Genome Engineering an IPSC Model of Alzheimer's Disease

阿尔茨海默病的基因组工程 IPSC 模型

• 批准号: 8756257
• 负责人: GEORGE M CHURCH
• 金额: $ 209.59万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-08-15 至 2019-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8756257
• 关键词: Affect; Age; Alzheimer' s Disease; Alzheimer' s Disease Pathway; Alzheimer' s disease risk; Amyloid beta-Protein; Area; Astrocytes; Brain; Cell Death; Cell Line; Cell model; Cells; Clustered Regularly Interspaced Short Palindromic Repeats; Data; Dermal; Disease Pathway; Down-Regulation; Fibroblasts; Gene Expression Profile; Generations; Genes; Genetic; Genetic Predisposition to Disease; Genome engineering; Genotype; Goals; Health; Human; Immune; Immune response; Individual; Inflammatory; Informatics; Laboratories; Late Onset Alzheimer Disease; Lead; Libraries; Mediator of activation protein; Metabolism; Microglia; Mutation; Neuronal Differentiation; Neurons; Pathogenesis; Pathology; Pathway interactions; Patients; Phenotype; Principal Investigator; Proteome; Proteomics; Religion and Spirituality; Resources; Role; Susceptibility Gene; System; Technology; Testing; Therapeutic; Variant; biological adaptation to stress; cohort; genetic variant; genome wide association study; human stem cells; in vivo; induced pluripotent stem cell; insight; interdisciplinary approach; mild cognitive impairment; nerve stem cell; network models; neuroregulation; novel; risk variant; stem cell technology; tau phosphorylation; therapeutic target; transcription factor REST; transcriptome sequencing

DESCRIPTION (provided by applicant): The identification of an expanding number of genetic susceptibility loci for Alzheimer's disease (AD) provides an opportunity for new mechanistic and therapeutic insights. The overall goal of this proposal is to bring together new genome engineering and stem cell technology to further our understanding of AD genetics and pathogenesis. The CRISPR-Cas9 system is a novel and facile platform for genome engineering that was described by us in the past year. We propose using the CRISPR-Cas system to generate a library of isogenic human IPSC lines with AD risk variants in each of the 10 susceptibility genes identified in multi-cohort GWAS studies. We have established 14 IPSC lines from dermal fibroblasts of late-onset AD (LOAD) and age-matched controls that have been differentiated to neurons and astrocytes. This led to the observation that IPSC-derived neural progenitors from LOAD patients differentiate to neurons prematurely, and differ in their transcriptional profiles when compared with controls. A similar phenotype was observed in AD vs control IPSC-derived neural progenitor lines from another laboratory. Informatic analysis implicated downregulation of a gene network controlled by the neural transcription factor REST/NRSF in AD neural progenitors. A similar change in this gene network was observed in vivo, in the brains of patients with mild cognitive impairment (MCI) and AD. Our preliminary studies suggest that REST also regulates genes involved in cell death pathways, AD pathology and inflammatory/immune responses. The generation of isogenic IPSC lines that differ only in the presence or absence of genetic variants associated with AD will enable us to explore the role of REST in a highly controlled manner, as well as investigate other pathogenic mechanisms involving Aβ metabolism, tau phosphorylation and neuronal stress responses. Furthermore, isogenic IPSCs will be differentiated into astrocytes and microglia to explore inflammatory phenotypes associated with risk alleles in the immune-related genes TREM- 2, CD33 and CR1. To elucidate the affected pathways, new high-sensitivity transcriptome sequencing and proteomic technology will be applied to IPSC-derived neurons and microglia with AD variants. Transcriptome data from isogenic cells will be integrated with RNA-seq analysis of the brain in subjects from the Religious Orders Study with the same genotypes. This approach will identify core regulatory mediators and pathways, with the goal of constructing a network model of AD risk variants. These studies will bring together two principal investigators and many collaborators with diverse but complementary areas of expertise in a multidisciplinary approach to understand genetic modifiers of AD.

描述（由申请人提供）：越来越多的阿尔茨海默病（AD）遗传易感位点的鉴定为新的机制和治疗见解提供了机会。该提案的总体目标是将新的基因组工程和干细胞技术结合在一起。为了进一步了解 AD 遗传学和发病机制，我们建议使用 CRISPR-Cas 系统来生成一个新颖且简便的基因组工程平台。在多队列 GWAS 研究中鉴定出的 10 个易感基因中每一个都具有 AD 风险变异的同基因人类 IPSC 系库 我们已经从迟发性 AD (LOAD) 的真皮成纤维细胞和年龄匹配的对照中建立了 14 个 IPSC 系。分化为神经元和星形胶质细胞这导致观察到来自 LOAD 患者的 IPSC 衍生的神经祖细胞过早分化为神经元，并且与 LOAD 患者相比，其转录谱有所不同。在来自另一个实验室的 AD 与对照 IPSC 衍生的神经祖细胞系中观察到类似的表型，表明 AD 神经祖细胞中由神经转录因子 REST/NRSF 控制的基因网络下调。我们的初步研究表明，REST 还可以调节参与细胞死亡途径、AD 病理学和炎症/免疫反应的基因的产生。仅在是否存在与 AD 相关的遗传变异方面存在差异的 IPSC 系将使我们能够以高度受控的方式探索 REST 的作用，并研究涉及 Aβ 代谢、tau 磷酸化和神经应激反应的其他致病机制。同基因 IPSC 将分化为星形胶质细胞和小胶质细胞，以探索与免疫相关基因 TREM-2、CD33 和 CR1 中的风险等位基因相关的炎症表型。高灵敏度转录组测序和蛋白质组技术将应用于具有 AD 变异的 IPSC 衍生神经元和小胶质细胞，来自同基因细胞的转录组数据将与宗教团体研究中具有相同基因型的受试者的大脑 RNA 测序分析相结合。这种方法将确定核心调节介质和途径，目标是构建 AD 风险变异的网络模型。这些研究将汇集两位主要研究人员和许多具有不同但互补的专业领域的合作者，以多学科方法来了解遗传。 AD 的修饰语。