Stem cell gene therapy for canine hemophilia

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DESCRIPTION (provided by applicant): Hemophilia can potentially be cured by ex vivo retrovirus-mediated transfer of the factor VIII or factor IX gene into autologous hematopoietic stem cells followed by infusion of the genetically corrected cells into the stem cell donor. Transduction efficiency into hematopoietic stem cells of large animals and humans using Moloney murine leukemia virus (MoMLV) vectors has been too low so far to achieve a therapeutic effect. One of the reasons is that hematopoietic stem cells are considered to be predominantly quiescent and MoMLV vectors are not able to transduce non-dividing target cells. In contrast, human immunodeficiency virus type 1 (HIV-1) based vectors have been shown to infect nondividing cells. We, therefore, wish to compare the efficiency of HIV-1 vectors containing the canine B-domain deleted factor VIII gene with corresponding MoMLV vectors to transduce stem cells of normal dogs. Using the most efficient vector system, we then wish to evaluate strategies with low toxicity to increase engraftment of transduced hematopoietic stem cells by partial marrow ablation with: a) cyclophosphamide, b) busulfan, or c) 100 cGy total body irradiation (TBI). We wish to compare these strategies with 200 cGy total body irradiation, shown to be most effective in enhancing engraftment of transduced marrow cells in our past studies. Another hurdle towards gene therapy is the possible induction of host immune responses to transduced cells and transgene encoded proteins. We propose to characterize in normal beagles the immune response to autologous marrow cells transduced with the "foreign" human factor VIII gene and to prevent or decrease this immune response by immunosuppressive treatment after transplantation. The most efficient vector system, the least toxic strategy to increase engraftment of transduced marrow cells and the most effective method to prevent a host immune response against factor VIII will then be used to investigate in hemophilia A dogs whether long-term correction of canine hemophilia can be achieved by retrovirus-mediated transfer of the canine factor VIII cDNA into hematopoietic stem cells. The outlined studies will address important problems of current gene transfer efforts and, if successful, will facilitate future gene therapy of hemophilia and other diseases in man.