Uch Rathviro, Gérolami René, Faivre Jamila, Hardwigsen Jean, Mathieu Sylvie, Mannoni Patrice, Bagnis Claude
Département de thérapie cellulaire et génique, Etablissement Français du Sang Alpes-Méditerranée, Marseille, France.
Cancer Gene Ther. 2003 Sep;10(9):689-95. doi: 10.1038/sj.cgt.7700621.
Suicide gene therapy combining herpes simplex virus thymidine kinase gene transfer and ganciclovir administration can be envisioned as a powerful therapeutical approach in the treatment of hepatocellular carcinoma; however, safety issues regarding transgene expression in parenchyma cells have to be addressed. In this study, we constructed LATKW, a lentiviral vector expressing the HSV-TkEGFP gene placed under the control of the promoter elements that control the expression of the rat alpha-fetoprotein, and assayed its specific expression in vitro in hepatocarcinoma and nonhepatocarcinoma human cell lines, and in epidermal growth factor stimulated human primary hepatocytes. Using LATKW, a strong expression of the transgene was found in transduced hepatocarcinoma cells compared to a very low expression in nonhepatocarcinoma human cell lines, as assessed by Northern blot, RT-PCR, FACS analysis and ganciclovir-mediated toxicity assay, and no expression was found in lentivirally transduced normal human hepatocytes. Altogether, these results demonstrate the possibility to use a lentivirally transduced expression unit containing the rat alpha-fetoprotein promoter to restrict the HSV-TK-mediated induced GCV sensitivity to human hepatocarcinoma cells.
将单纯疱疹病毒胸苷激酶基因转移与更昔洛韦给药相结合的自杀基因疗法，可以被设想为治疗肝细胞癌的一种强有力的治疗方法；然而，必须解决关于实质细胞中转基因表达的安全性问题。在本研究中，我们构建了LATKW，一种慢病毒载体，其表达受大鼠甲胎蛋白表达调控的启动子元件控制的HSV-TkEGFP基因，并在体外检测其在肝癌和非肝癌人类细胞系以及表皮生长因子刺激的人类原代肝细胞中的特异性表达。使用LATKW，通过Northern印迹、RT-PCR、流式细胞术分析和更昔洛韦介导的毒性试验评估，发现与非肝癌人类细胞系中极低的表达相比，转导的肝癌细胞中转基因有强烈表达，并且在慢病毒转导的正常人肝细胞中未发现表达。总之，这些结果证明了使用含有大鼠甲胎蛋白启动子的慢病毒转导表达单元来将HSV-TK介导的诱导的GCV敏感性限制于人类肝癌细胞的可能性。
