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.
Highlights
Genetic mapping of hereditary Parkinson’s disease (PD) over the last 12 years has revealed sixteen chromosomal ‘‘PARK’’ loci with linkage to PD
To investigate the potential of RNA interference (RNAi) as a therapeutic strategy for mutation carriers at the route of their disease, a panel of U6-transcribed short-hairpin RNAs (shRNAs) was designed which were fully complementary to the A30P mutant allele of a-synuclein and had a single G:G mismatch to the wild-type allele
The results demonstrate that allele-specific RNAi is possible for the a-synuclein A30P and leucine-rich repeat kinase 2 (LRRK2) G2019S PD-linked mutations, with alignments p10, p13, p14 and p1314 best for the A30P mutation and p4 best for the G2019S mutation, but that the success of this approach is highly dependent on the nature of the mutation
Summary
Genetic mapping of hereditary Parkinson’s disease (PD) over the last 12 years has revealed sixteen chromosomal ‘‘PARK’’ loci with linkage to PD. The precise function and role of each of these genes in non-familial PD remains unclear since only two of these candidates were identified in recent large-scale genome-wide association studies (GWAS) [2,3]. Collectively these hereditary cases account for 5–10% of all cases of PD and offer defined therapeutic targets for those patients bearing these genetic mutations. Many genes have essential or presently unknown roles which could be eliminated by a complete silencing, potentially leading to damaging effects [5] In such settings, allele-specific silencing has the obvious advantage that some of the wild-type gene product remains, whilst the pathogenic mutant is eliminated. This allele-specific silencing approach has been exploited to target disease-linked mutations linked to frontotemporal dementia [6], Alzheimer’s disease [7], Huntington’s disease [8], amyotrophic lateral sclerosis [9], spino-cerebellar ataxia type 7 [10] and pachyonychia congenital [11]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
