ADENOVIRUS MEDIATED NEURONAL GABA RECEPTOR ENGINEERING

描述：许多临床基本药物的治疗作用是 被认为部分是由大脑中的A型伽马氨基丁酸调节的。 (GABAA)受体(GABA)。这一知识帮助了 更好的针对大脑GABA能系统的药物。校长 研究人员认为，GABA能源的下一个概念前沿 治疗的关键在于对GABAR进行体内修饰。本论文是 基于大多数GABAR亚基广泛分布在 大脑对治疗特异度的理论限制 外源生物。相比之下，关于特定遗传基础的信息 与特定受体一起影响GABA能系统的疾病 药物作用的亚基基础还将继续积累。从网络开始 对大脑的任何药物作用的结果都是一个复杂的相互作用 药物和传递药物作用的受体，所需的 治疗目标可以通过改变受体以及 更传统的方法来创造新药。 在这项赠款申请中，我们建议朝着这一目标迈出第一步： 新型重组腺病毒介导的神经元型GABAA受体工程 在试管中。重组人将对大鼠海马神经元进行转导 表达全长正义表位的5型腺病毒 所需亚基的构建，或截断的反义构建 我们希望消除的亚单位。通过过度表达和/或反义 特定亚基的击倒，GABAA受体的亚基组成 都会经过精心设计。受体亚基在大肠杆菌中的成功表达 神经元胞体将被药理学和生理学证实 使用压电驱动快速药物应用技术进行表征。 成功地将所需的受体亚单位整合到突触中将 通过免疫细胞化学和定量分析证实自发性 利用全细胞膜片钳获得抑制性突触电流 技术。绿色荧光蛋白的病毒同步表达 (GFP)报告基因由第二个表达盒允许毫不含糊 从对照中鉴定病毒转导的荧光 电生理实验时的非荧光细胞。 如果成功，神经递质受体工程的概念 作为一种新的治疗方法将有广泛的应用前景 修复功能受损的患病大脑。这项拨款建议 描述了一种新的重组腺病毒介导的技术 这个目标。所提出的实验旨在验证该方法。 通过电生理学、药理学和免疫细胞化学 证实活神经元中的GABAA受体亚单位组成可以 是工程设计的。在未来的提案中，受体工程方法 这里首创的将应用于全动物体细胞基因治疗 可归因于癫痫等GABAR功能障碍的病理。

DESCRIPTION: The therapeutic actions of many clinically essential drugs are thought to be mediated, in part, by the brain gamma-aminobutyric acid type A (GABAA) receptors (GABARs). This knowledge has helped the development of better drugs targeting the brain GABAergic system. The principal investigator believes that the next conceptual forefront of GABAergic therapeutics lies in the in vivo modification of the Gabars. This thesis is based on the fact that most GABAR subunits are distributed diffusely in the brain posing a theoretical limit on the therapeutic specificity of xenobiotics. In contrast, information regarding the specific genetic basis for disease affecting the GABAergic system along with the specific receptor subunit basis for drug action will continue to accumulate. Since the net result of any drug action on the brain is a complex interaction between the drug and the receptors which transduce the action of that drug, the desired therapeutic goals could be attained by modifying the receptor as well as the more traditional approach of creating new drugs. In this grant application, we propose the first step towards this goal: a novel recombinant adenovirus mediated neuronal GABAA receptor-engineering, in vitro. Rat hippocampal neurons will be transduced with recombinant human adenovirus type 5 (hAd5) expressing epitope tagged full-length sense construct of the desired subunit, or a truncated antisense construct of the subunit we wish to eliminate. Through overexpression and / or antisense knock-down of given subunits, the subunit composition of the GABAA receptors will be engineered. Successful expression of the receptor subunits on the neuronal soma will be verified by pharmacological and physiological characterizations using the piezo-drive rapid drug application technique. Successful incorporation of the desired receptor subunits into synapses will be verified by immunocytochemical and quantitative analysis of spontaneous inhibitory synaptic currents obtained using the whole cell patch clamp technique. Simultaneous viral expression of the green fluorescent protein (GFP) reporter gene by a second expression cassette allows unambiguous identification of virally transduced fluorescent from control non-fluorescent cells at the time of electrophysiological experiments. If successful, the notion of neuronal neurotransmitter receptor engineering will have a wide range of application as a novel therapeutic approach for repairing the functionally impaired diseased brain. This grant proposal describes a novel recombinant adenovirus mediated technique for achieving this goal. The experiments proposed are designed to validate this approach through electrophysiological, pharmacological, and immunocytochemical confirmation that the GABAA receptor subunit composition in live neurons can be engineered. In future proposals, the receptor engineering methods pioneered here will be applied to whole animal somatic cell gene-therapy for pathology ascribable to the GABAR malfunction such as epilepsy.