New vector system for the gene therapy of abdominal cancers.

用于腹部癌症基因治疗的新载体系统。

• 批准号: 09557103
• 负责人: NIMURA Yuji
• 金额: $ 8.19万
• 依托单位: Nagoya University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 1997
• 资助国家: 日本
• 起止时间: 1997 至 1998
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09557103/
• 关键词: gene therapy; retroviral vector; peritoneal dessemination; transcriptional regulation; 腹膜播種癌; レトロウイルスベクター; ドキシサイクリン; on; off制御; GFP

Tight control of gene expression in temporal and quantitativemanner is an invaluable tool in both basic and clinical biology. The desired characteristics of the inducible system are as follows. (I) The regulator should be activated only upon administration of an exogenousmediator, and terminated when the exogenous stimulus is removed. (ii) The mediator should be non toxic and active upon oral administration. (iii) The system should not activate other endogenous cellular genes. We constricted a retroviral vector that has an autoregulatory cassette and express the gene of interest in response to oral administration of doxycycline (dox) invivo. The cassette contains the all components of the reversetetracycline-regulated (rtTA) system (Gossen M.et al. 1995), a drugselectable marker (BSD) with the internal ribosome entry site (IRES) and the gene of interest (GFP). The retroviral long term repeat enhancer and promoter elements driv-expression of the taransactivator (rTetR-VP16) and BSD, but … More the translation was terminated at the 3'-site of BSD. The expression of GFP was controlled under the tandem tet operator sequencesand the cytomegalovirus minimal promoter in a dose-dependent manner to dox. FACS analyses showed that GFP-fluoresence was induced in two-ordermagnitude in the retrovirus-infected 208F cells dependent on the amount of dox. Furthermore oral administration of dox could induce GFP protein in thetransplanted 208F cells in the peritoneal cavity of a nude mouse. Thus, this reverse tetracycline-regulated retroviral vector (RTRRV) system allowseasy delivery of controllable genes to cultured cells and transgenicanimals. However, the control system did not work in several human cancercells. This may be attributed to the species-dependent susceptibility toretorviruses. And we found that the introduction of a toxic gene into RTRRVkilled the PA317 retrovirus packaging cells, suggesting that RTRRV still suffer from problems of higher basal levels of gene expression undernoninduced condition and pleiotropic effects on host cell genes. If we will solve these problems mentions above, this inducible retroviral vector system may pave the way to the controlled gene expression during a particular window of time in gene therapy applications. Less

严格控制基因在时间和数量上的表达在基础生物学和临床生物学中都是非常有价值的工具。感应式系统的期望特性如下。(I)只有在使用外源性调节剂时才应激活调节器，并在移除外源性刺激时终止调节器。(Ii)调解人口服时应是无毒和活跃的。(3)该系统不应激活其他内源性细胞基因。我们构建了一个具有自我调节盒的逆转录病毒载体，并在体内表达目的基因，以响应口服多西环素(Dox)。该盒包含逆转四环素调节(RTTA)系统的所有组件(Gossen M.等人)。1995)，具有内部核糖体进入位点(IRES)和目的基因(GFP)的药物选择标记(BSD)。逆转录病毒长重复序列增强子和启动子元件驱动反式激活因子(rTetR-VP16)和BSD的表达，但…更多的翻译在BSD的3‘端终止。GFP的表达受控于串联的tet操纵子序列和巨细胞病毒最小启动子对DOX的剂量依赖关系。流式细胞仪分析显示，在逆转录病毒感染的208F细胞中，GFP-荧光被诱导出两个数量级的荧光，这取决于DOX的量。此外，口服DOX可诱导裸鼠腹膜移植208F细胞表达GFP蛋白。因此，这个反向四环素调控的逆转录病毒载体(RTRRV)系统可以将可控基因快速输送到培养细胞和转基因动物中。然而，控制系统在几个人类癌细胞中并不起作用。这可能归因于物种依赖的对逆转录病毒的易感性。我们还发现，将有毒基因导入RTRRV可以杀死PA317逆转录病毒包装细胞，这表明RTRRV在非诱导条件下仍存在基因表达的基础水平较高以及对宿主细胞基因的多效性的问题。如果我们能够解决上述问题，这种可诱导的逆转录病毒载体系统可能为基因治疗应用中在特定时间窗口内受控的基因表达铺平道路。较少

项目成果

Watanabe Y: "Characterization of a serum response factor-like protein in saccharomyces cerevisiae, Rlm1, which has transcriptional activity regulated by the Mpk 1 (Slt 2) mitogen-activated protein kinase pathway"Mol Cell Biol. 17. 2615-2623 (1997)

Watanabe Y：“酿酒酵母中血清反应因子样蛋白 Rlm1 的表征，其转录活性受 Mpk 1 (Slt 2) 丝裂原激活蛋白激酶途径调节”Mol Cell Biol。

Sakamoto E,et al.: "The pattern of infiltration at the proximal border of hilar bile duct carcinoma A histologic analysis of 62 resected cases"Ann Surg. 227. 405-411 (1998)

Sakamoto E 等：“肝门部胆管癌近端浸润模式对 62 例切除病例的组织学分析”Ann Surg。

Nimura Y,et al.: "Aggressive surgical treatment of hilar cholangiocarcinoma" J Hep Bil Pancr Surg. 5. 52-61 (1998)

Nimura Y 等人：“肝门部胆管癌的积极手术治疗”J Hep Bil Pancr Surg。

Watanabe Y,et al.: "Characterization of a serum response factor-like protein in saccharomyces cerevisiae,Rlml,which has transcriptional activity regulated by the Mpkl(Slt2)mitogen-activated protein kinase pathway" Mol Cell Biol. 17. 2615-1623 (1997)

Watanabe Y 等人：“酿酒酵母 Rlml 中血清反应因子样蛋白的表征，该蛋白具有受 Mpk1(Slt2) 丝裂原激活蛋白激酶途径调节的转录活性”Mol Cell Biol。

Sakamoto E: "The pattern of infiltration at the proximal border of hilar bile duct carcinoma. A histologic analysis of 62 resected cases"Ann Surg. 227. 405-411 (1998)

Sakamoto E：“肝门部胆管癌近端浸润模式。62 例切除病例的组织学分析”Ann Surg。