Establishment of Gene Therapy for Advanced Hepatocellular Carcinoma with Intra- and Extra- Hepatic Metastases

利用转录靶向治疗晚期肝癌肝内和肝外转移的新治疗载体。甲胎蛋白（AFP）在肝细胞癌中特异性表达是众所周知的。我们通过去除非功能部分和增强子序列来修饰AFP基因的启动子区域，并将新的AFP基因控制元件放入腺病毒载体中。研究了载体对裸鼠移植肝癌的治疗效果及表达分析。单纯疱疹。病毒胸苷激酶基因被用作治疗基因。由于启动子活性较弱，其抗肿瘤作用较低，其转录靶向作用主要体现在AFP控制元件上。因此，我们将cre-loxP系统引入到向量中。通过cre-loxP-AFP系统，可以在培养细胞和小鼠肿瘤（包括肝内和肝外病变）中获得特异性和高表达。但是，需要两个病毒同时感染一个细胞，并且需要超过25个MOI才能启用该系统。这些结果表明，大量的腺病毒应该被引入体内，这可能会造成严重的问题。将毒性较小的HVJ脂质体与AFP控制因子联合使用，在肝内转移模型中反复给药具有显著的抗肿瘤作用。因此，利用AFP启动子的转录靶向治疗伴有肝内和肝外转移的晚期肝细胞癌是有希望的。

New therapeutic vectors using transcriptional targeting were made for the treatment of advanced hepatocellular carcinoma with intra- and extra-hepatic metastases. Alpha-fetoprotein (AFP) is well known to be specifically expressed in hepatocellular carcinoma. We modified the promoter region of AFP gene by removal of non-functioning parts and enhancing the enhancer sequences, and put the new controlling element of AFP gene into an adenoviral vector. Therapeutic effect and expression analysis of the vectors was tested in transplanted hepatocellular carcinoma in athymic nude mice. Herpes simplex. virus thymidine kinase gene was used as a therapeutic gene.Effect of transcriptional targeting was demonstrated by the AFP controlling element, the anti-tumor effect was low due to the weak promoter activity. Thus, we introduced cre-loxP system into the vector. By the cre-loxP-AFP system, specific and high expression can be obtained in cultured cells and mouse tumors including intra- and extra-hepatic lesions.However, two viruses need to infect simultaneously a cell, and more than 25 MOI are necessary to enable the system. These results indicate large amount of adenovirus should be introduced in a body, which may cause serious problem in future.When HVJ liposome, which is known to have less toxicity, was used with AFP controlling element, repeated administration of the vector confer significant anti-tumor effect in intra-hepatic metastasis model. Transcriptional targeting using AFP promoter is, thus, promising for the treatment of advanced hepatocellular carcinoma with intra- and extra-hepatic metastases.