Development of testicular tumor gene therapy using human testicular tumor cell-specific promoters

肿瘤基因治疗最基本的问题之一是如何使相关基因在肿瘤细胞中高效、特异地表达。近年来，使用在癌细胞中特异性激活的器官特异性启动子的癌症基因治疗的基础研究和相关临床试验的热潮，特别关注涉及器官特异性启动子和自杀基因的组合的治疗。鉴于70%以上的晚期睾丸肿瘤患者血清β-HCG（human chorionic gonadotropin，人绒毛膜促性腺激素）水平升高，本研究旨在开发一种基于β-HCG启动子作为器官特异性启动子的基因治疗方法，并探讨其有效性。本实验旨在筛选一个DNA大小为729 bp的β-HCG启动子，但我们也克隆了其他不同大小的β-HCG启动子的DNA，并测定了每个启动子在睾丸肿瘤细胞中的活性，以确定β-HCG启动子在睾丸肿瘤细胞中的活性。 ...更多信息 er以确定用于基因治疗的β-HCG启动子的最佳大小。结果表明，克隆的729 bp的β-HCG启动子DNA具有最好的特异性，并将其插入到整合有荧光素酶基因的质粒载体中。将该载体基因导入多种细胞-绒毛膜癌细胞（CHR）、胚胎癌细胞（NEC 8、NEC 14）、前列腺癌细胞（PC-3、DU 145）和膀胱癌细胞（WH），并测量荧光素酶活性以确认β-HCG启动子中的细胞特异性活性。仅在产生β-HCG的绒毛膜癌细胞（CEA）和胚胎癌细胞（NEC 8、NEC 14）中观察到高水平的启动子活性。在此基础上，我们构建了Ad-β-HCG -TK，并进行了类似的实验，其中仅在绒毛膜癌细胞（CEA）和胚胎癌细胞（NEC 8，NEC 14）中发现特异性抗肿瘤活性。在正常组织中未观察到细胞损伤作用。接下来，为了探索使用可复制腺病毒载体的基因治疗的发展，我们创建了一个整合E1 A基因控制的一个？-HCG启动子。目前，我们正在使用该载体进行体内和体外治疗研究，以研究人类睾丸肿瘤的最佳基因治疗，最终目的是与使用自杀基因的基因治疗进行比较。少

One of the most basic issues in gene therapy for cancer is how to express the relevant genes efficiency and specifically in cancer cells. In recent years, there has been an upsurge in basic research into gene therapy for cancer using organ-specific promoters specifically activated in cancer cells and in related clinical trials, with particular attention focused on therapy involving a combination of organ-specific promoters and suicide genes. Motivated by the finding that serum β-HCG (human chorionic gonadotropin) is elevated in over 70% of patients with advanced testicular tumor resistant to chemotherapy, the present study was designed to develop a gene therapy based on the application of a β-HCG promoter as an organ-specific promoter and to investigate its usefulness. It is intended to select a β-HCG promoter with DNA size of 729 base pairs, but we also cloned the DNA of β-HCG promoters of various other sizes, and measured the activity of each promoter in testicular tumor cells in ord … More er to determine the optimal size of a β-HCG promoter for gene therapy. It was found that the cloned β-HCG promoter DNA with 729 base pairs had the best specificity, and this was inserted into a plasmid vector integrated into a luciferase gene. This vector was introduced genetically into a variety of cells-choriocarcinoma cells (JAR), embryonal cancer cells (NEC8, NEC14), prostate cancer cells (PC-3, DU145), and bladder cancer cells (WH), and luciferase activity measured to confirm cell-specific activity in the β-HCG promoter. High levels of promoter activity were observed only in the β-HCG producing choriocarcinoma cells (JAR) and embryonal cancer cells (NEC8, NEC14). Based on these findings, we created an Ad-β-HCG -TK and undertook a similar experiment, in which specific anti-tumor activity was found only in the choriocarcinoma cells (JAR) and the embryonal cancer cells (NEC8, NEC14). No cell-damaging action was observed in normal tissue. Next, in order to explore the development of gene therapy using replication-competent adenovirus vectors, we created one integrating an E1A gene controlled by a ? -HCG promoter. At present, we are engaged in in vivo and in vitro therapy studies using this vector to investigate the optimal gene therapy for human testicular tumors, with the eventual aim of conducting a comparison with gene therapy using suicide genes. Less