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

项目摘要 在这项提案中，我们的目标是确定基因调控元件，允许靶向和操纵大脑 人脑功能的电路模型。获得非转基因动物中特定神经元群体的遗传通路 动物和人类将能够实现用于假设检验的靶向电路调制，并提供一种手段， 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 这种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调节元件的鉴定（Joreans Dimidschstein）和AAV工程结合大规模筛选方法（Ben Deverman）。我们的努力 将受益于与索尔克研究所的约翰·雷诺兹在观测方面的持续合作， 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， 我们和其他人在识别基因调控元件，使细胞类型限制的基因表达， 当在重组腺相关病毒（AAV）载体内使用时。鉴定另外的增强子序列 在AAV运载能力有限的情况下，由于成功率有限， 以及这些努力的低吞吐量性质。在这里，我们的目标是应用一种新的高通量筛选方法， 用于快速鉴定一套增强子，从而能够研究和操纵基因定义的 细胞类型和跨物种的电路。我们的初步数据表明，我们的增强子识别 策略可以产生新的和高度特异性的增强子，其将表达限制于靶群体。此外，本发明还提供了一种方法， 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， 一次非侵入性注射就能注射进大脑这些成功突出表明，需要更迅速、 综合评估推定的增强子。在本提案的UH 3部分，我们将研究 在几个物种中对靶神经元群体内的神经元活性操纵的耐受性。我们还将 应用AAV增强子病毒，使用狂犬病追踪结合 光遗传学这一提议将在设计针对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。

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.