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

描述（由申请人提供）：在C9orf72基因的非编码区域扩展的GGGGCC六核苷酸重复最近被确定为肌萎缩性侧索硬化症（ALS）和额颞叶痴呆（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。这种扩增的致病机制尚不清楚，但初步观察表明，要么是内源性C9orf72基因的功能丧失，要么是扩增的RNA的功能毒性增加，这可能是通过RNA结合蛋白的隔离或通过重复相关的非atg依赖性（RAN）翻译产生异常多肽介导的。在这里，我建议使用转基因小鼠模型来确定rna介导的毒性增加和/或C9orf72功能丧失对由C9orf72重复扩增引起的ALS/FTD发病机制的贡献，并制定治疗策略。我已经建立了多株BAC转基因小鼠，表达含有重复序列的人类C9orf72基因，它们具有不同的重复长度和表达水平，包括RNA水平相似但六核苷酸重复数在~100到~450之间的小鼠，以及具有~450重复序列但RNA表达范围为4倍的小鼠。我还记录了到目前为止所有转基因小鼠（4系）的几个脑区和脊髓中含有义和反义重复RNA的异常病理RNA灶，类似于在人类C9orf72患者中观察到的情况，支持ALS/FTD发病机制至少部分是由RNA介导的毒性获得驱动的。在Aim 1A和1B中，我将确定表达C9orf72重复序列的转基因小鼠中是否存在任何年龄依赖性病理（RNA聚焦或重复序列相关的非atg （RAN）翻译）、RNA特征变化和/或表型，以及这种病理/表型是否与重复序列长度和/或表达相关。认识到C9orf72-ALS/FTD患者组织中C9orf72 mrna的减少支持了C9orf72单倍不全对ALS和FTD发病的贡献，我将确定C9orf72等位基因中断的小鼠中C9orf72表达减少的后果（如果有的话），以及转基因依赖性ALS/FTD相关表型是否因C9orf72功能丧失而加剧（Aim 1C）。最后，我将使用转基因小鼠来鉴定介导携带重复扩增的C9orf72 rna降解的反义寡核苷酸（ASOs），以开发一种治疗方法来减少任何毒性功能增加的后果（目标2）。我相信我在这里提出的Aims 1和2的结合有很大的希望从C9orf72的己核苷酸扩增中确定疾病机制，并促进ASO输注治疗作为人体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.