During hemostasis, the primary response to injury is mediated by activation of the platelet-specific receptor, glycoproteins (GP) IIb-IIIa, which assumes a conformation capable of binding its ligand, fibrinogen. GPIIb-IIIa (alphaIIbbeta3) is a member of the integrin family of receptors that mediate the adhesion of cells to each other and to the extracellular matrix. GPIIb-IIIa is missing or defective in platelets of individuals having the autosomal recessive bleeding disorder, Glanzmann Thrombasthenia (GT). Molecular defects in the sequences encoding either GPIIb or GPIIIa prevent thrombasthenic platelets from binding fibrinogen and forming platelet aggregates. GT is characterized by lifelong mucocutaneous bleeding that can lead to severe intracranial or gastrointestinal bleeding resulting in death. Although platelet transfusions are used therapeutically, no cure exists for GT. My laboratory has recently developed methods for lineage-targeted gene therapy by utilizing the promoter of the human GPIIb gene to direct transgene expression in megakaryocytes derived from retroviral transduced hematopoietic CD34+ cells. As a result, we demonstrated correction of the GT phenotype in cultured megakaryocytes from human patients and beta3-null mice. This application proposes to test the hypothesis that retrovirus-mediated transduction of CD34+ cells with a lineage-specific promoter of the GPIIb gene will result in a sustained therapeutic level of transgene expression in megakaryocytes in vivo. The foundation of this plan rests on the observation that the GPIIb promoter targets proviral-derived transgene expression preferentially in megakaryocytes and thus an immune response to proviral-transduced stem cells should be avoided. Accordingly, a line of Great Pyrenees dogs affected with GT due to a defect in GPIIb will serve as an animal model to test the feasibility of lineage- targeted gene expression. Specifically, I propose to 1) Examine megakaryocyte-targeted GPIIb-IIIa synthesis and function following transduction of gene expression system, """"""""pK9IIb"""""""", consisting of a retrovirus vector, the megakaryocyte-specific promoter of GPIIb, and cDNA encoding canine GPIIb into GT canine CD34+ hematopoietic cells, and 2) Transplant K9IIb-transduced GT canine CD34+ cells into immunocompromised mice and GT dogs to analyze the system's ability to correct the GT phenotype in platelets generated in these animals. This study should lay vital groundwork towards eventual lineage-targeted transgene expression with potential use for human gene therapy of hematological disorders.
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