Parouchev Alexandre, Nguyen Tuan Huy, Dagher Ibrahim, Mainot Sylvie, Groyer-Picard Marie-Thérèse, Branger Julie, Gonin Patrick, Di Santo James, Franco Dominique, Gras Gabriel, Weber Anne
INSERM EMI-020 and University Paris XI, IFR 93, Hôpital du Kremlin-Bicêtre, Le Kremlin-Bicêtre, France.
J Hepatol. 2006 Jul;45(1):99-107. doi: 10.1016/j.jhep.2006.03.014. Epub 2006 May 2.
BACKGROUND/AIMS: Lentivirus-mediated ex vivo gene therapy is becoming a promising approach for the treatment of liver metabolic disorders. However, the feasibility of this approach needs to be studied in large animal models. The purpose of this study was to evaluate the efficacy of ex vivo gene transfer into Macaca hepatocytes with two different HIV-1 derived lentiviral vectors.
A self-inactivating lentivector was constructed to express GFP under the control of the hepatic apolipoprotein A-II promoter. Freshly isolated and thawed hepatocytes were transduced in suspension with lentiviral vectors expressing the GFP gene under the control of a ubiquitous promoter (EF1-alpha) and the apolipoprotein A-II promoter. Transduced thawed hepatocytes were transplanted into the spleen of newborn mice, and livers analyzed 4 and 12 weeks after transplantation.
We show that lentivectors are efficient in transducing hepatocytes in suspension either freshly isolated or cryopreserved. We also show that thawed and transduced hepatocytes engrafted and participated in liver growth after transplantation into newborn mice and that the apolipoprotein A-II promoter is functional.
Our data show that transplantation of transduced hepatocytes into monkeys should allow to evaluate the fate of transplanted cells and transgene expression in a pre-clinical model of ex vivo gene therapy.
背景/目的：慢病毒介导的离体基因治疗正成为治疗肝脏代谢紊乱的一种有前景的方法。然而，这种方法的可行性需要在大型动物模型中进行研究。本研究的目的是评估用两种不同的源自HIV-1的慢病毒载体将基因离体导入猕猴肝细胞的效果。
构建一种自失活慢病毒载体，使其在肝载脂蛋白A-II启动子的控制下表达绿色荧光蛋白（GFP）。将新鲜分离和冻融的肝细胞与在遍在启动子（EF1-α）和载脂蛋白A-II启动子控制下表达GFP基因的慢病毒载体在悬浮液中进行转导。将转导后的冻融肝细胞移植到新生小鼠的脾脏中，并在移植后4周和12周对肝脏进行分析。
我们表明，慢病毒载体能够有效地转导新鲜分离或冻存的悬浮肝细胞。我们还表明，转导后的冻融肝细胞在移植到新生小鼠后能够植入并参与肝脏生长，并且载脂蛋白A-II启动子具有功能。
我们的数据表明，将转导后的肝细胞移植到猴子体内应该能够在离体基因治疗的临床前模型中评估移植细胞的命运和转基因表达。
