Long-term expression of murine activated factor VII is safe, but elevated levels cause premature mortality

Abstract

Intravenous infusion of recombinant human activated Factor VII (FVIIa) has been used for over a decade in the successful management of bleeding episodes in patients with inhibitory antibodies to Factor VIII or Factor IX. Previously, we showed that expression of murine FVIIa (mFVIIa) from an adeno-associated viral (AAV) vector corrected abnormal hemostatic parameters in hemophilia B mice. To pursue this as a therapeutic approach, we sought to define safe and effective levels of FVIIa for continuous expression. In mice transgenic for mFVIIa or injected with AAV-mFVIIa, we analyzed survival, expression levels, in vitro and in vivo coagulation tests, and histopathology for up to 16 months after birth/mFVIIa expression. We found that continuous expression of mFVIIa at levels at or below 1.5 μg/ml was safe, effective, and compatible with a normal lifespan. However, expression levels of 2 μg/ml or higher were associated with thrombosis and early mortality, with pathologic findings in the heart and lungs that were rescued in a low-factor X (low-FX) mouse background, suggesting a FX-mediated effect. The findings from these mouse models of continuous FVIIa expression have implications for the development of a safe gene transfer approach for hemophilia and are consistent with the possibility of thromboembolic risk of continuously elevated FVIIa levels.

Successful treatment of canine hemophilia by continuous expression of canine FVIIa

“…After a 3-minute incubation at 37°C, 50 L of 25 mM CaCl 2 was added and the time to clot was recorded. A typical standard curve had a correlation coefficient (r 2 ) of more than 0.97 (log 10 [concentration] vs log 10 [clot time]). In this assay, zymogen cFVII exhibited negligible activity (ϳ 3% relative to cFVIIa).…”

“…9,10 The cFVIIa transgene in this AAV vector did not contain the C-terminal epitope tag (HPC4). In preliminary experiments designed to test the functionality of the AAV vector, we performed tail vein vector administration in hemophilic mice at a dose of 0.3 to 1.2E12 vector genomes/mouse (1.2-4.8E13 vector genomes/kg).…”

“…Platelet counts were within the normal range throughout the study ( Figure 3A). Because mice continuously expressing mFVIIa at levels more than 2 g/mL exhibited premature mortality, 10 to further address the safety of this gene transfer approach, we used 3 different assays: levels of TAT complex, D-dimer levels (elevated levels of which are indicators of thromboembolic disease and disseminated intravascular coagulation [DIC] in dogs 29,30 ), and fibrinogen levels, elevated levels of which are associated with cardiovascular disease. 31 We did not detect any significant changes in TAT or D-dimer levels relative to pre-AAV administration (P Ͼ .2) in any of the vector doses used in AAV-treated dogs throughout the observation period ( Figure 3B and C, respectively).…”

“…10 However, a necessary step preceding human application is the demonstration of safety and efficacy in a large animal (canine) model. This important hemophilia model closely resembles the human disorder in terms of size, physiology, and the genetic determinants of immune response because it is outbred instead of inbred.…”

Engineered Factor VII, Factor IX, and Factor X Variants for Hemophilia Gene Therapy

“…Expression levels of the transgene reached up to 1000 ng/ml with the highest vector dose injected and didn't induce any thrombotic complications but still corrected phenotype [31,32]. In contrast, the overexpression at levels greater than 2 µg/ml caused mortality and pathological changes in heart and lung [33]. Gene transfer was also investigated in a large animal model (canine), which is a more related model for hemophilia and a good predictor of efficacy and safety of hemophilia treatments.…”