Systematic identification of enhancers to target the breadth of excitatory and inhibitory neuronal cell types in the cerebral cortex

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 操纵人脑功能的脑回路模型。获得特定的基因 非转基因动物和人类的神经元群体将使靶向电路 调节假设检验，并提供一种方法来评价安全性和有效性 用于治疗癫痫和精神疾病的电路调制。我们的方法 利用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和保拉Arlotta），识别功能的CIS调节元件 跨物种（王雅婷），AAV工程结合大规模筛选方法 (Ben Deverman）和结合RNA和染色质生物学的专业知识（Jason Pastrostro）。 我们的努力也将受益于与索尔克的约翰雷诺兹的持续合作 复杂视知觉过程中皮层回路的观察和操作研究所 任务这个项目将建立在我们和其他人在识别基因方面的成功基础上。 当在细胞内使用时能够实现细胞类型限制的基因表达的调控元件 重组腺相关病毒（AAV）载体。特别是，用于增强剂的管道 我们最近的UG 3资助的发现为我们提供了系统的识别方法， 另外的增强子序列，所述增强子序列在有限的携带能力的情况下起作用， AAV。在这里，我们的目标是应用我们在研究中设计的新的高通量筛选方法。 本UG 3的过程用于快速鉴定一套增强子，使研究和 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，我们已经证明， 有可能使用工程化的AAV-PHP.eB衣壳来筛选跨脑的增强子， 一次无创注射这些成功突出表明，需要更迅速地 和对推定的增强子的综合评估。此外，我们还将检查 在几个实验中，靶神经元群体内对神经元活动操纵的耐受性 物种这一提议将在设计方法方面具有变革性， 跨物种的特定神经元细胞群的脑活动，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 and Paola Arlotta), identification of CIS-regulatory elements that function across species (Yating Wang), AAV engineering combined with large-scale screening methods (Ben Deverman) and expertise at combining RNA and chromatin biology (Jason Buenrostro). Our efforts will also 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. In particular, the pipeline for enhancer discovery stemming from our recent UG3 grant provides us with the systematic for identifying additional enhancer sequences that function in the context of the limited carrying capacity of AAV. Here we aim to apply the novel high-throughput screening approach we devised in the course of this UG3 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 addition, we will examine the tolerance to neuronal activity manipulation within the target neuronal populations in several species. 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.