Glanzmann thrombasthenia (GT) is a rare autosomal-recessive bleeding disorder that is characterized by genetic defects of the platelet-specific integrin, αIIbβ3. Molecular defects in either the αIIb- or β3-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. To develop methods for gene therapy of inherited bleeding disorders, we targeted expression of the human integrin αIIb-subunit in megakaryocytes of dogs affected with GT due to a molecular defect in αIIb. cDNA encoding the human αIIb-subunit was subcloned into an HIV type-1 lentivirus-derived vector under the transcriptional control of the lineage-specific human αIIb gene promoter (nucleotides −889 to +35). The vector also contained a cassette encoding the P140K form of methylguanine methyltransferase (MGMT) DNA repair protein under the transcriptional control of the murine stem cell virus promoter to allow in vivo chemo-selection of transduced stem cells. CD34+ cells were isolated from the apheresis product of G-CSF/SCF mobilized peripheral blood cells (PBC), transduced with virions, and autologously transplanted into a sublethally-irradiated animal. One dog received 3 x 106 transduced CD34+ PBC/kg of body weight following 300 cGy of total body irradiation (TBI). Platelet counts decreased below 10,000/ul at two weeks following PBC transplant and the animal succumbed to an intracranial hemorrhage in the third week. Another animal received an autologous transplant of 0.5 x 106 transduced CD34+ PBC/kg and 3 x 108 CD34(−) PBC fraction/kg of body weight following 100 cGy of TBI. The platelet count decreased to 35,000/ul at three weeks post-transplant and recovered to 100,000/ul at week four without incident. Flow cytometric analysis using αIIbβ3 specific antibodies detected integrin on the surface of 3% of circulating platelets at 3–6 weeks post-transplant. Since the drug-resistance marker, P140K MGMT, was detectable within cultured PBC, the dog was treated with chemocytotoxic reagents (O6BG, BCNU) three times at six-week intervals post-transplant for in vivo enrichment of transduced-PBC. Flow cytometric analysis detected peak expression of the hybrid human/canine αIIbβ3 integrin complex on the surface of 34% circulating platelets following the third drug treatment at 6 months post-transplant. Similar to platelets from normal dogs, αIIb-transduced platelets could be induced to form aggregates and retract a fibrin blood clot upon activation with physiological agonists of platelet activation. Bruising was also reduced significantly on the animal’s legs, thus further indicating correction of the GT phenotype. These ongoing studies reveal the potential feasibility for utilization of a lentivirus vector encoding a drug-selectable gene in conjunction with a therapeutic gene for enabling platelets to deliver therapy at the site of a vascular injury in patients with inherited bleeding disorders.