In vivo liver-directed gene transfer using in-situ perfusion of the porcine liver

[目的]建立猪肝脏的体内基因转移模型，并利用该模型进行代谢紊乱（某些蛋白质或酶的缺陷或异常）的基因治疗。在这种情况下，预期转染的肝脏在成功的基因转移后产生靶蛋白。为了实现这些目标，将血管性血友病因子（vWF）基因转移到体内肝脏中，在患有和不患有血管性血友病（vWD）的猪中，然后评估血浆vWF水平。由于vWF是由内皮细胞产生和分泌的，因此vWF基因应该被转移到猪肝中的窦内皮细胞中。【结果】初步研究表明，无论是用腺病毒载体（AdV）还是HVJ-脂质体载体（HVJ），LacZ基因和人vWF在体外均能有效地转染猪血管内皮细胞（约90%）。另一方面，无论是全身注射， ...更多信息 使用相同的载体（AdV或HVJ），门静脉内注射也不能在体内猪肝中建立任何基因转移（LacZ，vWF）。我们接下来测试了原位肝脏灌注（ISP）作为体内基因转移的方法，其中在来自门静脉和下腔静脉的血流的泵驱动旁路下从体循环分离肝脏，并且在无血和冷缺血条件下用高滴度AdV或HVJ灌注肝脏。结果，在猪肝的肝细胞和窦状内皮细胞中证实了标志物LacZ的表达（X-gal染色和RT-PCR）。RT-PCR结果显示，转基因小鼠血浆中vWF（HVJ）的mRNA表达仅在ISP后才有表达，而转基因小鼠血浆中vWF水平无明显升高。[结论]通过肝ISP和HVJ-脂质体载体进行vWF的体内基因转移是可行的。然而，无法证实vWF的显著增加，因此表明需要进一步研究以确定临床相关的基因转移水平。少

【Aims】 The aim of this study was to establish the effective gene transfer to a porcine liver in-vivo, and to utilize this model for gene therapy for metabolic disorder (a deficiency or abnormality of certain proteins or enzymes). In this setting, the transfected liver was expected to produce a target protein after successful gene transfer. To accomplish these aims, the von Willebrand factor (vWF) gene was transferred into the livers in-vivo, in pigs with and without von Willebrand disease (vWD), and the plasma vWF levels were thereafter assessed. Since vWF is produced and secreted from the endothelial cells, the vWF gene should thus be transferred into the sinusoidal endothelial cells in the pig liver.【Results】 Preliminary studies have demonstrated that both the marker LacZ gene and human vWF were effectively transferred into the porcine endothelial cells in-vitro (≧ 90%), either with adenovirus vector (AdV) or HVJ-liposome vector (HVJ). On the other hand, neither the systemic injectio … More n nor the intraportal injection could establish any gene transfer (LacZ, vWF) in the in-vivo pig liver, using the same vectors (AdV or HVJ). We next tested in-situ perfusion of the liver (ISP) as a method for in-vivo gene transfer, in which the liver was isolated from systemic circulation under a pump driven bypass of the blood flow from the portal vein and inferior vena cava, and the liver was perfused with a high titer AdV or HVJ under bloodless and cold ischemic conditions. As a result, the marker LacZ expression was confirmed (X-gal staining ＆ RT-PCR) in both the hepatocytes and sinusoidal endothelial cells in the pig liver. The mRNA expression of transferred vWF (HVJ) was also confirmed by RT-PCR only after ISP.The plasma vWF levels, however, did not significantly increase after gene transfer.【Conclusion】 The in-vivo gene transfer of vWF was thought to be possible by an ISP of the liver and HVJ-liposome vector. A significant increase of vWF, however, could not be confirmed, thus suggesting that further investigations are thus needed to establish the clinically relevant levels of gene transfer. Less