The most common pathogenic mutation in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) is an intronic GGGGCC (G₄C₂) repeat in the chromosome 9 open reading frame 72 (C9orf72) gene. Cellular toxicity due to RNA foci and dipeptide repeat (DPR) proteins produced by the sense and antisense repeat-containing transcripts is thought to underlie the pathogenesis of both diseases.

RNA sequencing (RNA-seq) data of C9orf72-ALS patients and controls were analyzed to better understand the sequence conservation of C9orf72 in patients. MicroRNAs were developed in conserved regions to silence C9orf72 (miC), and the feasibility of different silencing approaches was demonstrated in reporter overexpression systems. In addition, we demonstrated the feasibility of a bidirectional targeting approach by expressing two concatenated miC hairpins. The efficacy of miC was confirmed by the reduction of endogenously expressed C9orf72 mRNA, in both nucleus and cytoplasm, and an ∼50% reduction of nuclear RNA foci in (G₄C₂)₄₄-expressing cells. Ultimately, two miC candidates were incorporated in adeno-associated virus vector serotype 5 (AAV5), and silencing of C9orf72 was demonstrated in HEK293T cells and induced pluripotent stem cell (iPSC)-derived neurons. These data support the feasibility of microRNA (miRNA)-based and AAV-delivered gene therapy that could alleviate the gain of toxicity seen in ALS and FTD patients.

肌萎缩侧索硬化症 (ALS) 和额颞叶痴呆 (FTD) 最常见的致病突变是9 号染色体开放阅读框 72 ( C9orf72 ) 基因中的内含子 GGGGCC (G ₄ C ₂ ) 重复序列。由含有正义和反义重复的转录物产生的RNA焦点和二肽重复(DPR)蛋白引起的细胞毒性被认为是这两种疾病的发病机制的基础。

对C9orf72 -ALS 患者和对照的 RNA 测序 (RNA-seq) 数据进行分析，以更好地了解患者中C9orf72的序列保守性。在保守区域开发了 MicroRNA 来沉默C9orf72 (miC)，并且在报告基因过表达系统中证明了不同沉默方法的可行性。此外，我们通过表达两个串联的 miC 发夹证明了双向靶向方法的可行性。miC 的功效通过细胞核和细胞质中内源表达的C9orf72 mRNA的减少以及 (G ₄ C ₂ ) ₄₄表达细胞中核 RNA 焦点减少约 50%得到证实。最终，两个 miC 候选物被整合到腺相关病毒载体血清型 5 (AAV5) 中，并且在 HEK293T 细胞和诱导多能干细胞 (iPSC) 衍生的神经元中证明了C9orf72的沉默。这些数据支持基于 microRNA (miRNA) 和 AAV 传递的基因疗法的可行性，该疗法可以减轻 ALS 和 FTD 患者的毒性增加。