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一个主要限制是，在大多数情况下，转导的肝细胞在移植后几乎没有增殖，严重限制了外源基因的表达水平。最近，我们开发了一种新的大鼠肝脏再生模型，其中99%以上的内源性肝细胞可以被移植细胞取代。广泛的肝脏再生可以用10000个移植细胞来实现，我们正在使用这个模型来研究胎儿肝细胞、肝细胞祖细胞和肝源性上皮细胞系的谱系和增殖潜力。我们已经开始使用该模型进行体外基因治疗的研究，通过将含有报告基因、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肝母细胞的原代培养物，然后将转导后的细胞移植到适当制备的宿主肝脏中。我们的第一个假设是肝细胞祖细胞或胎儿肝母细胞比初级成人具有更高的病毒转导效率。我们的第二个假设是，祖细胞或胎儿肝母细胞在移植到肝脏后，在维持转导基因的长期表达方面优于肝细胞。我们的第三个假设是，宿主肝脏中慢病毒转导细胞的扩增将导致整个器官中外源基因的高水平表达，从而达到适合有效基因治疗的表达水平。这些假设将在我们使用慢病毒-绿色荧光蛋白的新大鼠肝细胞移植模型中得到验证。一旦获得了有效的基因转移和表达的最佳条件，我们将使用克隆的威尔逊病基因，在威尔逊病模型Long Evans Cinnamon （LEC）大鼠中尝试纠正铜代谢缺陷。最终，我们希望将细胞移植和体外基因治疗方法应用于各种遗传性和获得性肝病患者，因为这些方法是互补的，但会有不同的临床应用。

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.