Faculty Collaboration Database

Targeting platelet GPIbalpha transgene expression to human megakaryocytes and forming a complete complex with endogenous GPIbbeta and GPIX. J Thromb Haemost 2004 Nov;2(11):1989-97

Date

11/20/2004

Pubmed ID

15550031

DOI

10.1111/j.1538-7836.2004.00961.x

Scopus ID

2-s2.0-13244262627 (requires institutional sign-in at Scopus site)   33 Citations

Abstract

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 (GPIbalpha, GPIbbeta, GPIX, and GPV). Mutations in either GPIbalpha, GPIbbeta, or GPIX can result in BSS with many of the known mutations occurring in GPIbalpha. In this study, we have developed a gene therapy strategy to express hemagglutinin (HA)-tagged GPIbalpha in megakaryocytes and potentially correct a hereditary deficiency. To direct GPIbalpha expression in megakaryocytic lineage cells, we designed a GPIbalpha cassette where human GPIbalpha cDNA was placed under control of the megakaryocytic/platelet-specific alphaIIb promoter and inserted into a lentiviral vector. Human CD34+ peripheral blood cells (PBC) and Dami cells were transduced with alphaIIb-HA-GPIbalpha-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 GPIbalpha protein. Immunoprecipitation with Western blot analysis demonstrated that transgene protein associated with endogenous GPIbbeta and GPIX proteins. To address further the lineage-specific expression of the alphaIIb-HA-GPIbalpha construct, three cell lines, Dami, AtT-20 and HepG2, were transfected with GPIbalpha expression plasmids and analyzed by confocal microscopy. The results demonstrated that among these three cell lines, the tissue-specific alphaIIb promoter was active only in Dami cells. Thus, GPIbalpha can be efficiently and specifically expressed in the megakaryocytic compartment of hematopoietic cells and the transgene product associates with endogenous GPIbbeta and GPIX forming a complete complex. This strategy could potentially be utilized for gene therapy of BSS.

Author List

Shi Q, Wilcox DA, Morateck PA, Fahs SA, Kenny D, Montgomery RR

Authors

Robert R. Montgomery MD Adjunct Professor in the Pediatrics department at Medical College of Wisconsin
Qizhen Shi MD, PhD Professor in the Pediatrics department at Medical College of Wisconsin
David A. Wilcox PhD Professor in the Pediatrics department at Medical College of Wisconsin

MESH terms used to index this publication - Major topics in bold

Antigens, CD34
Bernard-Soulier Syndrome
Cell Line
Cell Line, Tumor
Cells, Cultured
Genetic Therapy
Genetic Vectors
Hematopoietic Stem Cells
Humans
Megakaryocytes
Multiprotein Complexes
Platelet Glycoprotein GPIb-IX Complex
Promoter Regions, Genetic
Transduction, Genetic
Transgenes