Megakaryocyte-targeted synthesis of the integrin beta(3)-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, alp ha llb beta(3), As a result, alpha llb beta(3) cannot be activated and cann ot bind to fibrinogen, leading to a loss of platelet aggregation, Thrombast henia 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)-derive d vector, -889PI(A2)beta(3), was transduced into peripheral blood CD34(+) c ells from 2 patients with thrombasthenia with defects in the beta(3) gene. The human alpha llb promoter was used in this vector to drive megakaryocyte -targeted expression of the wild-type beta(3) subunit, Proviral DNA and alp ha llb beta(3) biosynthesis were detected after in vitro differentiation of transduced thrombasthenic CD34(+) cells with megakaryocyte growth and deve lopment factor. Flow cytometric analysis of transduced patient samples indi cated that 19% of megakaryocyte progeny expressed alpha llb beta(3) 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 alpha llb beta(3) 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 simil ar to megakaryocytes derived from a normal nonthrombasthenic individual. Th ese results demonstrate ex vivo phenotypic correction of Glanzmann thrombas thenia and support the potential use of hematopoietic CD34(+) cells as targ ets for alpha llb promoter-driven MuLV vectors for gene therapy of platelet disorders. (C) 2000 by The American Society of Hematology.