Efficacy of Zymogen Factor VII Is Comparable to Activated Factor VII for Nonviral Gene Therapy Treatment of Hemophilia A.

A major problem for clinical treatment of hemophilia A using factor replacement therapy is the high frequency formation of inhibitory antibodies against factor VIII. This problem is also predicted to occur following strategies currently aimed at targeted genetic correction of this disease. Recombinant activated factor VII (rFVIIa, NovoSeven^®, Novo Nordisk, Bagsvaerd, Denmark) has been successfully used as an effective alternative treatment for hemophilia patients who have developed inhibitors. In order to decrease the cost and fluctuation of FVIIa levels associated with frequent infusions of rFVIIa, nonviral gene transfer of factor VII (FVII) was attempted in a hemophilia A mouse model. In addition, to investigate the potential thrombotic risks associated with prolonged, high level of FVIIa expression following gene transfer, we compared the effects of gene transfer vectors encoding zymogen FVII with an engineered secreted FVIIa in hemophilia A mice. We inserted murine FVII (mFVII) cDNA into a liver-specific vector developed recently in our lab to generate pBS-HCRHPI-mFVII-A. The mFVII cDNA sequence was then modified by site-directed mutagenesis to insert a protease cleavage site in between Arg152-Ile153. The resulting construct of murine FVIIa (mFVIIa) encodes a modified protein, which can be cleaved by intracellular proteases of the furin family to secrete the activated form of mFVII. Fifty mg of either mFVII or mFVIIa the plasmid was delivered into hemophilia A mouse liver by a rapid, high volume (hydrodynamics-based) infusion method (n=8 mice/group). Phenotypic correction was evaluated using tail-clip assays by measuring the hemoglobin levels in the collected blood. This assay indicated that vectors carrying either mFVII or mFVIIa can partially correct clotting following gene transfer. Significant correction in PT and APTT was observed in mFVII or mFVIIa-treated mice. Plasma-based and platelet-based thrombin generation (TG) assays indicated that either mFVII or mFVIIa significantly shorten peak time and peak thrombin levels of TG. In addition, these effects lasted long-term in plasmid treated mice for at least three months (experimental duration). We also constructed vectors carrying human FVII (hFVII) or FVIIa (hFVIIa) to test their efficacy for correcting hemophilia A. Constructs produced supra-physiological levels (800–1200ng/ml) of hFVII or hFVIIa assayed by ELISA at days 1 and 4 post-injection. However, the levels of hFVII and hFVIIa dropped precipitously at day 14 post-injection shown by both ELISA and Western blot, most likely due to the formation of species-specific antibodies against hFVII. These results clearly demonstrate that nonviral gene therapy of either zymogene FVII or activated FVII can rescue bleeding in hemophilia A mice.