DEVELOPMENT OF GENE THERAPIES FOR HEMOPHILIA

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Hemophilia B is an X chromosome-linked bleeding disorder due to a deficiency of blood coagulation factor IX. Current protein replacement therapy is effective to temporarily relieve bleeding episodes, but the repeated applications required by hemophilia B patients expose them to the risk of several serious complications, notably infection of pathogenic viruses such as HIV-1 and hepatitis virus. In this proposal, we attempt to develop an alternative approach of an effective and safe treatment for hemophilia B based upon somatic cell gene therapy which utilizes skeletal myoblasts as a gene delivery vehicle for an efficient systemic delivery of recombinant factor IX. Replication-deficient recombinant retroviral vectors which can give high-level expressions of recombinant human factor IX in mouse skeletal myoblasts will be constructed. Mouse primary myoblasts transduced with an optimized retroviral vector(s) will then be injected into skeletal muscles of syngenic nude mice. Production and systemic delivery of biologically-active recombinant factor IX in the animals will be maximized by determining the optimal combination of variable conditions including purity of myoblasts, the total cell number to be injected, sites for injection, number of injections for each animal, and stimulation of myofiber regeneration. The transduced myoblasts injected into mouse skeletal muscles will be analyzed for their capability to produce a high-level recombinant factor IX into the systemic circulation. Any deleterious effect of the injected myoblasts to the animals, such as tumorigenicity and detrimental effects on the muscle strength, if it occurs, will also be extensively examined for a long period of time. After extensive testing and optimization in mice, the ex vivo approach will be tested on normal dogs preparing for its final application on hemophilia B dogs. Skeletal myoblasts isolated from normal dogs are transduced with the retroviral vector optimized in mice and tested for their full capability to express recombinant factor IX in vitro. The transduced dog myoblasts will then be injected back into the same dogs for further optimization of conditions involved in the cell transfer. The ex vivo approach, well tested and optimized in mice and normal dogs, will finally be applied to invaluable hemophilia B dogs. By employing an optimized retroviral vector containing a canine, instead of human, factor IX cDNA, its long-term effectiveness and safety will be demonstrated in a totally homogeneous system. The information obtained from these studies will provide us with a solid experimental base for developing an effective, safe and durable clinical protocol of somatic cell gene therapy for hemophilia B.