A novel mouse model of type 2N VWD was developed by CRISPR/Cas9 gene editing and recapitulates human type 2N VWD

Type 2N von Willebrand disease is caused by mutations in the factor VIII (FVIII) binding site of von Willebrand factor (VWF), resulting in dysfunctional VWF with defective binding capacity for FVIII. We developed a novel type 2N mouse model using CRISPR/Cas9 technology. In homozygous VWF(2N/2N) mice, plasma VWF levels were normal (1167 ± 257 mU/mL), but the VWF was completely incapable of binding FVIII, resulting in 53 ± 23 mU/mL of plasma FVIII levels that were similar to those in VWF-deficient (VWF(−/−)) mice. When wild-type human or mouse VWF was infused into VWF(2N/2N) mice, endogenous plasma FVIII was restored, peaking at 4 to 6 hours post-infusion, demonstrating that FVIII expressed in VWF(2N) mice is viable but short-lived unprotected in plasma due to dysfunctional 2N VWF. The whole blood clotting time and thrombin generation were impaired in VWF(2N/2N) but not in VWF(−/−) mice. Bleeding time and blood loss in VWF(2N/2N) mice were similar to wild-type mice in the lateral tail vein or ventral artery injury model. However, VWF(2N/2N) mice, but not VWF(−/−) mice, lost a significant amount of blood during the primary bleeding phase after a tail tip amputation injury model, indicating that alternative pathways can at least partially restore hemostasis when VWF is absent. In summary, we have developed a novel mouse model by gene editing with both the pathophysiology and clinical phenotype found in severe type 2N patients. This unique model can be used to investigate the biological properties of VWF/FVIII association in hemostasis and beyond.