Using RNA signatures for therapy development in neurodegeneration due to C9orf72 expansions

描述(由申请人提供)：越来越多的人认识到RNA加工改变在一系列疾病的发病机制中发挥关键作用，包括两种毁灭性的神经退行性疾病，肌萎缩侧索硬化症(ALS)和额叶颞叶痴呆(FTD)。2011年，C9orf72基因的六核苷酸扩增成为家族性ALS和FTD最常见的原因，这一开创性的发现极大地改变了我们对这些神经退行性疾病的看法。这种扩张的致病机制尚不清楚。然而，最近的一系列证据，包括我自己的工作，强烈支持通过积累从C9orf72基因座双向转录的含有重复序列的RNA来获得毒性属性。来自C9orf72患者的成纤维细胞、iPS细胞来源的神经元和运动皮质中RNA变化的独立报道提供了证据，表明RNA处理失调伴随着C9orf72疾病。然而，目前尚不清楚这些RNA改变是由一个或多个RNA结合蛋白的功能中断引起的，还是由另一种细胞毒机制引起的，例如最近在C9orf72患者中报道的从扩展RNA翻译的二肽重复蛋白的显著积累。在这个项目中，我提议定义一组mRNA处理中的变化，这些变化描述了疾病依赖的特征，并将作为功能读出，以区分当前对C9orf72 ALS/FTD潜在疾病机制的假说。通过使用为精确识别剪接和表达变化而优化的基因组方法，以及使用专门设计的人类神经元来单独探索目前提出的毒性机制，我的团队将确定哪个因素(S)导致C9orf72疾病出现RNA加工变化。特别是，我们将测试这一假设，即RNA改变是由含有正义或反义重复序列的RNA诱导的。我们还将使用功能筛查来确定C9orf72相关的RNA改变是否由一个或多个RNA结合蛋白的活性降低驱动，包括最近发现的与C9orf72扩展重复序列相互作用的蛋白质。最后，我的团队将使用C9orf72分子签名来筛选治疗性化合物。事实上，我们和其他人已经证实，针对C9orf72转录本的反义寡核苷酸(ASO)有效地减少了患者细胞中的病理性RNA焦点。我现在建议确定从正义或反义方向(或两者)转录的扩展RNA的降解是否对于恢复与C9orf72疾病相关的RNA处理改变是必要的，这一方法可能为设计一项在C9orf72 ALS/FTD患者中使用ASOS的临床试验提供关键信息。与疾病相关的RNA签名也将作为读数，在直接来自患者成纤维细胞的神经元中筛选新的治疗化合物。事实上，我们将不再使用单个靶标RNA来确定药物疗效，而是使用对一大批受到C9orf72扩展干扰的基因进行定量分析，以确定可以干预与疾病相关的途径的小分子，以恢复受影响最严重的RNA的水平。

DESCRIPTION (provided by applicant): RNA processing alterations are increasingly recognized to play a crucial role in the pathogenesis of a wide range of diseases including two devastating neurodegenerative conditions, amyotrophic lateral sclerosis (ALS) and frontal temporal dementia (FTD). The seminal discovery in 2011 of a hexanucleotide expansion in the C9orf72 gene as the most common cause of familial ALS and FTD significantly changed our perspective of these neurodegenerative diseases. The pathogenic mechanisms of this expansion are not yet understood. However recent lines of evidence, including from my own work, strongly support a gain of toxic property by accumulation of repeat-containing RNAs that are bidirectionally transcribed from the C9orf72 locus. Independent reports of RNA changes in fibroblasts, neurons derived from iPS cells and motor cortex from C9orf72 patients have offered evidence that RNA processing misregulation accompanies C9orf72 disease. However, it is still unresolved whether these RNA alterations are induced by the functional disruption of one or more RNA binding proteins sequestered into RNA foci or induced by another cytotoxic mechanism such as the striking accumulation of dipeptide repeat proteins translated from expanded RNAs that was recently reported in C9orf72 patients. In this project, I propose to define a set of alterations in mRNA processing that delineate a disease-dependent signature and that will serve as functional readouts to distinguish among current hypotheses for disease mechanism underlying C9orf72 ALS/FTD. By using genomic approaches optimized for precise identification of splicing and expression changes and human neurons specifically engineered to individually explore toxic mechanisms currently proposed, my group will determine which factor(s) drive the emergence of RNA processing alterations in C9orf72 disease. In particular, we will test the hypothesis that RNA alterations are induced either by sense or antisense repeat- containing RNAs. We will also use a functional screen to determine whether C9orf72-related RNA alterations are driven by reduced activity of one or more RNA binding proteins, including the proteins recently found to interact with the C9orf72 expanded repeats. Finally, my team will use the C9orf72 molecular signature to screen therapeutic compounds. Indeed, we and others have already established that antisense oligonucleotides (ASOs) targeting C9orf72 transcripts efficiently reduce pathological RNA foci in patient cells. I now propose to determine whether degradation of expanded RNAs transcribed from either the sense or antisense directions (or both), is necessary to restore RNA processing alterations linked to C9orf72 disease, an approach that may provide crucial information for the design of a clinical trial using ASOs in C9orf72 ALS/FTD patients. Disease-related RNA signatures will also serve as readouts to screen new therapeutic compounds in neurons directly derived from patient fibroblasts. Indeed, instead of using a single target RNA to determine drug efficacy, we will use quantitative analysis of a large panel of genes perturbed by C9orf72 expansions to identify small molecules that can intervene with disease-linked pathways to restore levels of the most affected RNAs.