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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 G2019S-targeting shRNAs incorporating secondary mismatches to wild-type LRRK2 against dual-luciferase targets.A, C and E) shRNAs were designed targeting the G2019S mutant allele of LRRK2 with the G2019S mutation aligned at stated positions in the 3′ region of the antisense species. Secondary mismatches to the wild-type allele were additionally made at indicated positions in the antisense species such that two mismatches are present to the wild-type target, and one mismatch to the G2019S mutant target. B, D and F) Dual-luciferase reporter assays at 48 hrs post-transfection with double mismatch shRNAs targeting the G2019S LRRK2 mutant following co-transfection with wild-type (dark bars) or mutant (light bars) luciferase targets. Values represent mean ratios of Renilla:Firefly luciferase +/− S.D. from n = 6. Values are normalized to cells transfected with non-specific shRNA and respective luciferase target. * = P<0.05 relative to respective normalising control.
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pone-0026194-g006: Screening of G2019S-targeting shRNAs incorporating secondary mismatches to wild-type LRRK2 against dual-luciferase targets.A, C and E) shRNAs were designed targeting the G2019S mutant allele of LRRK2 with the G2019S mutation aligned at stated positions in the 3′ region of the antisense species. Secondary mismatches to the wild-type allele were additionally made at indicated positions in the antisense species such that two mismatches are present to the wild-type target, and one mismatch to the G2019S mutant target. B, D and F) Dual-luciferase reporter assays at 48 hrs post-transfection with double mismatch shRNAs targeting the G2019S LRRK2 mutant following co-transfection with wild-type (dark bars) or mutant (light bars) luciferase targets. Values represent mean ratios of Renilla:Firefly luciferase +/− S.D. from n = 6. Values are normalized to cells transfected with non-specific shRNA and respective luciferase target. * = P<0.05 relative to respective normalising control.

Mentions: Despite allele-discrimination being evident with single-mismatch shRNAs targeting the G2019S mutation, particularly at p4, substantial silencing of the wild-type allele is also observed. Following the previous demonstration that secondary mismatches could improve the discrimination between mutant and wild-type targets in this and other reports [6], [17], a series of shRNAs containing one mismatch to the G2019S mutant allele and a secondary mismatch to the wild-type allele was initially designed to attempt to improve allele-specific discrimination of those constructs aligned in 3′ regions of the antisense species (Figs. 6A, C).


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

Sibley CR, Wood MJ - PLoS ONE (2011)

Screening of G2019S-targeting shRNAs incorporating secondary mismatches to wild-type LRRK2 against dual-luciferase targets.A, C and E) shRNAs were designed targeting the G2019S mutant allele of LRRK2 with the G2019S mutation aligned at stated positions in the 3′ region of the antisense species. Secondary mismatches to the wild-type allele were additionally made at indicated positions in the antisense species such that two mismatches are present to the wild-type target, and one mismatch to the G2019S mutant target. B, D and F) Dual-luciferase reporter assays at 48 hrs post-transfection with double mismatch shRNAs targeting the G2019S LRRK2 mutant following co-transfection with wild-type (dark bars) or mutant (light bars) luciferase targets. Values represent mean ratios of Renilla:Firefly luciferase +/− S.D. from n = 6. Values are normalized to cells transfected with non-specific shRNA and respective luciferase target. * = P<0.05 relative to respective normalising control.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3198729&req=5

pone-0026194-g006: Screening of G2019S-targeting shRNAs incorporating secondary mismatches to wild-type LRRK2 against dual-luciferase targets.A, C and E) shRNAs were designed targeting the G2019S mutant allele of LRRK2 with the G2019S mutation aligned at stated positions in the 3′ region of the antisense species. Secondary mismatches to the wild-type allele were additionally made at indicated positions in the antisense species such that two mismatches are present to the wild-type target, and one mismatch to the G2019S mutant target. B, D and F) Dual-luciferase reporter assays at 48 hrs post-transfection with double mismatch shRNAs targeting the G2019S LRRK2 mutant following co-transfection with wild-type (dark bars) or mutant (light bars) luciferase targets. Values represent mean ratios of Renilla:Firefly luciferase +/− S.D. from n = 6. Values are normalized to cells transfected with non-specific shRNA and respective luciferase target. * = P<0.05 relative to respective normalising control.
Mentions: Despite allele-discrimination being evident with single-mismatch shRNAs targeting the G2019S mutation, particularly at p4, substantial silencing of the wild-type allele is also observed. Following the previous demonstration that secondary mismatches could improve the discrimination between mutant and wild-type targets in this and other reports [6], [17], a series of shRNAs containing one mismatch to the G2019S mutant allele and a secondary mismatch to the wild-type allele was initially designed to attempt to improve allele-specific discrimination of those constructs aligned in 3′ regions of the antisense species (Figs. 6A, C).

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