Current efforts toward gene replacement therapy for the hemophilias using viral vectors show promise for long-term gene expression (over 1 year) of biologically active secreted proteins (i.e. > 1% of factor IX) in relevant animal models (hemophilia B mouse and dog models) without significant toxicity. Animal studies, in particular, underscore the importance of eliminating transient antibodies to the vector-expressed gene product and optimizing vector delivery and expression as the pressing challenges for assured success of human clinical trials. Generation of ideal animal models and more efficient vector cassettes could advance this phase of development immensely. Recently, we have been successful in developing Factor IX (FIX) molecules with higher specific activity due to increased affinity for Factor VIII or elevated catalytic activity. A single point mutant with threefold higher binding affinity for collagen IV is anticipated to maintain hemostasis at a lower concentration of plasma factor IX levels. Combining these viriants should generate FIX molecules with additional increases in activity. To effectively test these constructs in vivo, we have engineered a FIX deficient animal model using (""""""""knock-out"""""""") technology that allows for specific reinsertion (""""""""knock-out"""""""") of gene cassettes. With this model, we can assess the biological activity of the above proposed mutants which should provide a better understanding of FIX activity in vivo as well as assist in determining the potential lifelong efficacy and safety of these gene cassettes for viral vector delivery. An additional objective of this proposal is to generate normal as well as clinically relevant mutant human FIX mice using this approach. It is anticipated that we will be able to generate custom designed humanized FIX mouse models (CRM+/CRM-; inhibitor negative tolerant or inhibitor positive) thereby mimicking spontaneous mutants now seen in the clinic, for thorough characterization in the mouse. These animals will be important for studying Therapeutic levels required from vectors and potential immune response that may be generated in mutant human FIX mouse background. Therefore, the major focus of this proposal will be related to testing the molecular and biological consequences of human and variant factor IX gene products expressed in a """"""""knock-in"""""""" FIX deficient mouse model. The long-term objective is to better understand the molecular role of FIX in vivo with the hope of enhancing effective gene therapy in humans.
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