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：
9788222
负责人：
Martin Kampmann
金额：
$ 64.28万
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/9788222
关键词：
Address Affect Alleles Alzheimer&apos s Disease Alzheimer&apos s disease risk Amyloid Brain Brain Diseases 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 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/Corepressor Up-Regulation Variant amyloid precursor protein processing base cost functional genomics gain of function genetic makeup genome wide screen genome-wide induced pluripotent stem cell innovation innovative technologies novel novel therapeutic intervention protective effect recruit resilience risk variant therapeutic target trafficking unpublished works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.

项目摘要 /摘要对阿尔茨海默氏病（AD）进行有效的，改良疾病的治疗是一种紧急，未满足的需求。广告治疗以有效，它可能必须针对AD发病机理的早期事件。两条证据指向腹膜体贩运变化在AD发病机理中的中心和早期作用：首先，几种风险与内吞作用和内溶液性贩运中的迟发性AD（负载）功能相关的基因。有趣的是，Karch Lab最近共同发现的运输因子基因RAB10的一种变体降低Rab10的表达，赋予对AD的弹性。其次，内溶液体的病理变化系统，例如扩大的早期内体和溶酶体酶的上调，是最早的人类广告大脑的病理标志。因此，我们的中心假设是内溶性贩运是早期干预AD的治疗目标。拟议的研究的目的是阐明特定纠正与神经元中负载风险基因相关的内溶性缺陷的治疗靶标，以及在治疗上概括了由Rab10变体赋予的载荷的保护。坎普曼实验室共同开发了一个遗传筛查平台，可实现可诱导和可逆的抑制（CRISPRI）和激活（CRISPRA）在人类细胞中用于全基因组损失和功能获得筛查的基因，并将其实施人IPSC衍生的神经元。 Karch Lab已建立了大量患者衍生的成纤维细胞和IPSC，并产生了通过CRISPR校正的等源性控制线，使我们能够发现 IPSC衍生的神经元的表型与疾病变体有关，包括内溶性缺陷。我们建议将我们的创新方法结合在一起，以实现两个特定目标。目标1的目标是确定治疗靶标对于概括保护性RAB10变体机理的AD。我们假设保护性 Rab10中的变体抵消与AD相关的内溶性缺陷。我们将在 IPSC衍生的神经元和人类大脑。我们发现Rab10中的保护性变体降低了Rab10 在负载大脑中，表达和反相反的RAB10表达升高。我们将进行公正的基因组 - WT IPSC衍生的神经元中的宽CRISPRI/A屏幕，以识别控制RAB10水平的基因因此是治疗靶标。同时，我们将进行全基因组筛选以识别其他治疗性靶向Rab10中的表观复制保护变体。我们将专注于显示与rab10的上毒的命中保护性变体，最有可能表现为人类保护性Rab10的效果有AD风险的个人。目标2的目的是使用我们的遗传互动映射方法来阐明负载风险基因，内溶性途径和相关治疗靶标之间的连接。我们将验证一组AD患者衍生的IPSC衍生的鉴定的治疗靶标的潜力神经元和同源控制，用于广泛的内溶液体和应用程序处理表型。