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

Abstract

SummaryHaemophilia 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.

The treatment of hemophilia A: from protein replacement to AAV-mediated gene therapy

“…Using AAV-8, Sarkar et al (2004) obtained 100% correction of FVIII deficiency regardless of the vector type or route of administration. Rodriguez et al (2004) placed FVIII transgenes under the control of the human platelet glycoprotein IIb (GPIIb) promoter for stable transfection of the Dami megakaryocytic cell line and achieved high FVIII production which had biological activity. These results represent the first biochemical characterization of megakaryocyte-produced FVIII.…”

Methods for genetic modification of megakaryocytes and platelets

“…Shi et al [36] developed a system in which Dami cells were transfected with a Factor VIII expression cassette under the control of the megakaryocyte/plateletspecific GPIIb promoter, using the lipofectamine reagent (Invitrogen, Paisley, UK). This study was followed up by Rodriguez et al [37] who used the FuGENE transfection reagent (Roche Applied Science, West Sussex, UK) and also modified the construct to improve the levels of expression. Subsequent studies confirmed that expressed Factor VIII was biologically active.…”

“…Lipid-based transfection reagents Lipofectamine, Fugene/Dami cells/factor VIII cDNA/GPIIb or CMV promoter [36,37].…”