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

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 maturatio...

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Bibliographic Details
Published in:The Journal of biological chemistry 1990-05, Vol.265 (13), p.7318-7323
Main Authors: HOEBEN, R. C, VAN DER JAGT, R. M. C, SCHOUTE, F, VAN TIBURG, N. H, VERBEET, M. P, BRIET, E, VAN ORMONDT, H, VAN DER EB, A. J
Format: Article
Language:English
Subjects:
Animals
Base Sequence
Biological and medical sciences
Biotechnology
Blotting, Southern
Cell Line
Cells, Cultured
Chromosome Deletion
Cloning, Molecular
Factor VIII - biosynthesis
Factor VIII - genetics
Fibroblasts - enzymology
Fundamental and applied biological sciences. Psychology
Gene therapy
genes
Health. Pharmaceutical industry
Humans
Industrial applications and implications. Economical aspects
Mice
Molecular Sequence Data
Oligonucleotide Probes
Plasmids
Restriction Mapping
Retroviridae - genetics
Skin - enzymology
Transfection
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Summary: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.
ISSN:0021-9258
1083-351X
DOI:10.1016/S0021-9258(19)39116-1