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

描述（由申请人提供）：RNA加工改变越来越被认为在多种疾病的发病机制中起着至关重要的作用，包括两种破坏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基因中六核苷酸扩增的开创性发现是家族性ALS和FTD的最常见原因，显著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，最近的证据线，包括我自己的工作，强烈支持通过从C9orf72位点双向转录的含有重复的rna的积累获得毒性。在C9orf72患者的成纤维细胞、来自iPS细胞的神经元和运动皮层中RNA变化的独立报告提供了证据，表明RNA加工失调伴随着C9orf72疾病。然而，目前尚不清楚这些RNA改变是由隔离在RNA病灶中的一个或多个RNA结合蛋白的功能破坏诱导的，还是由另一种细胞毒性机制诱导的，例如最近在C9orf72患者中报道的扩增RNA翻译的二肽重复蛋白的惊人积累。在这个项目中，我建议在mRNA加工中定义一组改变，这些改变描述了疾病依赖的特征，并将作为功能读数来区分当前关于C9orf72 ALS/FTD的疾病机制假设。通过使用优化的基因组方法来精确识别剪接和表达变化，以及专门设计人类神经元来单独探索目前提出的毒性机制，我的团队将确定哪些因素驱动C9orf72疾病中RNA加工改变的出现。特别是，我们将测试假设，即RNA改变是由正义或反义重复RNA诱导的。我们还将使用功能筛选来确定C9orf72相关RNA改变是否由一个或多个RNA结合蛋白活性降低驱动，包括最近发现的与C9orf72扩增重复序列相互作用的蛋白质。最后，我的团队将使用C9orf72分子特征来筛选治疗性化合物。事实上，我们和其他人已经确定，靶向C9orf72转录物的反义寡核苷酸（ASOs）可以有效地减少患者细胞中的病理性RNA灶。我现在建议确定从正义方向或反义方向转录的扩增RNA的降解（或两者都有）对于恢复与C9orf72疾病相关的RNA加工改变是否必要，这一方法可能为设计在C9orf72 ALS/FTD患者中使用ASOs的临床试验提供重要信息。疾病相关的RNA特征也将作为读数来筛选直接来源于患者成纤维细胞的神经元中的新的治疗化合物。事实上，我们将不再使用单一的靶RNA来确定药物疗效，而是对大量受C9orf72扩增干扰的基因进行定量分析，以确定能够干预疾病相关途径的小分子，从而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.