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. Key words: Lentiviral vector, coagulation factor VIII, transfection, in vitro, gene therapy,
Highlights
Hemophilia A is an x-linked recessive bleeding disease which is characterized by deficiency of blood coagulation factor VIII (FVIII) and frequent hemarthrosis, leading to chronic crippling hemarthropathy if untreated early or
The present study aimed to investigate the transduction efficiency of human immunodeficiency virus-1 (HIV-1)-based lentiviral vector, and detect the expression of target gene in a variety of cell lines transfected with lenviral vector in vitro
The recombinant FVIII≥BD fragment was inserted into the downstream of CMV promoter to construct the recombinant vector pXZ208-FVIII≥BD
Summary
Hemophilia A is an x-linked recessive bleeding disease which is characterized by deficiency of blood coagulation factor VIII (FVIII) and frequent hemarthrosis, leading to chronic crippling hemarthropathy if untreated early or Abbreviations: HIV-1, Immunodeficiency virus-1; FVIII, factor VIII; FVIII≥BD, B-domain-deleted coagulation factor FVIII gene CMV, cytomegalovirus; SIN, self-inactivating; GFP, green fluorescent protein; VSV-G, vesicular stomotitis virus G; BMEC, brain microvascular endothelia cells; FCS, fetal calf serum; MSCs, mesenchymal stem cells; FCM, flow cytometry; RTPCR, reverse transcription polymerase chain reaction; mRNA, messager ribonucleic acid; RCLs, replication competent lentiviruses; LTR, long terminal repeat. Purified FVIII concentrates concentrated from human peripheral blood or produced by recombinant DNA and genetic engineering technology are available for replacement therapy and clinical effectiveness has been achieved (White et al, 1988). Recent advances in molecular biology and genetic engineering have made gene replacement therapy possible for patients with hemophilia A. Major progression has been achieved in the replacement therapy of hemophilia A using various gene delivery systems, critical limitations remain the major concerns Lentiviral vectors derived from human immunodeficiency virus-1 (HIV-1) have attracted attention of numerous investigators due to their ability to transfect non-dividing cells and low immunogenicity. The present study aimed to investigate the transduction efficiency of HIV-1-based lentiviral vector, and detect the expression of target gene in a variety of cell lines transfected with lenviral vector in vitro
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