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。我们之前报道过一种携带由0.3 kb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的肝癌细胞中提供更昔洛韦（GCV）介导的细胞毒性，与AFP的产生能力平行。在本研究中，研究了逆转录病毒介导的基因治疗对人肝癌细胞的体内效果。用LNAFO.3TK感染高产afp的人肝癌细胞HuH-7，并将其植入胸腺小鼠皮下。GCV处理可显著抑制病毒感染的小鼠HuH-7异种移植物的生长，但不影响亲代异种移植物的生长。为了提高逆转录病毒介导的基因治疗中低AFP产生肝癌细胞的效果，构建了新的重组逆转录病毒载体LNAFO.3E+TK，其人AFP增强子区与LNAEO.3TK的0.3 kb AFP启动子直接连接。在中低afp产生的人肝癌细胞PLC/PRF/5和huH/cl中。2 . LNAFO.3E+TK分别使这些细胞在体外对GCV增敏。在含有PLC/PRF/5细胞的胸腺小鼠体内模型中，GCV处理对LNAFO.3E+TK病毒感染细胞的生长抑制作用比LNAFO.3TK病毒感染细胞的生长抑制作用更明显。据报道，人类APP启动子中的G o a替换与APP的遗传持久性有关，因此在LNAFO.3TK中产生该替换，构建LNAFO.3MTK.LNAFO。3MTK感染PLQTPRF/5和huH/cl。与LNAFO.3TK感染相比，2细胞对GCV的敏感性更明显。

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.