期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：1999
卷号：96
期号：17
页码：9654-9659
DOI：10.1073/pnas.96.17.9654
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：Megakaryocyte-specific expression of the platelet-adhesion receptor, integrin IIb{beta}3, is caused by the presence of regulatory elements of the 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 IIb promoter (nucleotides -889 to +35). A naturally occurring cDNA encoding the PlA2 alloantigen form (Pro33) of the integrin {beta}3 subunit was subcloned into this construct (-889PlA2{beta}3) and transduced into cells that endogenously synthesized PlA1{beta}3 (Leu33) as a marker for detection of provirus-derived {beta}3. The ability of this vector to target expression of PlA2{beta}3 to megakaryocytes was first examined in cell lines. Immunoblot analysis with human anti-PlA2 alloserum detected synthesis of PlA2{beta}3 in transduced promegakaryocytic cells; however, PlA2{beta}3 protein was not detected in transduced epithelial cells. Human hematopoietic CD34+ cells were transduced with -889PlA2{beta}3 virions and induced to differentiate with megakaryocyte growth and development factor. A hybrid IIb{beta}3 complex was formed in progeny megakaryocytes where provirus-derived PlA2{beta}3 was detected associated with endogenous IIb subunit. Another IIb 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 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.
