A Factor VIII Minigene Comprising the Truncated Intron I of Factor IX Highly Improves the In Vitro Production of Factor VIII

Plantier, Jean‐Luc; Rodriguez, Marie-Hélène; Enjolras, Nathalie; Attali, Olivier; Négrier, Claude

doi:10.1055/s-0037-1616092

Cited by 33 publications

(37 citation statements)

References 41 publications

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“…These levels are substantially greater than in previously published reports of fVIII expression (8,18,19,32). No specific manipulations were made to either the cDNAs or the expression vector designed to enhance fVIII expression other than the incorporation of a linker sequence between the A2 and A3 domains that includes a PACE/furin recognition sequence.…”

Section: Discussionmentioning

confidence: 90%

High Level Expression of Recombinant Porcine Coagulation Factor VIII

Doering

Healey

Parker

et al. 2002

Journal of Biological Chemistry

154

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Recombinant human factor VIII expression levels, in vitro and in vivo, are significantly lower than levels obtained for other recombinant coagulation proteins. Here we describe the generation, high level expression and characterization of a recombinant B-domain-deleted porcine factor VIII molecule. Recombinant B-domaindeleted porcine factor VIII expression levels are 10-to 14-fold greater than recombinant B-domain-deleted human factor VIII levels by transient and stable expression in multiple cell lines. Peak expression of 140 units⅐10 6 cells ؊1 ⅐24 h ؊1 was observed from a baby hamster kidneyderived cell line stably expressing recombinant porcine factor VIII. Factor VIII expression was performed in serum-free culture medium and in the absence of exogenous von Willebrand factor, thus greatly simplifying protein purification. Real time reverse transcription-PCR analysis demonstrated that the differences in protein production were not caused by differences in steady-state factor VIII mRNA levels. The identification of sequence(s) in porcine factor VIII responsible for high level expression may lead to a better understanding of the mechanisms that limit factor VIII expression.Factor VIII (fVIII) 1 is a large (ϳ 300 kDa) glycoprotein that functions as an integral component of the intrinsic pathway of blood coagulation. Mutations in the fVIII gene that result in decreased or defective fVIII protein give rise to the genetic disease, hemophilia A, which is phenotypically characterized by recurrent bleeding episodes. Treatment of hemophilia A entails intravenous infusion of either human plasma-derived or recombinant fVIII material. Approximately 25% of all hemophilia A patients treated with fVIII products develop antibodies that inhibit fVIII activity and limit treatment efficacy (1). Patients with fVIII-inhibitory antibodies can be treated using porcine plasma-derived fVIII products, which generally display low cross-reactivity with the human fVIII antibodies (2, 3). Currently there is not a recombinant porcine fVIII product available for clinical use.Since the introduction of recombinant fVIII for the treatment of hemophilia A, commercial suppliers have struggled to keep up with high patient demand (4). The shortage of recombinant fVIII material has precluded prophylactic treatment of severely affected patients, limited the implementation of immune-tolerance regimens, and kept treatment costs high. Unfortunately, fVIII is expressed and recovered at low levels in the heterologous mammalian cell culture systems used for commercial manufacture. The importance of this problem has fueled significant research efforts to overcome the low fVIII expression barrier, and several basic mechanisms have been identified that limit fVIII expression (for review, see Kaufman et al. (5)) Despite these findings, fVIII expression levels remain low, and a product shortage persists.The porcine fVIII cDNA sequence has been reported and shown to encode the homology-defined internal protein domain structure, A1-A2-B-ap-A3-C1-C2 (6, 7). Porcin...

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Section: Discussionmentioning

confidence: 90%

High Level Expression of Recombinant Porcine Coagulation Factor VIII

Doering

Healey

Parker

et al. 2002

Journal of Biological Chemistry

154

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“…However, in these studies, the intronic sequence was placed in a location 5¢ to the FVIII cDNA. The expression-increasing effect of FIX intron 1 was demonstrated by several laboratories studying FIX cDNA (Jallat et al, 1990;Kurachi et al, 1995;Enjolras et al, 1999) but it was also found to be efficient when inserted in FVIII cDNA (Plantier et al, 2001). The present study demonstrated that the presence of the FIX intron in FVIII cDNA led to an improved production in MK cells, as in other cell lines like CHO and HepG2.…”

Section: Discussionmentioning

confidence: 93%

“…A similar digestion was used to isolate the GPIIb promoter from pcDNA3-GPIIb plasmid, to create pTracer/ GPIIb. The human FVIII cDNA cloned in our laboratory was a B domain-deleted sequence where TFIXI1 sequences were introduced at two locations corresponding to the sites of FVIII intron 1 and intron 13 (Plantier et al, 2001). cDNAs with or without introns were inserted in pcDNA3 expression vector leading to pcDNA3-FVIII and pcDNA3-FVIII I1 + 13.…”

Section: Fviii Expression Plasmidsmentioning

confidence: 99%

“…Therefore, we aimed to express FVIII in platelets using a FVIII cDNA expressed under the control of the GPIIb promoter. In a previous study, an original FVIII expression construct called FVIII I1 + 13 was generated using a B domain-deleted FVIII (FVIIIDB) cDNA containing two truncated intron 1 sequences of FIX (TFIXI1; Plantier et al, 2001). The present report demonstrates that the use of this modified transgene improves FVIII production in Dami cells.…”

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confidence: 99%

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

et al. 2004

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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.

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“…BDD-rFVIII remains the first and only modified human rFVIII molecule to come to commercialization. Other strategies are based on the extension of half-life in fact prolonging the half-life of FVIII could greatly reduce the frequency and dose of infusions, thereby improving the efficacy of prophylaxis through better compliance, as well as improve convenience and patient quality of life [8]. Some of the half-life extension strategies are sustained delivery through association of rFVIII with polyethylene glycol (PEG)ylated liposomes, chemical modification (PEGylation, polysialylation), bioengineering rFVIII through mutagenesis at putative binding sites for clearance receptors, or the generation of fusion proteins.…”

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confidence: 99%