Abstract

Hemophilia A, or Factor VIII (FVIII) deficiency, is the most common severe hereditary coagulation disorder, affecting 1 in 5000 male live births. Animal models in dog, mouse, and rabbit have been developed and used to study FVIII function and to evaluate new methods of treatment and prevention of inhibitor formation. Unfortunately, for unknown reasons, results obtained using these models didn’t always result in successful therapies when applied to humans. For new treatments to be safely and successfully translated from surrogate models to clinical trials, it is critical to develop an animal model that simultaneously and accurately parallels normal human physiology while mimicking human hemophilia’s physiopathological process. Due to its striking physiological and anatomical similarities to humans, sheep are considered an ideal model to study a vast array of pathologies. The aim of these studies was to re-establish, study, and characterize an extinct line of sheep with a spontaneous bleeding disorder that closely recapitulated human hemophilia A (

ThrombHaemost
68
:
618
,
1992
). Thus, we used frozen semen from an affected male to generate hemophilia A carriers. We obtained 20 females that when compared to pooled control sheep plasma, exhibited slightly increased PTT levels (38.1±1.1; N=30s), normal PT and platelet number, and slightly decreased FVIII:C (70±3%). Levels of Fibrinogen, FIX, vWF activity and vWF:ag were also normal. A second round of reproductive manipulations using the carriers’ oocytes and the affected semen produced 23 more animals, 16 of which were obligate carriers with a similar phenotype. The other 8 animals exhibited prolonged bleeding from the umbilical cord that promptly stopped upon administration of purified human FVIII concentrate using recommended dosing. Due to the unfeasibility of clamping the umbilical cord, therapy with human FVIII was continued each 12 hours until the umbilical cord dropped off. Blood collected prior to the administration of FVIII showed that these animals had almost non-existent levels of FVIIIc, and an extremely prolonged PTT (91.5±2.9, N=30.3) with normal levels of platelets, fibrinogen, FVII, FIX, and vWF. 2 of the animals died shortly after birth due to extensive hematomas related to lambing trauma. The other 6 animals, now 5 months old (maturity 6–9 months), developed clinical symptomatology closely mimicking that of human patients with severe hemophilia A. Each of these animals had between 2–6 episodes of severe bleeding including hemarthroses of the elbow, shoulder, hip, and knee, multiple muscle hematomas, including 1 hematoma of the tongue and 1 episode of mild hematuria. All of the bleeding episodes resolved upon administration of 1–2 treatments with human FVIII. Animals have thus far received between 964-4546U of human FVIII. Of interest is that low-titer inhibitors (1.3; 2.6; 3 BU) were detected in 3 of the animals showing that the nature of the mutation present in these sheep renders them prone to inhibitor development. Characterization of the mutation is currently underway. We hope that this large animal model will contribute to a better understanding of hemophilia and the development of novel treatments that can directly translate to human patients, such as stem cell transplantation and gene therapy-based approaches.

Author notes

Disclosure: No relevant conflicts of interest to declare.