Evolving CRISPR-based platforms for the discovery of Alzheimer relevant neurodegenerative pathways

不断发展的基于 CRISPR 的平台用于发现阿尔茨海默病相关的神经退行性通路

• 批准号: 10056618
• 负责人: Lennart Mucke
• 金额: $ 51.98万
• 依托单位: J. DAVID GLADSTONE INSTITUTES
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-01 至 2022-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10056618
• 关键词: Alzheimer associated neurodegeneration; Alzheimer' s Disease; Alzheimer' s disease brain; Amyloid; Animal Model; Biochemical; Biological Process; Brain; Brain Diseases; CRISPR screen; CRISPR/Cas technology; Cancer cell line; Cell Culture Techniques; Cells; Clinical Trials; Clustered Regularly Interspaced Short Palindromic Repeats; Communities; Complex; Diploidy; Disease; Engineering; Essential Genes; Functional disorder; Genes; Genetic; Genetic Screening; Glutamates; Guide RNA; Haploidy; Hippocampus (Brain); Human; Immune; Individual; Intercistronic Region; Knock-out; Knowledge; Lead; Libraries; Light; Literature; MAPT gene; Mediator of activation protein; Methodology; Modeling; Molecular Target; Mus; Nature; Nerve Degeneration; Neurodegenerative Disorders; Neurons; Organ; Organism; Pathogenesis; Pathologic; Pathway interactions; Peptides; Population; Prevention; Proteins; Public Health; Rattus; Rodent; Rodent Model; Small Interfering RNA; Technology; Testing; Vascular Dementia; adeno-associated viral vector; age related; amyloid peptide; apolipoprotein E-4; base; brain cell; effective therapy; excitotoxicity; experimental study; gene product; genetic risk factor; genome-wide; in vivo; insight; loss of function; mouse model; nervous system disorder; neuron loss; neuronal survival; neurotoxicity; new technology; new therapeutic target; novel therapeutics; pathogen; screening; success; targeted treatment; tau Proteins; tool

Project Summary/Abstract Alzheimer's disease (AD) is a major unresolved public health problem for which there are no effective treatments or means of prevention. Monotherapies aimed at individual molecular targets that have been implicated in the disease by pathological, biochemical or genetic evidence have so far had only minimal, if any, successes in clinical trials, despite reasonable evidence for target engagement. Diverse lines of evidence suggest that AD is a heterogenous disorder with a multifactorial pathogenesis involving diverse factors, including amyloid peptides, tau, aberrant immune cell activities, and in many cases also apolipoprotein (apo) E4, the major genetic risk factor for the disease. These and possibly other factors may “conspire” to cause the degeneration of vulnerable neuronal populations through complex interactions that are difficult to predict based on current knowledge. We hypothesize that broad and unbiased screens in which many different genes are experimentally disrupted could shed light on these interactions and on the mechanisms underlying AD-related neurodegeneration in general. Such screens have already yielded tremendous insights into diverse biological functions in simple organisms. We hypothesize that the recently established CRISPR/Cas9 technology should make it possible to carry out such screens also in what is widely considered to be the most complex organ of mammalian species, the brain. However, so far, most CRISPR screens have been carried out in cancer cell lines, which probably cannot faithfully recapitulate the specialized functions and selective vulnerabilities of brain cells, which are most relevant to neurodegenerative diseases such as AD. Here we propose to adapt this methodology to enable unbiased genetic screens in brains of AD-relevant rodent models. In Aim 1, we will use CRISPR screening to identify genes that can protect cultured rat neurons against glutamate-induced neurodegeneration, a form of neurotoxicity that is of likely relevance to AD, vascular dementia, and many other brain disorders. In Aim 2, we will establish mixed neuronal/glial brain cell cultures from genetically modified mice expressing human apoE4 and human tau, treat them with A, and use this model as a platform for a large-scale CRISPR screen to identify genes that can block the neurodegeneration that results from the interaction among these AD-relevant pathogens. In Aim 3, we will use adeno-associated viral vectors to advance this technology toward in vivo screens in brains of mouse models co-expressing human A, apoE4 and tau. We will determine whether knockout of specific genes can promote neuronal survival in this AD-relevant context. Adapting this technology to perform large-scale, unbiased genetic screens in primary neurons and rodent brains will provide valuable guidance to the scientific community. Beyond that, our studies could identify new mediators of neurodegeneration and open new therapeutic avenues for the treatment of AD and related conditions.

项目总结/摘要 阿尔茨海默病（AD）是一个尚未解决的重大公共卫生问题，目前尚无有效的治疗方法 或预防手段。单一疗法针对的是与癌症相关的单个分子靶点， 迄今为止，通过病理学、生物化学或遗传学证据发现的疾病在治疗方面的成功率即使有，也是微乎其微。 临床试验，尽管有合理的证据表明目标参与。各种证据表明，AD是 一种具有多因素发病机制的异质性疾病，涉及多种因素，包括淀粉样肽， tau、异常免疫细胞活性，在许多情况下还有载脂蛋白（apo）E4（主要遗传风险因素） 来治疗这种疾病。这些因素和其他可能的因素可能“合谋”造成脆弱群体的退化， 神经元群体通过复杂的相互作用，这是难以预测的基础上目前的知识。我们 假设广泛和无偏见的筛选，其中许多不同的基因被实验破坏， 揭示了这些相互作用和AD相关神经退行性变的机制。 这种筛选已经对简单生物体的各种生物功能产生了巨大的洞察力。 我们假设，最近建立的CRISPR/Cas9技术应该可以实现 这种屏障也存在于被广泛认为是哺乳动物物种中最复杂的器官--大脑中。 然而，到目前为止，大多数CRISPR筛选都是在癌细胞系中进行的， 忠实地概括了最相关的脑细胞的专门功能和选择性弱点 神经退行性疾病如AD。在这里，我们建议采用这种方法，使公正的 在AD相关啮齿动物模型的大脑中进行遗传筛查。在目标1中，我们将使用CRISPR筛选来识别 这些基因可以保护培养的大鼠神经元免受谷氨酸诱导的神经变性， 神经毒性，可能与AD、血管性痴呆和许多其他脑部疾病相关。在目标2中， 将从表达人apoE 4的转基因小鼠中建立混合神经元/神经胶质脑细胞培养物 和人类tau蛋白，用A β处理它们，并使用这个模型作为大规模CRISPR筛选的平台， 这些基因可以阻止神经退行性变，而神经退行性变是由这些AD相关基因之间的相互作用引起的。 病原体在目标3中，我们将使用腺相关病毒载体将该技术推向体内 在共表达人ApoE、apoE 4和tau的小鼠模型的脑中进行筛选。我们将决定 特定基因的敲除可以促进AD相关背景下的神经元存活。适应这项技术 在原代神经元和啮齿动物大脑中进行大规模、无偏见的基因筛选， 指导科学界。除此之外，我们的研究可以确定新的介质， 本发明的目的是提供一种治疗AD和神经变性的新方法，并为治疗AD和相关病症开辟新的治疗途径。