The techniques for efficient gene transfer into human cells are now being applied to the development of gene therapy for a variety of human diseases. Hemophilia B, an inborn error of metabolism resulting from a deficiency of clotting factor IX, is an attractive target for gene therapy, and it is the goal of these studies to develop methods for introducing the wild type cDNA for factor IX into hepatocytes and muscle cells to provide a stable and therapeutic level of factor IX to correct this disorder. The emphasis of our proposed studies is on direct in vivo delivery of factor IX with retroviral vectors, especially the VSV-G protein pseudotyped vectors recently developed in this laboratory. We propose to develop efficient methods for producing such pseudotypes through the use of stable packaging cells lines. In addition we plan to improve the retroviraI vectors by incorporating tissue-specific regulatory elements and an element designed to permit infection of non-replicating cells. We plan to determine the levels and stability of factor IX gene expression from these vectors and to identify optimum procedures for their delivery into liver and muscle in vivo. Ultimately, we will apply these optimized methods to correction of the bleeding disorder in factor IX- deficient dogs. Finally, we will examine one ex vivo approach to gene therapy for hemophilia B involving gene transfer into primary hepatocytes growing on supports of reticulated polyurethane followed by grafting to the liver of hemophilic dogs.
