Lentiviral vectors for targeted manipulation of amygdalar gene expression

描述（由申请人提供）：该项目的总体目标是开发大脑中细胞类型特异性基因表达调控的工具。大鼠脑杏仁核基底外侧区（ABL）将被用作模型系统。该项目将开发慢病毒载体，以ABL神经元亚群中的转基因过表达为目标，并选择性地敲除这些细胞群中的内源性基因表达。目标1-靶向过表达：提出的研究将验证这样的假设，即通过结合细胞类型特异性启动子，可以构建慢病毒载体来限制ABL神经元特定亚群的基因过表达。将慢病毒设计为靶向标记基因或c-myc标记的GABAA受体a1亚基到锥体细胞（CAM激酶II启动子）或表达VIP和calretinin （calretinin启动子）或parvalbumin（泛/白蛋白启动子）的GABAA能中间神经元的离散群体，将构建，测试和优化细胞类型靶向的特异性。目的2-靶向敲低表达：提出的研究将验证含有细胞类型特异性启动子的慢病毒可以通过表达反义RNA来选择性地降低ABL神经元中特定亚群的基因表达的假设。在培养细胞中鉴定出能有效减少NMDA受体亚基1和GABAA受体a1亚基mRNA的反义构建体后，在CaM kinase II、calretinin或parvalbumin启动子的控制下，将这些反义构建体插入慢病毒载体中。这些慢病毒载体将在立体定向递送到ABL后进行细胞类型特异性测试，以敲除靶基因表达。基因表达的细胞型选择性操作工具的开发将极大地促进确定特定基因产物在参与完整动物的特定神经回路的神经元亚群中的作用。反过来，这些知识将有助于开发针对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障碍以及癫痫的治疗特别相关。本项目开发的载体也有望用于研究包括皮层和海马在内的大脑其他区域的突触传递和神经元网络的调节。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。由于研究的是大脑区域，因此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针对这些和其他神经系统疾病的新药或基因疗法。

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.