Identification of Allele-Specific RNAi Effectors Targeting Genetic Forms of Parkinson's Disease

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.

帕金森病（Parkinson's disease, PD）是一种进行性神经系统疾病，全球范围内约有500万至1000万患者。近年研究证实，多种基因可直接引发帕金森病或增加其患病易感性。这些发现不仅加深了科研人员对帕金森病遗传病因学的认知，还为改变疾病进程提供了全新思路，部分路径可通过基因编辑手段实现。本研究构建了一系列经分步验证的、由RNA聚合酶III（RNA Pol III）表达的短发卡RNA（shRNAs），可同时靶向与帕金森病相关的α-突触核蛋白A30P（α-synuclein A30P）及富亮氨酸重复激酶2 G2019S（LRRK2 G2019S）两种致病突变位点。在两种模型系统（包括模拟疾病状态的杂合模型）中，通过在第10、13、14位进行序列比对，成功实现了对α-突触核蛋白A30P突变的等位基因特异性鉴别；同时借助5'末端快速扩增（5′RACE）技术验证了所采用的比对策略的有效性。针对临床最常见的帕金森病相关LRRK2 G2019S突变的鉴别效果，通过半合子双荧光素酶检测进行了系统评估。结果显示，将突变位点与反义链分子的第4位互补配对时，在所有检测时间点下均可实现稳定可靠的等位基因鉴别。后续通过小干扰RNA（siRNAs）进一步验证了该位点的鉴别效果，最高可实现10倍的等位基因区分效率。本研究结果表明，通过RNA干扰（RNAi）介导的帕金森病相关常染色体显性基因沉默，有望成为未来治疗对应临床亚型帕金森病的新型治疗策略。