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.

尽管长期基因转移和表达已经 在中枢神经系统中证明，重要的是 验证动物中特定的基因转移和表达策略 在尝试应用慢性治疗之前的模型 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We 已经表现出稳定的长期（3个月）表达 大鼠下丘的外国记者基因，癫痫发作部位 Genesis（McCown等，BrainRes。713：99，1996）。 因此，是 假设AAV-CMV向量将转移并表达 下丘中的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 反性构造的情况，破坏特定的组装 受体。为了检验该假设，AAV-CMV矢量将是 由三个GABA受体亚基盒之一构建，或 NMDA受体亚基盒。这些向量将被注入 进入大鼠的下丘。七天三个月后，我们 将确定载体介导的基因输送1） 定量RT-PGR的特定亚基mRNA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 使用氯化物的组装采取措施或功能性NMDA 使用NMDA特异性[3H]谷氨酸结合的受体组装。 因为下丘是癫痫发作的核心 模型，相同的向量将被注入下凸丘中 正常或遗传性癫痫易发的大鼠，体内癫痫发作 将评估灵敏度。这些研究代表了独特的 在体内评估基因输送策略的机会。而且， 由于我们针对调节癫痫发作敏感性的受体，因此 发现可能直接应用于局灶性癫痫发作的治疗 疾病。