Gene therapy for hepatocellular carcinoma

我们先前报道，在0.3kb的人甲胎蛋白(α)基因启动子(AF0.3)控制下，表达单纯疱疹病毒胸苷激酶(HSV-tk)基因的逆转录病毒载体对更昔洛韦(GCV)在高甲胎蛋白产生的人肝癌细胞中具有细胞毒作用，而在低甲胎蛋白产生的细胞中不具有细胞毒作用。因此，可能需要特异性地增强AFP启动子的活性，以在低AFP产生的肝癌细胞中诱导足够的细胞毒性。在本研究中，我们构建了一个含有缺氧反应元件(HRE)的人血管内皮生长因子(VEGF)5‘端0.4kb片段与AF0.3启动子融合的杂合启动子[HRE]AF。通过报告基因转染实验，在低AFP和不产生AFP的人肝癌细胞中检测到由[HRE]AF启动子介导的低氧诱导转录，而在非肝癌细胞中检测不到。用逆转录病毒载体将[HRE]AF启动子控制的HSV-tk基因导入肝癌和非肝癌细胞，1%O2可诱导肝癌细胞产生GCV-细胞毒作用。此外，在荷实体瘤移植瘤的裸鼠中，只有由感染病毒的肝癌细胞组成的肿瘤通过给予GCV逐渐消失。这些结果表明，低氧诱导的人血管内皮生长因子基因增强子与人AFP启动子直接相连，参与了肝癌基因治疗中选择性和增强的杀瘤活性。

We previously reported that the retroviral vector expressing the herpes simplex virus-thymidine kinase (HSV-tk) gene under the control of 0.3-kb human α-fetoprotein (AFP) gene promoter (AF0.3) provided the cytotoxicity to ganciclovir (GCV) in high-AFP-producing human hepatoma cells but not in low-AFP-producing cells. Therefore, specific enhancement of AFP promoter activity is likely to be required to induce enough cytotoxicity in low-AFP-producing hepatoma cells. In this study, we constructed a hybrid promoter, [HRE]AF, in which a 0.4-kb fragment of human vascular endothelial growth factor (VEGF) 5'-flanking sequences containing hypoxia responsive element (HRE) was fused to AF0.3 promoter. By means of the reporter gene transfection assay, hypoxia-inducible transcriptions that were mediated by [HRE]AF promoter were detected in low- and non-AFP-producing human hepatoma cells, but not in nonhepatoma cells. When the HSV-tk gene controlled by [HRE]AF promoter was transduced into hepatoma and nonhepatoma cells by a retroviral vector, the exposure to 1% O2 induced GCV-cytotoxicity specifically in the hepatoma cells. Moreover, in nude mice bearing solid tumor xenografts, only the tumors consisting of the virus-infected hepatoma cells gradually disappeared by GCV administration. These results indicate that the hypoxia-inducible enhancer of the human VEGF gene, which is directly linked to human AFP promoter, involves selective and enhanced tumoricidal activity in gene therapy for hepatocellular carcinoma.