856. Improvements on Safety and Efficacy of Nonviral Gene Therapy of Factor VII for Hemophilia A with Inhibitors

Top of pageAbstract Recombinant activated factor VII (rFVIIa) has been used as an effective alternative treatment for hemophilia patients who have developed inhibitors. We have successfully modeled a nonviral gene therapy approach to deliver factor VII (FVII) in a hemophilia A mouse model to decrease the cost and frequency of rFVIIa infusions. An engineered murine FVII (mFVIIa) cDNA was inserted into our gene transfer vector to generate a liver-specific construct pBS- HCRHPI-mFVIIa-A encoding a modified protein, which can be cleaved by intracellular proteases of the furin family to secrete the activated form of mFVII. Partial correction of bleeding was achieved following nonviral gene transfer of this construct into hemophilia A mice. To increase the safety and efficacy of FVII gene therapy, we devised two approaches. The first was to use a mFVII wild-type cDNA (mFVII) encoding the zymogen FVII to reduce the potential thrombotic risk associated with prolonged, high level FVIIa expression. The second was to use a mutant FVIIa or FVII molecule (mFVIIa-DVQ or mFVII-DVQ) encoding a superactive FVII molecule with amino-acid substitutions (V158D/E296V/M298Q; Persson et al., 2003) to increase the efficacy of nonviral gene transfer of FVII. Fifty mg of the respective plasmids encoding wild-type or modified proteins including mFVII, mFVIIa, mFVII-DVQ, and mFVIIa-DVQ, were delivered into 4 groups of hemophilia A mice by a rapid, high volume (hydrodynamics-based) infusion method (n=8 mice/group). Phenotypic correction was measured via tail-clip assays. A 30% reduction in hemoglobin loss was observed in mice treated with mFVII, and a 50% reduction in mice treated with mFVIIa. Significant and long-term correction in PT using 1:60 dilution of mouse plasma was observed in both mFVII and mFVIIa treated mice, with clotting values consistently lower than wild-type and hemophilia A controls (wild-type: 26.4 s, hemophilia A: 25.4 s, mFVII: 23.6 s, mFVIIa: 23.3 s). APTT performed on plasma diluted 1:3 similarly reflects the results of the PT assay, with lowered clotting times for both groups of treated mice (wild-type: 60.2, hemophilia A: 121.3, mFVII: 102.8, mFVIIa: 100.9). Both plasma- based and platelet-based thrombin generation (TG) assays indicated that both mFVII and mFVIIa have a positive effect on the three measured parameters with a lowered lag phase, lowered peak time and elevated peak thrombin levels. In addition, all these effects lasted long-term in plasmid treated mice for at least six months (experimental duration). Preliminary experiments using our most recently built constructs mFVII-DVQ and mFVIIa-DVQ showed reversion to wild-type phenotype in plasmid-treated mice as indicated by significantly reduced hemoglobin loss via tail clipping and substantial reduction in APTT. These results clearly demonstrate that nonviral gene therapy of either zymogen FVII or activated FVII can rescue bleeding in hemophilia A mice, and mFVII-DVQ/mFVIIa- DVQ may potentially recover wild-type clotting activity in treated mice.