Hemophilia B is a common X-chromosome-linked bleeding disorder which is due to a deficiency of biologically active blood coagulation factor IX in plasma. Current protein replacement therapy for hemophilia B is only effective to temporarily relieve bleeding episodes, but its 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. Here, we propose to establish a solid experimental base for a safe, effective, and practical gene therapy for hemophilia B based on primary myoblast-mediated gene transfer. A set of highly optimized new-generation retroviral vectors are constructed with a factor IX minigene, and a vector which can express factor IX at the highest level in mouse primary myoblasts and myotubes in culture is identified. Primary myoblast are transduced with the retroviral vector, and implanted into skeletal muscles of syngeneic mice. The systemic production of recombinant factor IX is analyzed, and maximized to reach a therapeutic level of recombinant factor IX (>5% the normal human plasma level) by optimizing individually and in combinations of various parameters and conditions involved in cell isolation and implantation procedures. Any deleterious effects to animals due to the injected myoblasts, such as tumorigenicity and detrimental effects on the muscle strength, are monitored and examined over a long period of time. After the extensive testing in mice, the optimized approach developed is then applied briefly to normal dogs, followed by the final, exhaustive testing with hemophilia B dogs. In the experiments with hemophilia dogs, a retroviral vector which is constructed with a canine factor IX minigene similar to the optimized human factor IX retroviral vector is used to transduce skeletal myoblasts isolated from hemophilia B dogs. The transduced cells are then implanted back into skeletal muscles of the same animals, permitting us to evaluate the effectiveness and safety to the procedure in a totally homogeneous system for at least several years. Finally, we submit the optimized human factor IX retrovirus to a series of extensive safety testings in culture as well as in monkeys which are required to certify it for its ultimate use in human applications in the next phase of this study. Information obtained from the proposed studies should provide us with a sound experimental base for developing a clinical protocol of durable ex vivo gene therapy for hemophilia B. Establishment of this effective, safe and practical gene therapy for hemophilia B should also open up an exciting avenue for its applications to many other diseases, not only various hematological and metabolic disorders which require an efficient systemic and local delivery of transgene products, but also muscular disorders.
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