340. Drug-Selectable as Well as Therapeutic Gene Transfer into Hematopoietic Stem Cells May Be Essential in Gene Therapy for Inherited Bleeding Disorders of Large Animals
Authors: David A. Wilcox, Juan Fang, Lily M. Du, Mary K. Boudreaux
Journal: Molecular Therapy (2005)

Glanzmann thrombasthenia (GT) is a rare autosomal-recessive bleeding disorder characterized by genetic defects of the platelet-specific integrin, |[alpha]|IIb|[beta]|3. Molecular defects in either the |[alpha]|IIb- or |[beta]|3-subunit can disrupt receptor synthesis, assembly, and/or function thereby preventing platelets from binding adhesive ligands (von Willebrand factor and fibrinogen) and forming aggregates as a primary response to vascular injury. We previously demonstrated that nonmyeloablative allogeneic bone marrow transplantation could be used to correct GT in dogs with a mutation in the gene encoding |[alpha]|IIb. To develop methods for hematopoietic stem cell gene therapy for hemorrhagic disorders, this large-animal model was treated with a lentivirus vector encoding the megakaryocyte-specific human |[alpha]|IIb gene promoter and cDNA cassette. 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 chemoselection of transduced hematopoietic 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. The first dog received 3 |[times]| 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. The second dog received an autologous transplant of 0.5 |[times]| 106 transduced CD34+ PBC/kg and 3 |[times]| 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 subunit and integrin complex-specific antibodies detected expression of a hybrid human/canine |[alpha]|IIb|[beta]|3 integrin complex on the surface of 1-3% of circulating platelets at 3-6 weeks post-transplant. Bruising was reduced significantly on the animal's legs indicating that a partial correction of the GT phenotype had occurred. Functional studies; however, failed to detect aggregation of agonist stimulated peripheral blood platelets at 4-6 weeks post-transplant indicating that a higher transduction efficiency is necessary to restore normal hemostasis. Immuno-histochemical analysis detected a low level of MGMT in cultured PBC at four days after transduction; therefore, the dog was treated with a low level of chemocytotoxic reagents (O6BG, BCNU) at six weeks post-transplant for in vivo enrichment of transduced PBC. These ongoing studies should reveal the potential feasibility for utilization of a lentivirus vector encoding a drug-selectable gene in conjunction with a therapeutic gene for genetic correction of inherited bleeding disorders.