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Targeting platelet GPIbα transgene expression to human megakaryocytes and forming a complete complex with endogenous GPIbβ and GPIX
Author(s) -
Shi Q.,
Wilcox D. A.,
Morateck P. A.,
Fahs S. A.,
Kenny D.,
Montgomery R. R.
Publication year - 2004
Publication title -
journal of thrombosis and haemostasis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.947
H-Index - 178
eISSN - 1538-7836
pISSN - 1538-7933
DOI - 10.1111/j.1538-7836.2004.00961.x
Subject(s) - microbiology and biotechnology , transfection , biology , haematopoiesis , transgene , platelet glycoprotein gpib ix complex , platelet , hek 293 cells , stem cell , cell culture , platelet membrane glycoprotein , immunology , glycoprotein , gene , genetics
Summary. Bernard–Soulier Syndrome (BSS) is a severe congenital platelet disorder that results from a deficiency of the platelet membrane glycoprotein (GP) Ib/IX complex that is composed of four subunits (GPIbα, GPIbβ, GPIX, and GPV). Mutations in either GPIbα, GPIbβ, or GPIX can result in BSS with many of the known mutations occurring in GPIbα. In this study, we have developed a gene therapy strategy to express hemagglutinin (HA)‐tagged GPIbα in megakaryocytes and potentially correct a hereditary deficiency. To direct GPIbα expression in megakaryocytic lineage cells, we designed a GPIbα cassette where human GPIbα cDNA was placed under control of the megakaryocytic/platelet‐specific αIIb promoter and inserted into a lentiviral vector. Human CD34+ peripheral blood cells (PBC) and Dami cells were transduced with αIIb‐HA‐GPIbα‐WPT virus. Flow cytometry analysis demonstrated that 50.1% of the megakaryocytes derived from CD34+ stem cells and 97.3% of Dami cells were transduced and expressed transgene GPIbα protein. Immunoprecipitation with Western blot analysis demonstrated that transgene protein associated with endogenous GPIbβ and GPIX proteins. To address further the lineage‐specific expression of the αIIb‐HA‐GPIbα construct, three cell lines, Dami, AtT‐20 and HepG2, were transfected with GPIbα expression plasmids and analyzed by confocal microscopy. The results demonstrated that among these three cell lines, the tissue‐specific αIIb promoter was active only in Dami cells. Thus, GPIbα can be efficiently and specifically expressed in the megakaryocytic compartment of hematopoietic cells and the transgene product associates with endogenous GPIbβ and GPIX forming a complete complex. This strategy could potentially be utilized for gene therapy of BSS.