Department of Internal Medicine (Thrombosis and Hemostasis), Einthoven Laboratory for Vascular and Regenerative Medicine, Leiden University Medical Center, Leiden, the Netherlands.
J Thromb Haemost. 2018 Jul;16(7):1357-1368. doi: 10.1111/jth.14140. Epub 2018 Jun 1.
Essentials Substitution therapy for von Willebrand (VW) disease leaves mutant VW factor (VWF) unhindered. Presence of mutant VWF may negatively affect phenotypes despite treatment. Inhibition of VWF by allele-specific siRNAs targeting single-nucleotide polymorphisms is effective. Allele-specific inhibition of VWF p.Cys2773Ser improves multimerization.
Background Treatment of the bleeding disorder von Willebrand disease (VWD) focuses on increasing von Willebrand factor (VWF) levels by administration of desmopressin or VWF-containing concentrates. Both therapies leave the production of mutant VWF unhindered, which may have additional consequences, such as thrombocytopenia in patients with VWD type 2B, competition between mutant and normal VWF for platelet receptors, and the potential development of intestinal angiodysplasia. Most cases of VWD are caused by dominant-negative mutations in VWF, and we hypothesize that diminishing expression of mutant VWF positively affects VWD phenotypes. Objectives To investigate allele-specific inhibition of VWF by applying small interfering RNAs (siRNAs) targeting common single-nucleotide polymorphisms (SNPs) in VWF. This approach allows allele-specific knockdown irrespective of the mutations causing VWD. Methods Four SNPs with a high predicted heterozygosity within VWF were selected, and siRNAs were designed against both alleles of the four SNPs. siRNA efficiency, allele specificity and siRNA-mediated phenotypic improvements were determined in VWF-expressing HEK293 cells. Results Twelve siRNAs were able to efficiently inhibit single VWF alleles in HEK293 cells that stably produce VWF. Transient cotransfections of these siRNAs with two VWF alleles resulted in a clear preference for the targeted allele over the untargeted allele for 11 siRNAs. We also demonstrated siRNA-mediated phenotypic improvement of the VWF multimerization pattern of the VWD type 2A mutation VWF p.Cys2773Ser. Conclusions Allele-specific siRNAs are able to distinguish VWF alleles on the basis of one nucleotide variation, and are able to improve a severe multimerization defect caused by VWF p.Cys2773Ser. This holds promise for the therapeutic application of allele-specific siRNAs in dominant-negative VWD.
目的 应用靶向血管性血友病因子（VWF）常见单核苷酸多态性（SNP）的小干扰 RNA（siRNA），研究 VWF 的等位基因特异性抑制。这种方法允许针对导致 VWD 的突变进行等位基因特异性敲低。
方法 选择 VWF 中具有高预测杂合性的 4 个 SNP，并针对这 4 个 SNP 的两个等位基因设计 siRNA。在稳定表达 VWF 的 HEK293 细胞中，测定 siRNA 的效率、等位基因特异性和 siRNA 介导的表型改善情况。
结果 在能够有效抑制稳定产生 VWF 的 HEK293 细胞中，有 12 个 siRNA 能够有效地抑制单个 VWF 等位基因。这些 siRNA 的瞬时共转染导致 11 个 siRNA 对靶向等位基因的偏好明显高于未靶向等位基因。我们还证明了 siRNA 介导的 VWF p.Cys2773Ser 所致 VWD 2A 突变的 VWF 多聚体化模式的表型改善。
结论 等位基因特异性 siRNA 能够基于一个核苷酸变异区分 VWF 等位基因，并能够改善由 VWF p.Cys2773Ser 引起的严重多聚体化缺陷。这为在显性负性 VWD 中应用等位基因特异性 siRNA 提供了希望。
