title: Megakaryocyte-targeted synthesis of the integrin beta(3)-subunit results in the phenotypic correction of Glanzmann thrombasthenia.
abstract: Glanzmann thrombasthenia is an inherited bleeding disorder characterized by qualitative or quantitative defects of the platelet-specific integrin, alphaIIbbeta(3). As a result, alphaIIbbeta(3) 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, -889Pl(A2)beta(3), was transduced into peripheral blood CD34(+) cells from 2 patients with thrombasthenia with defects in the beta(3) gene. The human alphaIIb promoter was used in this vector to drive megakaryocyte-targeted expression of the wild-type beta(3) subunit. Proviral DNA and alphaIIbbeta(3) 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 alphaIIbbeta(3) 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 alphaIIbbeta(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 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 alphaIIb promoter-driven MuLV vectors for gene therapy of platelet disorders. (Blood. 2000;95:3645-3651)
authors.affiliation: Center for Thrombosis and Hemostasis, Departments of Medicine and Pharmacology, Cystic Fibrosis/Pulmonary Research and Treatment Center, University of North Carolina, Chapel Hill, NC 27599, USA.
authors.forename: D A
authors.initials: DA
authors.lastname: Wilcox
authors.forename: J C
authors.initials: JC
authors.lastname: Olsen
authors.forename: L
authors.initials: L
authors.lastname: Ishizawa
authors.forename: P F
authors.initials: PF
authors.lastname: Bray
authors.forename: D L
authors.initials: DL
authors.lastname: French
authors.forename: D A
authors.initials: DA
authors.lastname: Steeber
authors.forename: W R
authors.initials: WR
authors.lastname: Bell
authors.forename: M
authors.initials: M
authors.lastname: Griffith
authors.forename: G C
authors.initials: GC
authors.lastname: White
mesh_terms: D015703:Antigens, CD / Q000235:genetics* / Q000502:physiology; D018952:Antigens, CD34 / Q000097:blood; D002460:Cell Line; D002478:Cells, Cultured; D005337:Fibrin / Q000378:metabolism; D005434:Flow Cytometry; D015316:Genetic Therapy; D006801:Humans; D039661:Integrin beta3; D016023:Integrins / Q000235:genetics; D008533:Megakaryocytes / Q000502:physiology*; D010641:Phenotype; D010980:Platelet Membrane Glycoproteins / Q000235:genetics* / Q000502:physiology; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000378:metabolism; D015398:Signal Transduction; D013915:Thrombasthenia / Q000097:blood / Q000235:genetics* / Q000628:therapy*; D014162:Transfection
chemical_list: D015703:Antigens, CD; D018952:Antigens, CD34; D039661:Integrin beta3; D016023:Integrins; D010980:Platelet Membrane Glycoproteins; D011994:Recombinant Proteins; D005337:Fibrin