Retrovirus-mediated gene therapy for hepatocellular cacrinoma

甲胎蛋白(AFP)基因在肝细胞癌中重新激活。我们先前已经报道，携带由0.3kb人AFP启动子调控的单纯疱疹病毒胸苷激酶基因的逆转录病毒载体(LNAFO.3TK)在产生AFP的肝癌细胞中具有更昔洛韦(GCV)介导的细胞毒作用，与产生AFP的能力平行。将LNAFO.3TK感染高甲胎蛋白产生的人肝癌细胞(HH-7)，接种于裸鼠皮下。为提高逆转录病毒载体LNAFO.3E+TK对中低产AFP肝癌细胞的基因治疗效果，构建了新的重组逆转录病毒载体(LNAFO.3E+TK)，将人AFP增强子区域直接连接到LNAE0.3TK的0.3kb的AFP启动子上，分别在中低产AFP的人肝癌细胞PLC/PRF/5和HH/Cl.2中对GCV增敏。在携带PLC/PRF/5细胞的裸鼠体内模型中，GCV对LNAFO.3E+TK病毒感染的细胞的生长抑制作用比LNAFO.3TK病毒感染的细胞更明显。据报道，人APP启动子上的G o A替换与APP的遗传持久性有关，因此在LNAFO.3TK中产生了这种替换来构建LNAFO.3MTK感染PLQTPRF/5和HH/CL.2细胞，并显示出比LNAFO.3TK感染更明显的GCV治疗敏感性。

The alpha-fetoprotein (AFP) gene is reactivated in hepatocellular carcinoma.. We have previously reported that a retrovirus vector (LNAFO.3TK) carrying a herpes simplex virus thymidine kinase gene regulated by the 0.3-kb human AFP promoter provides ganciclovir (GCV)-mediated cytotoxicity in AFP-producing hepatoma cells parallel with the ability of AFP production.In the present study, the in vivo effect of retrovirus-mediated gene therapy for human hepatoma cells was examined. High AFP-producing human hepatoma cells (HuH-7) was infected with LNAFO.3TK and implanted into subcutaneous of athymic mice. GCV treatment resulted in pronounced growth inhibition of the virus-infected HuH-7 xenograft in mice, but did not affect growth of parental xenograft.To improve the efficacy of retrovirus-mediated gene therapy for the intermediate and low AFP-producing hepatoma cells, new recombinant retrovirus vector (LNAFO.3E+TK) was constructed, in which human AFP enhancer region was directly linked to 0.3-kb AFP promoter of LNAEO.3TK.In the intermediate and low AFP-producing human hepatoma cells PLC/PRF/5 and huH/cl.2, respectively, LNAFO.3E+TK sensitized these cells to GCV in vitro. In vivo model using athymic mice harboring PLC/PRF/5 cells, GCV treatment resulted in more pronounced growth inhibition in the LNAFO.3E+TK virus-infected cells than in LNAFO.3TK virus-infected cells.It is reported that a G o A substitution in the human APP promoter is associated with hereditary persistence of APP, therefor this substitution was generated in LNAFO.3TK to construct LNAFO.3MTK.LNAFO.3MTK infection into PLQTPRF/5 and huH/cl.2 cells also showed more pronounced sensitivity to GCV treatment than LNAFO.3TK infection.