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

Megakaryocyte-specific expression of the platelet-adhesion receptor, integrin alpha IIb beta 3, is caused by the presence of regulatory elements of the alpha IIb 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 alpha IIb promoter (nucleotides -889 to +35). A naturally occurring cDNA encoding the P1(A2) alloantigen form (Pro33) Of the integrin beta 3 subunit was subcloned into this construct (-889P1(A2)beta 3) and transduced into cells that endogenously synthesized P1(A1)beta 3 (Leu(33)) as a marker for detection of provirus-derived beta 3, The ability of this vector to target expression of P1(A2)beta 3 to megakaryocytes was first examined in cell lines. Immunoblot analysis with human anti-P1(A2) alloserum detected synthesis of P1(A2)beta 3 in transduced promegakaryocytic cells; however, P1(A2)beta 3 protein was not detected in transduced epithelial cells, Human hematopoietic CD34+ cells were transduced with -889P(A2)beta 3 virions and induced to differentiate with megakaryocyte growth and development factor. A hybrid alpha IIb beta 3 complex was formed in progeny megakaryocytes where provirus-derived P1(A2)beta 3 was detected associated with endogenous alpha IIb subunit, Another alpha IIb promoter-driven MuLV vector (-889nIacZ) 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 alpha IIb 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.