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)、Fuchs内皮角膜营养不良和C9orf72 ALS/FTD(C9ALS/FTD)，其中 其他。消除或减少由这些扩展基因座产生的RNA和蛋白质的产生 治疗效果。在这里，我们将检验一种失活形式的Cas9酶会阻碍 在糖尿病和C9ALS/FTD的细胞和动物模型中，通过扩展的微卫星重复转录。我们 以前观察到重复序列的丰度随重复长度、PAM和链的变化而降低。 当直接以dCas9为靶点时，含有RNA的重复序列。肌盲依赖的异常剪接 在DM1细胞中挽救了模式，并产生了C9orf72 ALS/FTD细胞特有的RAN多肽 急剧减少。在DM1小鼠模型肌肉纤维中发现了病理性的含CUG的RNA灶。 由腺相关病毒携带的dCas9/gRNA还原。这些观察结果表明，转录 与其他RNA的转录相比，包含微卫星重复序列的RNA的部分对干扰更敏感， 表明潜在可行的治疗干预策略。在这项建议中，我们将评估以下程度 哪种病毒传递的dCas9/gRNA复合体可以挽救T细胞的分子、细胞和表型特征 建立了DM1和C9ALS/FTD模型。HSALR模型表现为肌强直、集中核、 以及人类DM1特有的改变的转录本，将被用于研究DM。C9-500BAC 转基因模型，表现出上下运动神经元变性，改变步态，瘫痪，和 过早死亡，将用于研究C9ALS/FTD。我们提议的实验将确定抑制 毒性重复转录可以挽救疾病表型，并定义了一个窗口，围绕着减少 大量的有毒核糖核酸具有治疗作用。

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.