ANTIEPILEPTIC EFFECT OF GENETICALLY MODIFIED FIBROBLASTS

申请者的长期职业目标是成为一名内科科学家 致力于神经科学。最终的方向是发展改进 治疗人类癫痫的方法。申请者计划使用 基因转移和神经移植相结合作为研究工具 癫痫的传播和作为一种潜在的癫痫治疗方法。 局部应用GABA受体激动剂(如麝香酚)或GABA 转氨酶抑制剂(例如，γ-乙烯基氨基丁酸)进入离散的大脑区域 (如黑质、内侧丘脑、杏仁核和下部 在不同的癫痫动物模型中，丘脑)可抑制癫痫发作。 此外，长期局部注射GABA完全进入运动皮质 在自然状态下阻断行为和脑电发作 对光敏感的狒狒。血脑屏障的不通透性和 然而，全球毒性排除了这些药物的系统性使用 抗癫痫药。因此，这项提案的总体目标是 为了测试这样一种想法：嫁接转基因的、分泌GABA的 成纤维细胞进入离散的大脑区域可以改变癫痫的表现。 为了解决这一目标，谷氨酸脱羧酶(GAD)基因将被 逆转录病毒作为报告基因，将成为基因传递的载体 原代皮肤成纤维细胞和成纤维细胞系都将是 捐献细胞。使用成纤维细胞作为供体细胞的原因是 (1)逆转录病毒需要分裂细胞才能感染；(2)成纤维细胞是 易于获得，易于体外培养和操作；(3) 潜在治疗的长期策略的关键是使用 原代皮肤成纤维细胞作为自体移植的供体细胞。 具体地说，将分离野生型逆转录病毒的前病毒基因 通过替换病毒所需的基因来构建质粒 使用GAD基因进行复制。可传播但可复制的- 携带GAD基因的无能逆转录病毒载体可通过 将构建的质粒导入含有GDV的细胞系 逆转录病毒辅助病毒前病毒基因。通过转染腺病毒 载体中，GAD基因将整合到成纤维细胞中。试管苗 GAD基因的表达将通过检测mRNA和分泌来定量 GABA。为了监测体内的功能，转基因的 成纤维细胞将被移植到海马体或侧脑室 测试对癫痫现象的影响(行为和 脑电惊厥)、惊厥阈值和惊厥诱发 神经元变性。用于这些目的的模型系统是一个长期的 穹隆海马伞损毁致癫痫模型及大鼠急性癫痫模型 杏仁核内注射海人酸致边缘惊厥。这个 移植物长期存活、体外基因表达与宿主免疫 反应将通过免疫组织化学方法进行评估 针对各种特定细胞标志物的抗体。

The applicant's long term career-goal is to become a physician scientist dedicated to neuroscience. The ultimate direction is to develop improved methods of treating human epilepsy. The applicant plans to use a combination of gene transfer and neural grafting as a tool to study seizure propagation and as a potential therapeutic approach to epilepsy. Local application of GABA receptor agonist (e.g. muscimol) or GABA transaminase inhibitor (e.g. gamma-vinyl GABA) into discrete brain regions (e.g. substantia nigra, entopeduncular nucleus, amygdala and inferior colliculus) could suppress seizure in various animal models of epilepsy. Moreover, chronic infusion of GABA locally into motor cortex completely blocked the behavioral and electrographical seizure in naturally photosensitive baboon. The impermeability to blood-brain-barrier and global toxicity, however, preclude the systemic use of these agents as antiepileptic drugs. Therefore, the overall objective of this proposal is to test the idea that grafting the genetically modified, GABA-secreting fibroblasts into discrete brain regions can modify seizure manifestations. To address this objective, glutamic acid decarboxylase (GAD) gene will be used as the reporter gene, retroviruses will be the gene delivery vectors and both primary skin fibroblasts and fibroblasts cell lines will be the donor cells. The reasons for the use of fibroblasts as donor cells are (1) retroviruses require dividing cells for infection; (2) fibroblasts are readily available and easily cultured and manipulated in vitro; (3) essential to the long-term strategy of potential therapy is the use of primary skin fibroblasts as donor cells for autograft. Specifically, the proviral gene of wild-type retrovirus will be isolated to construct the plasmid by replacing the genes necessary for viral replication with the GAD gene. The transmissible but replication- incompetent retroviral vectors carrying GAD gene can be produced by introducing the constructed plasmid to the cells lines containing the proviral gene of retroviral helper virus. By transfection with the vectors, GAD gene will be integrated into fibroblasts. The in vitro expression of GAD gene will be quantitated by measuring mRNA and secreted GABA. To monitor the function in vivo, the genetically modified fibroblasts will be grafted into hippocampus or lateral ventricle and tested for the effects on the seizure phenomenon (behavioral and electroencphalographical seizure), seizure threshold and seizure-induced neuronal degeneration. The model systems for these purposes are a chronic model of epilepsy induced by fimbria-fornix lesions and an acute model of limbic seizure induced by intraamygdala injection of kainic acid. The long-term graft survival, in vitro gene expression and host immune reaction will be assessed by immunohistochemical approaches using antibodies to various specific cell markers.