Majed N. Aljamali

Successful treatment of canine hemophilia by continuous expression of canine FVIIa

Continuous expression of activated factor VII (FVIIa) via gene transfer is a potential therapeutic approach for hemophilia patients with or without inhibitory antibodies to human factor VIII (FVIII) or IX (FIX). Here, we investigate whether gene transfer of an engineered canine FVIIa (cFVIIa) transgene can affect hemostasis in a canine model of hemophilia, a good predictor of efficacy of hemophilia treatments. Purified recombinant cFVIIa exhibited 12-fold higher tissue factor-dependent activity than purified recombinant zymogen cFVII. Subsequently, we generated a serotype 8 recombinant adeno-associated viral vector expressing cFVIIa from a liver-specific promoter. Vector delivery via the portal vein in hemophilia A and B dogs was well tolerated, and long-term expression of cFVIIa resulted in a shortening of the prothrombin time, partial correction of the whole blood clotting time and thromboelastography parameters, and a complete absence of spontaneous bleeding episodes. No evidence of hepatotoxicity, thrombotic complications, or inhibitory immune response was found. These data provide the first evidence for in vivo efficacy and safety of continuously expressed FVIIa as a FVIII/FIX-bypassing agent in a large animal model of hemophilia, avoiding the risk of inhibitor formation associated with bolus FVIII or FIX infusion.

Novel therapeutic approach for hemophilia using gene delivery of an engineered secreted activated Factor VII

Hemophilia is a bleeding disorder caused by mutations in the genes encoding coagulation Factor VIII (FVIII) or FIX. Current treatment is through intravenous infusion of the missing protein. The major complication of treatment is the development of neutralizing Abs to the clotting factor. Infusion of recombinant activated human Factor VII (rhFVIIa), driving procoagulant reactions independently of human FVIII (hFVIII) or hFIX, has been successful in such patients and could in theory provide hemostasis in all hemophilia patients. However, its high cost and short half-life have limited its use. Here, we report a novel treatment strategy with a recombinant adeno-associated virus vector delivering a modified FVII transgene that can be intracellularly processed and secreted as activated FVII (FVIIa). We show long-term expression, as well as phenotypic correction of hemophilia B mice following gene transfer of the murine FVIIa homolog, with no evidence of thrombotic complications at these doses. These data hold promise for a potential treatment for hemophilia and other bleeding disorders.

A novel missense mutation responsible for factor VII deficiency in research Beagle colonies

Summary.  Background: Canine factor VII (cFVII) deficiency, an autosomal recessive trait originally identified in research Beagles, is associated with a mild to moderate bleeding tendency. Objective: Our aim was to identify and characterize the mutation causing cFVII deficiency. Methods: In order to sequence the coding regions of the cFVII gene, we cloned the cFVII cDNA. Genomic DNA and plasma from FVII‐deficient Beagles and obligate carriers were utilized. Results: In all FVII‐deficient dogs, we identified a single causative G to A missense mutation in exon 5, encoding the second epidermal growth factor‐like domain, resulting in substitution of glycine 96 by glutamic acid, with plasma FVII coagulant activity of ≤ 4% in affected Beagles. In vitro expression indicated that the majority (96%) of cFVII‐G96E protein was retained intracellularly. In addition, analysis of purified recombinant wild‐type and mutant cFVII proteins demonstrated reduced activity of the mutant (< 2%) compared with wild‐type. Rotational thromboelastometry revealed a severe impairment of clotting activity in affected Beagles, and heterozygotes also exhibited changes in coagulation‐based assays. Using a mutation‐specific polymerase chain reaction/restriction digest that allows rapid identification of the G96E mutation, we surveyed a US research Beagle colony and identified a mutant allelic frequency of 31%. Conclusions: We have identified a single causative mutation for cFVII deficiency that may have implications for pharmacotoxicologic research, because reduced FVII coagulant activity may alter hemostatic and/or cardiovascular endpoints in this commonly used animal species.

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

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 microg/ml was safe, effective, and compatible with a normal lifespan. However, expression levels of 2 microg/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.