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

系统鉴定增强剂以靶向大脑皮层兴奋性和抑制性神经元细胞类型的广度

• 批准号: 10512459
• 负责人: Paola Arlotta
• 金额: $ 1067.93万
• 依托单位: BROAD INSTITUTE, INC.
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-01 至 2025-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10512459
• 关键词: Adult; Animals; Atlases; Automobile Driving; Bar Codes; Biology; Birth; Brain; Brain Diseases; Brain region; Callithrix; Capsid; Carrying Capacities; Cells; Cerebral cortex; Characteristics; Chromatin; Collaborations; Communities; Complement; Complex; Coupled; Data; Data Set; Dependovirus; Development; Developmental Gene; Engineering; Enhancers; Enterobacteria phage P1 Cre recombinase; Epilepsy; Etiology; Functional disorder; Funding; Gene Expression; Genes; Genetic; Genetic Enhancer Element; Grant; Histones; Human; Injections; Institutes; Interneurons; Laboratories; Libraries; Link; Maps; Mental disorders; Methods; Mining; Morphology; Motivation; Mus; Neurons; Organism; Organoids; Plant Roots; Plasmids; Population; Positioning Attribute; Property; RNA; Recombinant adeno-associated virus (rAAV); Recombinants; Regulator Genes; Regulatory Element; Reporter; Resected; Resolution; Rodent; Safety; Specificity; System; Target Populations; Technology; Testing; Transgenic Mice; Vertebrates; Viral; Visual Perception; Work; adeno-associated viral vector; base; brain cell; brain circuitry; brain tissue; cell type; excitatory neuron; high throughput screening; hippocampal pyramidal neuron; human model; improved; in vivo; interest; molecular marker; nonhuman primate; novel; screening; selective expression; sensor; single-cell RNA sequencing; stem; success; tool; transcriptome; transcriptomics

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.

项目总结 在这项提案中，我们的目标是确定允许靶向和 人脑功能的脑回路模型的操作。获得特定基因的访问 非转基因动物和人类中的神经元群体将使靶向回路 为假设检验提供了一种方法，并提供了一种评估 用于治疗癫痫和精神障碍的电路调制。我们的方法 利用我们在脑细胞类型和成熟的发展和成熟方面的综合专业知识 电路(Gord Fishell和Paola Arlotta)，识别起作用的独联体-调节元件 跨物种(王雅婷)，AAV工程结合大规模筛查方法 (Ben Deverman)和将RNA和染色质生物学相结合的专业知识(Jason Buenrostro)。 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 在复杂视觉知觉过程中观察和操纵大脑皮层回路研究所 任务。这个项目将建立在我们和其他人在鉴定基因方面取得的成功的基础上 当在体内使用时，允许细胞类型受限的基因表达的调节元件 重组腺相关病毒载体。特别是，增强剂的管道 来自我们最近的UG3资助的发现为我们提供了识别 额外的增强子序列在有限的携带能力的背景下发挥作用 AAV。在这里，我们的目标是应用我们在 UG3的课程，用于快速识别一套使研究和 跨物种对基因定义的细胞类型和回路的操纵。我们的初步数据 证明我们的增强子识别策略可以产生新颖和高度特异的 将表达限制在目标人群的增强剂。此外，我们已经证明了 有可能使用经过改造的AAV-PHP.eB衣壳在整个大脑中筛选增强剂 通过一次非侵入性注射。这些成功突显了需要更快地 以及对推定的增强剂的全面评估。此外，我们还将研究 几个靶神经元群体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，以瞄准和操纵 跨物种特定神经细胞群体的脑活动，包括人类细胞来源的 有机化合物。