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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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Screening of A30P-targeting shRNAs against full-length eGFP-tagged α-synuclein targets.A) shRNAs were designed targeting the A30P mutant allele of α-synuclein with the A30P mutation aligned at sequential positions in the 3′ region of the antisense species from positions 10–16 (P10-P16). In some shRNAs a secondary mismatch to the wild-type allele was additionally made immediately 3′ of the primary mismatch such that two mismatches are present to the wild-type target, and one mismatch to the A30P mutant target. B+D) Representative fluorescent images of HEK-293 cells co-transfected with stated eGFP-tagged α-synuclein targets and indicated single (B) or double (D) mismatch shRNA construct at 72 hrs post-transfection. C+E) Quantification of eGFP fluorescence at 72 hrs post-transfection following co-transfection of single (C) or double (E) mismatch shRNAs targeting the A30P α-synuclein mutant with wild-type (dark) or mutant (light) eGFP-tagged α-synuclein targets. Values represent mean ratios of normalized fluorescence +/− S.D. from n = 6. Values are normalized to cells transfected with non-specific shRNA and respective eGFP-tagged target. * = P<0.05 relative to respective normalising control.
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pone-0026194-g001: Screening of A30P-targeting shRNAs against full-length eGFP-tagged α-synuclein targets.A) shRNAs were designed targeting the A30P mutant allele of α-synuclein with the A30P mutation aligned at sequential positions in the 3′ region of the antisense species from positions 10–16 (P10-P16). In some shRNAs a secondary mismatch to the wild-type allele was additionally made immediately 3′ of the primary mismatch such that two mismatches are present to the wild-type target, and one mismatch to the A30P mutant target. B+D) Representative fluorescent images of HEK-293 cells co-transfected with stated eGFP-tagged α-synuclein targets and indicated single (B) or double (D) mismatch shRNA construct at 72 hrs post-transfection. C+E) Quantification of eGFP fluorescence at 72 hrs post-transfection following co-transfection of single (C) or double (E) mismatch shRNAs targeting the A30P α-synuclein mutant with wild-type (dark) or mutant (light) eGFP-tagged α-synuclein targets. Values represent mean ratios of normalized fluorescence +/− S.D. from n = 6. Values are normalized to cells transfected with non-specific shRNA and respective eGFP-tagged target. * = P<0.05 relative to respective normalising control.

Mentions: Mutations to the α-synuclein encoding SNCA gene have been reported to cause early-onset PD in an autosomal dominant manner. To investigate the potential of RNAi as a therapeutic strategy for mutation carriers at the route of their disease, a panel of U6-transcribed shRNAs was designed which were fully complementary to the A30P mutant allele of α-synuclein and had a single G:G mismatch to the wild-type allele. Based on previous reports [8], [10], shRNAs were designed with the A30P mutation aligned at sequential positions along the 3′-region of the antisense strand from p10-16, with P1 representing the most 5̀nt of the antisense species (Fig. 1A). Some studies have demonstrated that discrimination can be improved when secondary mismatches to the wild-type allele are incorporated despite this leading to a single mismatch with the targeted mutant allele [6], [17]. To test this hypothesis, a second pool of shRNAs was designed in which a single U→C swap to create a single A:C mismatch to the mutant A30P allele, and a secondary mismatch to the wild-type allele, was placed immediately 3′ of the mutation alignment in the antisense strand (Fig. 1A).


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

Sibley CR, Wood MJ - PLoS ONE (2011)

Screening of A30P-targeting shRNAs against full-length eGFP-tagged α-synuclein targets.A) shRNAs were designed targeting the A30P mutant allele of α-synuclein with the A30P mutation aligned at sequential positions in the 3′ region of the antisense species from positions 10–16 (P10-P16). In some shRNAs a secondary mismatch to the wild-type allele was additionally made immediately 3′ of the primary mismatch such that two mismatches are present to the wild-type target, and one mismatch to the A30P mutant target. B+D) Representative fluorescent images of HEK-293 cells co-transfected with stated eGFP-tagged α-synuclein targets and indicated single (B) or double (D) mismatch shRNA construct at 72 hrs post-transfection. C+E) Quantification of eGFP fluorescence at 72 hrs post-transfection following co-transfection of single (C) or double (E) mismatch shRNAs targeting the A30P α-synuclein mutant with wild-type (dark) or mutant (light) eGFP-tagged α-synuclein targets. Values represent mean ratios of normalized fluorescence +/− S.D. from n = 6. Values are normalized to cells transfected with non-specific shRNA and respective eGFP-tagged target. * = P<0.05 relative to respective normalising control.
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Related In: Results  -  Collection

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pone-0026194-g001: Screening of A30P-targeting shRNAs against full-length eGFP-tagged α-synuclein targets.A) shRNAs were designed targeting the A30P mutant allele of α-synuclein with the A30P mutation aligned at sequential positions in the 3′ region of the antisense species from positions 10–16 (P10-P16). In some shRNAs a secondary mismatch to the wild-type allele was additionally made immediately 3′ of the primary mismatch such that two mismatches are present to the wild-type target, and one mismatch to the A30P mutant target. B+D) Representative fluorescent images of HEK-293 cells co-transfected with stated eGFP-tagged α-synuclein targets and indicated single (B) or double (D) mismatch shRNA construct at 72 hrs post-transfection. C+E) Quantification of eGFP fluorescence at 72 hrs post-transfection following co-transfection of single (C) or double (E) mismatch shRNAs targeting the A30P α-synuclein mutant with wild-type (dark) or mutant (light) eGFP-tagged α-synuclein targets. Values represent mean ratios of normalized fluorescence +/− S.D. from n = 6. Values are normalized to cells transfected with non-specific shRNA and respective eGFP-tagged target. * = P<0.05 relative to respective normalising control.
Mentions: Mutations to the α-synuclein encoding SNCA gene have been reported to cause early-onset PD in an autosomal dominant manner. To investigate the potential of RNAi as a therapeutic strategy for mutation carriers at the route of their disease, a panel of U6-transcribed shRNAs was designed which were fully complementary to the A30P mutant allele of α-synuclein and had a single G:G mismatch to the wild-type allele. Based on previous reports [8], [10], shRNAs were designed with the A30P mutation aligned at sequential positions along the 3′-region of the antisense strand from p10-16, with P1 representing the most 5̀nt of the antisense species (Fig. 1A). Some studies have demonstrated that discrimination can be improved when secondary mismatches to the wild-type allele are incorporated despite this leading to a single mismatch with the targeted mutant allele [6], [17]. To test this hypothesis, a second pool of shRNAs was designed in which a single U→C swap to create a single A:C mismatch to the mutant A30P allele, and a secondary mismatch to the wild-type allele, was placed immediately 3′ of the mutation alignment in the antisense strand (Fig. 1A).

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