Gene therapy for hepatocellular carcinoma

我们以前报道过，在0.3kb人甲胎蛋白（AFP）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，在高AFP产生的人肝癌细胞中对更昔洛韦（GCV）具有细胞毒作用，而在低AFP产生的细胞中则没有。因此，可能需要特异性增强AFP启动子活性以在低AFP产生的肝癌细胞中诱导足够的细胞毒性。在本研究中，我们构建了一个杂合启动子，[HRE]AF，其中含有缺氧反应元件（HRE）的人血管内皮生长因子（VEGF）5 '侧翼序列的0.4-kb片段融合到AF 0.3启动子。通过报告基因转染实验，[HRE]AF启动子介导的低氧诱导转录在低AFP产生和非AFP产生的人肝癌细胞中被检测到，而在非肝癌细胞中未检测到。将[HRE]AF启动子控制的HSV-tk基因通过逆转录病毒载体导入肝癌细胞和非肝癌细胞后，1%O_2暴露可特异性诱导肝癌细胞的GCV细胞毒活性。此外，在荷实体瘤异种移植物的裸鼠中，只有由病毒感染的肝癌细胞组成的肿瘤通过GCV给药逐渐消失。这些结果表明，缺氧诱导的增强子的人VEGF基因，这是直接连接到人AFP启动子，涉及选择性和增强的杀肿瘤活性的基因治疗肝细胞癌。

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.