GENE THERAPY AND SEIZURES

尽管长期的基因转移和表达已经被证明是有效的。 在中枢神经系统中，重要的是 在动物中验证特定基因转移和表达策略 在尝试应用于治疗慢性 神经系统疾病，如癫痫。使用腺相关病毒 病毒（AAV）载体与巨细胞病毒启动子（CMV），我们 已经证明了稳定的、长期的（3个月）表达a 外源报告基因在大鼠下丘癫痫发作部位的表达 起源（McCown等人，713：99，1996）。 因此有 假设AAV-CMV载体将转移和表达 下丘中特异性受体亚单位蛋白的基因， 引起功能性神经递质受体的组装，或 反义构建体的情况下，破坏特异性 受体。为了检验这一假设，将使用AAV-CMV载体。 用三种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 植入大鼠的下丘7天零3个月后，我们 将确定载体介导的基因递送1）是否增加了 通过定量RT-PCR测定的特异性亚基mRNA的量，2） 增加受体亚单位蛋白的量， 免疫组化和3）增加功能性GABA受体 使用氯化物上取措施或功能性NMDA的组装 使用NMDA特异性[3 H]谷氨酸结合的受体组装。 因为下丘是很多癫痫发作的中枢 模型，将相同的载体注入下丘 正常或遗传性癫痫易感大鼠，和体内癫痫发作 将评估敏感性。这些研究代表了一种独特的 有机会在体内评估基因递送策略。此外，委员会认为， 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治疗局灶性癫痫发作 紊乱

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.