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Abstract 2198

While individual inherited platelet disorders are rare, in aggregate, these diseases can account for a bleeding diathesis in ~1:20,000 individuals. One classic disease is Glanzmann thrombasthenia (GT), a rare autosomal-recessive bleeding disorder that is characterized by genetic defects of the platelet-specific integrin, aIIbß3. Molecular abnormalities in either the aIIb- or ß3-subunit can disrupt receptor synthesis, assembly, and/or function thereby preventing platelets from binding the receptors major adhesive ligands (von Willebrand factor and fibrinogen) to form platelet aggregates as a primary response to vascular injury. As a model to develop methods for gene therapy of platelet defects, GT is especially challenging because platelet integrin level as well as genetic transfer efficiency play a crucial role in restoring normal hemostasis. Developing effective treatment for animal models for this disease should ensure that a similar strategy would be effective for the treatment of other inherited platelet disorders. In experiments to be presented, SCF/G-CSF mobilized CD34+ peripheral blood cells (PBC) from integrin aIIb-deficient dogs were transduced with a lentivirus vector encoding the human aIIb integrin subunit driven by the tissue-specific aIIb promoter. GT dogs received an autologous transplant of lentivirus-transduced PBC using low dose (100–300 cGy) irradiation as transplant preconditioning and three scheduled doses of post-transplant in vivo drug treatment to enrich for transduced hematopoietic stem cells. Fluorescent immunocytometric analysis detected a moderate level (10%) of circulating blood platelets expressing aIIbß3 following the initial transplant, which increased to 20–30% of normal levels following drug selection with O6BG and BCNU. Remarkably, we observed a significant improvement in a clinical sign of the GT phenotype with reduced bruising on the animals’ legs when aIIbß3 was restored to the surface of only a fraction of circulating platelets. Further analysis showed restored ability of lentivirus-transduced platelets to aggregate and retract a fibrin clot in vitro using peripheral blood collected from these animals. Treatment of two animals with intravenous immunoglobulin effectively diminished platelet clearance due to an antibody response to newly expressed aIIbß3 receptor. Reduced bleeding times were measured indicating the feasibility for targeting platelets with genetic therapies for better management of patients with inherited bleeding disorders. These studies should help us to define molecular and cellular events necessary for targeting therapeutics to platelets, which will contribute to eventual, safe, effective, and technologically viable strategies for correcting inherited bleeding disorders in humans.