GENE THERAPY AND SEIZURES

基因治疗和癫痫发作

• 批准号: 6188070
• 负责人: Thomas J. McCown
• 金额: $ 21.23万
• 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/6188070
• 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已 在中枢神经系统中得到证实，重要的是 验证动物中特定的基因转移和表达策略 在尝试将模型应用于慢性病的治疗之前 神经系统疾病，例如癫痫。使用腺相关 带有巨细胞病毒启动子（CMV）的病毒（AAV）载体 已证明a的稳定、长期（3个月）表达 大鼠下丘（癫痫发作部位）中的外源报告基因 (McCown et al., Brain Res. 713:99, 1996)。 因此，它是 假设 AAV-CMV 载体将转移并表达 下丘特定受体亚基蛋白的基因， 引起功能性神经递质受体的组装，或 在反义结构的情况下，破坏特定的组装 受体。为了检验这一假设，AAV-CMV 载体将是 用三个 GABA 受体亚基盒之一构建，或 NMDA 受体亚基盒。这些载体将被注入 进入大鼠下丘。 7天零3个月后，我们 将确定载体介导的基因传递 1) 是否增加 通过定量 RT-PCR 检测特定亚基 mRNA 的量，2) 使用定量增加受体亚基蛋白的量 免疫组织化学和 3) 增加功能性 GABA 受体 使用氯化物或功能性 NMDA 进行组装 使用 NMDA 特异性 [3H]谷氨酸结合进行受体组装。 因为下丘是许多癫痫发作的中心 模型中，相同的向量将被注入下丘 正常或有遗传性癫痫倾向的大鼠，以及体内癫痫发作 将评估敏感性。这些研究代表了独特的 评估体内基因传递策略的机会。而且， 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发作的治疗 失调。