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J. Biol. Chem., Vol. 265, Issue 13, 7318-7323, 05, 1990

Expression of functional factor VIII in primary human skin fibroblasts after retrovirus-mediated gene transfer

RC Hoeben, RC van der Jagt, F Schoute, NH van Tilburg, MP Verbeet, E Briet, H van Ormondt and AJ van der Eb
Department of Medical Biochemistry, Sylvius Laboratories, Leiden, The Netherlands.

We have developed a retroviral-vector system for the transfer and expression of a cloned blood clotting factor VIII cDNA. Since inclusion of the complete cDNA into existing vectors is precluded by its large size, we deleted most codons for the B-domain, which is also excised during in vivo maturation of factor VIII. When inserted into the retroviral vector M5-neoR (Laker, C., Stocking, C., Bergholtz, V., Hess, N., DeLamarter, J. F., and Ostertag, W. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 8458-8462), the sequence was shown to be efficiently expressed in murine fibroblast cell lines, as well as in primary human skin fibroblasts. Upon infection of murine fibroblast cell lines, clones containing only a single copy of the integrated vector-provirus secreted up to 125 milliunits of factor VIII antigen/10(6) cells/day. Equivalent amounts were found in a factor VIII activity assay, which signifies that the factor VIII protein secreted by the infected fibroblasts is fully functional. Primary human skin fibroblasts infected with the vector virus secreted up to 30 milliunits/10(6) cells/day.
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