• Allele-selective silencing of mutant VWF with endothelial-specific siRNAs is feasible in a VWD type 2B mouse model.

  • Treatment with allele-selective siRNAs in a VWD type 2B mouse model leads to normalization of VWF multimers and hemostatic response.

Abstract

Treatment options for the bleeding disorder von Willebrand disease type 2B (VWD2B) are insufficient and fail to address the negative effects of circulating mutant von Willebrand factor (VWF). The dominant-negative nature of VWD2B makes functionally defective VWF an interesting therapeutic target. Previous in vitro studies have demonstrated the feasibility of allele-selective silencing of mutant VWF using small interfering RNAs (siRNAs) targeting common single nucleotide polymorphisms (SNPs) in the human VWF gene, an approach that can be applied irrespective of the disease-causing VWF mutation. This study aims to extend this concept to a heterozygous VWD2B mouse model (c.3946G>A; p.Val1316Met) here using mouse strain-specific genetic differences as proxy for human SNPs. Homozygous VWD2B C57BL/6J (2B-B6) mice were crossed with homozygous wild-type 129S1/SvImJ (129S) mice to create heterozygous 2B-B6.129S F1 offspring. These 2B-B6.129S mice were intravenously injected with endothelial-specific lipid nanoparticles loaded with the allele-selective siVwf.B6 or control and 96 hours later, lung Vwf messenger RNA, plasma VWF levels, and phenotypic characteristics were evaluated. Treatment with siVwf.B6 reduced total VWF levels by 50%, with an expected selective reduction in mutant VWF protein. This coincided with normalization of multimeric structure, improved VWF collagen binding/VWF antigen ratio, and normalized bleeding times in two-thirds of heterozygous 2B-B6.129S mice. Being a novel approach in the field of hemostasis, we proved, for VWD, in mice, the concept of selectively inhibiting a mutant dominant-negative allele with siRNAs targeting a single nucleotide variation rather than the disease-causing mutation. For dominant-negative VWD, this offers potential for a customized therapeutic strategy.

Von Willebrand disease type 2B (VWD2B) is a hereditary bleeding disorder caused by dominant-negative gain-of-function mutations within exon 28 of the von Willebrand factor (VWF) gene.1 As a result, the A1 domain of VWF undergoes a conformational change allowing plasma multimeric VWF to spontaneously bind to platelet receptor glycoprotein Ibα without prior activation of VWF.2-4 This is followed by a rapid clearance of both VWF and platelets from the circulation. As a consequence, individuals with VWD2B experience a bleeding tendency along with a potential decrease in platelet count and a loss of high-molecular-weight multimers (HMWMs), the latter normally being the most efficient in facilitating platelet adhesion at sites of vascular injury.5-7 

The clinical management of VWD2B poses specific challenges. The mainstay of treatment is VWF replacement therapy, involving the administration of an exogenous source of plasma-derived VWF or recombinant VWF.8-10 Although desmopressin represents another treatment option, acting by releasing endogenous VWF from vascular endothelial cells, its use in VWD2B remains controversial because of the induced secretion of functionally defective VWF multimers into the circulation thereby causing or worsening thrombocytopenia.10-12 Consequently, desmopressin is generally considered contraindicated in VWD2B. Finally, despite substitution treatment with exogenous VWF concentrate, mutant 2B VWF remains in the circulation and may even increase and induce thrombocytopenia in certain clinical situations. In light of these challenges, there is consensus on the need for a customized approach to optimize treatment outcomes for patients with VWD, particularly those with VWD2B, to mitigate the aforementioned concerns.

Our group has previously published on a novel therapeutic approach using allele-selective small interfering RNAs (siRNAs) to modulate gene expression.13-16 In the context of VWD, we have designed siRNAs that specifically target a heterozygous single-nucleotide polymorphism (SNP) located on the diseased VWF allele, with the aim of selectively counteracting the dominant-negative effect of mutant VWF and, in doing so, leaving the synthesis of normal VWF intact thereby restoring the VWF multimeric composition (Figure 1A). By selecting 4 SNPs with high minor allele frequencies, this approach has the potential to target a substantial portion of the population, with up to 75% being heterozygous for at least 1 of these SNPs, as predicted for the Caucasian and African population.13 In vitro and ex vivo proof-of-concept studies demonstrated that selective inhibition of the mutant VWF allele led to correction of the VWD phenotype.13,14 More recently, we studied the feasibility of allele-selective inhibition in vivo in mice by exploiting the genetic variations within Vwf genes across different mouse inbred strains as a proxy for human SNPs. In both homozygous C57BL/6J (B6) and 129S1/SvImJ (129S) mice as well as the heterozygous wild-type (WT) offspring derived from a cross between these mouse strains, it was shown that allele-selective inhibition of endogenous endothelial Vwf is feasible and results in effective and selective inhibition of the targeted Vwf allele.15,16 

Figure 1.

Schematic overview of the experimental concept and in vivo approach to achieve allele-selective silencing of mutant VWF. (A) Allele-selective silencing as intended in a human setting. The siRNA is targeted against a common SNP that is located on the diseased VWF allele. This will result in degradation of the mutant VWF mRNA. A mismatch between the siRNA and normal VWF mRNA will prevent the degradation of normal VWF. (B) Allele-selective silencing as investigated in vivo in a mouse model. The siRNA is targeted against a strain-specific variation that is (co)located on the diseased mouse Vwf allele. In this study, the heterozygous offspring of a cross between homozygous WT 129S and homozygous 2B mutant B6 mice was used. (C) Experimental workflow: 96 hours after administration of LNP-encapsulated siRNAs, blood samples were collected to determine plasma VWF protein levels and VWD-related protein characteristics. Whole blood was used to assess platelet phenotype. Lung tissue was collected to measure Vwf lung mRNA expression. This figure was created using BioRender.

Figure 1.

Schematic overview of the experimental concept and in vivo approach to achieve allele-selective silencing of mutant VWF. (A) Allele-selective silencing as intended in a human setting. The siRNA is targeted against a common SNP that is located on the diseased VWF allele. This will result in degradation of the mutant VWF mRNA. A mismatch between the siRNA and normal VWF mRNA will prevent the degradation of normal VWF. (B) Allele-selective silencing as investigated in vivo in a mouse model. The siRNA is targeted against a strain-specific variation that is (co)located on the diseased mouse Vwf allele. In this study, the heterozygous offspring of a cross between homozygous WT 129S and homozygous 2B mutant B6 mice was used. (C) Experimental workflow: 96 hours after administration of LNP-encapsulated siRNAs, blood samples were collected to determine plasma VWF protein levels and VWD-related protein characteristics. Whole blood was used to assess platelet phenotype. Lung tissue was collected to measure Vwf lung mRNA expression. This figure was created using BioRender.

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This study builds upon previously established proof-of-concept findings by using a murine model of VWD2B, thereby extending our understanding of the therapeutic potential of allele-selective siRNAs that can be applied in all dominant-negative types of VWD irrespective of the disease-causing VWF mutation. The VWD2B mouse model was developed through knock in of the severe p.Val1316Met (c.3946G>A) mutation (Vwf/p.V1316M) as described by Adam et al.17 Importantly, this genetic alteration resulted in a distinct VWD2B human-like phenotype in mice fully homozygous for the mutation, with the additional key aspect of endogenous endothelial expression of mutant Vwf.17 Generation of heterozygous offspring, a prerequisite for our allele-selective siRNA approach, involved a crossing strategy between homozygous B6 Vwf/p.V1316M mice (2B-B6) and homozygous WT-129S mice. Consequently, these heterozygous mice possess 1 normal WT-129S allele and 1 diseased B6 allele, mimicking the heterozygous genetic configuration observed in patients with VWD2B (Figure 1B). We then used these heterozygous 2B-B6.129S F1 mice to study the effect of allele-selective siRNA treatment on protein and RNA level and disease phenotype. Our findings demonstrate the applicability of allele-selective silencing in this VWD2B model, in which targeted inhibition of the diseased allele leads to improvements in key VWD2B phenotypic characteristics, including VWF multimeric structure and hemostatic activity.

siRNAs

Three previously optimized and in vivo–validated siRNAs were used15: (1) an allele-selective siVwf.B6 designed to target a strain-specific variation present in the C57BL/6J (B6) allele; (2) a nonselective siVwf targeting Vwf of both B6 and 129S1/SvImJ (129S) mouse strains and; and (3) a scrambled control (siControl.B6) that was designed based on the sequence of the allele-selective siVwf.B6. All siRNAs were identically chemically modified as described and produced by Axolabs GmbH (Kulmbach, Germany).15 For endothelial delivery, siRNAs were encapsulated in lipid nanoparticles (LNPs) composed of an ionizable, low–molecular weight polymer, termed 7C1, and lipid poly(ethylene glycol; C14PEG2000), as previously described.18 

Animal procedures

Homozygous VWD2B male mice on a B6 background (2B-B6) (inhouse, engineered by Adam et al. using B6-derived embryonic stem cells17) were bred inhouse with homozygous WT 129S (WT-129S) female mice (no. 002448, The Jackson Laboratory, Bar Harbor, ME) generating a heterozygous (F1) offspring (2B-B6.129S). Similarly, homozygous WT-B6 (no. 000664, The Jackson Laboratory; obtained from Charles River, France) male mice and WT-129S female mice were bred inhouse to generate heterozygous offspring (WT-B6.129S). Both male and female mice were used in each treatment group because our previous research showed a comparable inhibitory effect on VWF across sexes.15 A single tail vein injection of LNP-encapsulated siRNA (1.5 mg siRNA per kg body weight) was administered to 8- to 10-week-old mice. After 96 hours, mice were anesthetized with a ketamine (150 mg/kg) and xylazine (10 mg/kg) intraperitoneal injection. Citrated blood was collected from the inferior vena cava, and plasma was obtained by centrifugation at 12 000g for 10 minutes at room temperature, aliquoted and stored at −80°C. Euthanasia of the anesthetized mice was followed by disruption of the aorta and inferior vena cava. Left lung lobules were removed, snap frozen, and stored at −80°C (Figure 1C). During experiments, mice were housed in ventilated cages with a 12:12 hour light-dark cycle with ad libitum access to food and water. A priori power analyses were performed to determine the appropriate sample size using G∗Power software version 3.1.9.719 (supplemental Methods). Randomization was done using RandoMice software.20 The primary researcher remained blinded until data analysis. Experimental setup and sample size calculations were in accordance with the Institute for Laboratory Animal Research Guide for the Care and Use of Laboratory Animals and had received approval from the ethical review boards for animal experimentation of the Leiden University Medical Center, Leiden, The Netherlands and INSERM (APAFIS #38228-202208021331789 v2), Le Kremlin-Bicêtre, France.

Plasma VWF quantification and VWF functional assays

Plasma VWF antigen (VWF:Ag) levels were measured according to a previously described enzyme-linked immunosorbent assay.15 Analysis of collagen binding (VWF:CB) was performed as previously described, with the only modification that incubation with detection antibodies was performed at 37°C at 100 RPM.14 To determine the VWF:CB/VWF:Ag ratio, both VWF:Ag and VWF:CB levels were normalized to VWF:Ag and VWF:CB levels of normal mouse plasma (pooled from WT-B6 mice). VWF multimeric structure was visualized by a 1.5% nonreducing agarose (SeaKem HGT Agarose, Lonza, Rockland, ME) gel electrophoresis followed by immunoblotting, as previously described.13,21 Plasma of 6 siControl.B6-treated WT-B6.129S mice was pooled and used as a reference. For densitometric analysis, ImageJ 2.54 software (National Institutes of Health) was used to quantify the intensity of multimers. Densitometry images were divided into small, intermediate, and large VWF multimers, as previously described.13,22 The VWF large multimer index was calculated by dividing the VWF large multimer ratio of siControl.B6 or siVwf.B6-treated 2B-B6.129S mice by the average VWF large multimer ratio of siControl.B6-treated WT-B6.129S mice, as measured twice in the same blot.

Quantitation of WT and mutant VWF protein in plasma of siRNA-injected mice

To quantitate the fraction of WT and mutant VWF protein in plasma VWF of 2B-B6.129S mice, a targeted quantitative protein mass spectrometry assay was developed based on methods used for human plasma antithrombin quantitation.23,24 A detailed description of experimental procedures can be found in the supplemental Methods. In brief, mouse VWF protein was captured from plasma using an anti-VWF–immunoglobulin G antibody (A0082-4.1 g/L, DAKO, Glostrup, Denmark; biotinylated inhouse using biotin N-hydroxysuccinimide ester, H1759, Sigma-Aldrich, St. Louis, MO) coupled to magnetic beads (Dynabeads MyOne Streptavidin T1 Beads, Invitrogen, Thermo Fisher Scientific, Carlsbad, CA). Initial studies using a mouse VWF enzyme-linked immunosorbent assay showed that this immunocapture was able to capture at least 80% of mouse plasma VWF. Captured VWF was subsequently enzymatically digested into peptides in the presence of consistent amounts of 2 stable isotope-labeled (SIL) “heavy” peptides VAVVEYHDGSR and MAVVEYHDGSR (synthesized by peptide synthesis facilities of the Department of Immunology, Leiden University Medical Center, The Netherlands). Addition of SIL heavy peptides to the sample is required to achieve precise quantification and the relative ratio of heavy and “light” peptides (the latter representing the target protein) represent the target protein concentration. Peptide VAVVEYHDGSR was used to quantify mouse WT VWF protein, and MAVVEYHDGSR was used to quantify mouse mutant VWF (supplemental Table 1). After trypsin digestion, quenching of trypsin activity, removal of magnetic beads, and solid-phase extraction, a targeted liquid chromatography–mass spectrometry (LC-MS) analysis was conducted targeting both VWF endogenous light and heavy SIL peptides for each peptide, and the relative ratio of the endogenous to heavy SIL peptide was calculated. For both peptides, the relative ratios were normalized to the median relative ratios observed in siControl.B6-injected 2B-B6.129S mice and expressed as the fraction of the sum of both peptides. Plasma of WT-B6.129S mice was pooled and used as a reference.

Figure 2.

Plasma VWF and FVIII activity levels and lung Vwf mRNA in 2B-B6.129S mice injected with siControl.B6, siVwf or siVwf.B6. After 96 hours, the following parameters were determined in siControl.B6- (circles), siVwf- (triangles), and siVwf.B6-treated mice (squares): (A) plasma VWF levels; (B) plasma FVIII activity levels; (C) total Vwf lung mRNA expression. (D) Allele-selectivity of the siRNA treatment using an allele-selective quantitative polymerase chain reaction in which B6 Vwf (blue) is a representation of the B6-allele and 129S Vwf (magenta) of the 129S-allele. Data from panels C-D were measured in the same quantitative polymerase chain reaction. Data are presented as individual data points (n = 12 per group) with indication of the median as horizontal line. Values are compared with the median of the corresponding scrambled siControl.B6. Statistical analysis was performed using a nonparametric Kruskal-Wallis test with Dunn multiple comparisons test (∗∗∗∗P ≤ 0.0001; ∗∗P ≤ 0.01; ∗P < 0.05; P > 0.05, is nonsignificant [ns]).

Figure 2.

Plasma VWF and FVIII activity levels and lung Vwf mRNA in 2B-B6.129S mice injected with siControl.B6, siVwf or siVwf.B6. After 96 hours, the following parameters were determined in siControl.B6- (circles), siVwf- (triangles), and siVwf.B6-treated mice (squares): (A) plasma VWF levels; (B) plasma FVIII activity levels; (C) total Vwf lung mRNA expression. (D) Allele-selectivity of the siRNA treatment using an allele-selective quantitative polymerase chain reaction in which B6 Vwf (blue) is a representation of the B6-allele and 129S Vwf (magenta) of the 129S-allele. Data from panels C-D were measured in the same quantitative polymerase chain reaction. Data are presented as individual data points (n = 12 per group) with indication of the median as horizontal line. Values are compared with the median of the corresponding scrambled siControl.B6. Statistical analysis was performed using a nonparametric Kruskal-Wallis test with Dunn multiple comparisons test (∗∗∗∗P ≤ 0.0001; ∗∗P ≤ 0.01; ∗P < 0.05; P > 0.05, is nonsignificant [ns]).

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Figure 3.

WT and mutant VWF protein in plasma of 2B-B6.129S mice injected with siControl.B6, siVwf, or siVwf.B6. (A) Schematic overview of a targeted quantitative protein MS assay for the targeted quantification of the fraction of WT and mutant mouse VWF protein in plasma of siRNA-treated 2B-B6.129S mice. This figure was created using BioRender. (B) Fraction of the sum of WT and mutant mouse VWF protein in plasma of siRNA-injected 2B-B6.129S mice expressed as percentage. Plasma from WT-B6.129S mice was pooled and used as a reference. Median plasma VWF levels (U/mL) of the WT-B6.129S plasma pool and siRNA-injected 2B-B6.129S mice are indicated below the figure with the following minimum and maximum plasma levels for siControl.B6 (0.82-1.29 U/mL), for siVwf (0.10-0.32 U/mL), and for siVwf.B6 (0.32-0.54 U/mL). Data are presented as individual data points (n = 12 per group) with indication of the median as horizontal line. Statistical analysis was performed using a nonparametric Kruskal-Wallis test with Dunn multiple comparisons test (∗∗∗∗P ≤ 0.0001; ∗∗P ≤ 0.01; ns = P > 0.05).

Figure 3.

WT and mutant VWF protein in plasma of 2B-B6.129S mice injected with siControl.B6, siVwf, or siVwf.B6. (A) Schematic overview of a targeted quantitative protein MS assay for the targeted quantification of the fraction of WT and mutant mouse VWF protein in plasma of siRNA-treated 2B-B6.129S mice. This figure was created using BioRender. (B) Fraction of the sum of WT and mutant mouse VWF protein in plasma of siRNA-injected 2B-B6.129S mice expressed as percentage. Plasma from WT-B6.129S mice was pooled and used as a reference. Median plasma VWF levels (U/mL) of the WT-B6.129S plasma pool and siRNA-injected 2B-B6.129S mice are indicated below the figure with the following minimum and maximum plasma levels for siControl.B6 (0.82-1.29 U/mL), for siVwf (0.10-0.32 U/mL), and for siVwf.B6 (0.32-0.54 U/mL). Data are presented as individual data points (n = 12 per group) with indication of the median as horizontal line. Statistical analysis was performed using a nonparametric Kruskal-Wallis test with Dunn multiple comparisons test (∗∗∗∗P ≤ 0.0001; ∗∗P ≤ 0.01; ns = P > 0.05).

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Allele-selective lung Vwf messenger RNA (mRNA) quantification

Lung RNA isolation and complementary DNA synthesis were performed as previously described.15,25 Total lung Vwf (ie, both B6 and 129S) transcript levels were determined by quantitative polymerase chain reaction as well as B6 Vwf-specific and 129S Vwf-specific transcript levels.16 Data were normalized against the median transcript level of siControl.B6-treated 2B-B6.129S or WT-B6.129S mice.

Quantification of plasma FVIII activity

Plasma coagulation factor VIII (FVIII) activity was measured using an activated partial thromboplastin time clotting assay as previously described,15 with the only modification that samples were diluted 1:10 in Owren-Koller buffer (Stago, Leiden, The Netherlands). Plasma of WT-B6.129S mice was pooled and used as a reference for calculating FVIII activity.

Whole-blood analysis

Platelet count and platelet volume were determined in citrated whole blood using a veterinary automatic cell counter (Scil Vet ABC Plus, Horiba Medical, France). Blood smears were prepared from citrated whole blood and stained using a Giemsa-Wright stain. For assessment of circulating platelet aggregates, stained blood smears were mounted using Entellan rapid mounting medium and imaged using the Zeiss Axio Scan.Z1 slide scanner using a Plan-Apochromat 40× objective. In total, 6 regions of interest (ROI) were captured (4.7 mm2 in size) for each 2B-B6.129S mouse treated with siRNA. Platelet and aggregate count, together with platelet size were measured by an automated quantification pipeline using CellProfiler (version 4.2.126; supplemental Methods). Only aggregates containing ≥4 platelets were considered true aggregates. The number of platelet aggregates was adjusted for the number of ROIs used per sample based on a quality control to limit the interference of red blood cell counts in actual data acquisition. Quality control images were generated per ROI, which were independently checked by 2 reviewers for validity of the object identification.

Figure 4.

Effect of allele-selective silencing of mutant Vwf on multimeric structure in 2B-B6.129S mice. (A) Representative multimeric pattern of 2B-B6.129S mice treated with either siControl.B6 or siVwf.B6. Each lane represents an individual mouse. Plasma of 6 siControl.B6-treated WT-B6.129S mice was pooled and used as a reference. Samples were diluted to a final sample concentration of 0.1 U/mL. (B) Representative densitometric images and area under the curve (in pixels2) of siControl.B6- (left) or siVwf.B6-treated (right) 2B-B6.129S mice. (C) VWF large multimer index of siControl.B6- and siVwf.B6-treated 2B-B6.129S mice. The VWF large multimer index is calculated by dividing the VWF large multimer ratio of siControl.B6 or siVwf.B6-treated 2B-B6.129S mice with the average VWF large multimer ratio of siControl.B6-treated WT-B6.129S mice as measured twice in the same blot. Data are presented as individual data points (n = 12 per group) with indication of the median as horizontal line. Statistical analysis was performed using a nonparametric Mann-Whitney U test (∗∗∗P ≤ 0.001). (D) Ratio of collagen binding (VWF:CB) over VWF antigen (VWF:Ag) levels in 2B-B6.129S mice treated with siRNAs. VWF:CB and VWF:Ag values were normalized to values of normal mouse plasma. Data are presented as individual data points (n = 12 per group) with indication of the median as horizontal line. Statistical analysis was performed using a nonparametric Kruskal-Wallis test with Dunn multiple comparisons test (∗∗∗P ≤0.001; ∗∗P ≤ 0.01; ns = P > 0.05).

Figure 4.

Effect of allele-selective silencing of mutant Vwf on multimeric structure in 2B-B6.129S mice. (A) Representative multimeric pattern of 2B-B6.129S mice treated with either siControl.B6 or siVwf.B6. Each lane represents an individual mouse. Plasma of 6 siControl.B6-treated WT-B6.129S mice was pooled and used as a reference. Samples were diluted to a final sample concentration of 0.1 U/mL. (B) Representative densitometric images and area under the curve (in pixels2) of siControl.B6- (left) or siVwf.B6-treated (right) 2B-B6.129S mice. (C) VWF large multimer index of siControl.B6- and siVwf.B6-treated 2B-B6.129S mice. The VWF large multimer index is calculated by dividing the VWF large multimer ratio of siControl.B6 or siVwf.B6-treated 2B-B6.129S mice with the average VWF large multimer ratio of siControl.B6-treated WT-B6.129S mice as measured twice in the same blot. Data are presented as individual data points (n = 12 per group) with indication of the median as horizontal line. Statistical analysis was performed using a nonparametric Mann-Whitney U test (∗∗∗P ≤ 0.001). (D) Ratio of collagen binding (VWF:CB) over VWF antigen (VWF:Ag) levels in 2B-B6.129S mice treated with siRNAs. VWF:CB and VWF:Ag values were normalized to values of normal mouse plasma. Data are presented as individual data points (n = 12 per group) with indication of the median as horizontal line. Statistical analysis was performed using a nonparametric Kruskal-Wallis test with Dunn multiple comparisons test (∗∗∗P ≤0.001; ∗∗P ≤ 0.01; ns = P > 0.05).

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Figure 5.

Effect of allele-selective silencing of mutant Vwf on platelet phenotype using blood smears in 2B-B6.129S mice. (A) Overview of data acquisition by an automated quantification pipeline using CellProfiler. For the purpose of this figure a smaller subset (0.05 mm2) of 1 ROI (4.7 mm2) was used. The signal of raw input (I) consisting of 6 ROIs per sample was inverted (II) to distinguish between platelets and red blood cells. Single objects (platelets) were then identified (III) and merged (IV) to distinguish between platelet aggregates and single platelets. For the final data output (V), objects consisting of ≥4 platelets were considered aggregates together with a BoundingBoxArea/AreaShapeArea ratio ranging from 2.0 to 6.0. The BoundingBoxArea measures the dimensions of a box that encloses the aggregate in pixels, whereas the AreaShapeArea represents the sum of pixels for each individual platelet within the aggregate. The sum of all ROIs per sample was calculated and corrected for the number of ROIs used per sample based on quality controls. (B) Platelet counts per ROI as determined with the automated quantification pipeline using CellProfiler in blood smears made from whole-blood samples of 2B-B6.129S mice, 96 hours after siRNA treatment. (C) Number of platelet aggregates containing ≥4 platelets per 100 platelets as determined with the automated quantification pipeline using CellProfiler in blood smears made from whole-blood samples of 2B-B6.129S mice 96 hours after siRNA treatment. Data are presented as individual data points (n = 12 per group, 1 sample was removed from the siVwf.B6 group as images did not pass quality control) with indication of the median as horizontal line. Statistical analysis was performed using a nonparametric Kruskal-Wallis test with Dunn multiple comparisons test.

Figure 5.

Effect of allele-selective silencing of mutant Vwf on platelet phenotype using blood smears in 2B-B6.129S mice. (A) Overview of data acquisition by an automated quantification pipeline using CellProfiler. For the purpose of this figure a smaller subset (0.05 mm2) of 1 ROI (4.7 mm2) was used. The signal of raw input (I) consisting of 6 ROIs per sample was inverted (II) to distinguish between platelets and red blood cells. Single objects (platelets) were then identified (III) and merged (IV) to distinguish between platelet aggregates and single platelets. For the final data output (V), objects consisting of ≥4 platelets were considered aggregates together with a BoundingBoxArea/AreaShapeArea ratio ranging from 2.0 to 6.0. The BoundingBoxArea measures the dimensions of a box that encloses the aggregate in pixels, whereas the AreaShapeArea represents the sum of pixels for each individual platelet within the aggregate. The sum of all ROIs per sample was calculated and corrected for the number of ROIs used per sample based on quality controls. (B) Platelet counts per ROI as determined with the automated quantification pipeline using CellProfiler in blood smears made from whole-blood samples of 2B-B6.129S mice, 96 hours after siRNA treatment. (C) Number of platelet aggregates containing ≥4 platelets per 100 platelets as determined with the automated quantification pipeline using CellProfiler in blood smears made from whole-blood samples of 2B-B6.129S mice 96 hours after siRNA treatment. Data are presented as individual data points (n = 12 per group, 1 sample was removed from the siVwf.B6 group as images did not pass quality control) with indication of the median as horizontal line. Statistical analysis was performed using a nonparametric Kruskal-Wallis test with Dunn multiple comparisons test.

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Bleeding time

WT-B6.129S and 2B-B6.129S mice, 8 to 10 weeks old, were subjected to a tail-clip bleeding assay 96 hours after siRNA injection, as previously described.16,27 Bleeding time was recorded from the moment of transection until first arrest of the bleeding. Additionally, blood loss was quantified during the entire tail-clip bleeding assay (600 seconds).

Statistical analysis

Statistically significant differences were assessed using Mann-Whitney U or Kruskal-Wallis tests, as indicated, using GraphPad Prism 9.3.1 (GraphPad Software, La Jolla, CA). Data are presented as individual data points with indication of the median as a horizontal line. Minimum and maximum values are included in the text for each relevant analysis. P < 0.05 was considered statistically significant.

Allele selectivity of siRNAs in a VWD2B mouse model

To explore feasibility of selective inhibition of the diseased Vwf allele in a VWD2B mouse model, heterozygous 2B-B6.129S mice were treated with LNP-encapsulated nonselective siVwf, allele-selective siVwf.B6 or scrambled siControl.B6. Previous investigations showed that endothelial-specific Vwf inhibition by siRNAs reduced plasma VWF for at least 10 days, with optimal inhibition 96 hours after a 1.5 mg/kg dose, which therefore served as the euthanasia time point and dose for all performed experiments.15 Treatment of 2B-B6.129S mice with the nonselective siVwf resulted in a robust inhibitory effect on plasma VWF (85.6% [69.6-90.6] inhibition; Figure 2A), FVIII activity (33.7% [−0.2 to 71.6]; Figure 2B), and total lung Vwf mRNA (77.5% [7-96]; Figure 2C). Injection with the allele-selective siVwf.B6 led to a variable effect on total lung Vwf mRNA level, with an anticipated more modest reduction of 24.5% (−97 to 93), leading to a 59.4% (49.3-69.3) decrease in plasma VWF levels, in line with previous findings.16 FVIII activity levels in siVwf.B6-treated mice (9.9% [−25.2 to 30.1] reduction) were not significantly different from siControl.B6-injected mice. To assess allele-selectivity at the mRNA level, lung transcript levels of both B6 Vwf and 129S Vwf were examined (Figure 2D). After treatment with siVwf, there was a respective median reduction of 54.5% (−68 to 94) of B6 Vwf and 39.5% (−47 to 83) of 129S Vwf. After siVwf.B6 treatment, a reduction of 42% (−275 to 92) of B6 Vwf lung mRNA was observed but no reduction in 129S Vwf (−29.0% [−438 to 75]) mRNA. Although the levels of B6 Vwf after both siVwf and siVwf.B6 treatment did not reach statistical significance compared with siControl.B6 treatment, the inhibition pattern observed was consistent with previous findings and displayed similarities to plasma VWF levels.16 As expected, 129S Vwf mRNA levels did not differ between the siControl.B6- and siVwf.B6-treated 2B-B6.129S mice, further corroborating the allele selectivity of siVwf.B6 in this VWD2B mouse model. Considering the potential variability stemming from RNA isolation and downstream processes and the clear effects observed for plasma VWF (Figure 2A), a novel targeted MS assay was developed to substantiate allele-selective silencing of mutant Vwf at the plasma protein level. In this method, the peptides VAVVEYHDGSR and MAVVEYHDGSR specific to WT and mutant mouse VWF, respectively, were quantified in VWF captured from mouse plasma (Figure 3A). As expected, VWF captured from plasma of WT-B6.129S mice (pooled plasma) harbored 100% of the WT peptide with full absence of mutant VWF (Figure 3B). In siControl.B6-treated 2B-B6.129S mice, VWF consisted of 49.7% (47.6-52.3) of WT and 50.3% (47.7-52.4) of mutant VWF protein, confirming expression of the WT and mutant allele in the heterozygous VWD2B mouse model. Similarly, although treatment with the nonselective siVwf resulted in a strong inhibitory effect on total plasma VWF levels, VWF consisted equally of WT (49.6% [47.2-51.7]) and mutant (50.4% [48.3-52.8]) protein. This result is consistent with the effect of siVwf, which targets and inhibits both alleles equally. In contrast, siVwf.B6 treatment led to a significant change in VWF protein composition reducing mutant VWF to 23% (16.9-31.9) of total, whereas favoring an increase in WT VWF to constitute 77% (68.1-83.1) of the captured plasma VWF protein. Thus, this MS method substantiates the selective inhibition of mutant VWF at the protein level by allele-selective silencing of the mutant Vwf allele.

VWF multimeric structure and collagen binding in siRNA-treated 2B-B6.129S mice

Given that VWD2B is characterized by an absence of HMWMs,28 we expected that the observed selective inhibition of mutant VWF would improve the multimeric profile. Qualitative analysis of VWF multimers revealed that siControl.B6-treated 2B-B6.129S mice exhibited a distinct VWD2B-specific phenotype, marked by a relative reduction of HMWMs and an increase of low-molecular-weight multimers (LMWMs) (Figure 4A; supplemental Figure 2). As anticipated, administration of the allele-selective siVwf.B6 to 2B-B6.129S mice led to an improvement in multimeric structure with an increase in HMWMs and a decrease of LMW proteolytic fragments. To quantitatively assess this increase in HMWMs, the large multimer index was calculated with densitometric analysis and related to reference values of siControl.B6-treated WT-B6.129S mice (Figure 4B). In line with qualitative analysis, siVwf.B6 treatment normalized the large multimer index, indicating increased HMWMs (Figure 4C). Because HMWMs exhibit greater collagen binding affinity than LMWMs, we conducted a VWF:CB assay and determined VWF:CB/VWF:Ag ratios. We observed a significant increase in VWF:CB/VWF:Ag ratio in siVwf.B6-treated 2B-B6.129S mice (median ratio of 1.51 [1.19-2.10]) as compared with those mice treated with siControl.B6 (median ratio of 1.02 [0.92-1.76]; Figure 4D), further supporting the increase in HMWMs structure after siVwf.B6 treatment.

Platelet count and circulating platelet aggregates in siRNA-treated 2B-B6.129S mice

To investigate the impact of allele-selective silencing on platelet phenotype, citrated whole-blood samples and blood smears were used to assess platelet count and the presence of circulating platelet aggregates using an automated image analysis (Figure 5A). Unexpectedly, no significant differences were identified in platelet counts across the siRNA-treated 2B-B6.129S mice (Figure 5B; supplemental Figure 3A) and the siRNA-treated WT-B6.129S and 2B-B6.129S mice after a tail-clip bleeding challenge (supplemental Figure 3C). Similarly, there were no discernable variations in mean platelet volume (supplemental Figure 3B,D). Further examination of the blood smears of 2B-B6.129S mice revealed the presence of platelet aggregates across all treatment groups. We calculated platelet aggregate number per 100 platelets because total platelet count and number of platelet aggregates may affect each other29 (Figure 5C). Compared with treatment with siControl.B6, treatment with both the nonselective siVwf and allele-selective siVwf.B6 resulted in a decrease in platelet aggregate number. Albeit not statistically significant, an effect in the expected direction was evident (P = 0.065), particularly in siVwf.B6-treated mice. Overall, the thrombocytopenic phenotype observed in 2B-B6.129S mice appeared relatively mild, with no significant differences in platelet aggregate number upon reduction of mutant VWF.

Effect of endothelial mutant Vwf inhibition on bleeding time

To evaluate whether the reduced fraction of mutant VWF protein in plasma, coinciding with improved multimerization and multimer function in siVwf.B6-treated 2B-B6.129S mice, also improved hemostasis in these mice, we conducted a tail-clip bleeding assay in both WT-B6.129S and 2B-B6.129S mice. Analysis of blood samples collected from the mice after the bleeding challenge confirmed that these mice had actually responded to siRNA treatment as expected (supplemental Figure 4A). In addition, FVIII activity was assessed after the bleeding challenge, indicating no differences between treatment groups (supplemental Figure 4B). In comparison with siControl.B6-injected WT-B6.129S mice, siControl.B6-injected 2B-B6.129S mice exhibited a significant threefold increase in bleeding time (median bleeding time 62 [50-78] vs 208 [106-517] seconds; Figure 6), thus confirming the VWD2B phenotype.17 In WT-B6.129S mice, treatment with the nonselective siVwf resulted in a notable increase in bleeding time (as compared with siControl.B6-injected WT-B6.129S mice, median bleeding time of 141 [73-600] seconds vs 62 seconds), confirming the phenotype associated with nearly complete VWF deficiency,30 whereas treatment with siVwf.B6 demonstrated no prolongation, measuring at 64 [49-114] seconds. Finally, in 2B-B6.129S mice, inhibition of the diseased Vwf allele with the allele-selective siVwf.B6 resulted in restoration of bleeding times approaching normal values in 4 out of 6 mice, with an overall median bleeding time of 88.5 [52-600] seconds after challenge. These findings suggest that hemostatic activity is beneficially influenced after siVwf.B6 treatment in 2B-B6.129S mice.

Figure 6.

Effect of allele-selective silencing of mutant Vwf on bleeding phenotype in 2B-B6.129S mice. Both WT-B6.129S (red symbols) and 2B-B6.129S (blue symbols) mice were subjected to a tail-clip bleeding assay 96 hours after siRNA treatment. The observation period lasted for a maximum of 600 seconds, after which bleeding was stopped by cauterization and bleeding time was set to 600 (indicated by dotted line). Data are presented as individual data points (n = 6 per group) with indication of the median as a horizontal line. Statistical analysis was performed using a nonparametric Kruskal-Wallis test with Dunn multiple comparisons test (∗∗P ≤ 0.01; ∗P < 0.05).

Figure 6.

Effect of allele-selective silencing of mutant Vwf on bleeding phenotype in 2B-B6.129S mice. Both WT-B6.129S (red symbols) and 2B-B6.129S (blue symbols) mice were subjected to a tail-clip bleeding assay 96 hours after siRNA treatment. The observation period lasted for a maximum of 600 seconds, after which bleeding was stopped by cauterization and bleeding time was set to 600 (indicated by dotted line). Data are presented as individual data points (n = 6 per group) with indication of the median as a horizontal line. Statistical analysis was performed using a nonparametric Kruskal-Wallis test with Dunn multiple comparisons test (∗∗P ≤ 0.01; ∗P < 0.05).

Close modal

In this study, we have demonstrated allele-selective inhibition of mutant Vwf in heterozygous VWD2B mice using LNP-encapsulated endothelial-specific siRNAs targeting a mouse strain-specific variation rather than the disease-causing Vwf mutation. Using a novel targeted quantitative protein MS assay, we substantiated this selective inhibition of mutant VWF at the protein level. Specifically, treatment with siVwf.B6 resulted in an improvement of VWF multimeric structure through an elevation in HMWMs as reflected by an increased affinity of VWF to collagen. Additionally, a substantial portion (two-thirds) of the heterozygous VWD2B mice exhibited a normalization of hemostatic response after a tail-clip bleeding assay. Our results provide in vivo proof of concept for selectively inhibiting a mutant dominant-negative allele with siRNAs targeting a single-nucleotide variation rather than the disease-causing mutation. Now tested in a VWD2B context, this approach holds significant potential for application in all dominant-negative VWD cases caused by heterozygous missense mutations.

Among the experimental mouse models reported in the literature for investigating VWF and VWD, the 2B-B6.129S mouse model used in this study prevails as the optimal choice for several reasons. Primarily, unlike in vivo models using hydrodynamic gene transfer,31 this VWD2B model permits specific endothelial expression and targeting of VWF. Furthermore, as a congenital Vwf/p.V1316M murine model, all hemostatic processes could be studied without issues of species incompatibility, particularly concerning the mouse-human glycoprotein Ibα–VWFA1 interaction critical for VWD2B. The homology between the p.Val1316Met mutation in this murine model and its human counterpart, along with the observed human-like VWD2B characteristics in these mice, adds additional support for this model. From a practical standpoint, the development of this VWD2B mouse model on a B6 genetic background provided a unique opportunity for the generation of a heterozygous offspring with 129S mice using strain-specific genetic differences between their Vwf alleles as a proxy for human SNPs. This approach facilitated the application of allele-selective siRNAs, now tested for the first time in vivo in a relevant mouse model for VWD.

Adam et al. demonstrated that manifestation of a severe platelet phenotype required a homozygous p.Val1316Met mutation, whereas heterozygous mice exhibited a milder and intermediate phenotype.17 Our measurements of platelet counts and number of platelet aggregates in the 2B-B6.129S murine model align with these findings because we were unable to reproduce a severe VWD2B platelet phenotype. However, our observations align with heterozygous human VWD2B cases, indicating a milder and heterogeneous phenotype.28,32 Although platelet counts were similar across treatment groups, we observed an expected trend toward fewer platelet aggregates after siVwf.B6 treatment.

A heterogeneous effect was also observed on bleeding phenotype, which may stem from the heterogeneous effects of the mutation within the murine context but might more likely be explained by the inherent variability of the performed tail-clip assay itself. Despite this variability, siVwf.B6 treatment effectively inhibited plasma VWF levels and normalized bleeding time in two-thirds of the 2B-B6.129S mice. Possibly, a more optimized siRNA and prolonged inhibition might result in a more pronounced beneficial change in composition of the circulating VWF protein and consequently a stronger impact on platelet and bleeding phenotype. These issues could be overcome15 and studied in future translational studies.

The need to shift VWD treatment from a “one-size-fits-all” approach to more personalized treatment has been of great interest.33-35 Although the siRNAs used in this study are specific for murine Vwf, the strategy of allele-selective silencing, relying on targeting a single-nucleotide difference, is applicable to human dominant-negative VWD cases as well. Similar to the mouse strain-specific genetic differences used in this study, siRNAs can be designed that target SNP sequences within the human VWF gene. When a patient is heterozygous for a specific SNP in the VWF coding sequence, an siRNA that targets the SNP allele located on the diseased VWF allele can silence the expression of mutant VWF (Figure 1A). Although in this study we have shown in vivo proof of concept for this allele-selective silencing and improvement of the VWD phenotype in mice, we have previously shown the possibility of SNP-based allele-selective targeting of human VWF in vitro.13,14 We are currently in the process of further developing those SNP-targeting siRNAs to improve their activity, selectivity, and biological persistence. These siRNA compounds will need to be tested in a human system for a wide range of different VWF mutations. Patient-derived endothelial colony forming cells will be of great use for this, because they can be directly harvested from peripheral blood from individuals with VWD, providing the genetic and cellular context as in a therapeutic setting. This allele-selective silencing approach offers an advantage for future clinical use, requiring only a limited set of siRNAs that target a few different SNPs which will be able to silence a broad spectrum of VWD mutations, and thereby be useful for a large number of patients with VWD.

In summary, we demonstrated the capability to effectively inhibit mutant Vwf in a heterozygous VWD2B mouse model. Our findings underscore that allele-selective Vwf silencing enhances VWF multimeric structure and function with a possibility to normalize bleeding phenotype. This work highlights the promise of this allele-selective siRNA approach, an approach targeting a single-nucleotide variation rather than the disease-causing mutation. For dominant-negative VWD, this offers potential for a customized treatment.

The authors thank Peter van Balen and Elizabeth C. van Weers for their assistance with the staining of blood smears, and Marie Clavel for her assistance during intermediate data analysis.

This work was supported by funding from The Netherlands Organization for Scientific Research, Domain Applied and Engineering Sciences (TTW/AES), “Connecting Innovators” Open Technology Programme, project no. 18712. The contribution of Y.K.J. was supported by The Netherlands Thrombosis Foundation grant no. 2018-01 and the contribution of S.N.J.L. by The Netherlands Organization for Scientific Research, Dutch Research Agenda-Research Along Routes grant 1160.18.038.

Contribution: N.A.L., B.J.M.v.V., and J.C.J.E. designed the study, analyzed and interpreted data, and wrote the manuscript; N.A.L., Y.K.J., and B.J.M.v.V. performed in vivo experiments; R.J.D., L.R.R., and B.J.M.v.V. designed and performed MS experiments and analyzed data; S.N.J.L. designed an automated quantification pipeline and analyzed data; R.A.C., N.D., and J.W.D. synthesized and contributed peptides used for MS; J.E.D. and E.S.E. designed, synthesized, and contributed 7C1 oligomeric lipid nanoparticles; C.V.D. and C.C. planned and supervised breeding of mice and supervised and assisted with in vivo experiments. R.B., F.W.G.L., J.V., and J.C.J.E. initiated, designed, and supervised the overall research project and obtained funding; and all authors contributed to revisions of the manuscript.

Conflict-of-interest disclosure: J.C.J.E. received research funding from CSL Behring, all funds to the institution. F.W.G.L. received unrestricted research grants from Takeda, CSL Behring, UniQure, and Sobi, and was consultant for Takeda, CSL Behring, and Biomarin, of which fees go to the university. J.V. is listed as an inventor on a patent on ADAMTS13 variants. The remaining authors declare no competing financial interests.

Correspondence: Jeroen C. J. Eikenboom, Department of Internal Medicine, Division of Thrombosis and Hemostasis, Einthoven Laboratory for Vascular and Regenerative Medicine, Leiden University Medical Center, Albinusdreef 2, P.O. Box 9600, 2300 RC Leiden, The Netherlands; email: [email protected].

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Author notes

Data are available on request from the corresponding author, Jeroen C. J. Eikenboom ([email protected]).

The full-text version of this article contains a data supplement.