A Novel Genome-Wide Screen to Identify and Characterize Regulators of ALS Disease Modifier Gene Ataxin-2

一种新型全基因组筛选，用于识别和表征 ALS 疾病修饰基因 Ataxin-2 的调节因子

基本信息

批准号：
10382981
负责人：
GARAM KIM
金额：
$ 4.68万
依托单位：
STANFORD UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-01-03 至 2023-01-02
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10382981
关键词：
ALS patients Action Potentials Alzheimer&apos s Disease Amyotrophic Lateral Sclerosis Antisense Oligonucleotides Autophagocytosis Behavior Biogenesis Biological Biological Models Blood - brain barrier anatomy Brain CRISPR screen CRISPR/Cas technology Cell model Cells Clinical Clinical Trials DNA-Binding Proteins Data Deposition Development Disease Dose Drosophila genus Drug Targeting Enhancers Enzymes FDA approved FRAP1 gene Fluorescence-Activated Cell Sorting Future Gene Targeting Genes Genetic Goals Health Human In Vitro Individual Induced pluripotent stem cell derived neurons Inherited Knock-out LY6E gene Lead Length Light Longevity Lysosomes Mammalian Cell Mediating Messenger RNA Methods Modeling Motor Neurons Mus Muscle Nature Neurodegenerative Disorders Neurons Parkinson Disease Pathogenesis Pathologic Pathology Pathway interactions Patients Pharmaceutical Preparations Pharmacology Physiological Protein Overexpression Proteins Regulation Role SCA2 protein Safety Signal Transduction Spinal Spinal Cord System Testing Therapeutic Therapeutic Intervention Toxic effect Writing Yeasts alpha synuclein amyotrophic lateral sclerosis therapy base cohort deletion library disease phenotype disorder risk druggable target fly genetic approach genetic risk factor genome wide screen genome-wide in vivo insight knock-down lifetime risk motor deficit motor impairment mouse model multicatalytic endopeptidase complex neuron loss new therapeutic target novel overexpression polyglutamine protein aggregation protein complex protein degradation risk variant screening small molecule small molecule inhibitor sporadic amyotrophic lateral sclerosis tau Proteins therapeutic target vacuolar H+-ATPase

项目摘要

Project Summary / Abstract Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder with an estimated lifetime risk of 1 in 400 individuals. ALS is clinically characterized by motor deficits, and pathologically by the selective loss of motor neurons in the brain and spinal cord, as well as deposition of ubiquitinated proteinaceous aggregates of TDP- 43. Despite the presence of TDP-43 pathology in nearly all (~97%) brains of ALS patients, the genetic underpinnings of the disease is highly heterogeneous, with ~90% being considered to be ‘sporadic,’ or having no known genetic cause. The variable nature of the underlying causes has made treatment of the disease historically difficult due to a lack of clear therapeutic targets. In the past decade, Ataxin-2 (ATXN2) has emerged as a promising therapeutic target for ALS, as a potent genetic modifier of TDP-43 aggregation and toxicity across multiple models of TDP-43 proteinopathy. Most excitingly, decreasing ATXN2 levels using anti-sense oligonucleotides (ASOs) in a mouse model of TDP-43 overexpression led to a marked rescue of motor impairments and dramatic extension of lifespan. Despite the promise of ASOs, having an orthogonal method to reduce ATXN2 levels—such as a small molecule drug that can target one of its regulators—could have immense practical benefit in the clinical context. Moreover, little remains known on how ATXN2 is normally regulated, as well as its true role in disease. To gain mechanistic insight as well as to identify additional therapeutic targets, I developed a novel FACS (fluorescence activated cell sorting)-based CRISPR/Cas9 genome-wide knockout screening strategy. The idea was to identify suppressors and enhancers of ATXN2 protein levels in a reliable and efficient way; genes that decrease ATXN2 levels upon knockout could serve as novel therapeutic targets for ALS, while those that increase ATXN2 levels upon knockout could potentially contribute to heightened risk for the disease. The screen yielded a multitude of promising hits, with many acting in same biological pathways, or sometimes encoding subunits of one protein complex. One example of this is the lysosomal vacuolar ATPase (v-ATPase), for which genes encoding nearly every subunit were found to be significant suppressors of ATXN2 protein levels in my screens. In addition to validating hits from the initial screens across multiple disease relevant systems—such as in mouse primary neurons and human iPSC-derived neurons—I will expand the analysis to delve deeper into the mechanism of how the v-ATPase is regulating ATXN2 protein levels. Moreover, given that several FDA-approved small molecule drugs are available that inhibit v-ATPase subunits, I will test their safety and efficacy in reducing ATXN2 levels and rescuing disease phenotypes in a mouse model of ALS in vivo. If this approach is successful, there are a multitude of exciting possibilities for this screening platform and overall target discovery approach that I believe could help to uncover regulators of many other neurodegenerative diseases genes (e.g., tau and Ab in FTD and Alzheimer’s Disease, a-synuclein in Parkinson’s Disease) to empower the discovery of novel therapeutic targets in contexts not limited to ALS.

项目概要/摘要肌萎缩侧索硬化症 (ALS) 是一种致命的神经退行性疾病，估计终生风险为 1 分之一 400 名 ALS 患者的临床特征为运动缺陷，病理特征为选择性运动丧失。大脑和脊髓中的神经元，以及 TDP- 泛素化蛋白质聚集体的沉积 43. 尽管几乎所有 (~97%) ALS 患者的大脑中都存在 TDP-43 病理，但遗传性该疾病的基础具有高度异质性，约 90% 被认为是“散发性”的，或者具有没有已知的遗传原因，导致该疾病的治疗方法多种多样。由于缺乏明确的治疗靶点，历史上一直很困难，在过去的十年中，Ataxin-2 (ATXN2) 出现了。作为 ALS 有前途的治疗靶点，作为 TDP-43 聚集和毒性的有效遗传修饰剂 TDP-43 蛋白病的多种模型最令人兴奋的是，使用反义降低 ATXN2 水平。 TDP-43 过表达小鼠模型中的寡核苷酸 (ASO) 显着挽救了运动能力尽管 ASO 有希望，但有一种正交方法可以减少损伤并显着延长寿命。降低 ATXN2 水平（例如可以针对其调节因子之一的小分子药物）可能会产生巨大的影响此外，对于 ATXN2 的正常调节方式仍知之甚少。及其在疾病中的真正作用，为了获得机制洞察并确定其他治疗靶点，我开发了一种基于 CRISPR/Cas9 的新型 FACS（荧光激活细胞分选）全基因组敲除筛选策略是确定可靠的 ATXN2 蛋白水平的抑制子和增强子。敲除后降低 ATXN2 水平的基因可以作为新的治疗靶点 ALS，而那些在敲除后增加 ATXN2 水平的药物可能会导致呼吸分析仪风险该疾病的筛选产生了许多有希望的成果，其中许多作用于相同的生物学途径，或者有时编码一种蛋白质复合物的亚基，其中一个例子是溶酶体液泡 ATP 酶。 (v-ATPase)，编码几乎每个亚基的基因被发现是 ATXN2 的显着抑制因子除了验证多种疾病相关的初始屏幕的命中率之外，我的屏幕中的蛋白质水平。系统——例如小鼠原代神经元和人类 iPSC 衍生神经元——我将把分析扩展到此外，考虑到 v-ATPase 如何调节 ATXN2 蛋白水平的机制。有几种 FDA 批准的小分子药物可以抑制 v-ATPase 亚基，我将测试它们的安全性以及在体内 ALS 小鼠模型中降低 ATXN2 水平和挽救疾病表型的功效。方法是成功的，这个筛选平台和总体目标有许多令人兴奋的可能性我相信这种发现方法可以帮助揭示许多其他神经退行性疾病的调节因子基因（例如 FTD 和阿尔茨海默病中的 tau 和 Ab、帕金森病中的 a-突触核蛋白）发现新的治疗靶点，不仅限于 ALS。