Pathogenic mechanisms of C9orf72 GGGGCC repeat expansions in amyotrophic lateral sclerosis and frontotemporaldementia and development of therapeutic strategies

描述(申请人提供)：C9orf72基因非编码区扩展的GGGGCC六核苷酸重复序列最近被确认为肌萎缩侧索硬化症(ALS)和额颞部痴呆(FTD)的最常见遗传原因，这两种神经退行性疾病具有遗传和病理重叠的特点。这种扩张的致病机制尚不清楚，但初步观察表明，要么是内源性C9orf72基因功能丧失，要么是由RNA结合蛋白的隔离或通过重复相关的非ATG依赖(RAN)翻译产生异常多肽(S)介导的。在这里，我建议使用转基因小鼠模型来确定RNA介导的毒性获得和/或C9orf72功能丧失在C9orf72重复扩增导致的ALS/FTD发病机制中的作用，并开发治疗策略。我已经建立了多个BAC转基因小鼠系，表达含有不同重复长度和表达水平的人C9orf72基因，包括RNA水平相似但六核苷酸重复数在~100到~450之间的BAC转基因小鼠，以及RNA表达范围为4倍的~450重复数的BAC转基因小鼠。我还记录了在到目前为止测试的所有转基因小鼠的几个大脑区域和脊髓中包含正义和反义重复RNA的异常病理RNA焦点(4个品系)，与在人类C9orf72患者中观察到的相似，支持ALS/FTD的发病机制至少部分是由RNA介导的毒性增加驱动的。在目标1A和1B中，我将确定在表达C9orf72重复的转基因小鼠中是否出现任何年龄相关的病理(RNA焦点或重复相关的非ATG(RAN)翻译)、RNA签名变化和/或表型，以及这些病理/表型是否依赖于重复长度和/或表达。认识到C9orf72单倍体不足在ALS和FTD发病机制中的作用是由C9orf72-ALS/FTD患者组织中C9orf72 mRNAs减少支持的，我将确定C9orf72等位基因中断的小鼠C9orf72表达减少的后果(如果有)，以及C9orf72功能丧失是否加剧了转基因依赖的ALS/FTD相关表型(目标1C)。最后，我将使用转基因小鼠来识别反义寡核苷酸(ASO)，这些反义寡核苷酸可以介导携带重复扩张的C9orf72 RNA的降解，以努力开发一种治疗方法，以减少任何有毒功能获得的后果(目标2)。我相信我在这里提出的目标1和2的组合在识别C9orf72中六核苷酸扩张的疾病机制方面有很大的希望，并促进ASO输注疗法作为人类临床试验的一种方法的发展。

DESCRIPTION (provided by applicant): Expanded GGGGCC hexanucleotide repeats in a non-coding region of the C9orf72 gene were recently identified as the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), two neurodegenerative conditions with genetic and pathological overlap. The pathogenic mechanisms of this expansion are not understood, but initial observations point to either a loss of function of the endogenous C9orf72 gene, and/or a toxic gain of function of the expanded RNA, mediated either by sequestration of RNA binding proteins or by production of aberrant polypeptide(s) through repeat-associated non-ATG-dependent (RAN) translation. Here, I propose to use genetically modified mouse models to determine contribution of RNA-mediated gain of toxicity and/or C9orf72 loss of function to ALS/FTD pathogenesis caused by C9orf72 repeat expansions and to develop therapeutic strategies. I have already established multiple lines of BAC transgenic mice expressing a repeat-containing human C9orf72 gene with different repeat lengths and expression levels, including ones with a similar RNA level but with hexanucleotide repeats between ~100 and ~450, and those that have ~450 repeats with a 4-fold range of RNA expression. I have also documented abnormal, pathologic RNA foci containing both sense and antisense repeat RNAs in several brain regions and spinal cords of all transgenic mice tested so far (4 lines), similar to what have been observed in human C9orf72 patients, supporting that ALS/FTD pathogenesis is driven, at least in part, by RNA-mediated gain of toxicity. In Aim 1A & 1B, I will determine if any age-dependent pathology (RNA foci or repeat-associated non-ATG (RAN) translation), RNA signature change and/or phenotype is developed in C9orf72 repeat expressing transgenic mice and if such pathology/phenotype is repeat length and/or expression dependent. Recognizing that a contribution of C9orf72 haploinsufficiency to ALS and FTD pathogenesis is supported by reduced C9orf72 mRNAs in tissues from C9orf72-ALS/FTD patients, I will determine the consequence (if any) of diminished C9orf72 expression in mice with a disrupted C9orf72 allele and if the transgene-dependent ALS/FTD related phenotype is exacerbated by C9orf72 loss of function (Aim 1C). Finally, I will use transgenic mice to identify antisense oligonucleotides (ASOs) that mediate degradation of C9orf72 RNAs carrying repeat expansions in an effort to develop a therapeutic approach to diminish the consequences of any toxic gain of function (Aim 2). I believe that the combination of Aims 1 and 2 that I propose here have substantial promise to identify disease mechanism from hexanucleotide expansion in C9orf72 and facilitate development of ASO infusion therapy as an approach for human clinical trial.