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

使用基于 CRISPR 的功能基因组学系统阐明内体运输作为 AD 治疗机会

基本信息

批准号：
10220769
负责人：
Martin Kampmann
金额：
$ 64.63万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-09-30 至 2023-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10220769
关键词：
Address Affect Alleles Alzheimer&apos s Disease Alzheimer&apos s disease brain Alzheimer&apos s disease patient Alzheimer&apos s disease risk Amyloid Brain CRISPR interference Cells Clustered Regularly Interspaced Short Palindromic Repeats Collection Defect Disease Early Endosome Early Intervention Economic Burden Endocytosis Enzymes Event Family Fibroblasts Genes Genetic Genetic Epistasis Genetic Screening Genome Genomic approach Goals Human Individual Induced pluripotent stem cell derived neurons Late Onset Alzheimer Disease Link Maps Nerve Degeneration Neurons Pathogenesis Pathologic Pathology Pathway interactions Patients Phenocopy Phenotype Public Health Repression Research Risk Role System Technology Testing Therapeutic Transcription Coactivator Transcription Repressor Up-Regulation Variant amyloid precursor protein processing autosomal dominant Alzheimer&apos s disease base cost functional genomics gain of function genetic makeup genetic variant genome wide screen genome-wide induced pluripotent stem cell innovation innovative technologies novel novel therapeutic intervention protective allele protective effect recruit resilience risk variant therapeutic target trafficking unpublished works

项目摘要

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.

项目概要/摘要阿尔茨海默氏病 (AD) 的有效、疾病缓解疗法是一项迫切且尚未得到满足的需求。对于广告治疗要想有效，就必须针对 AD 发病机制的早期事件。两条证据线指出内溶酶体运输变化在 AD 发病机制中的核心和早期作用：首先，几个风险与迟发性 AD (LOAD) 内吞作用和内溶酶体运输功能相关的基因。有趣的是，Karch 实验室最近共同发现了贩运因子基因 RAB10 的一个变体，该变体降低 RAB10 表达，赋予 AD 恢复能力。二、内溶酶体的病理变化系统，例如扩大的早期内体和溶酶体酶的上调，是最早的一些系统人类 AD 大脑的病理特征。因此，我们的中心假设是内溶酶体运输是AD早期干预的治疗靶点。拟议研究的目标是阐明具体的治疗目标是纠正与神经元中 LOAD 风险基因相关的内溶酶体缺陷，并治疗上概括了 RAB10 变体所赋予的 LOAD 保护作用。坎普曼实验室合作开发了一个基因筛选平台，可实现诱导和可逆抑制（CRISPRi）和激活（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 衍生的神经元。卡奇实验室建立了大量患者来源的成纤维细胞和 iPSC，并生成了 CRISPR 校正的同基因对照线，使我们能够发现 iPSC 衍生神经元的表型与疾病变异相关，包括内溶酶体缺陷。我们建议将我们的创新方法结合起来实现两个具体目标。目标 1 的目标是确定治疗靶点 AD 概括了保护性 RAB10 变体的机制。我们假设保护性 RAB10 的变异可以抵消与 AD 相关的内溶酶体缺陷。我们将在 iPSC 衍生的神经元和人脑。我们发现 RAB10 中的保护性变异会降低 RAB10 表达，相反，RAB10 表达在 LOAD 大脑中升高。我们将进行公正的基因组- 在 WT iPSC 衍生的神经元中进行广泛的 CRISPRi/a 筛选，以识别控制 RAB10 水平的基因，并可能因此成为治疗靶点。与此同时，我们将进行全基因组筛选，以确定其他治疗方法目标是 RAB10 中的表型保护性变异。我们将重点关注与 RAB10 表现出上位性的热门作品保护性变体，最有可能复制人类保护性 RAB10 等位基因的作用有 AD 风险的个体。目标 2 的目标是使用我们的遗传相互作用作图方法来阐明 LOAD 风险基因、内溶酶体途径和相关治疗靶点之间的联系。我们将验证一组 AD 患者衍生的 iPSC 衍生的治疗靶点的潜力神经元和广泛的内溶酶体和 APP 处理表型的同基因对照。