Prolonged bleeding can be life-threatening for individuals with inherited molecular defects in platelet proteins that play an intricate role in maintaining hemostasis. Transfusion with normal platelets or cryopreserved plasma components has been used for temporary relief of intermittent bleeding episodes, while bone marrow transplant has been used effectively for long-term correction of some patients with inherited bleeding disorders. Although, multiple transfusions are costly and can lead to the immune mediated destruction or inactivation of foreign platelets and coagulation factors. In addition, it can be difficult to find suitable marrow donors and recipients may also suffer deleterious effects from graft-vs-host disease and graft rejection. We have recently shown a potentially feasible solution for this problem by transferring a therapeutic gene with a lentivirus vector into hematopoietic stem cells. This strategy proved useful for correction of bleeding in the murine model for Glanzmann Thrombasthenia (GT). Individuals with GT have genetic defects in a member of the integrin family of adhesion receptors, allbbeta3. Molecular abnormalities in the sequences encoding either the allb or beta3 subunit prevent GT platelets from binding fibrinogen and forming platelet aggregates. Our gene transfer vector utilizes the allb gene promoter to confine transgene expression specifically to the platelet lineage. The current proposal will test the hypothesis that this platelet-specific lentivirus system can be utilized for therapeutic correction of bleeding in large animals affected with inherited platelet defects. A line of Great Pyrenees dogs affected with GT due to an abnormality in allb will serve as a model to test the feasibility for platelet-targeted gene therapy in large animals.
Specific Aim 1 will test novel strategies for their potential to increase transgene expression to 50% of normal levels on platelets derived from hematopoietic stem cells transduced with an allb gene promoter controlled lentivirus vector.
Specific Aim 2 proposes to develop a feasible approach for human gene therapy for inherited platelet bleeding disorders using a clinically relevant genetic transfer protocol in a canine model for GT.
In Specific Aim 3, an allb-driven lentivirus will be used to explore strategies to correct canine Hemophilia A via targeted synthesis and storage of coagulation factor VIII to platelet a-granules. This study should lead to the development of safe and effective methods for human gene therapy for inherited platelet bleeding disorders and other diseases of hemostasis.
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