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Biosynthesis of FVIII in megakaryocytic cells: improved production and biochemical characterization
Author(s) -
Rodriguez MarieHélène,
Plantier JeanLuc,
Enjolras Nathalie,
Réa Muriel,
Leboeuf Marylène,
Uzan Georges,
Négrier Claude
Publication year - 2004
Publication title -
british journal of haematology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.907
H-Index - 186
eISSN - 1365-2141
pISSN - 0007-1048
DOI - 10.1111/j.1365-2141.2004.05244.x
Subject(s) - transgene , haematopoiesis , microbiology and biotechnology , haemophilia a , recombinant dna , megakaryocyte , transfection , genetic enhancement , platelet , gene , biology , chemistry , stem cell , immunology , biochemistry , haemophilia , genetics
Summary Haemophilia A is an attractive target for gene therapy. We designed a haemophilia A gene therapy strategy involving the genetic modification of haematopoietic stem cells to achieve tissue‐specific expression of a factor VIII (FVIII) transgene in the megakaryocytic lineage. Platelets would then serve as vehicles to store the expressed FVIII and deliver the coagulation factor at the site of vascular injury. A local correction of the haemostasis defect could, therefore, be expected following platelet activation and secretion. In this study, we demonstrated that a model of haematopoietic cell lines (Dami cells) could produce a correctly processed FVIII. FVIII transgenes were placed under the control of the human platelet glycoprotein IIb (GPIIb) promoter and used for stable transfection of the Dami megakaryocytic cell line. The highest FVIII production was obtained when the FVIII transgene contained a factor IX intron 1 gene sequence inserted in the FVIII intron 1 and 13 sites. Reverse transcription polymerase chain reaction demonstrated that the splicing of these introns was complete. Recombinant FVIII (rFVIII) produced in Dami cells was a biologically active molecule (specific activity: 5664 IU/mg) that was correctly glycosylated and sulphated. This recombinant FVIII protein exhibited biochemical characteristics after deglycosylation or thrombin activation that were comparable to a commercially available B‐domainless rFVIII. These results demonstrate the advantages of a modified FVIII transgene and represent the first biochemical characterization of megakaryocyte‐produced FVIII.