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Identification of allele-specific RNAi effectors targeting genetic forms of Parkinson's disease.

Sibley CR, Wood MJ - PLoS ONE (2011)

Bottom Line: Here we generated a 'walk-through' series of RNA Pol III-expressed shRNAs targeting both the α-synuclein A30P and LRRK2 G2019S PD-associated mutations.Discrimination at this position was subsequently confirmed using siRNAs, where up to 10-fold discrimination was seen.The results suggest that RNAi-mediated silencing of PD-associated autosomal dominant genes could be a novel therapeutic approach for the treatment of the relevant clinical cases of PD in future.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, United Kingdom.

ABSTRACT
Parkinson's disease (PD) is a progressive neurological disorder affecting an estimated 5-10 million people worldwide. Recent evidence has implicated several genes that directly cause or increase susceptibility to PD. As well as advancing understanding of the genetic aetiology of PD these findings suggest new ways to modify the disease course, in some cases through genetic manipulation. Here we generated a 'walk-through' series of RNA Pol III-expressed shRNAs targeting both the α-synuclein A30P and LRRK2 G2019S PD-associated mutations. Allele-specific discrimination of the α-synuclein A30P mutation was achieved with alignments at position 10, 13 and 14 in two model systems, including a heterozygous model mimicking the disease setting, whilst 5'RACE was used to confirm stated alignments. Discrimination of the most common PD-linked LRRK2 G2019S mutation was assessed in hemizygous dual-luciferase assays and showed that alignment of the mutation opposite position 4 of the antisense species produced robust discrimination of alleles at all time points studied. Discrimination at this position was subsequently confirmed using siRNAs, where up to 10-fold discrimination was seen. The results suggest that RNAi-mediated silencing of PD-associated autosomal dominant genes could be a novel therapeutic approach for the treatment of the relevant clinical cases of PD in future.

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Determination of A30P mutation alignment with siRNA and 5′RACE.A) siRNA design with A30P mutation aligned opposite P13 of the antisense species, and secondary mismatch to the wild-type α-synuclein allele at P14. B) Representative merged fluorescent images of HEK-293 cells co-transfected with the Het-A30P plasmid siRNA-1314 at 48 hrs post-transfection. C) Quantification of wild-type α-synuclein eGFP (green bars) or A30P mutant α-synuclein mCherry (red bars) fluorescence at 48 hrs post-transfection following co-transfection of siRNA-1314 with the Het-A30P plasmid. Values represent mean ratios of normalized fluorescence +/− S.D. from n = 6. Values are normalized to respective fluorescence in cells transfected with non-specific siRNA. * = P<0.05 relative to respective normalising control. D) Visualisation of PCR products following 5′RACE using RNA from cells transfected with mCherry-tagged A30P mutant α-synuclein and stated constructs. Products were run on a 2% agarose gel. E) Expected target cleavage site for siRNA-1314. F) Sequencing of siRNA-1314 PCR product following 5′RACE. G) Mapping of 5′ adaptor ligations sites, determination of target cleavage sites and determination of A30P mutation alignments in stated constructs.
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pone-0026194-g003: Determination of A30P mutation alignment with siRNA and 5′RACE.A) siRNA design with A30P mutation aligned opposite P13 of the antisense species, and secondary mismatch to the wild-type α-synuclein allele at P14. B) Representative merged fluorescent images of HEK-293 cells co-transfected with the Het-A30P plasmid siRNA-1314 at 48 hrs post-transfection. C) Quantification of wild-type α-synuclein eGFP (green bars) or A30P mutant α-synuclein mCherry (red bars) fluorescence at 48 hrs post-transfection following co-transfection of siRNA-1314 with the Het-A30P plasmid. Values represent mean ratios of normalized fluorescence +/− S.D. from n = 6. Values are normalized to respective fluorescence in cells transfected with non-specific siRNA. * = P<0.05 relative to respective normalising control. D) Visualisation of PCR products following 5′RACE using RNA from cells transfected with mCherry-tagged A30P mutant α-synuclein and stated constructs. Products were run on a 2% agarose gel. E) Expected target cleavage site for siRNA-1314. F) Sequencing of siRNA-1314 PCR product following 5′RACE. G) Mapping of 5′ adaptor ligations sites, determination of target cleavage sites and determination of A30P mutation alignments in stated constructs.

Mentions: First, a siRNA bearing a primary mutation at p13 of the antisense species, and secondary mismatch at p14 was tested for allele-specific silencing ability to compare activity with the previously identified shRNA construct p1314 (Fig. 3A). The double mismatch siRNA-p1314 was chosen over a single mismatch variant since it was clear from results in the previous models that only one double mismatch construct was capable of allele-specific silencing whilst all other double mismatch variants failed to silence the wild-type and mutant transcripts. Thus, if siRNA-p1314 was capable of allele-specific discrimination then it is highly likely that shRNA p1314 has the primary mutation aligned correctly at p13 whilst other constructs have the mutation aligned accordingly. Transfection of siRNA-p1314 with the HetA30P plasmid demonstrated that the mutant A30P target was silenced by 79% and the wild-type target by 31% to produce a 3.4-fold (p<0.001) allele-specific discrimination (Figs. 3B, C). These results correlate closely with the 81% silencing of the mutant target and 30% reduction in wild-type target observed with shRNA p1314 at 48 hrs which strongly suggests that the alignments of mutations in the shRNAs are as predicted.


Identification of allele-specific RNAi effectors targeting genetic forms of Parkinson's disease.

Sibley CR, Wood MJ - PLoS ONE (2011)

Determination of A30P mutation alignment with siRNA and 5′RACE.A) siRNA design with A30P mutation aligned opposite P13 of the antisense species, and secondary mismatch to the wild-type α-synuclein allele at P14. B) Representative merged fluorescent images of HEK-293 cells co-transfected with the Het-A30P plasmid siRNA-1314 at 48 hrs post-transfection. C) Quantification of wild-type α-synuclein eGFP (green bars) or A30P mutant α-synuclein mCherry (red bars) fluorescence at 48 hrs post-transfection following co-transfection of siRNA-1314 with the Het-A30P plasmid. Values represent mean ratios of normalized fluorescence +/− S.D. from n = 6. Values are normalized to respective fluorescence in cells transfected with non-specific siRNA. * = P<0.05 relative to respective normalising control. D) Visualisation of PCR products following 5′RACE using RNA from cells transfected with mCherry-tagged A30P mutant α-synuclein and stated constructs. Products were run on a 2% agarose gel. E) Expected target cleavage site for siRNA-1314. F) Sequencing of siRNA-1314 PCR product following 5′RACE. G) Mapping of 5′ adaptor ligations sites, determination of target cleavage sites and determination of A30P mutation alignments in stated constructs.
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Related In: Results  -  Collection

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pone-0026194-g003: Determination of A30P mutation alignment with siRNA and 5′RACE.A) siRNA design with A30P mutation aligned opposite P13 of the antisense species, and secondary mismatch to the wild-type α-synuclein allele at P14. B) Representative merged fluorescent images of HEK-293 cells co-transfected with the Het-A30P plasmid siRNA-1314 at 48 hrs post-transfection. C) Quantification of wild-type α-synuclein eGFP (green bars) or A30P mutant α-synuclein mCherry (red bars) fluorescence at 48 hrs post-transfection following co-transfection of siRNA-1314 with the Het-A30P plasmid. Values represent mean ratios of normalized fluorescence +/− S.D. from n = 6. Values are normalized to respective fluorescence in cells transfected with non-specific siRNA. * = P<0.05 relative to respective normalising control. D) Visualisation of PCR products following 5′RACE using RNA from cells transfected with mCherry-tagged A30P mutant α-synuclein and stated constructs. Products were run on a 2% agarose gel. E) Expected target cleavage site for siRNA-1314. F) Sequencing of siRNA-1314 PCR product following 5′RACE. G) Mapping of 5′ adaptor ligations sites, determination of target cleavage sites and determination of A30P mutation alignments in stated constructs.
Mentions: First, a siRNA bearing a primary mutation at p13 of the antisense species, and secondary mismatch at p14 was tested for allele-specific silencing ability to compare activity with the previously identified shRNA construct p1314 (Fig. 3A). The double mismatch siRNA-p1314 was chosen over a single mismatch variant since it was clear from results in the previous models that only one double mismatch construct was capable of allele-specific silencing whilst all other double mismatch variants failed to silence the wild-type and mutant transcripts. Thus, if siRNA-p1314 was capable of allele-specific discrimination then it is highly likely that shRNA p1314 has the primary mutation aligned correctly at p13 whilst other constructs have the mutation aligned accordingly. Transfection of siRNA-p1314 with the HetA30P plasmid demonstrated that the mutant A30P target was silenced by 79% and the wild-type target by 31% to produce a 3.4-fold (p<0.001) allele-specific discrimination (Figs. 3B, C). These results correlate closely with the 81% silencing of the mutant target and 30% reduction in wild-type target observed with shRNA p1314 at 48 hrs which strongly suggests that the alignments of mutations in the shRNAs are as predicted.

Bottom Line: Here we generated a 'walk-through' series of RNA Pol III-expressed shRNAs targeting both the α-synuclein A30P and LRRK2 G2019S PD-associated mutations.Discrimination at this position was subsequently confirmed using siRNAs, where up to 10-fold discrimination was seen.The results suggest that RNAi-mediated silencing of PD-associated autosomal dominant genes could be a novel therapeutic approach for the treatment of the relevant clinical cases of PD in future.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, United Kingdom.

ABSTRACT
Parkinson's disease (PD) is a progressive neurological disorder affecting an estimated 5-10 million people worldwide. Recent evidence has implicated several genes that directly cause or increase susceptibility to PD. As well as advancing understanding of the genetic aetiology of PD these findings suggest new ways to modify the disease course, in some cases through genetic manipulation. Here we generated a 'walk-through' series of RNA Pol III-expressed shRNAs targeting both the α-synuclein A30P and LRRK2 G2019S PD-associated mutations. Allele-specific discrimination of the α-synuclein A30P mutation was achieved with alignments at position 10, 13 and 14 in two model systems, including a heterozygous model mimicking the disease setting, whilst 5'RACE was used to confirm stated alignments. Discrimination of the most common PD-linked LRRK2 G2019S mutation was assessed in hemizygous dual-luciferase assays and showed that alignment of the mutation opposite position 4 of the antisense species produced robust discrimination of alleles at all time points studied. Discrimination at this position was subsequently confirmed using siRNAs, where up to 10-fold discrimination was seen. The results suggest that RNAi-mediated silencing of PD-associated autosomal dominant genes could be a novel therapeutic approach for the treatment of the relevant clinical cases of PD in future.

Show MeSH
Related in: MedlinePlus