Our work is based on the premise that the ability to target retroviral vectors to specific cell surface receptors will increase the efficiency of gene transfer, and that this along with other advances will lead to significant therapeutic benefits to patients. To target vectors specifically to the hepatocytes' asialo-glycoprotein receptor, (ASG-R), we have constructed chimeric envelope glycoproteins, in which the native receptor binding domain of gp70 (ecotropic), has been replaced by peptide sequences derived from alpha-1-acid glycoprotein (AGP). Once we can target HepG2 cells in vitro these vectors will be tested in vivo. Our ultimate goal is to transfer genes of therapeutic value to the liver. The c-DNAs encoding two isoenzymes with (human) bilirubin-UDP-glucuronyl transferase activity, have been subcloned into retroviral vectors, and their expression has been tested in vitro. The Gunn rat animal model, for Crigler-Najjar syndrome type I, will be our test system. Optimizing gene transfer efficiency, by comparing alternate routes of virus delivery, and increasing the titer and stability of vector formulations, will represent major ongoing efforts in this system. Protein C deficiency represents another hereditary liver disease, which for homozygotes is invariably fatal in the neonatal period. Our near-term application for vectors producing this key anticoagulant is to optimize its production in vitro. Another possible goal is to test its ability to prevent intra-vascuar thrombosis, particularly in conjunction with tPA. Finally, new retroviral packaging cell lines based on hepatocytes, bone marrow stromal and thymic epithelial cells, are under development. These may have certain advantages over the current cell lines which are derived from 3T3 cells.
