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

核苷酸重复扩增突变引起多种遗传性疾病，包括1型和2型肌强直性营养不良（DM），以及大部分额颞叶痴呆（FTD）和肌萎缩性侧索硬化症（ALS）。我们开发了一种基于细胞的筛选和二次验证方法来鉴定Dm1的小分子。我们筛选了多种小分子，并鉴定了60种新的hit化合物（肌强直营养不良抑制剂- mdis），这些化合物在二级分析中选择性地分散了DM1患者衍生细胞中易于形成核聚集体的扩增CUG重复rna，并隔离了关键的rna结合蛋白。值得注意的是，我们发现这些化合物还能消除DM2患者衍生细胞中含有CCUG重复序列的扩增RNA病灶，这表明它们可能具有治疗作用

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.