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

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.