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.

项目摘要 在此提案中，我们旨在确定允许靶向和目标的基因调节元素 操纵人脑功能的脑电路模型。获得特定的遗传获取 非转基因动物和人类的神经元种群将实现目标电路 对假设检验的调节，并提供了一种评估安全性和功效的方法 电路调节，用于治疗癫痫和精神疾病。我们的方法 利用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和Paola Arlotta），识别功能的顺式调节元素 跨物种（Yating Wang），AAV工程与大规模筛选方法结合 （Ben Deverman）和结合RNA和染色质生物学（Jason Buenrostro）的专业知识。 我们的努力还将受益于与约翰·雷诺兹（John Reynolds）在Salk的持续合作 在复杂的视觉感知期间观察和操纵皮质电路的研究所 任务。这个项目将基于我们和其他人在识别基因方面所拥有的成功 在内部使用时，可以启用细胞类型限制基因表达的调节元素 重组腺相关病毒（AAV）载体。特别是增强器的管道 来自我们最近的UG3赠款的发现，为我们提供了识别的系统 在有限的承载能力的背景下起作用的其他增强器序列 AAV。在这里，我们旨在应用我们在 该UG3的过程，用于快速识别一套增强子，以使研究和 操纵跨物种遗传定义的细胞类型和电路。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且高度具体的 将表达限制为目标人群的增强剂。此外，我们已经证明了 可以使用工程化的AAV-PHP.EB CAPSID来筛选大脑的增强剂 单个无创注射。这些成功强调了需要更快的 以及对推定增强剂的全面评估。此外，我们将研究 在几个目标神经元种群中对神经元活动操纵的耐受性 物种。该提案将在设计方法和操纵方法方面具有变革性 跨物种的特定神经元细胞群的大脑活动，包括人类细胞衍生 器官。