Hemophilia-A is an X-linked genetic disorder caused by a deficiency in coagulation factor VIII. Current therapy consists of factor VIII replacement when bleeding occurs. However, this therapy cannot prevent bleeding. Because hemophilia is caused by a single gene defect, it is an excellent disorder for gene therapy. However, current methods for ex vivo gene therapy have been unsuccessful because of lack of suitable target cells, and poor long-term production of factor VIII following transplantation of target cells due in part to poor target cell engraftment and problems with gene transfer and gene expression. We have recently characterized human post natal bone marrow cells that differentiate into most cells of mesodermal origin, including myocytes and endothelium, as well as neuroectodermal cells and cytokeratin-8,18,l9 and cMET positive epithelial cells, termed Multipotent Adult Stem Cells, or MAPC. MAPC can undergo >70 cell doublings in an undifferentiated state, can readily be transduced with retroviral vectors and can be detected, in multiple organs following transplantation in xenogeneic animals. MAPC are therefore ideal target cells for gene therapy of hemophilia-A. Several groups, including our won, have shown that immune recognition of the "neoprotein" may lead to the production of antibodies against fVIIl. We have extensive experience ion characterizing the immune response against fVIII which will allow us to test in murine models how we may circumvent immune rejection of transgenically produced fVIII. We propose the following specific aims: Spedfic Aim 1. We will test the transduction efficiency in MAPC and long-term expression of factor VIII in by undifferentiated MAPC and their differentiated progeny following transduction with a B-domain deleted human fVIII (hfVIII) containing vector. Specific Aim 2. We will transplant hfVIII transduced human MAPC in NOD-SCID mice and test (1) engraftment of the undifferentiated cells in vivo, (2) organ specific differentiation of MAPC in vivo, (3) longevity of MAPC or differentiated progeny in vivo and (4) production of factor VIII in vivo. Specific Aim 3. We will transplant hfVIll transduced murine MAPC (mMAPC) in syngeneic murine recipients which should allow us to determine long-term engraftment of MAPC and long-term expression of fVIII in vivo. Specific Aim 4: We will characterize the immune rejection fVIII following transplantation hfVIII transduced mMAPC in hemophilic mice.