Megakaryocyte-targeted synthesis of the integrin β3-subunit results in the phenotypic correction of Glanzmann thrombasthenia

Glanzmann thrombasthenia is an inherited bleeding disorder characterized by qualitative or quantitative defects of the platelet-specific integrin, IIbβ3. As a result, IIbβ3 cannot be activated and cannot bind to fibrinogen, leading to a loss of platelet aggregation. Thrombasthenia is clinically characterized by mucocutaneous hemorrhage with episodes of intracranial and gastrointestinal bleeding. To develop methods for gene therapy of Glanzmann thrombasthenia, a murine leukemia virus (MuLV)-derived vector, −889PlA2β3, was transduced into peripheral blood CD34+ cells from 2 patients with thrombasthenia with defects in the β3 gene. The human IIb promoter was used in this vector to drive megakaryocyte-targeted expression of the wild-type β3 subunit. Proviral DNA and IIbβ3 biosynthesis were detected after in vitro differentiation of transduced thrombasthenic CD34+ cells with megakaryocyte growth and development factor. Flow cytometric analysis of transduced patient samples indicated that 19% of megakaryocyte progeny expressed IIbβ3 on the surface at 34% of normal receptor levels. Treatment of transduced megakaryocytes with a combination of agonists including epinephrine and the thrombin receptor-activating peptide induced the IIbβ3 complex to form an activated conformation capable of binding fibrinogen as measured by PAC-1 antibody binding. Transduced cells retracted a fibrin clot in vitro similar to megakaryocytes derived from a normal nonthrombasthenic individual. These results demonstrate ex vivo phenotypic correction of Glanzmann thrombasthenia and support the potential use of hematopoietic CD34+ cells as targets for IIb promoter-driven MuLV vectors for gene therapy of platelet disorders.