Gene therapy for inherited neurodegenerative disease using fetal bone marrows stem cell transplantation.

本实验旨在阐明人C型尼曼-匹克病的发病机制，为鼻咽癌缺陷基因的克隆及逆转录病毒载体对大鼠胎儿的基因治疗提供基础。1）观察到C型尼曼-匹克病成纤维细胞摄取低密度脂蛋白后胞浆钙离子浓度的降低。此外，我们发现钙通道激动剂（YC-170）可改善钙浓度的升高和胆固醇酯化不足，分别为正常对照的40%和90%。这些数据表明，胞浆钙浓度的急剧升高与人类尼曼-匹克病C型的病因密切相关。2）我们证实了来源缺陷型猴病毒40转化的NPC成纤维细胞中细胞内胆固醇的合成强烈加速，并且我们显示了由SV 40转化的NPC细胞系对HMG-CoA还原酶抑制剂（洛伐他汀，Sinvastatin）的超敏性。因此，这些细胞系和HMG-CoA还原酶抑制剂可用于NPC中缺陷基因的克隆。3）利用新开发的逆转录病毒载体成功地将阿萨基因导入人MLD成纤维细胞中并表达。这是评估先天性代谢紊乱基因治疗潜力的第一步。并利用该逆转录病毒载体对大鼠胚胎进行阿萨基因治疗。将该载体经洋葱静脉注射后，我们尝试在肝、脾、肾等器官中发现阿萨基因的表达，但用人阿萨抗体进行免疫组化染色，我们在这些器官中没有发现任何蛋白表达。目前，我们正在尝试利用转α-葡萄糖醛酸酶基因的腺相关载体对MPS小鼠胎儿进行基因治疗。

In this experiment, we tried to elucidate the mechanism of pathogenesis of humann Niemann-Pick disease type C and to have a basic approach for cloning of the defective gene in NPC and to create gene therapy for rat fetus by using of retrovirus vector.1) We detected the attenuated elevation of cytoplasmic calcium concentration following the uptaking of Low Density Lipoprotein in type C Niemann-Pick fibroblast. Moreover, we found that calcium channel agonist (YC-170) improve the attemuated elevation of calcium concentration and the deficient cholesterol esterification by 40% and 90% of normal control, respectively. These data indicate that the attemuated elevation of cytoplasmic calcim concentration was strongly related to the etiology of humann Niemann-Pick disease type C.2) We confirmed the synthesis of cholesterol within cells was strongly accelerated in transformed NPC fibroblast by origin defective simian virus 40 and we showed the hypersensitivity of NPC cell lines transformed by SV40 to HMG-CoA reductase inhibitor (Lovastatin, Sinvastatin). Therefor, these cell lines and HMG-CoA reductase inhibitor could be useful for the cloning of the defective gene in NPC.3) We have the succesful transduction and expression of ASA gene in human MLD fibroblast using a newly developed retroviral vector. This is a first step in evaluating the potential of gene therapy for congenital metabolic disorder. And we tried to have the gene theraly for rat fetus by using this developed retroviral vector transduced with ASA gene. After injection of this vector into amonion vein, we tried to find the expression of ASA gene in several organs such as liver, spleen, and kidny but we could not find any protein expression in these organs by immunohistochemical staining of human ASA antibody. Now we are trying to have gene therapy of MPS mouse fetus by using adeno associated vector which was transduced by alpha-glucuronidase gene.