Development and validation of AAV vectors to manipulate specific neuronal subtypes and circuits involved in epilepsy and psychiatric disorders across mammalian species.

开发和验证 AAV 载体，以操纵哺乳动物物种中与癫痫和精神疾病有关的特定神经元亚型和回路。

• 批准号: 10001022
• 负责人: Benjamin E Deverman
• 金额: $ 224.04万
• 依托单位: BROAD INSTITUTE, INC.
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-08-22 至 2022-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10001022
• 关键词: Animals; Biological Models; Blood; Blood - brain barrier anatomy; Brain; Callithrix; Capsid; Carrying Capacities; Cells; Collaborations; Complex; Corpus striatum structure; Data; Dependovirus; Development; Disease; Engineering; Enhancers; Enterobacteria phage P1 Cre recombinase; Epilepsy; Evolution; Exhibits; Functional disorder; Gene Expression; Genes; Genetic; Genetic Enhancer Element; Human; Individual; Injections; Institutes; Interneurons; Intravenous; Macaca; Maps; Mental disorders; Methods; Modeling; Mus; Nature; Neurons; Organoids; Performance; Phase; Population; Prosencephalon; Rabies; Reagent; Recombinant adeno-associated virus (rAAV); Recombinants; Regulator Genes; Regulatory Element; Safety; Specificity; Target Populations; Technology; Testing; Transgenic Mice; Validation; Viral; Virus; Visual Perception; adeno-associated viral vector; base; brain cell; brain circuitry; cell type; cholinergic; expression vector; high throughput screening; human disease; human model; in vitro Model; in vivo; induced pluripotent stem cell; neuroregulation; nonhuman primate; novel; optogenetics; screening; selective expression; sensor; success; transgene expression; vector

PROJECT SUMMARY In this proposal we aim to identify gene regulatory elements that permit the targeting and manipulation of brain circuit models of human brain function. Gaining genetic access to specific neuron populations in nontransgenic animals and humans would enable targeted circuit modulation for hypothesis testing and provide a means to evaluate the safety and efficacy of circuit modulation for the treatment of epilepsy and psychiatric disorders. Our approach capitalizes on our combined expertise in the development and maturation of brain cell-types and circuits (Gord Fishell), identification of CIS-regulatory elements that function across species (Jordane Dimidschstein) and AAV engineering combined with large-scale screening methods (Ben Deverman). Our efforts will benefit from an ongoing collaboration with John Reynolds at the Salk Institute on observation and manipulation of cortical circuits during complex visual perception tasks. This project will build upon success that we and others have had in identifying gene regulatory elements that enable cell type-restricted gene expression when used within recombinant adeno-associated virus (AAV) vectors. Identifying additional enhancer sequences that function in the context of the limited carrying capacity of AAV has been slow due to the limited success rate and low throughput nature of these efforts. Here we aim to apply a novel high-throughput screening approach for the rapid identification of a suite of enhancers that enable the study and manipulation of genetically defined cell types and circuits across species. Our preliminary data demonstrates that our enhancer identification strategy can yield novel and highly specific enhancers that restrict expression to target populations. In addition, we have demonstrated that it is possible to use the engineered AAV-PHP.eB capsid to screen enhancers across the brain with a single noninvasive injection. These successes have highlighted the need for more rapid and comprehensive assessment of putative enhancers. In the UH3 portion of this proposal we will examine the tolerance to neuronal activity manipulation within the target neuronal populations in several species. We will also apply the AAV-enhancer viruses for querying disease-related circuits using Rabies tracing in conjunction with optogenetics. This proposal will be transformative in devising methods to target and manipulate the brain activity of specific neuronal cell populations across species, including human cell-derived organoids.

项目摘要 在此提案中，我们旨在确定允许大脑靶向和操纵的基因调节元素 人脑功能的电路模型。在非转基因中获得对特定神经元种群的遗传获取 动物和人类将使有针对性的电路调制进行假设检验，并提供一种手段 评估电路调节治疗癫痫和精神疾病的安全性和功效。我们的 方法利用了我们在脑细胞类型的发展和成熟方面的综合专业知识和 电路（Gord Fishell），识别跨物种（Jordane）运作的顺式调节元件 Dimidschstein）和AAV工程结合了大规模筛选方法（Ben Deverman）。我们的努力 将受益于与萨尔克研究所（Salk Institute）的约翰·雷诺兹（John Reynolds）的持续合作。 在复杂的视觉感知任务中对皮层电路的操纵。这个项目将以成功为基础 我们和其他人在识别能够启用细胞类型限制基因表达的基因调节元件方面所拥有的 当在重组腺相关病毒（AAV）载体中使用时。识别其他增强器序列 由于成功率有限，在AAV的承载能力有限的情况下，该功能很慢 这些努力的低通量性质。在这里，我们的目标是采用一种新颖的高通量筛选方法 为了快速识别一组增强子，这些增强子可以使研究和操纵遗传定义 跨物种的细胞类型和电路。我们的初步数据表明，我们的增强子识别 策略可以产生新颖和高度特定的增强子，这些增强剂将表达限制在目标人群中。此外， 我们已经证明，可以使用工程的AAV-PHP.EB CAPSID来筛选跨越 带有单个无创注射的大脑。这些成功强调了需要更快的需求 对推定增强剂的全面评估。在本提案的UH3部分中，我们将研究 靶神经元种群中几种物种中神经元活性操纵的耐受性。我们也会 将AAV增强剂病毒应用于与疾病相关的电路，并使用狂犬病跟踪与 光遗传学。该建议将在设计方法和操纵大脑活动方面具有变革性 跨物种（包括人类细胞衍生的类器官）的特定神经元细胞群体的。