The goal of this study is to develop a safe, efficient, and clinically feasible nonviral gene therapy strategy for the treatment of hemophilia A. Currently hemophilia A patients are treated with repeated infusions of protein concentrates, which is both costly and inconvenient. Furthermore, ~30% of hemophilia A patients developed inhibitory antibodies against factor VIII (FVIII) following protein replacement therapy. We have previously demonstrated that nonviral gene transfer of a liver-specific FVIII plasmid produced persistent, supra-physiological/therapeutic levels of FVIII in immunodeficient mice using a hydrodynamics-based delivery method. However, robust FVIII-specific immune responses occurred in immunocompetent HemA mice and eliminated functional FVIII two weeks following gene transfer. This murine model permitted us to develop an effective immunosuppressive regimen in combination with nonviral gene therapy to achieve a long-term therapeutic effect. Nine single or combined immunosuppressive regimens have been tested, and the best strategy of blocking the co-stimulation pathway using a combination of Ctla4-Ig and anti-murine CD40 ligand mAb (MR1) induced long- term tolerance to factor VIII in mice. Furthermore, since hydrodynamic gene delivery is unsuitable for use in humans in its current form, we have also pursued and gained preliminary success in the development of ultrasound (US)-mediated gene delivery in vivo and cell permeable peptide (CPP)-mediated gene transfer in vitro. To facilitate direct translation of these methods to human applications, we propose to further optimize the efficiency of the US-mediated gene delivery of plasmid DNA or CPP/DNA complexes, and evaluate immunomodulation therapy for the most effective and least toxic immunosuppressive regimen in the hemophilia A mouse model. The optimized delivery method will also be tested in preliminary scale-up experiments in normal dogs in preparation for further evaluation in the hemophilia A dog model. We will test the hypotheses that: 1) Safe and clinically feasible nonviral gene delivery methods, mainly US-mediated gene therapy, can be established to allow efficient plasmid DNA transfer into the mouse liver;2) Minimally toxic immunosuppressive regimens using immunosuppressive agents alone or in combination can be developed to prevent and/or modulate transgene-specific immune responses following gene therapy; 3) These combined approaches will lead to long-term correction of disease in a hemophilia A murine model;and 4) The ultrasound-mediated gene delivery method can be scaled up in normal dog experiments.
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