Expression of human factor VIII under control of the platelet-specific alphaIIb promoter in megakaryocytic cell line as well as storage together with VWF. Mol Genet Metab 2003 May;79(1):25-33
Date
05/27/2003Pubmed ID
12765843DOI
10.1016/s1096-7192(03)00049-0Scopus ID
2-s2.0-0038522536 (requires institutional sign-in at Scopus site) 43 CitationsAbstract
Hemophilia A, which results in defective or deficient factor VIII (FVIII) protein, is one of the genetic diseases that has been addressed through gene therapy trials. FVIII synthesis does not occur in normal megakaryocytes. In hemophilia patients who have inhibitors to FVIII activity, megakaryocytes could be a protected site of FVIII synthesis and subsequent release. Since von Willebrand factor (VWF) is a carrier protein for FVIII, we hypothesize that by directing FVIII synthesis to megakaryocytes, it would traffick together with VWF to storage in megakaryocyte alpha-granules and the platelets derived from these cells. Such synthesis would establish a protected, releasable alpha-granule pool of FVIII together with VWF. When platelets are activated in a region of local vascular damage, FVIII and VWF could potentially be released together to provide improved local hemostatic effectiveness. To direct FVIII expression to the megakaryocyte lineage, we designed a FVIII expression cassette where the human B-domain deleted FVIII cDNA was placed under the control of the megakaryocytic/platelet-specific glycoprotein IIb (alphaIIb) promoter. We demonstrated by means of a functional FVIII activity assay that the biosynthesis of FVIII occurred normally in Dami cells transfected with FVIII. FVIII production was higher when driven by the alphaIIb promoter compared to the CMV promoter, and was increased about 8-fold following PMA treatment of the transfected Dami cells. Immunofluorescence staining of the transfected cells showed that FVIII stored together with VWF in the granules. The data indicate that the megakaryocytic compartment of hematopoietic cells may represent a potential target of gene therapy for hemophilia A-especially in those patients who have developed inhibitors to plasma FVIII.
Author List
Shi Q, Wilcox DA, Fahs SA, Kroner PA, Montgomery RRAuthors
Robert R. Montgomery MD Adjunct Professor in the Pediatrics department at Medical College of WisconsinQizhen 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
AnimalsBlood Platelets
Carcinoma, Hepatocellular
Cells, Cultured
Cytomegalovirus
Factor VIII
Fluorescent Antibody Technique
Gene Expression
Humans
Liver Neoplasms
Megakaryocytes
Mice
Pituitary Neoplasms
Platelet Membrane Glycoprotein IIb
Promoter Regions, Genetic
Transfection
von Willebrand Factor
