Abstract
Background:
Lentiviral vectors have the capacity to transduce stably non-dividing, differentiated and undifferentiated cells of various tissues, including liver. To obtain high-level expression of transgenes, vectors often rely on viral promoters. However, recent data suggest that the supraphysiologic expression from ubiquitous viral promoters may not be beneficial and harbor the risk of oncogene activation. Therefore this study explored the lentiviral-mediated expression of human coagulation factor VIII (FVIII) driven by the physiologic FVIII gene promoter (FVIII-p), the liver-specific human α-1-antitrypsin gene promoter (hAAT-p), the ubiquitous but non-viral EF1α promoter (EF1α-p) and the viral CMV promoter.
Methods:
Hepatic and non-hepatic cell lines were stably transduced with lentiviral vectors encoding FVIIIdelB and EGFP. To compare the different promoters, lentiviral vectors were cloned to drive FVIII expression from FVIII-p, EF1α-p, hAAT-p and CMV-p.
Results:
As expected, the strong viral CMV-p and the ubiquitous EF1α-p resulted in the highest FVIII expression in all cell lines tested (CMV-p 1.85 IU/mL/106 cells for 293T, 3.15 for HepG2, 5.03 for SK-Hep, 0.91 for Hepa1-6; EF1-α promoter 0.30 IU/mL/106 cells for 293T, 0.04 for HepG2, 2.75 for SK-Hep, 0.46 for Hepa1-6). While the hAAT-p resulted in low FVIII levels (0.10 IU/mL/106cells in HepG2 and 0.04 in Hepa1-6), the FVIII promoter gave reasonable expression levels in hepatic cells (0.47 IU/mL/106cells in Hepa1-6 and 0.44 in SK-Hep).
Discussion:
These results indicate the potential usefulness of the FVIII-p for hemophilia A gene therapy.