED), wild-type (atx-10-G-eGFP) and shR-NS (non-specific shRNA); mutant, wild-type and shR-P16. Cells were counted separately in the red and the green filter by collecting 3 representative images from each well and combining the total number. This was performed for each indicated combination in biological triplicate, yielding standard deviations. The bars comprise the total number of cells counted in each transfection; separated according to whether they contained aggregates (blue) or a dispersed pattern of expression (grey). Note that the decrease in expression from target vectors transfected alone to co-transfected cells is due to a promoter occlusion effect of co-expression of targets and not due to the addition of shR-NS which has no effect upon the target vectors (data not shown). Statistically significant differences in % of aggregate containing cells are indicated by corresponding p values.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0007232-g005: Investigation of aggregate formation.(A) Representative confocal images of cells transfected with (i, ii) mutant (atx7-100-A-DsRED) alone; (iii, iv) wild-type (atx7-10-G-eGFP) alone. (i) Image visualized under red fluorescence, (ii) red fluorescence merged with the bright field, (iii) green fluorescence, and (iv) green fluorescence merged with the bright field. (B) Representative confocal images of cells co-transfected with wild-type and mutant expression plasmids in addition to (i–iii) shR-NS or (iv–vi) shR-P16. (i) and (iv) show images under green fluorescence to reveal wild-type protein, (ii) and (v) show images under red fluorescence to reveal mutant protein, and (iii) and (vi) show images then merged under green and red fluorescence and bright field. Scale bar represents 10 µm. C. Cells expressing eGFP and/or DsRED were counted according to whether they contained aggregates or a dispersed pattern of expression of mutant and wild-type ataxin-7. Cells were transfected with wild-type target (atx-10-G-eGFP) alone; mutant target (atx7-100-A-DsRED) alone; mutant (atx7-100-A-DsRED), wild-type (atx-10-G-eGFP) and shR-NS (non-specific shRNA); mutant, wild-type and shR-P16. Cells were counted separately in the red and the green filter by collecting 3 representative images from each well and combining the total number. This was performed for each indicated combination in biological triplicate, yielding standard deviations. The bars comprise the total number of cells counted in each transfection; separated according to whether they contained aggregates (blue) or a dispersed pattern of expression (grey). Note that the decrease in expression from target vectors transfected alone to co-transfected cells is due to a promoter occlusion effect of co-expression of targets and not due to the addition of shR-NS which has no effect upon the target vectors (data not shown). Statistically significant differences in % of aggregate containing cells are indicated by corresponding p values.
Mentions: It is well documented that mutant polyQ proteins form intracellular aggregates which are associated with, if not directly causative of, disease pathogenesis [27]. In addition, it has been shown that mutant ATXN7 protein recruits wild-type ATXN7 into intracellular inclusion-like aggregates [28]. In our studies, confocal microscopy showed that while the mutant protein (CAG100) forms large distinct aggregates, expression of the wild-type protein (CAG10) alone resulted in minimal aggregate formation (Figure 5A). However, heterozygous co-expression of both wild-type and mutant atxn7 genes reproduces previous reports [28] in which the wild-type ATXN7 protein is found to co-localize with aggregates of mutant protein (Figure 5Bi–iii). Transfection of mutation-specific shR-P16 into these cells resulted not only in decreased numbers of cells with mutant protein aggregates, but also in wild-type protein normalised to a non-aggregated, more diffuse pattern of cellular localisation seen when the wild-type is transfected alone and typical of the non-disease state (Figure 5Biv–vi). To quantify these findings, the number of cells containing detectable aggregates or showing a dispersed pattern of wild-type protein localisation was determined following shR-P16 treatment and compared to that seen with the non-specific control (Figure 5C). This data shows a highly significant decrease in the number of cells expressing mutant protein aggregates in the presence of shR-P16, corroborating data from the fluorescent heterozygous assay. More importantly, these results further support the original observation that, not only does expression of both mutant and wild-type protein result in a significantly increased percentage of aggregates containing wild-type protein (p<0.05), but that treatment with shR-P16 promotes restoration of the wild-type protein expression pattern.

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