Recombinant expression of coagulation factor VIII in hepatic and non-hepatic cell lines stably transduced with third generation lentiviral vectors comprising the minimal factor VIII promoter.
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 alpha-1-antitrypsin gene promoter (hAAT-p), the ubiquitous but non-viral EF1alpha promoter (EF1alpha-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, EF1alpha-p, hAAT-p and CMV-p.


RESULTS
As expected, the strong viral CMV-p and the ubiquitous EF1alpha-p resulted in the highest FVIII expression in all cell lines tested (CMV-p 1.85 IU/mL/10(6) cells for 293T, 3.15 for HepG2, 5.03 for SK-Hep, 0.91 for Hepa1-6; EF1-alpha promoter 0.30 IU/mL/10(6) 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/10(6)cells in HepG2 and 0.04 in Hepa1-6), the FVIII promoter gave reasonable expression levels in hepatic cells (0.47 IU/mL/10(6)cells in Hepa1-6 and 0.44 in SK-Hep).


CONCLUSIONS
These results indicate the potential usefulness of the FVIII-p for hemophilia A gene therapy.
Picanço
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Institute for Transfusion Medicine and Immunohematology, Red Cross Blood Donor Service Baden-Wuerttemberg-Hesse, Johann Wolfgang Goethe University Clinics, Frankfurt/Main, Germany.
Heinz
S
S
Bott
D
D
Behrmann
M
M
Covas
D T
DT
Seifried
E
E
Tonn
T
T
D002460:Cell Line; D005169:Factor VIII / Q000096:biosynthesis / Q000235:genetics*; D015316:Genetic Therapy / Q000379:methods; D005822:Genetic Vectors; D006467:Hemophilia A / Q000235:genetics / Q000628:therapy; D006801:Humans; D016086:Lentivirus; D008099:Liver / Q000378:metabolism / Q000473:pathology; D009928:Organ Specificity; D011401:Promoter Regions, Genetic; D015533:Transcriptional Activation; D014161:Transduction, Genetic; D019076:Transgenes
D005169:Factor VIII