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

本实验试图阐明人类C型尼曼-匹克病的发病机制，为克隆鼻咽癌缺陷基因和利用逆转录病毒载体建立大鼠胚胎基因治疗奠定基础。1)检测C型尼曼-皮克病成纤维细胞摄取低密度脂蛋白后胞浆内钙离子浓度的轻度升高。此外，我们还发现，钙通道激动剂(YC-170)可使正常对照组的钙浓度升高和胆固醇酯化不足的发生率分别提高40%和90%。这些数据表明，胞浆内钙浓度的升高与人尼曼-匹克病C.2的病因密切相关。我们证实了原始缺陷猴病毒40转化的鼻咽癌成纤维细胞内胆固醇的合成被强烈地加速，并且我们发现SV40转化的鼻咽癌细胞对HMG-CoA还原酶抑制剂(洛伐他丁，Sinvastatin)具有超敏反应。因此，这些细胞系和HMG-CoA还原酶抑制剂可用于NPC.3缺陷基因的克隆。我们利用新开发的逆转录病毒载体成功地在人MLD成纤维细胞中转导和表达了ASA基因。这是评估先天性代谢紊乱基因治疗潜力的第一步。并利用所研制的转导AsA基因的逆转录病毒载体对大鼠胚胎的基因进行了初步研究。将此载体注射到羊膜静脉后，我们试图发现AsA基因在肝、脾、肾等器官中的表达，但通过人AsA抗体的免疫组织化学染色，我们没有发现这些器官中有任何蛋白表达。目前，我们正在尝试利用α-葡萄糖苷酸酶基因转导的腺相关载体对MPS小鼠胚胎进行基因治疗。

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.