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

Abstract

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.