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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 the heterozygous eGFP-tagged wild-type and mCherry-tagged A30P mutant α-synuclein expression plasmid.A) eGFP-tagged wild-type α-synuclein and mCherry tagged A30P mutant α-synuclein were expressed in anti-parallel directions to one another in the Het A30P plasmid. B) Expression of the Het A30P plasmid resulted in robust eGFP fluorescence and robust mCherry fluorescence at 48 hrs post-transfection in HEK-293 cells. C+E) Representative merged fluorescent images of HEK-293 cells co-transfected with the Het-A30P plasmid and indicated single (C) or double (E) mismatch shRNA construct at 48 hrs post-transfection. D+F) 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 single (D) or double (F) mismatch shRNAs targeting the A30P α-synuclein mutant 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 shRNA. * = P<0.05 relative to respective normalising control.
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pone-0026194-g002: Screening of A30P-targeting shRNAs against the heterozygous eGFP-tagged wild-type and mCherry-tagged A30P mutant α-synuclein expression plasmid.A) eGFP-tagged wild-type α-synuclein and mCherry tagged A30P mutant α-synuclein were expressed in anti-parallel directions to one another in the Het A30P plasmid. B) Expression of the Het A30P plasmid resulted in robust eGFP fluorescence and robust mCherry fluorescence at 48 hrs post-transfection in HEK-293 cells. C+E) Representative merged fluorescent images of HEK-293 cells co-transfected with the Het-A30P plasmid and indicated single (C) or double (E) mismatch shRNA construct at 48 hrs post-transfection. D+F) 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 single (D) or double (F) mismatch shRNAs targeting the A30P α-synuclein mutant 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 shRNA. * = P<0.05 relative to respective normalising control.

Mentions: The autosomal dominant A30P mutation will result in both wild-type and mutant alleles being transcribed in patient cells. The silencing of either mRNA transcript will therefore be competitive under these conditions, and it is unknown how this will affect the allele-specific outcome. To investigate this a more complex model of α-synuclein overexpression was generated in which a eGFP-tagged wild-type and a mCherry-tagged A30P mutant transcript were expressed from the same plasmid using identical promoters located side-by-side, but directing anti-parallel transcription (Fig. 2A). This heterozygous A30P (HetA30P) arrangement was considered the most favourable for transcribing two constructs from one plasmid at equal levels with minimal interference of promoter activity on neighboring transcription units. Fluorescence microscopy at 48 hrs post-transfection revealed strong eGFP and mCherry fluorescence in HEK-293 cells to create a merged yellow pattern of expression (Fig. 2B), viability assays demonstrated that this plasmid presented no detectable toxicity relative to mock-transfections (Fig. S3) whilst qPCR confirmed that levels of both wild-type and mutant transcripts were comparable (data not shown).


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 the heterozygous eGFP-tagged wild-type and mCherry-tagged A30P mutant α-synuclein expression plasmid.A) eGFP-tagged wild-type α-synuclein and mCherry tagged A30P mutant α-synuclein were expressed in anti-parallel directions to one another in the Het A30P plasmid. B) Expression of the Het A30P plasmid resulted in robust eGFP fluorescence and robust mCherry fluorescence at 48 hrs post-transfection in HEK-293 cells. C+E) Representative merged fluorescent images of HEK-293 cells co-transfected with the Het-A30P plasmid and indicated single (C) or double (E) mismatch shRNA construct at 48 hrs post-transfection. D+F) 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 single (D) or double (F) mismatch shRNAs targeting the A30P α-synuclein mutant 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 shRNA. * = P<0.05 relative to respective normalising control.
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Related In: Results  -  Collection

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pone-0026194-g002: Screening of A30P-targeting shRNAs against the heterozygous eGFP-tagged wild-type and mCherry-tagged A30P mutant α-synuclein expression plasmid.A) eGFP-tagged wild-type α-synuclein and mCherry tagged A30P mutant α-synuclein were expressed in anti-parallel directions to one another in the Het A30P plasmid. B) Expression of the Het A30P plasmid resulted in robust eGFP fluorescence and robust mCherry fluorescence at 48 hrs post-transfection in HEK-293 cells. C+E) Representative merged fluorescent images of HEK-293 cells co-transfected with the Het-A30P plasmid and indicated single (C) or double (E) mismatch shRNA construct at 48 hrs post-transfection. D+F) 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 single (D) or double (F) mismatch shRNAs targeting the A30P α-synuclein mutant 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 shRNA. * = P<0.05 relative to respective normalising control.
Mentions: The autosomal dominant A30P mutation will result in both wild-type and mutant alleles being transcribed in patient cells. The silencing of either mRNA transcript will therefore be competitive under these conditions, and it is unknown how this will affect the allele-specific outcome. To investigate this a more complex model of α-synuclein overexpression was generated in which a eGFP-tagged wild-type and a mCherry-tagged A30P mutant transcript were expressed from the same plasmid using identical promoters located side-by-side, but directing anti-parallel transcription (Fig. 2A). This heterozygous A30P (HetA30P) arrangement was considered the most favourable for transcribing two constructs from one plasmid at equal levels with minimal interference of promoter activity on neighboring transcription units. Fluorescence microscopy at 48 hrs post-transfection revealed strong eGFP and mCherry fluorescence in HEK-293 cells to create a merged yellow pattern of expression (Fig. 2B), viability assays demonstrated that this plasmid presented no detectable toxicity relative to mock-transfections (Fig. S3) whilst qPCR confirmed that levels of both wild-type and mutant transcripts were comparable (data not shown).

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