Integrin αIIb promoter-targeted expression of gene products in megakaryocytes derived from retrovirus-transduced human hematopoietic cells

摘要

Megakaryocyte-specific expression of the platelet-adhesion receptor, integrin αIIbβ3, is caused by the presence of regulatory elements of the αIIb 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 αIIb promoter (nucleotides -889 to +35). A naturally occurring cDNA encoding the Pl~(A2) alloantigen form (Pro_(33)) of the integrin β3 subunit was subcloned into this construct (-889Pl~(A2)β3) and transduced into cells that endogenously synthesized Pl~(A1)β3 (Leu_(33)) as a marker for detection of provirus-derived β3. The ability of this vector to target expression of Pl~(A2)β3 to megakaryocytes was first examined in cell lines. Immunoblot analysis with human anti-Pl~(A2) alloserum detected synthesis of Pl~(A2)β3 in transduced promegakaryocytic cells; however, Pl~(A2)β3 protein was not detected in transduced epithelial cells. Human hematopoietic CD34+ cells were transduced with -889Pl~(A2)β3 virions and induced to differentiate with megakaryocyte growth and development factor. A hybrid αIIbβ3 complex was formed in progeny megakaryocytes where provirus-derived Pl~(A2)β3 was detected associated with endogenous αIIb subunit. Another αIIb promoter-driven MuLV vector (-889nlacZ) encoding Escherichia coli β-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 αIIb 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.