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 阿尔茨海默病(AD)是一个尚未解决的重大公共卫生问题，目前还没有有效的治疗方法 或者是预防的手段。针对单个分子靶点的单一疗法，这些分子靶点已经牵涉到 病理、生化或遗传学证据显示的疾病迄今只取得了很小的成功，如果有的话。 临床试验，尽管有合理的证据表明目标参与。不同的证据表明，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，异常的免疫细胞活动，在许多情况下还包括载脂蛋白(Apo)E4，这是主要的遗传风险因素 治疗这种疾病。这些因素以及可能的其他因素可能会“合谋”导致脆弱人群的退化。 神经元群体通过复杂的相互作用，这是很难预测的现有知识。我们 假设许多不同基因被实验干扰的广泛而公正的筛查可能 阐明了这些相互作用和阿尔茨海默病相关神经变性的一般机制。 这样的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技术应该能够实现 这样的屏蔽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筛查都是在癌细胞系中进行的，这可能无法实现 忠实地概括了脑细胞的特殊功能和选择性弱点，这是最相关的 神经退行性疾病，如阿尔茨海默病。在这里，我们建议采用此方法，以实现无偏见 阿尔茨海默病相关啮齿动物模型大脑中的基因筛选。在目标1中，我们将使用CRISPR筛查来识别 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，谷氨酸是一种 可能与阿尔茨海默病、血管性痴呆和许多其他大脑疾病有关的神经毒性。在目标2中，我们 将从表达人apoE4的转基因小鼠中建立神经元/神经胶质细胞混合培养 和人类tau，用A对待它们，并以这个模型为平台进行大规模的CRISPR屏幕识别 可以阻止AD相关基因之间相互作用所导致的神经退化的基因 病原体。在目标3中，我们将使用腺相关病毒载体将这项技术推向体内 共表达人A、apoE4和tau的小鼠模型的大脑屏幕。我们将确定是否 在AD相关背景下，特定基因的敲除可以促进神经元存活。采用这项技术 在初级神经元和啮齿类动物大脑中进行大规模、无偏见的遗传筛选将提供有价值的 对科学界的指导。除此之外，我们的研究可以确定新的介体 神经退行性变，为治疗AD和相关疾病开辟了新的治疗途径。