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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novel novel therapeutic intervention protective effect recruit resilience risk variant therapeutic target trafficking unpublished works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