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大脑中细胞类型特异性基因表达调节的工具。 大鼠大脑杏仁核基底外侧区域（ABL）将用作模型系统。 该项目将开发慢病毒载体，以针对已识别的 ABL 神经元亚群中的转基因过度表达，并选择性地敲低这些相同细胞群中的内源基因表达。 目标 1 - 定向过度表达：拟议的研究将测试以下假设：可以构建慢病毒载体，通过掺入细胞类型特异性启动子来将基因过度表达限制在特定的 ABL 神经元亚群中。 慢病毒设计用于将标记基因或 c-myc 标记的 GABAA 受体 a1 亚基靶向锥体细胞（CAM 激酶 II 启动子）或表达 VIP 和钙调蛋白（钙调蛋白启动子）或小白蛋白（泛/白蛋白启动子）的 GABA 能中间神经元的离散群体。针对细胞类型靶向的特异性进行构建、测试和优化。 目标 2 靶向表达敲低：拟议的研究将检验这样的假设：掺入细胞类型特异性启动子的慢病毒可用于通过反义 RNA 的表达选择性降低特定 ABL 神经元亚群中的基因表达。 在鉴定出可有效减少培养细胞中 NMDA 受体亚基 1 和 GABAA 受体 a1 亚基 mRNA 的反义构建体后，将反义构建体插入 CaM 激酶 II、钙视网膜蛋白或小清蛋白启动子控制下的慢病毒载体中。 这些慢病毒载体将在立体定位递送至 ABL 后测试靶基因表达敲低的细胞类型特异性。 开发用于细胞类型选择性操纵基因表达的工具将极大地有助于确定特定基因产物在参与完整动物的确定神经元回路的神经元亚群中的作用。 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。 预计该项目开发的载体将有助于研究突触传递和其他大脑区域（包括皮层和海马体）神经元网络的调节。 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，特别是在确定特定脑细胞群中发现的单个蛋白质在整个生物体行为中的作用方面。 由于所研究的大脑区域，这项工作与压力、焦虑和癫痫的理解和治疗特别相关。 预计这项工作最终将带来针对这些和其他神经系统疾病的新药或基因疗法。