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 FVIII9s plasma half-life from 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 VWF2N mice (termed VWF2N1/2N1 and VWF2N2/2N2) 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 VWF2N1/2N1 mice and refer to them as wt VWF+/+, heterozygous VWF2N/+ or homozygous VWF2N/2N mice. The plasma VWF in VWF+/+, VWD2N/+ or VWF2N/2N mice was 109±31, 100±27, and 115±29 U/dL. The plasma FVIII levels in homozygous VWF2N/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 To assess the clinical phenotype of VWF2N/2N mice, tail bleeding times were determined demonstrating prolongation to 3.1±0.4 hrs in VWF2N/2N mice in contrast to 2.4±0.4 in VWF2N/+ 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 VWF2N/2N mice compared to 8.3±0.5 min in VWF2N/+ heterozygotes. In wbTGA, the Lag Time and Peak Time in the VWF2N/2N group were 18.53±5.57 and 34.15±8.37 min, respectively, which were significantly longer than those obtained in the VWF2N/+ group (8.3±0.5 and 17.83±2.33 min, respectively). Peak Thrombin, Endogenous Thrombin Potential, and Thrombin Generation rate in the VWF2N/2N group were significantly lower than those obtained in the VWF2N/+ 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 VWF2N/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 VWF2N/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. Disclosures Montgomery:BCW: Patents & Royalties: GPIbM assay patent to the BloodCenter of Wisconsin.