Hemophilia B is an X-linked hemorrhagic disorder due to factor IX (FIX) deficiency. Hemophilia B patients suffer from recurrent spontaneous bleeding episodes and the severely affected patients become crippled resulting from recurrent bleeding in the major joints. Current treatment for hemophilia B consists of infusion of FIX concentrates in response to the bleeding episode. This approach is non-prophylactic and up to 5 percent of hemophilia B patients develop anti-FIX antibody after exposure to FIX. Continuous infusion of FIX concentrates can induce FIX tolerance, although the underlying mechanism is not clear. However, the huge cost of continuous infusion therapy makes it prohibitive to apply this treatment to the majority of hemophilia patients, even in developed western countries. Gene therapy based on direct intramuscular injection of recombinant adeno-associated virus serotype 2 (AAV2) can provide sustained expression of FIX, but is complicated by generation of anti-FIX inhibitory antibodies in immune competent animals. The applicants recently reported that direct intramuscular injections of AAV serotype one vectors (AAV1) effected sustained and complete hemophilia B phenotype correction without formation of anti-FIX inhibitory antibody in immunocompetent hemophilia B mice. Understanding the mechanism accounting for immune tolerance to FIX is crucial for the development of successful hemophilia therapy. We hypothesize that sustained expression of high levels of FIX expressed from AAV1 in skeletal muscle induces FIX tolerance by mechanism(s) of helper T lymphocyte energy or clonal deletion, and/or generation of regulatory T lymphocytes. The hypothesis will be tested by completing two specific aims: 1) investigating the effects of sustained high level FIX expression on induction of FIX tolerance, and 2) investigating the state of maturation of antigen presenting cells and helper T cell responses after antigen presentation in an AAV1- based gene transfer protocol. The applicant's long-term goal is to develop a successful gene therapy approach for hemophilia B. The objective of this proposal is to elucidate the mechanism(s) accounting for FIX tolerance after direct intramuscular injection of AAV1, developing a novel approach not only to prevent generation of inhibitory FIX antibody but also to induce tolerance to pre-existing inhibitory FIX antibody in hemophilia B patients.
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