Gene Therapy for Advanced Hepatocellular Carcinoma using Transcriptional Control

我们已经证明了肿瘤特异性启动子作为癌症基因治疗工具的有效性。然而，这些启动子的表达水平普遍较低，因此我们开发了一种新的通用方法来提高组织特异性启动子的表达水平，同时保持特异性。我们构建了一个在肝癌特异性α(AFP)启动子控制下产生位点特异性重组Cre的调节性重组腺病毒(Rad)。将Rad与含有Cre激活的有效表达单位的靶向Rad一起感染AFP产生细胞。在体外实验中，双重感染方法比由AFP启动子直接驱动的单一Rad感染方法获得了约50倍的高表达，同时保持了对AFP产生细胞的严格特异性。在体内肿瘤模型中也观察到了增强的和特异性的表达。该方法不仅有助于建立特异性的基因治疗方法，而且为阐明细胞型特异性基因的功能提供了基础研究。下一步，我们将表达单纯疱疹病毒胸苷激酶基因的Rad作为靶向Rad，并开始检测其对甲胎蛋白产生细胞系和裸鼠移植瘤的细胞毒作用。

We have demonstrated the usefullness of tumor specific promoter as a tool of cancer gene therapy. The expression levels of such promoters, however, are generally low, and we have therefore develop a novel and general method to enhance the expression level of a tissuespecific promoter while maintaining specificity. We constructed a regulatory recombinant adenovirus (rAd) producing the site-specific recombinant Cre under the control of the hepatocellular carcinoma-specific a (AFP) promoter. The rAd was infected to AFP-producing cells together with a target rAd containing a Cre-activating potent expression unit. In in vitro experiments, the duble infection method gave about 50-fold higher expression than the single rAd infection directly driven by the AFP promoter, while maintaining strict specificity to AFP- producing cells. The enhanced and specific expression was also observed in in vivo tumor models. This method may contribute not only to the establishement of specific gene therapies but also to basic study for elucidating cell-type specific gene functions. As a next step, we have just made a rAd expressing herpes simplex virus thymidine kinase gene as a target rAd, and started testing the cytotoxicity in AFP producing cell lines and transplanted tumor on nude mice.