Expression of Factor IX cDNA Introduced into Human Marrow Stromal Cells by Electroporation

Management of hemophilia B with gene therapy is an attractive and potentially feasible goal since stringent regulation of the recombinant protein is not required and low circulating levels may be sufficient to prevent symptoms. We are investigating the potential of gene transfer by electroporation for a role in human gene therapy. In this study, we used electroporation to physically co-transfer human factor IX cDNA under the influence of the potent human CMV-IE promoter and a second plasmid containing a neomycin resistance gene into human bone marrow stromal cells. Following electroporation, stromal cells were selected for neomycin resistance as co-transfection of both plasmids into the cells was expected from the results of previous studies. Analysis of genomic DNA from transfected stromal cells showed stable integration of factor IX cDNA at several sites in the genome. Following electroporation, the stromal cells were shown to secrete factor IX for three weeks in culture at a maximum concentration of 17ng/10(6) cells/day. As is the case with normal, functionally active, endogenous factor IX, the glutamic acid residues in the Gla domain of the factor IX protein were found to be post-translationally modified. Our results demonstrate the feasibility of gene transfer by electroporation and the successful post-translational modification and secretion of the human factor IX protein by stromal cells. This study provides evidence of the feasibility of electroporation and the use of stromal cells for the potential correction of hemophilia B in human gene therapy.