ALS/FTD mutant C9orf72-induced genetic and nuclear pathology in iPS cell models

ALS/FTD 突变体 C9orf72 在 iPS 细胞模型中诱导遗传和核病理学

• 批准号: 8989628
• 负责人: Jeffrey D Rothstein
• 金额: $ 4.05万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-09-01 至 2018-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8989628
• 关键词: 9p21; Address; Antisense Oligonucleotide Therapy; Antisense Oligonucleotides; Astrocytes; Autopsy; Binding; Biological Markers; C9ORF72; Cell Line; Cell Nucleus; Cell model; Cells; Chromosomes; Code; DNA-Binding Proteins; Data; Development; Disease; Disease model; Down-Regulation; Drug effect disorder; Event; Fibroblasts; Functional disorder; Future; Gene Expression; Genes; Genetic; Genetic study; Genomics; Health; Human; Human Cell Line; Laboratories; Microarray Analysis; Modeling; Molecular; Motor Neurons; Mutation; Nerve Degeneration; Neuroglia; Neurons; Nuclear; Nuclear RNA; Oligodendroglia; Oligonucleotide Probes; Open Reading Frames; Pathologic; Pathology; Pathway interactions; Patients; Phenotype; Process; RNA; RNA Editing; RNA Splicing; RNA-Binding Proteins; Small Interfering RNA; Specificity; Technology; Tissue Sample; Tissues; Toxic effect; Untranslated RNA; Validation; Work; brain tissue; cell type; crosslinking and immunoprecipitation sequencing; design; drug discovery; experience; genetic profiling; human disease; human tissue; induced pluripotent stem cell; loss of function; molecular phenotype; mutant; novel; novel therapeutics; protein expression; success; therapeutic development; tool

DESCRIPTION (provided by applicant): The overall aim of this proposal is to study the pathology of the expanded hexanucleotide repeats in the C9orf72 gene using human iPS differentiated neurons and glia cells. The expanded GGGGCC hexanucleotide repeat in the non-coding region of the C9orf72 gene on chromosome 9p21 has been discovered as the cause of approximately 30-50% of familial and up to 10% of sporadic ALS cases as well as 12% of familial FTD cases, making this the most common known genetic cause of ALS/FTD to date. Expanded repeats are highly unstable and potentially yield toxic RNAs that accumulate in the nucleus and are hypothesized to cause cellular dysfunction via aberrant DNA and protein binding. Preliminary data from our laboratory confirm (GGGGCC)n nuclear RNA foci, aberrant gene expression and nuclear retention of RNA binding proteins (RBPs) in C9orf72 patient derived fibroblasts and iPS differentiated neurons. We therefore propose to elaborate on these early findings by generating an extensive genetic profile of ALS/FTD human iPS neurons and glia cells through the use of microarray and validate whether the iPS changes are relevant by comparing to human autopsy C9orf72 brain tissues. Furthermore, we will use C9orf72 iPS cell lines to investigate the RNA toxicity/pathology thru the identification of aberrant accumulation and binding of RNA binding proteins. Finally we will determine if we can abrogate C9orf72 genomic toxicity and pathology with antisense oligonucleotides already designed and validated in our laboratory. The likelihood of success will be greatly enhanced through a collaborative working relationship, the availability and experience of using iPS cells and human tissues. The extensive use of iPS cells to model disease, to cross correlate with human tissues and their use to validate ameliorative antisense therapy provides an important and possibly new direction for understanding disease pathophysiology and therapeutics development.

描述（由申请人提供）：本提案的总体目标是使用人类 iPS 分化的神经元和神经胶质细胞来研究 C9orf72 基因中扩展的六核苷酸重复的病理学。已发现染色体 9p21 上 C9orf72 基因非编码区中扩展的 GGGGCC 六核苷酸重复是约 30-50% 的家族性、高达 10% 的散发性 ALS 病例以及 12% 的家族性 FTD 病例的病因，使其成为迄今为止最常见的 ALS/FTD 遗传原因。扩展的重复序列高度不稳定，可能会产生有毒的 RNA，这些 RNA 会在细胞核中积累，并推测会通过异常的 DNA 和蛋白质结合导致细胞功能障碍。我们实验室的初步数据证实了 C9orf72 患者来源的成纤维细胞和 iPS 分化神经元中的 (GGGGCC)n 核 RNA 灶、异常基因表达和 RNA 结合蛋白 (RBP) 的核保留。因此，我们建议通过使用微阵列生成 ALS/FTD 人类 iPS 神经元和神经胶质细胞的广泛遗传谱来详细阐述这些早期发现，并通过与人类尸检 C9orf72 脑组织进行比较来验证 iPS 变化是否相关。此外，我们将使用 C9orf72 iPS 细胞系通过鉴定 RNA 结合蛋白的异常积累和结合来研究 RNA 毒性/病理学。最后，我们将确定是否可以使用我们实验室已经设计和验证的反义寡核苷酸消除 C9orf72 基因组毒性和病理学。通过协作工作关系、使用 iPS 细胞和人体组织的可用性和经验，成功的可能性将大大提高。 iPS 细胞广泛用于疾病建模、与人体组织交叉关联以及用于验证改善性反义疗法，为理解疾病病理生理学和治疗方法的开发提供了重要且可能是新的方向。