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

【目的】本研究旨在建立有效的猪肝脏基因转移模型，并利用该模型对代谢紊乱（某些蛋白质或酶的缺乏或异常）进行基因治疗。在这种情况下，转染的肝脏有望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）基因转移到患有和不患有血管性血友病（vWD）的猪体内肝脏，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窦内皮细胞中。【结果】初步研究表明，用腺病毒载体（AdV）或HVJ脂质体载体（HVJ）将标记物LacZ基因和人vWF在体外均能有效转入猪内皮细胞（≧90%）。另一方面，使用相同的载体（AdV或HVJ），系统注射和门静脉内注射均不能在活体猪肝中建立任何基因转移（LacZ, vWF）。接下来，我们测试了原位肝脏灌注（ISP）作为体内基因转移的一种方法，在泵驱动的门静脉和下腔静脉血流旁路下，将肝脏从体循环中分离出来，在无血和冷缺血条件下向肝脏灌注高滴度的AdV或HVJ。结果，通过X-gal染色和RT-PCR证实了LacZ在猪肝肝细胞和肝窦内皮细胞中的表达。转染后的vWF (HVJ) mRNA的表达也仅在ISP后用RT-PCR进行了证实。然而，基因转移后血浆vWF水平没有显著升高。【结论】通过肝脏的ISP和hvj脂质体载体可以实现vWF的体内基因转移。然而，vWF的显著增加尚未得到证实，这表明需要进一步的研究来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