Factor VIII (FVIII) is the plasma protein deficient or functionally defective in hemophilia A, an X chromosome- linked bleeding disorder affecting 1/5,000 males. Affected patients experience significant morbidity and mortality related to repeated and/or life-threatening bleeding events. Presently, the preferred treatment is protein replacement with recombinant-derived FVIII produced in mammalian cells. Generally, patients are treated on demand to correct bleeding episodes and not by prophylaxis, a regimen demonstrated to limit morbidity later in life. In addition, the high cost of recombinant FVIII, inconvenient access to peripheral veins, and the frequent development of antibodies that inhibit FVIII function remain significant problems. Our previous studies demonstrated that FVIII production in mammalian cells is limited due to inefficient transport through the secretory pathway. The long-term goal of the proposed research is to provide fundamental insight into mechanisms that limit FVIII secretion with the ultimate goal of developing improved therapies for hemophilia A.
The specific aims of this proposal are to test the following three hypotheses (*): FVIII accumulation within the secretory pathway activates a cell stress response that leads to cell death. FVIII trafficking through the secretory pathway is regulated by the modification of oligosaccharide structures on the FVIII polypeptide. Combined deficiency of FVIII and another clotting factor, factor V(FV), results from defective production of either ERGIC53 or MCFD2, two proteins that function together in a molecular complex to facilitate FVIII and FV transport through the cell. The approach will use a combination of biochemical, cell biological, and novel genetic mouse models to dissect fundamental processes that direct and limit FVIII trafficking through the secretory pathway. The findings will identify intracellular folding pathways for FVIII, improve FVIII secretion efficiency and elucidate how FVIII expression is toxic to cells. In addition, these studies will uncover how deficiencies in either ' ERGIC-53 or MCFD2 cause combined deficiency of FV and FVIII. The findings will provide fundamental mechanistic insight into the processes that regulate protein folding and trafficking in the secretory pathway. The information will be vital to the future development of improved gene therapy protocols for hemophilia A.
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