Impeding transcription of expanded microsatellite repeats using deactivated Cas9

扩增的微卫星重复序列的转录与许多人类疾病有关，包括 强直性肌营养不良（DM）、Fuch角膜内皮营养不良和C9 orf 72 ALS/FTD（C9 ALS/FTD），其中 他人消除或减少这些扩增基因座产生的RNA和蛋白质的产生， 治疗益处。在这里，我们将测试一个假设，即Cas9酶的失活形式阻碍了 在DM和C9 ALS/FTD的细胞和动物模型中，我们 先前已经观察到重复序列长度，PAM和链依赖性的重复序列丰度减少， 在将dCas 9直接靶向重复序列后，异常肌盲依赖性剪接 在DM 1细胞中挽救了模式，并且产生C9 orf 72 ALS/FTD细胞特征性的RAN肽 急剧下降。DM 1小鼠模型肌纤维中病理性含CUG的RNA灶， 通过腺相关病毒递送的dCas 9/gRNA减少。这些观察表明，转录 含有微卫星重复序列的RNA的转录比其他RNA的转录对扰动更敏感， 表明潜在可行的治疗干预策略。在本建议中，我们将评估 其中病毒递送的dCas 9/gRNA复合物可以拯救细胞中的分子、细胞和表型特征， 建立了DM 1和C9 ALS/FTD模型。HSALR模型表现出肌强直、核集中， 和人DM 1的改变的转录组特征，将用于研究DM。C9-500 BAC 转基因模型，其表现出上和下运动神经元变性、步态改变、瘫痪， 将用于研究C9 ALS/FTD。我们提出的实验将确定抑制是否 毒性重复转录的减少可以挽救疾病表型，并定义一个窗口， 有毒的RNA丰度是治疗性的。

Transcription of expanded microsatellite repeats is associated with a number of human diseases, including myotonic dystrophy (DM), Fuch's endothelial corneal dystrophy, and C9orf72 ALS/FTD (C9ALS/FTD), among others. Eliminating or reducing production of RNA and proteins arising from these expanded loci holds therapeutic benefit. Here, we will test the hypothesis that a deactivated form of the Cas9 enzyme impedes transcription across expanded microsatellite repeats, in cell and animal models of DM and C9ALS/FTD. We have previously observed a repeat length-, PAM-, and strand-dependent reduction in the abundance of repeat- containing RNAs upon targeting dCas9 directly to repeat sequences. Aberrant Muscleblind-dependent splicing patterns were rescued in DM1 cells, and production of RAN peptides characteristic of C9orf72 ALS/FTD cells was drastically decreased. Pathological CUG-containing RNA foci in DM1 mouse model muscle fibers was reduced by dCas9/gRNA delivered by adeno-associated virus. These observations suggest that transcription of microsatellite repeat-containing RNAs is more sensitive to perturbation than transcription of other RNAs, indicating potentially viable strategies for therapeutic intervention. In this proposal, we will assess the extent to which virally delivered dCas9/gRNA complexes can rescue molecular, cellular, and phenotypic features in to established models of DM1 and C9ALS/FTD. The HSALR model, which exhibits myotonia, centralized nuclei, and altered transcriptomes characteristic of human DM1, will be used to study DM. The C9-500 BAC transgenic model, which exhibits upper and lower motor neuron degeneration, altered gait, paralysis, and premature death, will be used to study C9ALS/FTD. Our proposed experiments will establish whether inhibition of toxic repeat transcription can rescue disease phenotypes, and define a window around which reduction of toxic RNA abundance is therapeutic.