Lentiviral vectors for targeted manipulation of amygdalar gene expression

用于靶向操纵杏仁核基因表达的慢病毒载体

• 批准号: 7514944
• 负责人: Marlene A. Wilson
• 金额: $ 15.8万
• 依托单位: UNIVERSITY OF SOUTH CAROLINA AT COLUMBIA
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-12-15 至 2009-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7514944
• 关键词: Albumins; Amygdaloid structure; Anatomy; Animals; Antisense RNA; Anxiety; Anxiety Disorders; Area; Base Pairing; Behavior; Behavior Disorders; Biological Models; Brain; Brain region; Calcium; Calmodulin; Cells; Confocal Microscopy; Cultured Cells; Development; Epilepsy; Gene Expression; Gene Expression Alteration; Gene Expression Regulation; Gene Targeting; Genes; Genetic Transcription; Glutamate Receptor; Goals; Green Fluorescent Proteins; Hippocampus (Brain); Immunofluorescence Immunologic; Individual; Injection of therapeutic agent; Interneurons; Knowledge; Lead; Learning; Lentivirus Vector; Messenger RNA; N-Methyl-D-Aspartate Receptors; N-Methylaspartate; Neuraxis; Neurologic; Neurons; Pan Genus; Parvalbumins; Pharmaceutical Preparations; Phosphoglycerate Kinase; Phosphotransferases; Population; Proteins; Pyramidal Cells; Range; Rattus; Regulation; Relative (related person); Reverse Transcriptase Polymerase Chain Reaction; Role; Specificity; Standards of Weights and Measures; Stress; Subfamily lentivirinae; Synaptic Transmission; Testing; Time; Toxic effect; Transfection; Transgenes; Whole Organism; Work; abl Oncogene; base; brain cell; c-myc Genes; calmodulin-dependent protein kinase II; calretinin; cell type; cellular transduction; design; experience; gene therapy; glycoprotein G; hippocampal pyramidal neuron; in vivo; knock-down; nervous system disorder; promoter; receptor; tool; tool development; vector

DESCRIPTION (provided by applicant): The overall goal of this project is to develop tools for cell-type-specific regulation of gene expression in the brain. The basolateral region of the amygdala (ABL) of the rat brain will be used as a model system. The project will develop lentiviral vectors to target transgene over-expression in identified subpopulations of ABL neurons and to selectively knock-down endogenous gene expression in these same cell groups. Aim 1- Tarqeted Over-expression: The proposed studies will test the hypothesis that lentiviral vectors can be constructed to restrict gene over-expression to defined subpopulations of ABL neurons by incorporating cell- type-specific promoters. Lentiviruses designed to target a marker gene or c-myc-tagged GABAA receptor a1 subunit to pyramidal cells (CAM kinase II promoter) or discreet populations of GABAergic interneurons expressing VIP and calretinin (calretinin promoter) or parvalbumin (pan/albumin promoter) will be constructed, tested and optimized for specificity of cell-type targeting. Aim 2-Targeted Knock-down of Expression: The proposed studies will test the hypothesis that lentiviruses incorporating cell-type-specific promoters can be used to selectively reduce gene expression in defined subpopulations of ABL neurons by expression of antisense RNA. After identifying antisense constructs that effectively reduce mRNA for the NMDA receptor subunit 1 and for GABAA receptor a1 subunit in cultured cells, the antisense constructs will be inserted into lentiviral vectors under control of the CaM kinase II, calretinin or parvalbumin promoters. These lentiviral vectors will be tested for cell-type specificity in knock-down of target gene expression following stereotaxic delivery to the ABL. Development of tools for cell-type selective manipulation of gene expression will greatly facilitate determining the roles of specific gene products in subpopulations of neurons participating in defined neuronal circuits of intact animals. This knowledge will, in turn, permit development of new drugs and genetic therapies for neurological and behavioral disorders. In the ABL, the development of these tools is particularly relevant to treatments for stress and anxiety disorders and for epilepsy. It is also expected that the vectors developed in this project will be useful to study synaptic transmission and the regulation of neuronal networks in other brain areas including the cortex and the hippocampus. These tools will have wide applicability in elucidating the functional anatomy of the central nervous system, particularly in determining the roles of individual proteins found in specific groups of brain cells in the behavior of the whole organism. Because of the brain region being studied, this work is of particular relevance to the understanding and treatment of stress, anxiety and epilepsy. It is expected that this work will ultimately lead to new drugs or genetic therapies for these and other disorders of the nervous system.

描述（由申请人提供）：该项目的总体目标是开发用于脑中基因表达的细胞类型特异性调节的工具。 大鼠大脑杏仁核（ABL）的基底外侧区域将被用作模型系统。 该项目将开发慢病毒载体，以靶向ABL神经元亚群中的转基因过表达，并选择性地敲低这些相同细胞群中的内源性基因表达。 目标1-靶向过表达：所提出的研究将检验以下假设：可以构建慢病毒载体，以通过掺入细胞类型特异性启动子将基因过表达限制于ABL神经元的限定亚群。 将构建、测试和优化慢病毒的细胞类型靶向特异性，所述慢病毒设计为将标记基因或c-myc标记的GABAA受体α 1亚基靶向锥体细胞（CAM激酶II启动子）或表达VIP和钙视网膜蛋白（钙视网膜蛋白启动子）或小清蛋白（泛/白蛋白启动子）的GABA能中间神经元的离散群体。 目标2-靶向敲低表达：所提出的研究将检验以下假设：掺入细胞类型特异性启动子的慢病毒可用于通过表达反义RNA选择性降低ABL神经元的限定亚群中的基因表达。 在鉴定出有效减少培养细胞中NMDA受体亚基1和GABAA受体α 1亚基的mRNA的反义构建体后，将反义构建体插入到CaM激酶II、钙视黄蛋白或小清蛋白启动子控制下的慢病毒载体中。 这些慢病毒载体将进行测试的细胞类型特异性敲低靶基因表达后，立体定位交付的ABL. Development的工具，细胞类型选择性操纵基因表达将大大有助于确定特定的基因产物的作用，在亚群的神经元参与定义的神经元回路的完整动物。 这些知识反过来将有助于开发治疗神经和行为障碍的新药和基因疗法。 在ABL中，这些工具的开发与压力和焦虑症以及癫痫的治疗特别相关。 我们也期望本计画所开发的载体，将可用于研究突触传递及其他脑区（包括皮质及海马）神经元网络的调控。 这些工具将在阐明中枢神经系统的功能解剖学方面具有广泛的适用性，特别是在确定在整个生物体的行为中在特定脑细胞群中发现的单个蛋白质的作用方面。 由于正在研究的大脑区域，这项工作与理解和治疗压力，焦虑和癫痫特别相关。 预计这项工作将最终导致这些和其他神经系统疾病的新药或基因疗法。