Transfection, viii

E) Retroviral vector expressing B-domain deleted factor VIII transfected into dermal fibroblasts that are then reimplanted... 

Clinical trials have demonstrated excellent efficacy with recombinant human factor VIII concentrates available as Recombinate and Kogenate. These recombinant factor VIII products are purified from the cell culture of plasmids, not viral DNA-transfected hamster cells and therefore do not express viral sequences. The addition of human serum albumin for stabilization, constitutes the sole possible source for human viral contamination. More recently recombinant factor IX has been genetically engineered by insertion of the human factor IX gene into a Chinese hamster ovary cell line. It has been proved to be safe and effective in the treatment of patients with hemophilia B. 

The investigators evaluated the safety of their nonviral somatic-cell gene therapy system, which they call transkaryotic implantation, in six patients with severe hemophilia. The procedure involved isolation of dermal fibroblasts from the patients upper arms. The fibroblasts were then transfected with a factor VIII genebearing plasmid. Cells that expressed factor VIII were cloned, propagated, and implanted into the patients abdomens. This technique can be considered as a less invasive form of ex vivo gene therapy. 

In transfected mammalian cells inhibition of N-glycan addition by treatment with tunicamycin prevented secretion of factor VIII, whereas treatment with an inhibitor of complex oligosaccharide biosynthesis, deoxymannojirimycin, did not affect secretion, although the specific activity of factor VIII was slightly increased [45]. Thus, the presence of complex oligosaccharide was not required for secretion or functional activity of factor VIII. A23187 treatment inhibited addition of serine/threonine (O)-linked glycans to factor VIII. 

The complete 7 kb protein-coding sequence of human factor VIII was assembled from portions of overlapping cDNA and genomic clones and Introduced into appropriate mammalian expression vectors (9,10). Following transformation into either hamster kidney cells (9) or COS-1 monkey cells (11) factor VIII expression in transfected cell lines was characterized. The recombinant protein was shown to be biologically active, demonstrating the ability to activate factor IX and to reduce clotting time in plasma derived from patients affected with hemophilia A. Additionally, factor VIII activity of transfected cells was inhibited by a factor Vlll-specific antibody. 

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