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

基本信息

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
项目类别：
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

项目摘要

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.

项目概要在本提案中，我们的目标是确定基因调控元件，以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启用目标电路假设检验的调制并提供评估安全性和有效性的方法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 Paola Arlotta），识别起作用的 CIS 调节元件跨物种（王亚婷），AAV工程结合大规模筛选方法 (Ben Deverman) 以及结合 RNA 和染色质生物学的专业知识 (Jason Buenrostro)。我们的努力也将受益于与索尔克约翰·雷诺兹的持续合作复杂视觉感知过程中皮层回路的观察和操纵研究所任务。该项目将建立在我们和其他人在识别基因方面所取得的成功的基础上调节元件，在使用时能够实现细胞类型限制的基因表达重组腺相关病毒（AAV）载体。特别是，增强剂的管道我们最近 UG3 资助的发现为我们提供了系统地识别在有限承载能力的情况下发挥作用的附加增强子序列腺病毒。在这里，我们的目标是应用我们在该 UG3 课程用于快速识别一套增强子，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颖且高度特异性的将表达限制于目标人群的增强子。此外，我们还证明了可以使用工程化的 AAV-PHP.eB 衣壳来筛选大脑中的增强子通过一次无创注射。这些成功凸显了需要更快速并对假定的增强剂进行综合评估。此外，我们将检查在一些目标神经元群体中对神经元活动操纵的耐受性物种。该提案将在设计目标和操纵的方法方面具有变革性跨物种特定神经元细胞群的大脑活动，包括人类细胞衍生的类器官。