Megakaryocyte-targeted synthesis of the integrin b3-subunit results in the phenotypic correction of Glanzmann thrombasthenia

Glanzmann thrombasthenia is an inher- ited bleeding disorder characterized by qualitative or quantitative defects of the platelet-specific integrin, aIIbb3 .A s a result, aIIbb3 cannot be activated and cannot bind to fibrinogen, leading to a loss of platelet aggregation. Thrombasthe- nia is clinically characterized by mucocu- taneous hemorrhage with episodes of intracranial and gastrointestinal bleed- ing. To develop methods for gene therapy of Glanzmann thrombasthenia, a murine leukemia virus (MuLV)-derived vector, 2889PlA2b3, was transduced into periph- eral blood CD341 cells from 2 patients with thrombasthenia with defects in the b3 gene. The human aIIb promoter was used in this vector to drive megakaryo- cyte-targeted expression of the wild-type b3 subunit. Proviral DNA and aIIbb3 biosynthesis were detected after in vitro differentiation of transduced thrombas- thenic CD341 cells with megakaryocyte growth and development factor. Flow cytometric analysis of transduced patient samples indicated that 19% of megakaryo- cyte progeny expressed aIIbb3 on the surface at 34% of normal receptor levels. Treatment of transduced megakaryo- cytes with a combination of agonists including epinephrine and the thrombin receptor-activating peptide induced the aIIbb3 complex to form an activated conformation capable of binding fibrino- gen as measured by PAC-1 antibody binding. Transduced cells retracted a fibrin clot in vitro similar to megakaryo- cytes derived from a normal nonthrom- basthenic individual. These results dem- onstrate ex vivo phenotypic correction of Glanzmann thrombasthenia and support the potential use of hematopoietic CD341 cells as targets for aIIb promoter-driven MuLV vectors for gene therapy of platelet disorders. (Blood. 2000;95:3645-3652)