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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 siRNAs against dual-luciferase targets.A) Dual-luciferase assays at 48 hrs with stated siRNAs targeting the G2019S LRRK2 mutant following co-transfection with wild-type (blue lines) or mutant (red lines) luciferase targets. B) Dual-luciferase assays at 48 hrs with siRNA p4 targeting the G2019S LRRK2 mutant at stated concentration 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-g005: Screening of LRRK2 G2019S-targeting siRNAs against dual-luciferase targets.A) Dual-luciferase assays at 48 hrs with stated siRNAs targeting the G2019S LRRK2 mutant following co-transfection with wild-type (blue lines) or mutant (red lines) luciferase targets. B) Dual-luciferase assays at 48 hrs with siRNA p4 targeting the G2019S LRRK2 mutant at stated concentration 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: In order to verify the sequence alignment of the G2019S mutation in the generated antisense species of the p4 construct, siRNAs with alignment of the mutations at p3, p4 and p5 were screened against the luciferase targets. At 48 hrs post-transfection siRNA p4 displayed a 7.7-fold (p<0.001) discrimination that was improved upon that seen with shRNA p4 at this time point (Fig 5A). In contrast siRNAs p3 and p5 displayed limited, if any, discrimination between the two alleles, again agreeing with the trends from previous shRNA data which showed alignment at p4 to be superior to these two constructs. Further, discrimination by siRNA p4 was evident using siRNA concentrations as low as 0.1 nM, and was increased to >8-fold by concentrations of siRNA greater than 1 nM in separate experiments (Fig 5B). The greatest discrimination was 10.8-fold (p<0.001) when using 10 nM siRNA, and at this concentration a 96% silencing of the mutant target was seen which was accompanied by a modest 58% silencing of the wild-type target. However, the greatest difference in target silencing was the 61% difference between the 92% silencing of the mutant target and 31% silencing of the wild-type target when using 1 nM siRNA. Collectively this data strongly suggests that the alignment of the G2019S mutation in shRNA p4 was as stated, whilst additionally demonstrating that siRNA p4 has impressive and potent discriminating ability that could be useful in future pre-clinical models of G2019S associated pathology.


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 siRNAs against dual-luciferase targets.A) Dual-luciferase assays at 48 hrs with stated siRNAs targeting the G2019S LRRK2 mutant following co-transfection with wild-type (blue lines) or mutant (red lines) luciferase targets. B) Dual-luciferase assays at 48 hrs with siRNA p4 targeting the G2019S LRRK2 mutant at stated concentration 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-g005: Screening of LRRK2 G2019S-targeting siRNAs against dual-luciferase targets.A) Dual-luciferase assays at 48 hrs with stated siRNAs targeting the G2019S LRRK2 mutant following co-transfection with wild-type (blue lines) or mutant (red lines) luciferase targets. B) Dual-luciferase assays at 48 hrs with siRNA p4 targeting the G2019S LRRK2 mutant at stated concentration 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: In order to verify the sequence alignment of the G2019S mutation in the generated antisense species of the p4 construct, siRNAs with alignment of the mutations at p3, p4 and p5 were screened against the luciferase targets. At 48 hrs post-transfection siRNA p4 displayed a 7.7-fold (p<0.001) discrimination that was improved upon that seen with shRNA p4 at this time point (Fig 5A). In contrast siRNAs p3 and p5 displayed limited, if any, discrimination between the two alleles, again agreeing with the trends from previous shRNA data which showed alignment at p4 to be superior to these two constructs. Further, discrimination by siRNA p4 was evident using siRNA concentrations as low as 0.1 nM, and was increased to >8-fold by concentrations of siRNA greater than 1 nM in separate experiments (Fig 5B). The greatest discrimination was 10.8-fold (p<0.001) when using 10 nM siRNA, and at this concentration a 96% silencing of the mutant target was seen which was accompanied by a modest 58% silencing of the wild-type target. However, the greatest difference in target silencing was the 61% difference between the 92% silencing of the mutant target and 31% silencing of the wild-type target when using 1 nM siRNA. Collectively this data strongly suggests that the alignment of the G2019S mutation in shRNA p4 was as stated, whilst additionally demonstrating that siRNA p4 has impressive and potent discriminating ability that could be useful in future pre-clinical models of G2019S associated pathology.

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