Ex vivo Improvement of a von Willebrand Disease Type 2A Phenotype Using an Allele-Specific Small-Interfering RNA

Abstract

Von Willebrand disease (VWD) is the most common inherited bleeding disorder and is mainly caused by dominant-negative mutations in the multimeric protein von Willebrand factor (VWF). These mutations may either result in quantitative or qualitative defects in VWF. VWF is an endothelial protein that is secreted to the circulation upon endothelial activation. Once secreted, VWF multimers bind platelets and chaperone coagulation factor VIII in the circulation. Treatment of VWD focuses on increasing VWF plasma levels, but production and secretion of mutant VWF remain uninterrupted. Presence of circulating mutant VWF might, however, still affect normal hemostasis or functionalities of VWF beyond hemostasis. We hypothesized that inhibition of the production of mutant VWF improves the function of VWF overall and ameliorates VWD phenotypes. We previously proposed the use of allele-specific small-interfering RNAs (siRNAs) that target frequent VWF single nucleotide polymorphisms to inhibit mutant VWF. The aim of this study is to prove the functionality of these allele-specific siRNAs in endothelial colony-forming cells (ECFCs). We isolated ECFCs from a VWD type 2A patient with an intracellular multimerization defect, reduced VWF collagen binding, and a defective processing of proVWF to VWF. After transfection of an allele-specific siRNA that specifically inhibited expression of mutant VWF, we showed amelioration of the laboratory phenotype, with normalization of the VWF collagen binding, improvement in VWF multimers, and enhanced VWF processing. Altogether, we prove that allele-specific inhibition of the production of mutant VWF by siRNAs is a promising therapeutic strategy to improve VWD phenotypes.

Quantitative 3D microscopy highlights altered von Willebrand factor α‐granule storage in patients with von Willebrand disease with distinct pathogenic mechanisms

“…Heterozygous mutations at this position 40,59,60 and at other cysteine residues within this domain 12 have been previously linked to type 2A VWD, a subtype that is characterized by low platelet binding activity of VWF due to loss of HMW multimers (also Figure 6). Lower multimers in type 2A VWD arise from either intracellular trafficking and/or multimerization defects during VWF biosynthesis (group I) or an increased sensitivity to extracellular proteolysis by ADAMTS-13 (group II).…”

“…Regardless, both this previous and our current study suggest that multimerization per se is not a prerequisite for VWF to enter platelet α-granules.Despite the reduction of HMW VWF multimers in their plasma, patients heterozygous for p.C1190R or p.C1190Y had platelet VWF multimers that were indistinguishable from healthy controls. De Jong et al59 recently tested a single nucleotide polymorphism-based approach to specifically silence expression of an autosomal dominant VWD allele in blood outgrowth endothelial cells of the same patient with type 2A with p.C1190Y included in our study. VWF multimers from heterozygous p.C1190Y endothelial cells showed an overabundance of dimeric VWF and pronounced retention of VWF in the endoplasmic reticu-…”

A Rollercoaster Plunge into 2022

“…In 2020, we introduced optional “Visual Summaries” to convey the main message of the manuscript in a clear and concise graphic manner so that readers can grasp its contents and relevance at a glance. In the past two years, we have received an increasing number of such graphical abstracts which greatly improved the reading quality of the articles published 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 some of which have even been selected for the T&H Issue Cover 4 5 11 15 33 38 ( Fig. 2 ).…”

von Willebrand disease: what does the future hold?

“…71 To find a broader application for this technology, de Jong et al designed siRNAs targeting common single-nucleotide polymorphisms in the VWF mutant allele, while leaving expression of the wild-type allele unaffected. 72 Alternatively, another option could be to correct the VWF gene itself, using gene-editing approaches such as Crispr-Cas9. Little information on this approach in view of VWD currently exists, with the exception of a study investigating the bi-allelic deletion of the VWF gene in endothelial colony forming cells (ECFCs, also known as blood outgrowth endothelial cells).…”