GENE DELIVERY TO THE CNS TO MODULATE EPILEPSY

描述：（改编自申请人摘要）常规药物治疗 对于中枢神经系统疾病，无论是外科手术还是药理学，通常都有广泛的 对与所治疗的疾病无关的大脑区域产生不必要的影响。在 对此，研究人员正在开发“分子神经外科学”， 使用转基因病毒治疗神经系统疾病的新方法 调节神经系统细胞的载体。该项目的目标是 递送编码谷氨酸的工程化转录单位（转基因 通过缺陷型单纯疱疹病毒（HSV）载体将脱羧酶（GAD）导入细胞 以调节神经元的活动。GAD直接将 谷氨酸，大脑中主要的兴奋性氨基酸，γ氨基丁酸 氨基酸（GABA），主要抑制氨基酸。有缺陷的HSV载体是 在体外和体内将转基因递送至CNS细胞的有效手段 vivo.含有来自大鼠的GAD转基因（GAD 65和67同种型）的载体和 将产生具有不同启动子的植物以靶向GAD表达， 调节GAD活性。GAD转基因表达对GABA的影响 将在原代CNS细胞培养物中检查代谢。小说能力 将确定GABA合成和释放以抑制癫痫样活动 在体外“癫痫发作”模型中，使用皮层培养物和大鼠脑切片。在 GAD转基因在脑中的体内递送和表达将允许我们 开始评估这种定点基因治疗的功能影响。 通过直接调节神经递质的局部水平的能力 在大脑中原位引入相关基因可以应用于 许多神经系统疾病。GABA水平的改变在以下方面发挥作用： 癫痫、亨廷顿氏病、帕金森氏病、疼痛和精神分裂症。 超过200万美国人患有癫痫，其中约25 %的患者药物治疗无效， 疗法目前的神经外科手术是高度侵入性的，并且通常涉及 切除大面积的正常大脑这份提案结合了 神经外科的高度定位载体应用，分子生物学的 缺陷型载体构建，GAD表征的细胞生物学，以及 用于新型GABA合成功能分析的生理学。

DESCRIPTION: (adapted from applicant's abstract) 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.