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Correction of a dominant‐negative von Willebrand factor multimerization defect by small interfering RNA ‐mediated allele‐specific inhibition of mutant von Willebrand factor
Author(s) -
Jong A.,
Dirven R. J.,
Oud J. A.,
Tio D.,
Vlijmen B. J. M,
Eikenboom J.
Publication year - 2018
Publication title -
journal of thrombosis and haemostasis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.947
H-Index - 178
eISSN - 1538-7836
pISSN - 1538-7933
DOI - 10.1111/jth.14140
Subject(s) - von willebrand disease , small interfering rna , allele , von willebrand factor , gene knockdown , mutant , single nucleotide polymorphism , microbiology and biotechnology , biology , genetics , chemistry , immunology , gene , platelet , genotype , transfection
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.Summary 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 RNA s (si RNA s) targeting common single‐nucleotide polymorphisms ( SNP s) in VWF . This approach allows allele‐specific knockdown irrespective of the mutations causing VWD . Methods Four SNP s with a high predicted heterozygosity within VWF were selected, and si RNA s were designed against both alleles of the four SNP s. si RNA efficiency, allele specificity and si RNA ‐mediated phenotypic improvements were determined in VWF ‐expressing HEK 293 cells. Results Twelve si RNA s were able to efficiently inhibit single VWF alleles in HEK293 cells that stably produce VWF. Transient cotransfections of these si RNA s with two VWF alleles resulted in a clear preference for the targeted allele over the untargeted allele for 11 si RNA s. We also demonstrated si RNA ‐mediated phenotypic improvement of the VWF multimerization pattern of the VWD type 2A mutation VWF p.Cys2773Ser. Conclusions Allele‐specific si RNA s 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 si RNA s in dominant‐negative VWD .