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

C9orf72 GGGGCC重复扩增在肌萎缩侧索硬化症和额颞叶痴呆中的致病机制及治疗策略的开发

• 批准号: 8835911
• 负责人: Jie Jiang
• 金额: $ 5.33万
• 依托单位: LUDWIG INSTITUTE FOR CANCER RES LTD
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-12-01 至 2015-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8835911
• 关键词: A Mouse; Alleles; Amyotrophic Lateral Sclerosis; Antisense Oligonucleotides; Brain region; C9ORF72; Clinical Trials; Development; Dipeptides; Disease; Frontotemporal Dementia; Functional RNA; Genes; Genetic; Genetic Transcription; High-Throughput Nucleotide Sequencing; Human; Individual; Infusion procedures; Length; Mediating; Messenger RNA; Mus; Nerve Degeneration; Neurodegenerative Disorders; Pathogenesis; Pathologic; Pathology; Patients; Pattern; Phenotype; Production; RNA; RNA Processing; RNA Splicing; RNA-Binding Proteins; Spinal Cord; Testing; Therapeutic; Tissues; Toxic effect; Transgenes; Transgenic Mice; Translations; age related; efficacy testing; gain of function; loss of function; mouse model; polypeptide; protein B; public health relevance; therapeutic development

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.

描述（由申请人提供）：C9 orf 72基因的非编码区中的扩增GGGGCC六核苷酸重复序列最近被确定为肌萎缩侧索硬化症（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，但初步观察表明内源性C9 orf 72基因功能丧失，和/或扩增RNA功能的毒性获得，其通过RNA结合蛋白的螯合或通过重复相关的非ATG依赖性（RAN）翻译产生异常多肽介导。 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C9 orf 72功能丧失对C9 orf 72重复扩增引起的ALS/FTD发病机制的贡献，并制定治疗策略。我已经建立了多个BAC转基因小鼠品系，这些小鼠表达具有不同重复长度和表达水平的含重复的人C9 orf 72基因，包括具有相似RNA水平但具有约100和约450之间的六核苷酸重复的小鼠，以及具有约450个重复且RNA表达范围为4倍的小鼠。我还记录了迄今为止检测的所有转基因小鼠（4系）的几个脑区和脊髓中含有正义和反义重复RNA的异常病理性RNA病灶，与在人C9 orf 72患者中观察到的结果相似，支持ALS/FTD发病机制至少部分由RNA介导的毒性获得驱动。在目标1A和1B中，我将确定在C9 orf 72重复表达转基因小鼠中是否出现任何年龄依赖性病理（RNA病灶或重复相关的非ATG（RAN）翻译）、RNA特征改变和/或表型，以及这种病理/表型是否是重复长度和/或表达依赖性的。认识到C9 orf 72-ALS/FTD患者组织中C9 orf 72 mRNA减少支持C9 orf 72单倍不足对ALS和FTD发病机制的作用，本人将确定C9 orf 72等位基因破坏小鼠中C9 orf 72表达减少的后果（如有），以及C9 orf 72功能丧失是否加重转基因依赖性ALS/FTD相关表型（目的1C）。最后，我将使用转基因小鼠来鉴定介导携带重复扩增的C9 orf 72 RNA降解的反义寡核苷酸（ASO），以努力开发一种治疗方法来减少任何毒性功能获得的后果（目的2）。 我相信，我在此提出的目标1和2的组合具有从C9 orf 72中的六核苷酸扩增中确定疾病机制的实质性希望，并促进阿索输注疗法作为人类临床试验方法的发展。