Basic research of malignant glioma-specific gene therapy by using very high titer retrovirus vectors

1992年，美国首次进行了恶性胶质瘤的基因治疗。他们将产生逆转录病毒载体的PA317(小鼠)包装细胞植入恶性胶质瘤患者的肿瘤床(Ram Z等人：自然医学，1997)。这一过程不仅会引起免疫异种移植反应，而且会对正常分裂的细胞产生严重的副作用。为此，我们对启动子表达髓鞘碱性蛋白(MBP)基因的脑特异性基因治疗自杀基因(单纯疱疹病毒胸苷激酶；HSVtk)进行了基础研究(J Neurosci res 36,1993)。为了获得高滴度的逆转录病毒载体(Hum gene Ther 9,1998)，我们将多瘤基因ORI早期区域导入产生逆转录病毒的包装细胞中。小鼠脑肿瘤模型被这些高滴度载体完全治愈(基因Ther 5,1998；基因Ther 8,2001；人类细胞14,2001)。The Sa…2002年12月2日，文部科学大臣批准了用普通绒猴(灵长类)更快速地测试这些媒介的确认实验。现在，我们对这些高滴度的脑特异性逆转录病毒载体产生(包装)细胞(母细胞)进行了几项体外安全性研究，并将这些母细胞冷冻保存，然后利用普通的绒猴进行体内安全性检查。此外，由于这些恶性胶质瘤表现出MBP高表达和低表达的基因，我们利用SAGE(基因表达系列分析)方法确定MAGE-E1基因是胶质瘤相关基因的候选基因之一(癌症资源61,2001；基因277,2001)。现在，我们寻找表达MAGE-E1基因的启动子，然后尝试一种新的针对胶质瘤的基因治疗，该新的启动子被转导到哪些载体上。此外，我们还将根据《关于人类基因组和基因分析研究的伦理指示》，在患者本人同意的情况下，研究MAGE-E1基因的出现与脑胶质瘤患者主要组织相容性复合体(MHC)的相关性。较少

Gene therapy for malignant glioma was done for the first time in the United States in 1992. They implanted PA317 (mouse) packaging cells, which produced the retrovirus vectors transduced the non-specific SV40 promoter, into the tumor beds of malignant glioma patients (Ram Z, et al : Nature Med, 1997). This procedure may induce not only the immunological xeno-transplantation reactions but also the serious side-effects into the normal divided cells. Therefore, we have repeated some basic research on brain-specific gene therapy controlled suicide gene (Herpes Simplex Virus thymidine kinase ; HSVtk) with promoter expressed Myelin basic protein (MBP) gene (J Neurosci Res 36, 1993). We transduced the Polyoma ori early region into the packaging cells produced retrovirus in order to obtain the high-titer retroviral vectors (Hum Gene Ther 9, 1998). And mouse brain tumor models were completely cured by these high-titer vectors (Gene Ther 5, 1998 ; Gene Ther 8, 2001 ; Human Cell 14, 2001). The sa … More fety test these vectors by using a common marmoset (primates) was approved as a confirmation experiment of the Minister of Education, Culture, Sports, Science and Technology on December 2, 2002. Now, we perform several in vitro safety studies of these high-titer brain-specific retroviral vector-producing (packaging) cells (master cells) and cryopreserve these master cells, and then take a plan of in vivo safety examinations by using common marmosets. Finally, we are planning clinical phase I studies of gene therapy for brain tumor patients in 2004.In addition, as these malignant gliomas showed high and low MBP-expressed gene, we identified MAGE-E1 gene, as one of the candidates for glioma-related genes, by using the SAGE(serial analysis of gene expression) method (Cancer Res 61, 2001 ; Gene 277, 2001). Now, we search the promoter expressing MAGE-E1 gene, and then will try a new glioma-specific gene therapy, which vectors were transduced this new promoter. Moreover, we will research the correlation between this MAGE-E1 gene appearance and major histocompatibility complexes (MHC) of glioma patients under agreement of the patient himself, according to "Ethics indication concerning human genome and gene analysis research". Less