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Generation of viral vectors that use alternative splicing to drive cell type-specific gene expression in the nervous system

生成使用选择性剪接驱动神经系统中细胞类型特异性基因表达的病毒载体

基本信息

批准号：
10012468
负责人：
Seth Blackshaw
金额：
$ 153.45万
依托单位：
JOHNS HOPKINS UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-08-01 至 2023-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10012468
关键词：
Afferent Neurons Alternative Splicing Animal Model Animals Archives Astrocytes Auditory Behavior Calcium Cells Cerebral cortex Chimera organism Cognition Communities Coupling Data Data Set Databases Development Event Exons Ferrets Funding Gene Expression Gene Transfer Techniques Generations Genes Human Individual Introns Investigation Label Length Link Mammals Molecular Monitor Motor Neurons Mus Muscle Muscle Cells Nervous Mouse Nervous system structure Neuraxis Neuroglia Neurons Neurosciences Research Olfactory Pathways Oligodendroglia Organoids Pattern Peripheral Photoreceptors Promoter Regions RNA Splicing Rattus Reagent Reporter Retinal Photoreceptors Specificity Subfamily lentivirinae System Techniques Testing Viral Viral Vector base calcium indicator cell type design design and construction designer receptors exclusively activated by designer drugs excitatory neuron experimental study genetic manipulation in vivo evaluation induced pluripotent stem cell inhibitory neuron interest neural circuit novel optogenetics promoter selective expression sensor single-cell RNA sequencing somatosensory tool vector

项目摘要

Project Summary New tools are urgently needed to selectively target constructs that monitor and manipulate the activity of individual cell types without having to rely on genetic manipulation. This proposal aims to develop viral tools that use cell type-specific alternative splicing events to drive cell type-specific gene expression in the nervous system independent of genetic manipulation, an approach we term splicing-linked expression design (SLED). SLED-based vectors use evolutionarily conserved, highly cell type-specific exons, identified using the ASCOT database developed by our group, to drive expression of reporter and effector constructs. We have demonstrated feasibility of this approach using constructs that selectively target retinal photoreceptors, muscle cells, and cortical neurons. We propose to extend this by combining cell-specific alternative exon/intron sequences with appropriate promoter sequences to generate a toolbox of AAV and lentiviral SLED vectors that selectively target multiple cell types of interest to the neuroscience research community. We will first generate SLED vectors that target primary sensory and motor neurons, as well as multiple subtypes of cortical neurons and glia, and validate the specificity of these reagents in mice. We will next test the cell specificity of SLED reagents that are validated in mice in rats, ferrets, as well as human cortical organoids and rat-human chimeras. Finally, highly specific SLED fluorescent reporter constructs will be converted to drive expression of calcium indicators, as well as optogenetic and chemogenetic constructs. We anticipate that SLED-based reagents will allow highly cell type-specific expression of a broad range of molecular tools useful for analysis of neural circuitry in multiple mammalian species.

项目摘要迫切需要新的工具来选择性地针对监视和操纵活动的构造单独的细胞类型，而不必依赖于基因操作。该提案旨在开发病毒式工具使用细胞类型特定的选择性剪接事件来驱动神经中细胞类型特定的基因表达独立于基因操作的系统，我们称之为剪接连锁表达设计(SLED)。基于SLED的载体使用进化上保守的、高度细胞类型特异的外显子，通过ASCOT鉴定由我们团队开发的数据库，用于驱动报告和效应器结构的表达。我们有使用选择性靶向视网膜光感受器、肌肉的构建物证明了这种方法的可行性细胞和大脑皮层神经元。我们建议通过结合细胞特定的替代外显子/内含子来扩展这一点带有适当启动子序列的序列，以产生AAV和慢病毒SLED载体工具箱，该工具箱选择性地针对神经科学研究社区感兴趣的多种细胞类型。我们将首先生成针对初级感觉神经元和运动神经元以及多种亚型皮质神经元的SLED载体和神经胶质细胞，并在小鼠身上验证了这些试剂的特异性。接下来我们将测试雪橇的细胞特异性。在大鼠、雪貂、人类皮质类器官和大鼠-人身上得到验证的试剂嵌合体。最后，高度特异的雪橇荧光报告构建体将被转化为驱动表达钙指示剂，以及光发生和化学发生结构。我们预计基于雪橇的试剂将允许高度细胞类型特异性表达对分析有用的广泛的分子工具多种哺乳动物的神经回路。