Discrimination of Parkinson-associated LRRK2 alleles by introduction of a single nucleotide mismatch into siRNA

Abstract

The protein leucine-rich repeat kinase 2 (LRRK2) is a key player in the pathogenesis of Parkinson's disease (PD). Mutations in the LRRK2 gene account for up to 10% of all autosomal dominant forms of familiar and for approximately 1–3% of sporadic PD patients. Among these mutations, G2019S is the most frequent pattern. Allele-specific gene silencing by RNA interference (RNAi) (ASP-RNAi) is therapeutically useful for specifically inhibiting the expression of disease-associated alleles without suppressing the expression of corresponding wild-type alleles. Artificial 19 siRNAs, which fully matched LRRK2 gene G2019S mutation, but contained a U:G mismatch with the wild-type allele, were examined in two eukaryotic expression systems which contained the LKKR2 full-length with either the wild-type sequence or the G2019S mutation. The changes of LRRK2 mRNA levels and protein levels in HEK-293 cells were evaluated. Our data showed that P10, P11, P14 and P16 displayed high discrimination between the mutant and wild-type. P16 inhibitory potency was the highest, causing the reductions of 84.59% in mRNA level and of 81.36% in protein level of the G2019S mutation. Our results indicated that nucleotide mismatches at the central and 3′ region of antisense-strand, which possibly increased the assembly of guide strand into RNA-induced silencing complexes (RISCs), could enhance discrimination ability of siRNA duplexes. The data showed that some siRNAs we designed could discriminate the LRRK2-G2019S mutant from the wild-type allele and provide a new insight to the development of a potent strategy in PD therapy.