GENE THERAPY AND SEIZURES

基因治疗和癫痫发作

• 批准号: 2038496
• 负责人: Thomas J. McCown
• 金额: $ 19.88万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-04-01 至 2002-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2038496
• 关键词: GABA receptor; NMDA receptors; adeno associated virus group; cytomegalovirus; epilepsy; gene therapy; genetic promoter element; inferior colliculus; laboratory rat; nonhuman therapy evaluation; protein biosynthesis; transfection /expression vector

Although long term gene transfer and expression have been demonstrated within the central nervous system, it is important to validate specific gene transfer and expression strategies in animal models before attempting application to the treatment of chronic neurological disorders, such as epilepsy. Using an adeno-associated virus (AAV) vector with a cytomegalovirus promoter (CMV) we have demonstrated stable, long term (3 months) expression of a foreign reporter gene in the rat inferior colliculus, a site of seizure genesis (McCown et al., Brain Res. 713:99, 1996). Therefore, it is hypothesized that AAV-CMV vectors will transfer and express the genes for specific receptor subunit proteins in the inferior colliculus, causing the assembly of functional neurotransmitter receptors, or in the case of anti sense constructs, disrupt assembly of specific receptors. To test this hypothesis, AAV-CMV vectors will be constructed with one of three GABA receptor subunit cassettes, or an NMDA receptor subunit cassette. These vectors will be infused into the inferior colliculus of rats. Seven days and 3 months later, we will determine if the vector-mediated gene delivery 1) increases the amount of specific subunit mRNA by quantitative RT-PGR, 2) increases the amount of receptor subunit protein using quantitative immunohistochemistry and 3) increases functional GABA receptor assembly using a chloride up take measure or functional NMDA receptor assembly using NMDA specific [3H]glutamate binding. Because the inferior colliculus is central to a number of seizure models, the same vectors will be infused into the inferior colliculus of normal or genetic epilepsy-prone rats, and in vivo seizure sensitivity will be evaluated. These studies represent a unique opportunity to evaluate gene delivery strategies in vivo. Moreover, since we are targeting receptors that modulate seizure sensitivity, the findings may have direct application to the treatment of focal seizure disorders.

尽管长期的基因转移和表达一直是 在中枢神经系统内表现出来，重要的是 在动物体内验证特定的基因转移和表达策略 在尝试应用于慢性阻塞性肺疾病治疗之前的模型 神经系统疾病，如癫痫。使用腺相关的 携带巨细胞病毒启动子的AAV载体 已表现出稳定、长期(3个月)的 外源记者基因在大鼠下丘癫痫发作中的作用 Genesis(McCown等人，Brain Res.713：99,1996)。因此，它是 假设AAV-CMV载体将转移和表达 下丘特定受体亚单位蛋白的基因， 导致功能性神经递质受体的组装，或者在 在反义构建的情况下，扰乱特定的组装 感受器。为了检验这一假设，AAV-CMV载体将是 用三个GABA受体亚单位盒中的一个构建，或 一种NMDA受体亚单位盒。这些载体将被注入 进入大鼠的下丘。7天零3个月后，我们 将决定载体介导的基因传递是否增加了 定量RT-PGR检测特异性亚基mRNA的量，2) 使用定量方法增加受体亚单位蛋白质的数量 免疫组织化学和3)增加功能性GABA受体 使用氯化物上升采取措施或功能性NMDA的组件 受体组装使用NMDA特异性的[~3H]谷氨酸结合。 因为下丘是许多癫痫发作的中枢 模型中，相同的载体将被注入下丘 正常或遗传性癫痫易感大鼠，以及体内癫痫发作 将评估敏感度。这些研究代表了一种独特的 评估体内基因传递策略的机会。此外， 由于我们的目标是调节癫痫敏感性的受体， 研究结果可能直接应用于局灶性癫痫的治疗。 精神错乱。