Conventional medical therapies for CNS disorders, both surgical and pharmacological, often have widespread unwanted effects on brain areas not involved in the disorder being treated. In response to this the investigators are developing """"""""molecular neurosurgery"""""""", a novel approach to nervous system disorders that uses genetically modified viral vectors to modulate cells in the nervous system. The goal of this project is to deliver an engineered transcription unit (transgene) encoding glutamic acid decarboxylase (GAD) via defective herpes simplex virus (HSV) vectors to cells of the CNS, in order to modulate neuronal activity. GAD directly converts glutamate, the major excitatory amino acid in the brain, to gamma aminobutyric acid (GABA), the major inhibitory amino acid. Defective HSV vectors are an efficient means to deliver transgenes to cells of the CNS, both in vitro and in vivo. Vectors containing GAD transgenes from rat (GAD65 and 67 isoforms) and plant, with different promoters will be generated to target GAD expression and regulate GAD activity. The effect of GAD transgene expression on GABA metabolism will be examined in primary CNS cell cultures. The ability of novel GABA synthesis and release to inhibit epileptiform activity will be determined in in vitro 'seizure' models, using cortical cultures and rat brain slices. In vivo delivery and expression of the GAD transgene in the brain will allow us to begin to evaluate the functional impact of this site-directed gene therapy. The ability to modulate localized levels of neurotransmitters by directly introducing the relevant genes in situ in the brain has applications to numerous nervous system disorders. Alterations in GABA levels play a role in epilepsy, Huntington's disease, Parkinson's disease, pain and schizophrenia. Over 2 million Americans suffer from epilepsy, of which approximately 25 percent are refractory to drug treatment and are candidates for surgical therapy. Current neurosurgical procedures are highly invasive and often involve the removal of large areas of normal brain. This proposal combines the skills of neurosurgery for highly localized vector application, molecular biology for defective vector construction, cell biology for GAD characterization, and physiology for functional analysis of novel GABA synthesis.
