Campioni Matteo, Legendre Paulette, Loubiere Cécile, Lunghi Barbara, Pinotti Mirko, Christophe Olivier D, Lenting Peter J, Denis Cécile V, Bernardi Francesco, Casari Caterina
Department of Life Science and Biotechnology, University of Ferrara, Ferrara, Italy.
Institut National de la Santé et de la Recherche Médicale, Unité Mixte de Recherche Scientifique, Université Paris-Sud, Université Paris-Saclay, Le Kremlin-Bicêtre, France.
J Thromb Haemost. 2021 Jan;19(1):139-146. doi: 10.1111/jth.15131. Epub 2020 Nov 3.
Essentials Treatment options for von Willebrand disease (VWD) patients are limited. The p.P1127_C1948delinsR deletion/variant is a useful model to study VWD in vitro and in vivo. Counteracting dominant-negative effects restores von Willebrand factor multimerization in mice. This is the first siRNA-based treatment applied to a mouse model of VWD-type 2A. ABSTRACT: Background Treatment options for patients suffering from von Willebrand disease (VWD) are limited. Von Willebrand factor (VWF) is a polymeric protein that undergoes regulated dimerization and subsequent multimerization during its biosynthesis. Numerous heterozygous variants within the VWF gene display a dominant-negative effect and result in severe VWD. Previous studies have suggested that preventing the assembly of wild-type and mutant heteropolymers using siRNAs may have beneficial effects on VWF phenotypes in vitro. Objectives To study heterozygous dominant-negative variants in vivo, we developed a mouse model of VWD-type 2A and tested two independent strategies to modulate its detrimental effect. Methods The p.P1127_C1948delinsR deletion/variant, causing defective VWF multimerization, was expressed in mice as a model of VWD-type 2A variant. Two corrective strategies were applied. For the first time in a mouse model of VWD, we applied siRNAs selectively inhibiting translation of the mutant transcripts and we combined the VWD-type 2A deletion with the Cys to Arg substitution at position 2773, which is known to prevent dimerization. Results The RNA silencing approach induced a modest but consistent improvement of the VWF multimer profile. However, due to incomplete efficiency, the dominant-negative effect of the original variant could not be completely prevented. In contrast, the DNA approach resulted in increased antigen levels and restoration of a normal multimer profile. Conclusions Our data showed that preventing the detrimental impact of dominant-negative VWF variants by independent molecular mechanisms has beneficial consequences in vivo, in mouse models of dominant VWD.
血管性血友病（VWD）患者的治疗选择有限。p.P1127_C1948delinsR缺失/变体是在体外和体内研究VWD的有用模型。抵消显性负效应可恢复小鼠体内血管性血友病因子多聚体的形成。这是首次将基于小干扰RNA（siRNA）的治疗应用于2A型VWD小鼠模型。摘要：背景 血管性血友病（VWD）患者的治疗选择有限。血管性血友病因子（VWF）是一种聚合蛋白，在其生物合成过程中经历受控的二聚化和随后的多聚化。VWF基因内的许多杂合变体表现出显性负效应，并导致严重的VWD。先前的研究表明，使用siRNA阻止野生型和突变杂聚物的组装可能对体外VWF表型有有益影响。目的 为了在体内研究杂合显性负变体，我们开发了一种2A型VWD小鼠模型，并测试了两种独立的策略来调节其有害效应。方法 p.P1127_C1948delinsR缺失/变体导致VWF多聚化缺陷，在小鼠中作为2A型VWD变体的模型表达。应用了两种纠正策略。在VWD小鼠模型中，我们首次应用siRNA选择性抑制突变转录本的翻译，并将2A型VWD缺失与2773位的半胱氨酸到精氨酸替换相结合，已知该替换可阻止二聚化。结果 RNA沉默方法使VWF多聚体谱有适度但一致的改善。然而，由于效率不完全，原始变体的显性负效应无法完全消除。相比之下，DNA方法导致抗原水平升高和正常多聚体谱的恢复。结论 我们的数据表明，在显性VWD小鼠模型中，通过独立的分子机制防止显性负VWF变体的有害影响在体内具有有益后果。
