An imbalance in von Willebrand factor (VWF) may either lead to bleeding (von Willebrand disease, VWD) or thrombosis. Both disorders have shortcomings in the currently available treatments. VWF... Show moreAn imbalance in von Willebrand factor (VWF) may either lead to bleeding (von Willebrand disease, VWD) or thrombosis. Both disorders have shortcomings in the currently available treatments. VWF itself could be a potential therapeutic target because of its role in both bleeding and thrombosis. Inhibiting VWF gene expression through allele-selective silencing of VWF with small interfering RNAs (siRNAs) could be a personalized approach to specifically inhibit mutant VWF in VWD or to normalize increased VWF levels in thrombotic disorders without complete VWF knockdown. Therefore, we investigated a method to allele-selectively silence the VWF gene in mice as a therapeutic strategy. Fourteen candidate siRNAs targeting murine Vwf of either the C57BL/6J (B6) or the 129S1/SvImJ (129S) strain were tested in vitro in cells expressing B6- and 129S-Vwf for inhibitory effect and allele-selective potential. Together with a nonselective siVwf, 2 lead candidate siRNAs, siVwf.B6 and siVwf.129S, were further tested in vivo in B6 and 129S mice. Efficient endothelial siRNA delivery was achieved by siRNA encapsulation into 7C1 oligomeric lipid nanoparticles. Treatment with the nonselective siVwf resulted in dose-dependent inhibition of up to 80% of both lung messenger RNA and plasma VWF protein in both mouse strains. In contrast, the allele-selective siVwf.B6 and siVwf.129S were shown to be effective in and selective solely for their corresponding mouse strain. To conclude, we showed efficient endothelial delivery of siRNAs that are highly effective in allele-selective inhibition of Vwf in mice, which constitutes an in vivo proof of principle of allele-selective VWF silencing as a therapeutic approach. Show less
Murine atherosclerosis models are key for investigation of atherosclerosis pathophysiology and drug development. However, they do not feature spontaneous atherothrombosis as a final stage of... Show moreMurine atherosclerosis models are key for investigation of atherosclerosis pathophysiology and drug development. However, they do not feature spontaneous atherothrombosis as a final stage of atherosclerosis. Transgenic mice expressing both the human mutant apolipoprotein E form APOE*3-Leiden and human cholesteryl ester transfer protein (CETP), i.e. APOE*3-Leiden.CETP mice, feature a moderate hyperlipoproteinemia and atherosclerosis phenotype. In contrast to apolipoprotein E deficient (Apoe(-/-)) mice, APOE*3-Leiden.CETP mice respond well to lipid-lowering and anti-atherosclerotic drugs. The aim of the study was to investigate whether silencing of anticoagulant Protein C (Proc) allows APOE*3-Leiden.CETP mice to feature thrombosis as a final stage of atherosclerosis. Female APOE*3-Leiden.CETP mice were fed a Western-type diet to induce advanced atherosclerosis, followed by an injection with a small interfering RNA targeting Proc (siProc). Presence of atherosclerosis and atherothrombosis was determined by histologic analysis of the aortic root. Atherosclerosis severity in the aortic root area of APOE*3-Leiden.CETP mice varied from type "0" (no lesions) to type "V" lesions (advanced and complex lesions). Atherothrombosis following siProc injection was observed for 4 out of 21 APOE*3-Leiden.CETP mice (19% incidence). The atherothrombosis presented as large, organized, fibrin- and leukocyte-rich thrombi on top of advanced (type "V") atherosclerotic plaques in the aortic root. This atherothrombosis was comparable in appearance and incidence as previously reported for Apoe(-/-) mice with a more severe atherosclerosis (19% incidence). APOE*3-Leiden.CETP mice with modest hyperlipidemia and atherosclerosis can develop atherothrombosis upon transient Proc-silencing. This further extends the use of these mice as a test model for lipid-lowering and anti-atherosclerotic drugs. Show less
