The goal of this project is to develop gene therapy approaches as potential treatments for both hemophilia A and B. Although these disease are similar clinically, they are caused by distinct genes whose proteins display different challenges for effective gene expression. In the case of factor VIII, it is a large protein/cDNA that has proved difficult to express in any system and must be produced by cells that have ready access to the circulation. For factor IX, gene expression has not been problematic, but because it has a higher steady state concentration and larger volume distribution, effective gene therapy strategies will require 50-100 fold higher gene expression than for factor VIII. Retroviruses and AAV are the only stable gene transfer systems in current clinical trials. These vectors are being investigated as delivery systems for both factor IX and factor VIII. Specific research accomplishments for this period were as follows. For Factor VIII: isolation of high titer ecotropic and amphotropic factor VIII producers cell clones demonstration of gene transfer into murine bone marrow, establishment of a factor VIII knock-out mouse colony, initial in vivo experimentation using growth factor stimulation of hepatocyte division followed by administration of concentrated retroviral vectors, demonstration of gene transfer and expression in intestinal epithelial cells in vitro, and preliminary evidence for the induction of immune tolerance to factor VIII administration following transplant of factor VIII transduced bone marrow. Research on factor IX included: obtained factor IX knock-out mouse embryos and beginning establishment self-sufficient colony, demonstration of factor IX gene transfer and expression in intestinal epithelial cells in vitro, construction of muscle-specific AAV-based expression vectors, demonstration of efficient engineering of human bone marrow stromal cells in vitro, initial construction of new class of SV40-based factor IX gene transfer vectors, and demonstration in rhesus monkeys that administration of human factor IX does not elicit a humoral immune response. - gene transfer, retroviral vector, adenovirus vector, AAV, factor VIII, factor IX, inhibitor
