A NEW MODEL FOR LIVER GENE THERAPY USING THE LIVER

目前使用肝脏的离体基因治疗方案的主要限制是，在大多数情况下，转导的肝细胞在移植后几乎不发生增殖，严重限制了外源基因表达的水平。最近，我们开发了一种新的大鼠肝再生模型，其中超过99%的内源性肝细胞可以被移植细胞替代。广泛的肝脏再生可以用10,000个移植细胞来实现，我们正在使用这个模型来研究胎肝细胞，肝细胞祖细胞和肝源性上皮细胞系的谱系和增殖潜力。我们已经开始了使用该模型进行离体基因治疗的研究，通过用含有报告基因绿色荧光蛋白（GFP）的慢病毒转导肝细胞、肝细胞祖细胞和胎儿成肝细胞的原代培养物，然后将转导的细胞移植到适当制备的宿主的肝脏中。我们的第一个假设是，肝细胞祖细胞或胎儿成肝细胞将具有比原代成人更高的病毒转导效率。我们的第二个假设是，祖细胞或胎儿成肝细胞在移植到肝脏后，在维持转导基因的长期表达方面上级肝细胞。我们的第三个假设是，慢病毒转导的细胞在宿主肝脏中的扩增将导致整个器官中高水平的外源基因表达，从而达到适合于有效基因治疗的表达水平。这些假设将在我们的新的大鼠肝细胞移植模型中使用慢病毒-GFP进行测试。一旦达到有效基因转移和表达的最佳条件，我们将使用这种方法，试图纠正缺陷的铜代谢在长埃文斯肉桂（LEC）大鼠，威尔逊病的模型，使用克隆的威尔逊病基因。最终，我们希望将细胞移植和离体基因治疗方法用于患有各种遗传性和获得性肝病的人，因为这些方法是互补的，但将具有不同的临床应用。

A major limitation of current ex vivo gene therapy protocols using the liver is that under most circumstances, little if any proliferation of transduced hepatocytes occurs after their transplantation, severely limiting levels of foreign gene expression. Recently, we developed a new model for liver repopulation in the rat in which more than 99% of endogenous hepatocytes can be replaced with transplanted cells. Extensive liver repopulation can be achieved with as little as 10,000 transplanted cells, and we are using this model to study the lineage and proliferative potential of fetal liver cells, hepatocyte progenitor cells, and liver-derived epithelial cell lines. We have initiated studies using this model for ex vivo gene therapy by transducing primary cultures of hepatocytes, hepatocyte progenitor cells and fetal hepatoblasts with lentivirus containing a reporter gene, green fluorescent protein (GFP), and then transplanting the transduced cells into the liver of suitably prepared hosts. Our fist hypothesis is that hepatocyte progenitor cells or fetal hepatoblasts will have a higher efficiency of viral transduction than primary adult. Our second hypothesis is that progenitor cells or fetal hepatoblasts will be superior to hepatocytes in maintaining long-term expression of transduced genes after they are transplanted into the liver. Our third hypothesis is that amplification of lentivirus transduced cells in the host liver will result in a high level of foreign gene expression throughout the organ, and thus achieve a level of expression suitable for effective gene therapy. These hypotheses will be tested in our new rat hepatic cell transplantation model using lentivirus-GFP. Once optimal conditions for effective gene transfer and expression are achieved, we will use this approach in an attempt to correct defective copper metabolism in the Long Evans Cinnamon (LEC) rat, a model for Wilson's disease, using a cloned Wilson's disease gene. Ultimately, we hope to adapt both cell transplantation and ex vivo gene therapy methods to humans with various inherited and acquired liver diseases, as these approaches are complementary but will have different clinical applications.