A rat model of severe VWD by elimination of the VWF gene using CRISPR/Cas9

Jessica Garcia, Veronica H. Flood, Sandra L. Haberichter, Scot A. Fahs, Jeremy G. Mattson, Aron M. Geurts, Mark Zogg, Hartmut Weiler, Qizhen Shi, Robert R. Montgomery

Research output: Contribution to journalArticlepeer-review

Abstract

Background: Von Willebrand Disease (VWD) is the most common inherited bleeding disorder, caused by quantitative and qualitative changes in von Willebrand factor (VWF). The biology of VWD, studied in canine, porcine, and murine models, differ in species-specific biology of VWF and the amenability to experimental manipulations such as phlebotomy. The factor VIII (FVIII) levels in these models are higher than in humans with type 3 VWD, suggesting functional differences between FVIII and VWF. Objectives. To develop a VWF knock out (VWF–/–) rat by excision of all 52 exons of the VWF locus. Methods: The entire VWF gene was eliminated in Sprague-Dawley (Crl:SD) rats via CRISPR/Cas9-mediated gene editing. VWF antigen (VWF:Ag), VWF propeptide, and VWF collagen IV binding (VWF:CB4) levels were determined by ELISA assays and FVIII chromogenic activity (FVIII:C) levels by chromogenic FVIII assays. Lateral tail veins were transected to measure bleeding time. VWF–/– rats were infused with FVIII–/– rat platelet poor plasma (PPP) to determine response of plasma FVIII. Results: Breeding of VWF ± rats yielded VWF–/– offspring at normal Mendelian ratios. VWF:Ag, VWF propeptide, VWF:CB4, and FVIII:C plasma levels were undetectable in VWF–/– rats. VWF–/– rats bled longer and more than VWF+/– and VWF+/+ rats when challenged. Transfusion of FVIII-deficient platelet-poor plasma induced a rapid rise in endogenous FVIII:C in VWF–/– rats. Conclusion: This rat model of severe VWD due to elimination of the entire VWF gene recapitulates the severe secondary deficiency of FVIII seen in human type 3 VWD and facilitates the study of VWF and FVIII and their interactions.

Original languageEnglish (US)
Pages (from-to)64-71
Number of pages8
JournalResearch and Practice in Thrombosis and Haemostasis
Volume4
Issue number1
DOIs
StatePublished - Jan 1 2020

Keywords

  • CRISPR
  • factor VIII
  • rat model
  • severe von Willebrand disease
  • von Willebrand factor

ASJC Scopus subject areas

  • Hematology

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