ADENOVIRUS MEDIATED NEURONAL GABA RECEPTOR ENGINEERING

描述：许多临床必需药物的治疗作用是 被认为部分是由大脑γ-氨基丁酸A型介导的 （GABAA）受体（GABAR）。 这些知识有助于发展 更好的针对大脑GABA能系统的药物。 校长 研究人员认为，GABA能的下一个概念前沿 治疗方法在于Gabar的体内修饰。 本文 基于大多数GABAR亚基在细胞中弥散分布的事实， 大脑对治疗特异性的理论限制 异生物质 相反，关于特定遗传基础的信息 用于影响GABA能系统沿着特定受体的疾病 药物作用的亚基基础将继续积累。 因为网络 任何药物对大脑作用的结果是大脑与大脑之间的复杂相互作用。 药物和受体的作用，该药物，所需的 治疗目标可以通过修饰受体以及 更传统的方法来制造新药。 在这个拨款申请中，我们提出了实现这一目标的第一步： 新型重组腺病毒介导的神经元GABAA受体工程， 体外 大鼠海马神经元将转导重组人 表达表位标记的全长正义的5型腺病毒（hAd 5） 所需亚基的截短的反义构建体，或所需亚基的截短的反义构建体。 我们希望消除的亚单位。 通过过表达和/或反义 给定亚基的敲低，GABAA受体的亚基组成 将被设计。 受体亚单位在细胞膜上的成功表达， 神经元索马将通过药理学和生理学验证 使用压电驱动快速药物施加技术进行表征。 成功地将所需受体亚单位整合到突触中， 通过免疫细胞化学和定量分析证实自发 使用全细胞膜片钳获得抑制性突触电流 法 绿色荧光蛋白的同时病毒表达 (GFP)报告基因通过第二表达盒的表达允许明确的 从对照中鉴定病毒转导荧光 在电生理学实验时无荧光细胞。 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， 修复功能受损的患病大脑 这份拨款申请 描述了一种新的重组腺病毒介导的技术， 这个目标 设计了实验来验证这种方法 通过电生理学、药理学和免疫细胞化学 证实了活神经元中GABAA受体亚单位的组成可以 被设计。 在未来的建议中，受体工程方法 在这里开创的将应用于整个动物体细胞基因治疗， 归因于GABAR功能障碍的病理学，如癫痫。

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.