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

使用人睾丸肿瘤细胞特异性启动子开发睾丸肿瘤基因治疗

• 批准号: 12671530
• 负责人: GOTOH Akinobu
• 金额: $ 1.09万
• 依托单位: Kobe University School of Medicine
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2000
• 资助国家: 日本
• 起止时间: 2000 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-12671530/
• 关键词: testicular tumor; gene therapy; cell specific; β-HCG; promoter; adenovirus; TK; Replication Competent Adenovirus

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

肿瘤基因治疗中最基本的问题之一就是如何在肿瘤细胞中高效表达相关基因。近年来，利用肿瘤细胞中特异激活的器官特异性启动子进行癌症基因治疗的基础研究和相关临床试验掀起了一股热潮，尤其是器官特异性启动子和自杀基因的联合治疗。由于发现70%以上的晚期睾丸肿瘤化疗耐药患者血清β-hCG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。本研究拟筛选β-hcg启动子，dna大小为729个碱基对，同时克隆了不同大小的β-hcg启动子，并测定了各启动子在睾丸肿瘤细胞中的…活性。进一步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β-hCG启动子具有最好的特异性，并将其插入到整合有荧光素酶基因的载体中。该载体被基因导入多种细胞-绒毛膜癌细胞(JAR)、胚胎癌细胞(NEC8、NEC14)、前列腺癌细胞(PC-3、DU145)和膀胱癌细胞(WH)，并检测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(JAR)和胚胎癌细胞(NEC8、NEC14)中观察到高水平的启动子活性。基于这些发现，我们构建了Ad-β-hCG-TK，并进行了类似的实验，在该实验中，仅在绒毛癌细胞(JAR)和胚胎癌细胞(NEC_8、NEC_(14))中发现了特异性的抗肿瘤活性。在正常组织中未观察到细胞损伤作用。接下来，为了探索利用复制型腺病毒载体进行基因治疗的发展，我们创建了一个整合了由？-hCG启动子控制的E1a基因的重组腺病毒载体。目前，我们正在利用该载体进行体内和体外治疗研究，以探索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。较少

项目成果

Wada Y., Gotoh A., Shirakawa T., Hara I., Hamada K., Ko SC., Kao C., Chung LWK., Kamidono S.: "Gene therapy for bladder cancer using adenoviral vector"Molecular Urology. 5. 47-52 (2001)

Wada Y.、Gotoh A.、Shirakawa T.、Hara I.、Hamada K.、Ko SC.、Kao C.、Chung LWK.、Kamidono S.：“使用腺病毒载体进行膀胱癌的基因治疗”分子泌尿学。

Akinobu Gotoh, Toshiro Shirakawa, Yoshitake Wada, Nobuyuki Hinata, Isao Hara, Masato Fujisawa, Hiroshi Okada, Soichi Arakawa, Sadao Kamidono: "Prospects for molecular research in urological oncology: Gene therapy"Acta Urol. Jpn.. 47. 829-832 (2001)

Akinobu Gotoh、Toshiro Shirakawa、Yoshitake Wada、Nobuyuki Hinata、Isao Hara、Masato Fujisawa、Hiroshi Okada、Soichi Arakawa、Sadao Kamidono：“泌尿肿瘤分子研究的前景：基因治疗”Acta Urol。

Zhang ZJ., Shirakawa T., Hinata N., Matsumoto A., Fujisawa M., Okada H., Kamidono S, Matsuo M., Gotoh A.: "Combination with CD/5-FC gene therapy enhances killing of human bladder cancer cells by radiation"Journal of Gene Medicine. in press.

张 ZJ.、Shirakawa T.、Hinata N.、Matsumoto A.、Fujisawa M.、Okada H.、Kamidono S、Matsuo M.、Gotoh A.：“与 CD/5-FC 基因治疗相结合可增强对人类膀胱的杀伤作用

後藤章暢: "泌尿器科腫瘍学における分子研究の展望:遺伝子治療"泌尿紀要. 47. 829-832 (2001)

Akinori Goto：“泌尿肿瘤学分子研究的前景：基因治疗”《泌尿通报》47. 829-832 (2001)。

Shirakawa T., Sasaki R., Gardner TA., Kao C., Zang ZJ., Sugimura K., Matsuo M., Kamidono S., Gotoh A.: "Drug resistant human bladder cancer cells are more sensitive to the adenoviral-mediated wt-p53 gene therapy compared to the drug-sensitive human bladde

Shirakawa T.、Sasaki R.、Gardner TA.、Kao C.、Zang ZJ.、Sugimura K.、Matsuo M.、Kamidono S.、Gotoh A.：“耐药人类膀胱癌细胞对腺病毒更敏感 -