title: Biosynthesis of FVIII in megakaryocytic cells: improved production and biochemical characterization.
abstract: 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.
authors.affiliation: Laboratoire de Thérapie Génique de l'Hémophilie, EA3735, Faculté de Médecine RTH Laennec, 8 rue Guillaume Paradin, 69372 Lyon cedex 08, France. mhrodrig@sante.univ-lyon.fr
authors.forename: Marie-Hélène
authors.initials: MH
authors.lastname: Rodriguez
authors.forename: Jean-Luc
authors.initials: JL
authors.lastname: Plantier
authors.forename: Nathalie
authors.initials: N
authors.lastname: Enjolras
authors.forename: Muriel
authors.initials: M
authors.lastname: Réa
authors.forename: Marylène
authors.initials: M
authors.lastname: Leboeuf
authors.forename: Georges
authors.initials: G
authors.lastname: Uzan
authors.forename: Claude
authors.initials: C
authors.lastname: Négrier
mesh_terms: D019149:Bioreactors; D015153:Blotting, Western / Q000379:methods; D002460:Cell Line; D002704:Chlorates / Q000494:pharmacology; D005169:Factor VIII / Q000032:analysis / Q000096:biosynthesis* / Q000235:genetics; D015944:Factor VIIIa / Q000032:analysis; D015316:Genetic Therapy / Q000379:methods*; D006467:Hemophilia A / Q000628:therapy*; D006801:Humans; D047468:Immunoprecipitation; D039421:Integrin alpha2; D008533:Megakaryocytes / Q000378:metabolism*; D008562:Membrane Glycoproteins / Q000235:genetics; D011401:Promoter Regions, Genetic; D014162:Transfection / Q000379:methods; D019076:Transgenes
chemical_list: D002704:Chlorates; C056564:ITGA2B protein, human; D039421:Integrin alpha2; D008562:Membrane Glycoproteins; D015944:Factor VIIIa; D005169:Factor VIII; C032706:sodium chlorate