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.

项目概要 在本提案中，我们的目标是确定允许靶向和操纵大脑的基因调控元件 人脑功能的电路模型。在非转基因中获得特定神经元群体的遗传途径 动物和人类将为假设检验启用有针对性的电路调制，并提供一种方法 评估电路调制治疗癫痫和精神疾病的安全性和有效性。我们的 该方法利用了我们在脑细胞类型的发育和成熟方面的综合专业知识， 电路（Gord Fishell），跨物种功能的 CIS 调节元件的识别（Jordane Dimidschstein）和 AAV 工程与大规模筛选方法相结合（Ben Deverman）。我们的努力 将受益于与索尔克研究所约翰·雷诺兹在观察和 在复杂的视觉感知任务中操纵皮层回路。该项目将建立在成功的基础上 我们和其他人已经确定了能够实现细胞类型限制的基因表达的基因调控元件 当在重组腺相关病毒 (AAV) 载体中使用时。识别额外的增强子序列 在 AAV 运载能力有限的情况下，由于成功率有限，该功能一直进展缓慢 以及这些工作的低吞吐量性质。在这里，我们的目标是应用一种新颖的高通量筛选方法 用于快速鉴定一套增强子，从而能够研究和操作基因定义的 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鉴定 策略可以产生新颖且高度特异性的增强子，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化的 AAV-PHP.eB 衣壳来筛选增强子 通过一次无创注射进入大脑。这些成功凸显了需要更加快速和 对假定的增强剂进行综合评估。在本提案的 UH3 部分中，我们将研究 几个物种的目标神经元群体对神经元活动操纵的耐受性。我们也会 应用 AAV 增强病毒，结合狂犬病追踪来查询疾病相关回路 光遗传学。该提案将在设计目标和操纵大脑活动的方法方面具有变革性 跨物种的特定神经元细胞群，包括人类细胞衍生的类器官。