Retrovirus-mediated gene therapy for hepatocellular cacrinoma

甲胎蛋白（AFP）基因在肝细胞癌中被重新激活。我们以前曾报道过一种逆转录病毒载体（LNAFO.3TK），该载体携带由0.3kb人AFP启动子调控的单纯疱疹病毒胸苷激酶基因，在产生AFP的肝癌细胞中提供更昔洛韦（GCV）介导的细胞毒作用，与产生AFP的能力平行。用LNAFO.3TK感染高AFP产生的人肝癌细胞（HuH-7）并植入无胸腺小鼠的皮下。为提高逆转录病毒介导的基因治疗中、低AFP水平肝癌细胞的疗效，我们研制了一种新型的重组逆转录病毒载体，（LNAF0.3E +TK），其中人AFP增强子区直接连接到LNAE0.3TK的0.3kb AFP启动子。5和huH/cl.2的作用下，LNAF0.3E +TK在体外使这些细胞对GCV敏感。在PLC/PRF/5细胞的裸鼠体内模型中，GCV处理导致LNAFO.3E+TK病毒感染的细胞比LNAFO.3TK病毒感染的细胞更明显的生长抑制。因此，在LNAFO.3TK中产生这种取代以构建LNAFO.3MTK.LNAFO.3MTK感染PLQTPRF/5和huH/cl.2细胞也显示出比LNAFO.3TK感染对GCV处理更显著的敏感性。

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.