Development and validation of a cell-based assay system to identify novel therapeutics for C9ALS/FTD

核苷酸重复序列扩增突变导致多种遗传疾病，包括1型和2型强直性肌营养不良(DM)，以及很大一部分额颞痴呆(FTD)和肌萎缩侧索硬化症(ALS)。我们开发了一种基于细胞的筛选和二次验证分析来识别DM1的小分子。我们筛选了一系列不同的小分子，并确定了60种新的HIT化合物(强直性肌营养不良抑制剂-MDI)，这些化合物在二次检测中选择性地分散易于形成核聚集的扩展的包含CUG重复序列的RNA，并在DM1患者的衍生细胞中隔离关键的RNA结合蛋白。值得注意的是，我们发现这些化合物还分散了DM2患者来源细胞中扩展的CCUG重复序列RNA灶，这表明它们可能具有治疗作用 跨越不同的富含GC的重复扩张性疾病的可能性。以我们在DM1中获得的知识为基础 在这个项目中，我们设计了一种筛选策略，以确定C9orf72基因(c9FTD/ALS)的六核苷酸扩展引起的FTD/ALS的潜在小分子疗法。在这个项目中，我们建议开发、优化和验证一个基于细胞的检测平台来测量C9FTD/ALS疾病的关键分子参数。接下来，我们将利用这一平台来测试影响毒性CUG RNA焦点稳定性的化合物是否可以分散扩大的G4C2重复RNA焦点和减少二肽蛋白质的积累，这是导致C9FTD/ALS患者来源细胞毒性的两个原因。最后，我们将对一组不同的定制小分子化合物进行概念验证筛选，以最好地评估我们筛选平台的价值和有效性，以确定C9FTD/ALS的潜在治疗剂。就像现在一样 FTD或ALS无有效治疗，鉴定具有潜在治疗价值的小分子 为患有这些衰弱和致命疾病的人带来了希望。

Nucleotide repeat expansion mutations cause a variety of genetic disorders including myotonic dystrophy (DM) types 1 and 2, and a large fraction of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). We developed a cell-based screen and secondary validation assays to identify small molecules for Dm1. We screened a diverse collection of small molecules and identified 60 novel hit compounds (myotonic dystrophy inhibitors-MDIs) that in secondary assays selectively disperse expanded CUG repeat- containing RNAs prone to form nuclear aggregates and sequester critical RNA-binding proteins in DM1 patient’s derived cells. Significantly, we showed that these compounds also dissipate expanded CCUG repeat-containing RNA foci in DM2 patient’s derived cells, suggesting that they may hold therapeutic potential across distinct GC-rich repeat expansion diseases. Building on the knowledge gained in our DM1 project, we have designed a screening strategy to identify potential small molecule therapeutics for FTD/ALS caused by an hexanucleotide expansion in the C9orf72 gene (C9FTD/ALS). In this project, wepropose to develop, optimize and validate a cell-based assay platform to measure critical molecular parameters of C9FTD/ALS disease. Next, we will use this platform to test whether compounds that affect the stability of toxic CUG RNA foci can disperse expanded G4C2 repeat RNA foci and decrease dipeptide proteins accumulation, two causes of toxicity in C9FTD/ALS patient’s derived cells. Lastly, we will perform a proof-of-concept screen of a diverse custom set of small-molecule compounds to best assess the value and efficacy of our screening platform to identify potential therapeutic agents for C9FTD/ALS. As there is no effective treatment for FTD or ALS, the identification of small molecules of potential therapeutic value offers hope for individuals with these debilitating and fatal diseases.