Generation of Transgenic Mice Expressing High Levels of Activated Murine Coagulation Factor VII.

Treatment of acute bleeding episodes in hemophilic patients with inhibitors can be successfully managed by the infusion of recombinant human factor VIIa (rhFVII, NovoSeven“). We have recently shown the efficacy of a gene transfer approach to treat hemophilia B (HB) mice by adeno-associated virus (AAV) expressing activated murine FVII (mFVIIa) (J Clin Invest. 2004 Apr; 113(7): 1025–31). To assess the consequences of long-term expression of different levels of mFVIIa, we generated transgenic mice expressing mFVIIa driven by a liver-restricted (transthyretin) promoter. Results from four founders have been analyzed. The levels of mFVII antigen in both founders and their offspring were 3.5–7.5 microgram/ml, about 2.5–5 fold the baseline compared to their non-transgenic littermates. Moreover, the expressed protein retains its coagulation activity in the extrinsic pathway as demonstrated by shortening of the prothrombin time (PT) from 22.3±0.6 sec in the non-transgenic mice to 12.3±1.6 sec in the transgenic littermates. We found two male HB mice that were also transgenic for mFVIIa, resulting from the breeding of one male founder and an HB heterozygote female. The high levels of mFVII antigen (7.5 and 5.5 microgram/ml) were accompanied by significantly shorter PTs (9.8 and 12.3, respectively) compared to wild-type baseline of 22 sec, and most importantly, by a shorter activated partial thromboblastin time (aPTT) compared to their two HB littermates (36.3 and 28.8 versus 61.3 and 61.8 sec, respectively), i.e., mice transgenic for mFVIIa show aPTT similar to their wild type littermates. Additionally, kinetics and general characteristics of in vivo clot formation after laser-induced injuries to the arteries of the cremaster muscle were similar in an HB-mFVIIa transgenic mouse (the only one tested) and in normal mice, while clots were absent in HB control mice. On the other hand, thrombin anti-thrombin (TAT) levels in the transgenic mice were comparable to their HB and wild type littermates. These findings support the efficacy and safety of a gene therapy approach for the expression of mFVIIa and should further allow us to assess the risk of continuous expression of elevated levels of mFVIIa in mouse plasma.