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.

项目概要/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，目前尚无有效的治疗方法 或针对与该疾病有关的单个分子靶标的单一疗法。 迄今为止，通过病理、生化或遗传证据来诊断疾病，在这方面取得的成功（如果有的话）也是微乎其微的。 尽管有合理的证据表明目标参与，但临床试验表明 AD 是一种疾病。 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，在许多情况下还有载脂蛋白 (apo) E4，这是主要的遗传风险因素 这些因素以及可能的其他因素可能“共同”导致弱势群体的退化。 通过复杂的相互作用来形成神经群体，而根据当前的知识很难预测这些相互作用。 带来了广泛且公正的筛选，其中许多不同的基因被实验破坏 揭示了这些相互作用以及 AD 相关神经变性的一般机制。 这样的屏幕已经对简单生物体的多种生物功能产生了巨大的洞察力。 我们加强了最近建立的 CRISPR/Cas9 技术应该能够进行 这种筛选还存在于被广泛认为是哺乳动物最复杂的器官——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，这可能无法 忠实地概括了最相关的脑细胞的特殊功能和选择性脆弱性 在这里，我们建议调整这种方法以实现公正。 在 AD 相关啮齿类动物模型的大脑中进行遗传筛选 在目标 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（一种形式）的基因 在目标 2 中，我们发现可能与 AD、血管性痴呆和许多其他脑部疾病相关的神经毒性。 将从表达人类 apoE4 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，用 A 处理它们，并使用该模型作为大规模 CRISPR 筛选的平台来识别 可以阻止这些 AD 相关基因之间相互作用导致的神经退行性变的基因 在目标 3 中，我们将使用腺相关病毒载体将这项技术推进到体内。 我们将在共表达人类 A、apoE4 和 tau 的小鼠模型的大脑中进行筛选。 敲除特定基因可以促进 AD 相关环境中的神经元存活。 在初级神经元和啮齿类动物大脑中进行大规模、公正的遗传筛选将提供有价值的信息 除此之外，我们的研究还可以确定新的介导因素。 神经退行性变并为 AD 及相关疾病的治疗开辟新的治疗途径。