Genome Engineering an IPSC Model of Alzheimer's Disease

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。本提案的总体目标是将新的基因组工程和干细胞技术结合起来，以进一步了解阿尔茨海默病的遗传学和发病机制。CRISPR-Cas9系统是我们在过去一年中描述的一种新颖而便捷的基因组工程平台。我们建议使用CRISPR-Cas系统生成一个多队列GWAS研究中确定的10个易感基因中每个都有AD风险变异的等基因人类IPSC系文库。我们从迟发性AD （LOAD）和年龄匹配对照的真皮成纤维细胞中建立了14个IPSC系，这些细胞已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地分化为神经元，并且与对照组相比，其转录谱不同。在来自另一个实验室的ipsc衍生的神经祖系中，也观察到类似的表型。信息学分析表明，在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。在体内轻度认知障碍（MCI）和AD患者的大脑中也观察到类似的基因网络变化。我们的初步研究表明，REST还调节参与细胞死亡途径、AD病理和炎症/免疫反应的基因。仅存在或不存在与AD相关的遗传变异的等基因IPSC系的产生将使我们能够以高度控制的方式探索REST的作用，以及研究涉及a β代谢，tau磷酸化和神经元应激反应的其他致病机制。此外，等基因IPSCs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风险等位基因相关的炎症表型。为了阐明受影响的通路，新的高灵敏度转录组测序和蛋白质组学技术将应用于ipsc衍生的神经元和AD变体的小胶质细胞。来自等基因细胞的转录组数据将与来自宗教秩序研究的具有相同基因型的受试者的大脑RNA-seq分析相结合。该方法将识别核心调控介质和途径，目的是构建AD风险变异的网络模型。这些研究将汇集两位主要研究者和许多具有不同但互补的专业领域的合作者，以多学科方法了解AD的遗传修饰因子。

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.