Construction of a novel lentiviral vector carrying human B-domain-deleted factor VIII gene

Abstract

Vectors derived from human immunodeficiency virus-1 (HIV-1) are highly efficient vehicles for gene delivery. The present study aimed to establish a potent expression system of human factor VIII (FVIII) with lentiviral vectors. FVIII³BD gene was obtained by enzyme digestion and inserted into lentiviral vector pXZ208 driven by cytomegalovirus (CMV) promoter/enhancer. Recombinant viral particles were prepared by cotransfection of 293T cells with packaging plasmids ³NRF and VSV-G through calcium phosphate precipitation. A variety of cell lines including 293T, HLF, NIH3T3, BMEC, Chang-Liver cells and MSCs were infected with recombinant virus containing FVIII³BD. The expression of FVIII³BD
mRNA, FVIII procoagulant activity and genomic DNA integration were detected. All the above cell lines were successfully transfected by recombinant lentiviruses. The transfection efficiencies in 293T, HLF,
NIH3T3, BMEC, Chang-Liver cells and MSCs were 59.57 ± 5.24, 74.52 ± 7.57, 41.33 ± 5.82, 42.34 ± 5.84, 14.38 ± 2.73% and 27.24 ± 6.53, respectively. All the cell lines expressed FVIII after infection to different
extents and the activity of FVIII in 293T, HLF, NIH3T3, mBMEC, Chang-Liver cells and MSCs was 43.2 ± 3.2, 54.1 ± 5.6, 14.2 ± 2.8, 8.7 ± 1.3, 22.5 ± 2.9 and 12.5 ± 2.7%, respectively. In addition, FVIII³BD mRNA
and genomic DNA integration were detected in all cell lines after  transfection. A novel lentiviral vector carrying human FVIII³BD was constructed, which was able to transfect different mammalian cell types
accompanied by high-level activity. This lentiviral vector may provide a theoretical basis for the gene therapy of patients with hemophilia A.