Systematic elucidation of endosomal trafficking as a therapeutic opportunity in AD using CRISPR-based functional genomics

项目摘要/摘要 治疗阿尔茨海默病(AD)的有效的疾病修正疗法是一种迫切的、尚未得到满足的需求。对于广告 治疗要有效，很可能要针对AD发病机制中的早期事件。两条证据 指出内溶酶体转运变化在AD发病机制中的中心和早期作用：第一，几个风险 与晚发性AD(LOAD)相关的基因在内吞作用和内溶酶体转运中起作用。 有趣的是，卡奇实验室最近共同发现了贩运因子基因RAB10的一个变体，它 降低RAB10的表达，增强对AD的韧性。第二，内溶酶体的病理变化 系统，如扩大的早期内噬菌体和上调的溶酶体酶是最早的 人类阿尔茨海默病大脑的病理特征。因此，我们的中心假设是内溶酶体贩运 是AD早期干预的治疗靶点。这项拟议研究的目标是阐明具体的 治疗的目标是纠正与神经元负载风险基因相关的内溶酶体缺陷，并 从治疗上概括保护免受RAB10变种所带来的负荷。坎普曼实验室联合- 开发了一种可诱导和可逆抑制(CRISPRi)和激活的基因筛选平台 (CRISPRa)对人类细胞中的基因进行全基因组功能丧失和功能获得筛选，并在 人类IPSC来源的神经元。卡奇实验室已经建立了大量患者来源的成纤维细胞 和IPSCs，并产生了CRISPR校正的等基因控制线，使我们能够发现 IPSC来源的神经元的表型与疾病变异有关，包括内溶酶体缺陷。我们建议 将我们的创新方法结合起来，以实现两个具体目标。目标1的目标是确定治疗靶点 对于AD，它概括了保护性RAB10变体的机制。我们假设保护性的 RAB10的变异可以抵消与AD相关的内溶酶体缺陷。我们将在以下方面测试这一假设 IPSC衍生的神经元和人脑。我们发现RAB10中的保护性变异体降低了RAB10 相反，RAB10的表达在负荷脑中升高。我们将进行无偏见的基因组- 在WT iPSC来源的神经元中进行广谱CRISPRi/a筛查以识别控制RAB10水平的基因，并可能 因此成为治疗的靶点。同时，我们将进行全基因组筛查，以确定其他治疗方法 目标是RAB10中的表观复制保护性变体。我们将重点关注显示RAB10上位的热门歌曲 保护性变异体，最有可能复制人类保护性RAB10等位基因的影响 有患AD风险的个人。目标2的目标是使用我们的遗传互作图谱方法来阐明 负荷风险基因、内溶酶体途径和相关治疗靶点之间的联系。我们 将在AD患者派生的IPSC派生的小组中验证已确定的治疗靶点的潜力 大量内溶酶体和APP加工表型的神经元和等基因对照。

PROJECT SUMMARY / ABSTRACT An effective, disease-modifying treatment for Alzheimer's Disease (AD) is an urgent, unmet need. For an AD treatment to be effective, it will likely have to target early events in AD pathogenesis. Two lines of evidence point to a central and early role for changes in endolysosomal trafficking in AD pathogenesis: First, several risk genes associated with late-onset AD (LOAD) function in endocytosis and endolysosomal trafficking. Intriguingly, a variant in the trafficking factor gene RAB10 recently co-discovered by the Karch lab, which lowers RAB10 expression, confers resilience to AD. Second, pathological changes in the endolysosomal system, such as enlarged early endosomes and upregulation of lysosomal enzymes, are some of the earliest pathological hallmarks of human AD brains. Therefore, our central hypothesis is that endolysosomal trafficking is a therapeutic target for early intervention in AD. The goal of the proposed research is to elucidate specific therapeutic targets to correct endolysosomal defects associated with LOAD risk genes in neurons, and to therapeutically recapitulate protection from LOAD conferred by variants of the RAB10. The Kampmann lab co- developed a genetic screening platform enabling inducible and reversible repression (CRISPRi) and activation (CRISPRa) of genes in human cells for genome-wide loss- and gain-of-function screens, and implemented it in human iPSC-derived neurons. The Karch lab has established a large collection of patient-derived fibroblasts and iPSCs, and generated CRISPR-corrected isogenic control lines that have enabled us to uncover phenotypes in iPSC-derived neurons linked to disease variants, including endolysosomal defects. We propose to combine our innovative approaches for two Specific Aims. The goal of Aim 1 is to identify therapeutic targets for AD that recapitulate the mechanism of protective RAB10 variants. We hypothesize that the protective variants in RAB10 counteract the endolysosomal defects associated with AD. We will test this hypothesis in iPSC-derived neurons and human brains. We found that protective variants in RAB10 reduce RAB10 expression, and conversely RAB10 expression is elevated in LOAD brains. We will conduct unbiased genome- wide CRISPRi/a screens in WT iPSC-derived neurons to identify genes that control RAB10 levels and may therefore be therapeutic targets. In parallel, we will conduct genome-wide screens to identify other therapeutic targets that phenocopy protective variants in RAB10. We will focus on hits that show epistasis with the RAB10 protective variant, which are most likely to phenocopy the effect of the protective RAB10 allele in human individuals at risk for AD. The goal of Aim 2 is to use our genetic interaction mapping approach to elucidate connections between LOAD risk genes, the endolysosomal pathway, and associated therapeutic targets. We will validate the potential of the identified therapeutic targets in a panel of AD patient-derived iPSC-derived neurons and isogenic controls for an extensive array of endolysosomal and APP processing phenotypes.