A Murine Model of Type 2N VWD Was Developed By CRISPR/Cas9 Gene Editing and Recapitulates Human Type 2N VWD

Von Willebrand Disease (VWD) is caused by either an inherited deficiency of von Willebrand factor (VWF) protein or synthesis of a dysfunctional von Willebrand factor (VWF) that is referred to as a type 2 variant. One of the important functions of VWF is to serve as a carrier protein for FVIII - prolonging FVIII's plasma half-life from <2 hrs without VWF to 10-12 hrs with VWF. A variant form of VWF was identified in 1982 in which VWF did not bind FVIII resulting in FVIII's rapid clearance and a phenotype similar to moderate hemophilia and currently referred to as type 2N VWD. Type 2N VWD is caused by an autosomal recessive variant of VWF in which mutations in the D'D3 region of VWF cause decreased or absent binding of FVIII to VWF resulting in rapid FVIII clearance similar to that seen in the absence of VWF. These individuals may have a type 2N mutation on each of their VWF alleles or, more commonly, have 1 allele with a 2N mutation and their 2^nd allele contains a null mutation (producing no VWF). In each of these scenarios, only functionally abnormal VWF is synthesized, resulting in reduced or absent FVIII binding, rapid FVIII clearance, and a marked reduction in plasma FVIII. While Lillicrap and coworkers have demonstrated 2N VWF expression by hydrodynamic transfection in a VWF^-/- mouse, their approach represents a model of plasma 2N VWF dysfunction but does not recapitulate human 2N VWD where the abnormal VWF is not only in plasma, but also in endothelial cells and platelets.

We selected 8 different human type 2N mutations, and mutated murine VWF at these positions and expressed the mutant VWF in HEK293T cells in vitro. Binding of each of the 2N mutants of mouse VWF to both murine and human FVIII was tested. The G785E 2N mutation had the most severe defect with negligible binding to both human (rhF8) and mouse FVIII (mF8) and was therefore chosen for further study. Using CRISPR/Cas9 gene editing, we generated two lines of VWF^2N mice (termed VWF^2N1/2N1 and VWF^2N2/2N2) each with a 2N causative 2354G>A (G785E) mutation on the C57BL6 background. Plasma VWF levels were measured by ELISA and FVIII was measured by chromogenic assay. VWF binding to FVIII was determined by FVIII binding to monoclonal captured VWF or by VWF binding to monoclonal captured FVIII. For the remainder of this abstract, we will only discuss the VWF^2N1/2N1 mice and refer to them as wt VWF^+/+, heterozygous VWF^2N/+ or homozygous VWF^2N/2N mice. The plasma VWF in VWF^+/+, VWD^2N/+ or VWF^2N/2N mice was 109±31, 100±27, and 115±29 U/dL. The plasma FVIII levels in homozygous VWF^2N/2N mice were markedly reduced to 4.9±2.2 U/dL compared to either wt (84±21U/dL) or heterozygous (82±18U/dL) controls. VWF binding to rhF8 was reduced to <2% in VWF^2N/2N mice and 57±9% in VWF^2N/+ mice when compared to wt controls.

To assess the clinical phenotype of VWF^2N/2N mice, tail bleeding times were determined demonstrating prolongation to 3.1±0.4 hrs in VWF^2N/2N mice in contrast to 2.4±0.4 in VWF^2N/+ and 2.1±1.0 hrs in wt controls. ROTEM assessment of whole blood clotting time (wbCT) and whole blood thrombin generation assays (wbTGA) were performed. The wbCT was 10.7±1.1 min in VWF^2N/2N mice compared to 8.3±0.5 min in VWF^2N/+ heterozygotes. In wbTGA, the Lag Time and Peak Time in the VWF^2N/2N group were 18.53±5.57 and 34.15±8.37 min, respectively, which were significantly longer than those obtained in the VWF^2N/+ group (8.3±0.5 and 17.83±2.33 min, respectively). Peak Thrombin, Endogenous Thrombin Potential, and Thrombin Generation rate in the VWF^2N/2N group were significantly lower than those obtained in the VWF^2N/+ group.

In human type 2N VWD, FVIII synthesis is normal but plasma levels are reduced because of increased FVIII clearance in the absence of VWF binding. Infusion of rhVWF (no FVIII) into the VWF^2N/2N mice demonstrated rhVWF peaking by 30 minutes with a rescue of the endogenous mouse FVIII (44±1.7 U/dL) at 4-6 hrs post infusion. This demonstrates improved FVIII survival in the presence of normal VWF.

In summary, we have developed a novel mouse model by gene editing with both the pathophysiology and clinical phenotype found in Type 2N patients. Plasma levels of VWF are normal in our VWF^2N/2N mice. This VWF is incapable of binding FVIII at neutral pH, but is otherwise fully functional. This is a unique model of 2N VWD that can be used to investigate the biological properties of VWF/FVIII association.