Abstract
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.
Highlights
IntroductionPrimary human skin fibroblasts infected with the vector virus secreted up to 30 milliunits/lO* cells/day
From the Department o/Medical Biochemistry, Sylvius Laboratories, P. 0
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
Summary
Primary human skin fibroblasts infected with the vector virus secreted up to 30 milliunits/lO* cells/day. Factor VIII deficiency or hemophilia A is a X-linked genetic disorder in humans, occurring in about lo-20 males in every 100,000 For those afflicted, it is potentially life-threatening and crippling. Since the introduction of factor VIII-enriched concentrates derived from pooled human plasma, effective protein replacement therapy has resulted in a dramatic increase in both quality of life and life expectancy (Smit et ul., 198% Brettler and Levine, 1989). This therapy has a number of serious drawbacks.
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