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    Oral Communication

    Platelet Disorders, von Willebrand Disease and Thrombotic Microangiopathies

    Von Willebrand Disease and Thrombotic Thrombocytopenic Purpura (TTP) Novel Treatments

    OC 08.5 - Allele-selective inhibition of mutant von Willebrand factor with small interfering RNAs to ameliorate a von Willebrand disease type 2B phenotype in vivo in mice

    Saturday, June 24, 2023
    14:00 – 14:15 ET
    Room: 518
    Session Available As: On-Demand

      Presenting Author(s)

    • Noa A. Linthorst, MSc

      PhD candidate
      Department of Internal Medicine, Division of Thrombosis and Hemostasis, Einthoven Laboratory for Vascular and Regenerative Medicine, Leiden University Medical Center, Leiden, The Netherlands.
      Leiden, Zuid-Holland, Netherlands

      • Co-Author(s)

    • Yvonne K. Jongejan, MSc

      PhD candidate
      Department of Internal Medicine, Division of Thrombosis and Hemostasis, Einthoven Laboratory for Vascular and Regenerative Medicine, Leiden University Medical Center, Leiden, The Netherlands.
      Leiden, Zuid-Holland, Netherlands

    • Ruben Bierings, PhD

      Principal investigator
      Department of Hematology, Erasmus MC, University Medical Center Rotterdam, the Netherlands
      Rotterdam, Zuid-Holland, Netherlands

    • JD

      James E. Dahlman

      Associate professor
      Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University School of Medicine, Atlanta, Georgia, United States.
      Atlanta, Georgia, United States

    • Cécile V. Denis, PhD

      Principal investigator, Director of Research
      Laboratory for Hemostasis, Inflammation and Thrombosis, Unité Mixte de Recherche S1176, Institut National de la Santé et de la Recherche Médicale, Université Paris-Saclay, Le Kremlin-Bicêtre, France
      Le Kremlin-Bicêtre, Ile-de-France, France

    • Frank W.G. Leebeek, MD, PhD

      Professor and Head of Department of Hematology
      Erasmus MC, University Medical Center Rotterdam, The Netherlands
      Rotterdam, Zuid-Holland, Netherlands

    • ES

      Elisa Schrader

      PhD candidate
      Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University School of Medicine, Atlanta, Georgia, United States.
      Atlanta, Georgia, United States

    • JV

      Jan Voorberg

      Professor, Principal investigator
      Department of Molecular Hematology, Sanquin Research and Landsteiner Laboratory
      Amsterdam, Noord-Holland, Netherlands

    • Bv

      Bart J.M. van Vlijmen

      Associate professor
      Department of Internal Medicine, Division of Thrombosis and Hemostasis, Einthoven Laboratory for Vascular and Regenerative Medicine, Leiden University Medical Center, Leiden, The Netherlands.
      Leiden, Zuid-Holland, Netherlands

    • Jeroen C.J Eikenboom, MD. PhD

      Professor, Head of Division of Thrombosis and Hemostasis
      Leiden University Medical Center
      Leiden, Zuid-Holland, Netherlands

    Background: Von Willebrand disease (VWD) is the most common inherited bleeding disorder caused by defects in von Willebrand factor (VWF). Current therapies do not inhibit mutant VWF expression. Inhibition of mutant VWF through allele-selective silencing using small interfering RNAs (siRNAs) could potentially improve VWF function and alleviate VWD phenotype.

    Aims: Investigate the feasibility of allele-selective inhibition of mutant VWF with siRNAs and its effect on disease phenotype in a VWD-type 2B mouse model.

    Methods: Three siRNAs were used: 1) siVwf.B6, a strong selective inhibitor of mouse Vwf expressed from the C57BL/6J (B6) allele, and not from the 129S1/SvImJ (129S) allele, 2) a non-selective siVwf and 3) a scrambled control (siControl.B6). siRNAs were encapsulated in nanoparticles for endothelial targeting in vivo. Homozygous 2B-B6 mice were crossed with 129S mice to create 2B-B6.129S F1 offspring. 96 hours after siRNA injection, citrated blood and lung tissue were collected for determining VWF plasma protein and lung mRNA. In parallel, 2B-B6.129S and WT-B6.129S F1 mice were subjected to a tail-clip bleeding assay.

    Results: Treatment of 2B-B6.129S mice with siVwf resulted in a strong median 86% reduction in plasma VWF levels, while upon treatment with siVwf.B6 a reduction limited to 59% was observed (Figure 1). These findings were comparable to that observed for siRNA-treated WT-B6.129S mice (not shown). Upon tail-clipping, 2B-B6.129S mice showed prolonged bleeding compared to WT-B6.129S mice (siControl.B6-treated, Figure 2). While treatment with siVwf coincided with prolonged bleeding in both WT-B6.129S and 2B-B6.129S mice, siVwf.B6 coincided with normal bleeding times for all WT-B6.129S mice as well as 4 out of 6 2B-B6.129S mice.

    Conclusion(s): First results regarding allele-selective inhibition of mutant VWF in a VWD-type 2B mouse model are promising. Evaluation of platelet phenotype, mRNA expression, VWF activity and multimer distribution is currently ongoing.

    Funding was received from NWO-AES (#18712), Dutch Thrombosis Foundation (#2018-1) and EHA Research Mobility Grant.

    Learning Objectives:

    • understand the concept of allele-selective silencing in dominant-negative von Willebrand disease.
    • translate the concept of allele-selective silencing to an in vivo experimental setting.
    • interpret the different assays and corresponding results to understand the effect of siRNA treatment on disease phenotype.