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.

项目摘要/摘要 阿尔茨海默氏病（AD）是一个主要未解决的公共卫生问题，没有有效的治疗方法 或预防手段。针对与已涉及的单个分子靶标的单疗法 到目前 临床试验，任务合理的目标参与证据。各种证据表明广告是 具有多因素发病机理的异质疾病，涉及不同的因子，包括淀粉样蛋白肽， tau，异常免疫细胞活性，在许多情况下，载脂蛋白（APO）E4，这是主要的遗传危险因素 为了疾病。这些和可能的其他因素可能会“共谋”导致脆弱的变性 神经元人群通过复杂的相互作用，这些相互作用难以根据当前知识进行预测。我们 假设广泛而公正的屏幕可以实验许多不同的基因可以 阐明了这些相互作用以及一般与广告相关的神经变性的机制。 这种筛选已经对简单生物的潜水员生物学功能产生了深刻的见解。 我们假设最近建立的CRISPR/CAS9技术应该使得 这些筛选也被广泛认为是哺乳动物物种的最复杂器官，即大脑。 但是，到目前为止，大多数CRISPR屏幕都是在癌细胞系中进行的，这可能无法 忠实地概括了最相关的脑细胞的专业功能和选择性漏洞 致神经退行性疾病，例如AD。在这里，我们建议适应这种方法以实现公正 与广告相关的啮齿动物模型的大脑中的遗传筛选。在AIM 1中，我们将使用CRISPR筛选来识别 可以保护培养的大鼠神经元免受谷氨酸诱导的神经变性的基因，一种形式 可能与AD，血管痴呆和许多其他脑部疾病有关的神经毒性。在AIM 2中，我们 将建立来自表达人ApoE4的转基因小鼠的混合神经元/神经胶质脑细胞培养 和人类tau，用A对其进行处理，并使用此模型作为大规模CRISPR屏幕的平台来识别 可以阻止神经退行性的基因，这些神经退行性因素与这些相关的相互作用导致 病原体。在AIM 3中，我们将使用与腺相关的病毒媒介来推进这项技术朝体内 在小鼠模型的大脑中筛选，共表达人A，apoe4和tau。我们将确定是否 特定基因的敲除可以在这种与广告相关的情况下促进神经元存活。适应这项技术 要进行大规模的，在原发性神经元和啮齿动物大脑中的公正遗传筛选将提供有价值的 对科学界的指导。除此之外，我们的研究还可以确定 神经变性和开放新的治疗途径，用于治疗AD和相关疾病。