Proteomics Unit, CIC bioGUNE, Derio, Bizkaia, Spain.
PLoS One. 2011;6(6):e21352. doi: 10.1371/journal.pone.0021352. Epub 2011 Jun 21.
Since RNA interference (RNAi) has the potential to discriminate between single nucleotide changes, there is growing interest in the use of RNAi as a promising therapeutical approach to target dominant disease-associated alleles. Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene have been linked to dominantly inherited Parkinson's disease (PD). We focused on three LRRK2 mutations (R1441G/C and the more prevalent G2109S) hoping to identify shRNAs that would both recognize and efficiently silence the mutated alleles preferentially over the wild-type alleles. Using a luciferase-based reporter system, we identified shRNAs that were able to specifically target the R1441G and R1441C alleles with 80% silencing efficiency. The same shRNAs were able to silence specifically mRNAs encoding either partial or full-length mutant LRRK2 fusion proteins, while having a minimal effect on endogenous wild-type LRRK2 expression when transfected in 293FT cells. Shifting of the mutant recognition site (MRS) from position 11 to other sites (4 and 16, within the 19-mer window of our shRNA design) reduced specificity and overall silencing efficiency. Developing an allele-specific RNAi of G2019S was problematic. Placement of the MRS at position 10 resulted in efficient silencing of reporters (75-80%), but failed to discriminate between mutant and wild-type alleles. Shifting of the MRS to positions 4, 5, 15, 16 increased the specificity of the shRNAs, but reduced the overall silencing efficiency. Consistent with previous reports, these data confirm that MRS placement influences both allele-specificity and silencing strength of shRNAs, while further modification to hairpin design or MRS position may lead to the development of effective G2019S shRNAs. In summary, the effective shRNA against LRRK2 R1441 alleles described herein suggests that RNAi-based therapy of inherited Parkinson's disease is a viable approach towards developing effective therapeutic interventions for this serious neurodegenerative disease.
由于 RNA 干扰 (RNAi) 具有区分单核苷酸变化的潜力，因此人们越来越感兴趣地将 RNAi 用作针对显性疾病相关等位基因的有前途的治疗方法。富含亮氨酸重复激酶 2 (LRRK2) 基因的突变与显性遗传性帕金森病 (PD) 有关。我们专注于三种 LRRK2 突变（R1441G/C 和更常见的 G2109S），希望找到既能识别又能有效地沉默突变等位基因，而对野生型等位基因选择性沉默的 shRNA。我们使用基于荧光素酶的报告系统，鉴定了能够特异性靶向 R1441G 和 R1441C 等位基因的 shRNA，其沉默效率达到 80%。相同的 shRNA 能够特异性沉默编码部分或全长突变 LRRK2 融合蛋白的 mRNA，而在转染 293FT 细胞时对内源性野生型 LRRK2 表达的影响最小。突变识别位点 (MRS) 从位置 11 转移到其他位置（4 和 16，在我们 shRNA 设计的 19 个碱基窗口内）降低了特异性和整体沉默效率。开发 G2019S 的等位基因特异性 RNAi 存在问题。将 MRS 放置在位置 10 会导致报告器的有效沉默（75-80%），但无法区分突变型和野生型等位基因。将 MRS 移至位置 4、5、15、16 会提高 shRNA 的特异性，但降低整体沉默效率。与先前的报道一致，这些数据证实 MRS 位置既影响 shRNA 的等位基因特异性和沉默强度，进一步修饰发夹设计或 MRS 位置可能会导致开发有效的 G2019S shRNA。总之，本文描述的针对 LRRK2 R1441 等位基因的有效 shRNA 表明，基于 RNAi 的遗传性帕金森病治疗是开发针对这种严重神经退行性疾病的有效治疗干预措施的可行方法。
