A factor VIII minigene comprising the truncated intron I of factor IX highly improves the in vitro production of factor VIII.
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.
Plantier
J L
JL
INSERM U331, Laboratoire d'Hemobiologie-Faculté de Médecine RTH Laënnec, Lyon, France.
Rodriguez
M H
MH
Enjolras
N
N
Attali
O
O
Négrier
C
C
D000818:Animals; D001665:Binding Sites; D016466:CHO Cells / Q000378:metabolism; D003001:Cloning, Molecular / Q000379:methods; D006224:Cricetinae; D005164:Factor IX / Q000235:genetics*; D005169:Factor VIII / Q000096:biosynthesis* / Q000235:genetics*; D005818:Genetic Engineering; D006801:Humans; D007438:Introns / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000235:genetics; D012097:Repressor Proteins; D014158:Transcription, Genetic; D014162:Transfection; D014407:Tumor Cells, Cultured / Q000378:metabolism
D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D005169:Factor VIII; D005164:Factor IX