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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 LRRK2 G2019S-targeting shRNAs against dual-luciferase targets.A) shRNAs were designed targeting the G2019S mutant allele of LRRK2 with the G2019S mutation aligned at sequential positions from position 1–16 (P1–P16) of the antisense species. B) Dual-luciferase assays at indicated time-points with stated shRNAs targeting the G2019S LRRK2 mutant following co-transfection with wild-type (blue lines) or mutant (red lines) 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-g004: Screening of LRRK2 G2019S-targeting shRNAs against dual-luciferase targets.A) shRNAs were designed targeting the G2019S mutant allele of LRRK2 with the G2019S mutation aligned at sequential positions from position 1–16 (P1–P16) of the antisense species. B) Dual-luciferase assays at indicated time-points with stated shRNAs targeting the G2019S LRRK2 mutant following co-transfection with wild-type (blue lines) or mutant (red lines) 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: Following the successful allele-specific discrimination achieved with shRNAs targeting the A30P α-synuclein mutation, a second PD candidate mutation was chosen for shRNA screening. The LRRK2 G2019S mutation is the most common PD-linked mutation currently known and therefore represents the most attractive mutation for allele-specific silencing. This mutation leads to a G:A conversion in the LRRK2 mRNA which results in a G:U mismatch between the antisense species of targeting RNAi effectors and the wild-type allele. A pool of shRNAs was initially designed which was fully complementary to the G2019S mutant allele of LRRK2 resulting in a single G:U mismatch to the wild-type allele mutation aligned at sequential positions from p10-16 of the antisense arm (Fig. 4A). However, kinetic studies on RNAi suggest that alignments in the 5′ region of the antisense species could lead to improved discrimination of the G2019S mutation. Specifically, G:U wobbles between the targeting shRNA and wild-type allele, have been reported to strongly interfere with pairings of antisense species to mRNA when placed either 5′ or centrally in the RNAi effector sequence [22], [23]. Accordingly, shRNAs were subsequently designed with mutations at sequential positions from P1-9 of the antisense arm.


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

Sibley CR, Wood MJ - PLoS ONE (2011)

Screening of LRRK2 G2019S-targeting shRNAs against dual-luciferase targets.A) shRNAs were designed targeting the G2019S mutant allele of LRRK2 with the G2019S mutation aligned at sequential positions from position 1–16 (P1–P16) of the antisense species. B) Dual-luciferase assays at indicated time-points with stated shRNAs targeting the G2019S LRRK2 mutant following co-transfection with wild-type (blue lines) or mutant (red lines) 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-g004: Screening of LRRK2 G2019S-targeting shRNAs against dual-luciferase targets.A) shRNAs were designed targeting the G2019S mutant allele of LRRK2 with the G2019S mutation aligned at sequential positions from position 1–16 (P1–P16) of the antisense species. B) Dual-luciferase assays at indicated time-points with stated shRNAs targeting the G2019S LRRK2 mutant following co-transfection with wild-type (blue lines) or mutant (red lines) 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: Following the successful allele-specific discrimination achieved with shRNAs targeting the A30P α-synuclein mutation, a second PD candidate mutation was chosen for shRNA screening. The LRRK2 G2019S mutation is the most common PD-linked mutation currently known and therefore represents the most attractive mutation for allele-specific silencing. This mutation leads to a G:A conversion in the LRRK2 mRNA which results in a G:U mismatch between the antisense species of targeting RNAi effectors and the wild-type allele. A pool of shRNAs was initially designed which was fully complementary to the G2019S mutant allele of LRRK2 resulting in a single G:U mismatch to the wild-type allele mutation aligned at sequential positions from p10-16 of the antisense arm (Fig. 4A). However, kinetic studies on RNAi suggest that alignments in the 5′ region of the antisense species could lead to improved discrimination of the G2019S mutation. Specifically, G:U wobbles between the targeting shRNA and wild-type allele, have been reported to strongly interfere with pairings of antisense species to mRNA when placed either 5′ or centrally in the RNAi effector sequence [22], [23]. Accordingly, shRNAs were subsequently designed with mutations at sequential positions from P1-9 of the antisense arm.

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