title: Integrin alphaIIb promoter-targeted expression of gene products in megakaryocytes derived from retrovirus-transduced human hematopoietic cells.
abstract: Megakaryocyte-specific expression of the platelet-adhesion receptor, integrin alphaIIbbeta3, is caused by the presence of regulatory elements of the alphaIIb promoter that direct high-level, selective gene transcription early in megakaryocytopoiesis. To develop methods for targeted expression of transgenes, we transduced human CD34+ peripheral blood cells with a murine leukemia virus (MuLV) vector controlled by the human integrin alphaIIb promoter (nucleotides -889 to +35). A naturally occurring cDNA encoding the Pl(A2) alloantigen form (Pro(33)) of the integrin beta3 subunit was subcloned into this construct (-889Pl(A2)beta3) and transduced into cells that endogenously synthesized Pl(A1)beta3 (Leu(33)) as a marker for detection of provirus-derived beta3. The ability of this vector to target expression of Pl(A2)beta3 to megakaryocytes was first examined in cell lines. Immunoblot analysis with human anti-Pl(A2) alloserum detected synthesis of Pl(A2)beta3 in transduced promegakaryocytic cells; however, Pl(A2)beta3 protein was not detected in transduced epithelial cells. Human hematopoietic CD34+ cells were transduced with -889Pl(A2)beta3 virions and induced to differentiate with megakaryocyte growth and development factor. A hybrid alphaIIbbeta3 complex was formed in progeny megakaryocytes where provirus-derived Pl(A2)beta3 was detected associated with endogenous alphaIIb subunit. Another alphaIIb promoter-driven MuLV vector (-889nlacZ) encoding Escherichia coli beta-galactosidase was used to demonstrate that transgene expression was selectively targeted to the megakaryocyte progeny of transduced CD34+ cells. These studies demonstrate the feasibility of using alphaIIb promoter-driven MuLV vectors for gene transfer of hematopoietic CD34+ cells to target transgene expression in developing megakaryocytes and platelets and indicate potential applications toward human gene therapy for platelet disorders.
authors.affiliation: Department of Medicine, 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: M
authors.initials: M
authors.lastname: Griffith
authors.forename: G C
authors.initials: GC
authors.lastname: White
mesh_terms: D018952:Antigens, CD34 / Q000032:analysis; D002460:Cell Line; D005786:Gene Expression Regulation; D015316:Genetic Therapy; D005822:Genetic Vectors; D006412:Hematopoietic Stem Cells / Q000378:metabolism / Q000502:physiology* / Q000821:virology; D006801:Humans; D009052:Leukemia Virus, Murine; D008533:Megakaryocytes / Q000378:metabolism / Q000502:physiology*; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000235:genetics*; D011401:Promoter Regions, Genetic; D014162:Transfection; D019076:Transgenes; D001616:beta-Galactosidase / Q000235:genetics
chemical_list: D018952:Antigens, CD34; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D001616:beta-Galactosidase