Impact of allele-selective silencing of von Willebrand factor in mice based on a single nucleotide allelic difference in von Willebrand factor
Yvonne K. Jongejan a), Noa A. Linthorst a), Elisa Schrader Echeverri b), Sebastiaan N.J. Laan a), Richard J. Dirven a), , James E. Dahlman b), Bart J.M. van Vlijmen a), Cécile V. Denis c), Jeroen C. J. Eikenboom a), *
a) Department of Internal Medicine, Division of Thrombosis and Hemostasis, Einthoven Laboratory for Vascular and Regenerative Medicine, Leiden University Medical Center, Leiden, the Netherlands
b) Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University School of Medicine, Atlanta, GA, United States
c) Laboratory for Hemostasis, Inflammation and Thrombosis, Unité Mixed de Recherche S1176, Institut National de la Santé et de la Recherche Médicale, Université Paris-Saclay, Le Kremlin-Bicêtre, France
Abstract
Introduction
Von Willebrand factor (VWF) plays a pathophysiological role in hemostatic disorders. Partial inhibition of the VWF gene through small interfering RNA (siRNA)-mediated allele-selective silencing could be a promising therapeutic strategy. For von Willebrand disease, allele-selectively inhibiting dominant-negative VWF-alleles might ameliorate the phenotype. For thrombotic disorders, partial VWF reduction can lower thrombotic risk, while avoiding bleeding. Previously, we demonstrated the feasibility of Vwf-silencing in homozygous C57BL/6J (B6) or 129S1/SvImJ (129S) mice. The present study investigated allele-selective Vwf-silencing in a complex heterozygous setting of crossed B6 and 129S mice and its subsequent hemostatic impact.
Materials and methods
Heterozygous B6.129S mice were treated with siRNAs targeting Vwf expressed from either B6- (siVwf.B6) or 129S-alleles (siVwf.129S). Plasma VWF and lung Vwf mRNA were determined. siVwf.B6-treated B6.129S mice were subjected to ferric chloride-induced mesenteric vessel thrombosis and tail-bleeding.
Results
In B6.129S mice, siVwf.B6 reduced Vwf mRNA of the targeted B6-allele by 72% vs. only 12% of the non-targeted 129S-allele (41% total mRNA reduction), lowering plasma VWF by 46%. Oppositely, siVwf.129S reduced Vwf mRNA by 45%, now selectively inhibiting the 129S-allele over the B6-allele (58% vs. 9%), decreasing plasma VWF by 43%. The allele-selective VWF reduction by siVwf.B6 coincided with decreased thrombus formation in mesenteric arterioles, without prolonging tail-bleeding times.
Conclusions
This study demonstrates the feasibility of allele-selective Vwf-silencing in a heterozygous setting, achieving a controlled close to 50% reduction of plasma VWF. The observed thromboprotection and absence of prolonged bleeding times underline the potential of allele-selective Vwf-silencing as a therapeutic strategy in hemostatic disorders.