ADENOVIRUS MEDIATED NEURONAL GABA RECEPTOR ENGINEERING

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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.