Gilbert James W, Kennedy Zachary, Godinho Bruno M D C, Summers Ashley, Weiss Alexandra, Echeverria Dimas, Bramato Brianna, McHugh Nicholas, Cooper David, Yamada Ken, Hassler Matthew, Tran Hélène, Gao Fen Biao, Brown Robert H, Khvorova Anastasia
RNA Therapeutic Institute, Worcester, MA 01655, USA.
Department of Neurology, University of Massachusetts Chan Medical School, Worcester, MA 01655, USA.
Mol Ther Nucleic Acids. 2024 Jul 31;35(3):102291. doi: 10.1016/j.omtn.2024.102291. eCollection 2024 Sep 10.
A hexanucleotide (GC) repeat expansion (HRE) within intron one of is the leading genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). haploinsufficiency, formation of RNA foci, and production of dipeptide repeat (DPR) proteins have been proposed as mechanisms of disease. Here, we report the first example of disease-modifying siRNAs for driven ALS/FTD. Using a combination of reporter assay and primary cortical neurons derived from a C9-ALS/FTD mouse model, we screened a panel of more than 150 fully chemically stabilized siRNAs targeting different transcriptional variants. We demonstrate the lack of correlation between siRNA efficacy in reporter assay versus native environment; repeat-containing mRNA variants are found to preferentially localize to the nucleus, and thus mRNA accessibility and intracellular localization have a dominant impact on functional RNAi. Using a C9-ALS/FTD mouse model, we demonstrate that divalent siRNAs targeting mRNA variants specifically or non-selectively reduce the expression of mRNA and significantly reduce DPR proteins. Interestingly, siRNA silencing all mRNA transcripts was more effective in removing intranuclear mRNA aggregates than targeting only HRE-containing mRNA transcripts. Combined, these data support RNAi-based degradation of as a potential therapeutic paradigm.
内含子1内的六核苷酸（GC）重复扩增（HRE）是肌萎缩侧索硬化症（ALS）和额颞叶痴呆（FTD）的主要遗传病因。半合子不足、RNA病灶的形成以及二肽重复（DPR）蛋白的产生已被提出作为疾病机制。在此，我们报告了首个针对C9-ALS/FTD的疾病修饰性小干扰RNA（siRNA）实例。利用报告基因检测和源自C9-ALS/FTD小鼠模型的原代皮质神经元相结合的方法，我们筛选了一组超过150种针对不同C9ORF72转录变体的完全化学稳定的siRNA。我们证明了报告基因检测中的siRNA效力与天然环境之间缺乏相关性；发现含重复序列的C9ORF72 mRNA变体优先定位于细胞核，因此C9ORF72 mRNA的可及性和细胞内定位对功能性RNA干扰具有主导影响。利用C9-ALS/FTD小鼠模型，我们证明靶向C9ORF72 mRNA变体的二价siRNA特异性或非选择性地降低C9ORF72 mRNA的表达，并显著减少DPR蛋白。有趣的是，沉默所有C9ORF72 mRNA转录本的siRNA在去除核内mRNA聚集体方面比仅靶向含HRE的C9ORF72 mRNA转录本更有效。综合来看，这些数据支持基于RNA干扰的C9ORF72降解作为一种潜在的治疗模式。
