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

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

doi:10.1055/s-0037-1616092

Thrombosis and Haemostasis, Table of Contents

Thromb Haemost 2001; 86(02): 596-603
DOI: 10.1055/s-0037-1616092

Review Article

Schattauer GmbH

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

Jean-Luc Plantier

¹INSERM U331, Laboratoire d’Hémobiologie-Faculté de Médecine RTH Laënnec, Lyon, France

,

Marie-Hélène Rodriguez

¹INSERM U331, Laboratoire d’Hémobiologie-Faculté de Médecine RTH Laënnec, Lyon, France

,

Nathalie Enjolras

¹INSERM U331, Laboratoire d’Hémobiologie-Faculté de Médecine RTH Laënnec, Lyon, France

,

Olivier Attali

¹INSERM U331, Laboratoire d’Hémobiologie-Faculté de Médecine RTH Laënnec, Lyon, France

,

Claude Négrier

¹INSERM U331, Laboratoire d’Hémobiologie-Faculté de Médecine RTH Laënnec, Lyon, France

› Author Affiliations

Abstract

Summary

The biosynthesis of coagulation factor VIII (FVIII) is hampered by successive controls that limit its production. To improve this production, a truncated intron I sequence of factor IX (TFIXI1) was inserted in FVIII cDNA in place of FVIII introns 1, 12 and 13 and also as a combination between introns 1 and 12, and introns 1 and 13. The intron 12 and 13 locations were targeted because this region was previously shown to contain a transcriptional silencer. The expression of FVIII in CHO and HepG2 cells revealed important variations in the properties of the minigenes depending on the TFIXI1 insertion sites. In FVIII intron 13 location the TFIXI1 seemed to diminish the transcriptional silencer activity, whereas it was poorly spliced in intron 12 position. Among the five constructs, FVIII I1+13 leaded to a significant improvement in FVIII secretion (13 times) that was associated with a dramatic intracellular accumulation in cells. Therefore, the FVIII I1+13 minigene could represent a particular interest to produce recombinant FVIII in vitro as well as in the aim of gene therapy of haemophilia A.

Keywords

Factor VIII - intron - transgene - protein production

Full Text

References

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