Biosynthesis of FVIII in megakaryocytic cells: improved production and biochemical characterization

Autor:	Jean-Luc Plantier, Nathalie Enjolras, Muriel Réa, Marie-Hélène Rodriguez, Georges Uzan, Marylène Leboeuf, Claude Negrier
Rok vydání:	2004
Předmět:	congenital hereditary and neonatal diseases and abnormalities Genetic enhancement Transgene Hematology Biology Molecular biology law.invention Haematopoiesis law Cell culture hemic and lymphatic diseases Recombinant DNA Cancer research Platelet Platelet activation Stem cell
Zdroj:	British Journal of Haematology. 127:568-575
ISSN:	0007-1048
DOI:	10.1111/j.1365-2141.2004.05244.x
Popis:	SummaryHaemophilia A is an attractive target for gene therapy. We designed ahaemophilia A gene therapy strategy involving the genetic modification ofhaematopoietic stem cells to achieve tissue-specific expression of a factor VIII(FVIII) transgene in the megakaryocytic lineage. Platelets would then serve asvehicles to store the expressed FVIII and deliver the coagulation factor at thesite of vascular injury. A local correction of the haemostasis defect could,therefore, be expected following platelet activation and secretion. In thisstudy, we demonstrated that a model of haematopoietic cell lines (Dami cells)could produce a correctly processed FVIII. FVIII transgenes were placedunder the control of the human platelet glycoprotein IIb (GPIIb) promoterand used for stable transfection of the Dami megakaryocytic cell line. Thehighest FVIII production was obtained when the FVIII transgene contained afactor IX intron 1 gene sequence inserted in the FVIII intron 1 and 13 sites.Reverse transcription polymerase chain reaction demonstrated that thesplicing of these introns was complete. Recombinant FVIII (rFVIII) producedin Dami cells was a biologically active molecule (specific activity: 5664 IU/mg) that was correctly glycosylated and sulphated. This recombinant FVIIIprotein exhibited biochemical characteristics after deglycosylation orthrombin activation that were comparable to a commercially availableB-domainless rFVIII. These results demonstrate the advantages of a modifiedFVIII transgene and represent the first biochemical characterization ofmegakaryocyte-produced FVIII.Keywords: factor VIII, haemophilia, glycoprotein IIb promoter, haemato-poietic cell line, megakaryocytes.
Databáze:	OpenAIRE
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