A hexanucleotide GGGGCC expansion in intron 1 of chromosome 9 open reading frame 72 (C9orf72) gene is the most frequent cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The corresponding repeat-containing sense and antisense transcripts cause a gain of toxicity through the accumulation of RNA foci in the nucleus and deposition of dipeptide-repeat (DPR) proteins in the cytoplasm of the affected cells. We have previously reported on the potential of engineered artificial anti-C9orf72-targeting miRNAs (miC) targeting C9orf72 to reduce the gain of toxicity caused by the repeat-containing transcripts. In the current study, we tested the silencing efficacy of adeno-associated virus (AAV)5-miC in human-derived induced pluripotent stem cell (iPSC) neurons and in an ALS mouse model. We demonstrated that AAV5-miC transduces different types of neuronal cells and can reduce the accumulation of repeat-containing C9orf72 transcripts. Additionally, we demonstrated silencing of C9orf72 in both the nucleus and cytoplasm, which has an added value for the treatment of ALS and/or FTD patients. A proof of concept in an ALS mouse model demonstrated the significant reduction in repeat-containing C9orf72 transcripts and RNA foci after treatment. Taken together, these findings support the feasibility of a gene therapy for ALS and FTD based on the reduction in toxicity caused by the repeat-containing C9orf72 transcripts.

染色体9开放阅读框72（C9orf72）基因的内含子1中的六核苷酸GGGGCC扩增是肌萎缩性侧索硬化症（ALS）和额颞痴呆（FTD）的最常见原因。相应的包含重复序列的有义和反义转录物通过在细胞核中积累RNA病灶以及在受影响细胞的细胞质中沉积二肽重复（DPR）蛋白而引起毒性增加。我们对人工合成的抗潜力先前报道C9orf72靶向的miRNA（MIC）针对C9orf72减少因含有重复序列的转录本而引起的毒性增加。在当前的研究中，我们测试了腺相关病毒（AAV）5-miC在人源性诱导多能干细胞（iPSC）神经元和ALS小鼠模型中的沉默效果。我们证明了AAV5-miC可以转导不同类型的神经元细胞，并且可以减少含有重复序列的C9orf72转录物的积累。此外，我们证明了C9orf72在细胞核和细胞质中均沉默，这对于治疗ALS和/或FTD患者具有附加价值。ALS小鼠模型中的概念验证证明了包含重复序列的C9orf72的显着减少治疗后的转录本和RNA病灶。综上所述，这些发现基于减少由重复的C9orf72转录物引起的毒性，支持了ALS和FTD基因治疗的可行性。