In this video lecture, Dr. Jeroen Eikenboom discusses:
- How dominant-negative VWF variants disrupt multimer formation and limit the effectiveness of current VWD treatments.
- Allele-selective strategies using siRNA and CRISPR-Cas to silence pathogenic VWF alleles while preserving normal expression.
- Emerging gene transfer and gene-editing approaches that may correct specific molecular defects across different VWD subtypes.
Jeroen Eikenboom (MD, PhD) received his MD degree (1988) at the Erasmus University Rotterdam, the Netherlands. He obtained his PhD (1994) at the Leiden University and was subsequently trained in internal medicine, hematology and vascular medicine at the Leiden University Medical Centre. He was appointed full professor of Internal Medicine/Haemostasis and Thrombosis in 2011. His clinical work is focused on bleeding disorders, thrombophilia and vascular medicine. His research is mainly focused on the molecular genetics of von Willebrand disease, genotype-phenotype associations in von Willebrand disease, and the pathophysiology of von Willebrand factor. The current research focus is on development of genomic editing approaches to treat von Willebrand disease. Dr. Eikenboom (co)authored over 300 papers. He is former chairman of the Scientific Subcommittee on von Willebrand factor of the ISTH and the former chair of the Dutch Society of Haemophilia Treaters. He is chair of the scientific advisory board of the Landsteiner Foundation for Blood Transfusion Research.
(Video Lecture Summary)
Introduction
Dr. Jeroen Eikenboom reviews the current status of genomic editing approaches for treating von Willebrand disease (VWD). He begins by outlining the normal function and assembly of von Willebrand factor (VWF), then explains why existing treatments do not fully correct the underlying molecular defects in many patients.
The lecture focuses especially on dominant-negative VWF variants and the possibility of selectively silencing or editing the pathogenic allele. Dr. Eikenboom also discusses broader gene transfer and gene-correction strategies for forms of VWD that cannot be addressed through allele-selective silencing.
Limitations of Current Treatments
Desmopressin releases endogenous VWF from endothelial storage sites and can increase circulating VWF levels. However, response varies according to VWD subtype and may be inadequate in many patients. Repeated treatment can produce tachyphylaxis as endogenous stores become depleted. Desmopressin may also cause adverse effects such as hyponatremia and can worsen thrombocytopenia in type 2B VWD.
Plasma-derived and recombinant VWF concentrates provide an external source of normal VWF and can be used across VWD subtypes. Their effects are temporary because of the limited half-life of VWF. Some forms of bleeding, particularly gastrointestinal bleeding associated with angiodysplasia, may respond incompletely. In addition, the patient’s mutant VWF remains present and may continue to exert harmful effects. Patients with severe type 3 VWD may also develop alloantibodies against infused VWF.
These limitations have encouraged the development of therapies that act at the molecular or genetic level.
Antibody and Aptamer Approaches
Before turning to genomic strategies, Dr. Eikenboom briefly reviews investigational therapies designed to prolong the survival of endogenous VWF.
A pegylated aptamer that binds the VWF A1 domain was initially developed to inhibit VWF activity in thrombotic disorders. Its binding also prolongs the VWF half-life and increases circulating levels. However, because it may interfere with platelet binding, its broader hemostatic effectiveness remains uncertain. It may have a more selective role in type 2B VWD by reducing thrombocytopenia.
He also discusses KB-V13A12, a bispecific nanobody that binds both VWF and albumin, thereby prolonging VWF survival through albumin-associated recycling. A separate monovalent antibody, HMB-002, binds the VWF CK domain and similarly extends its half-life. These approaches may be especially relevant to type 1 VWD, where functional VWF is present but reduced in quantity.
Dominant-Negative VWF Variants
VWF is produced as monomers that form dimers and then assemble into large multimers. In a healthy individual, both VWF alleles produce normal subunits that combine into functional multimers.
In many forms of VWD, one allele produces a mutant VWF subunit. Mutant and normal subunits then assemble together into the same dimers and multimers. As a result, a mutation in only one allele can disrupt much of the circulating VWF. This is known as a dominant-negative effect.
Dr. Eikenboom explains that this mechanism is common in many patients with missense variants, including forms of type 1 VWD and type 2A, 2B, and 2M disease. Current treatments do not remove these abnormal multimers. Desmopressin releases more of the patient’s own VWF, including mutant protein, while concentrate replacement adds normal VWF without eliminating the abnormal protein already present.
The Rationale for Allele-Selective Silencing
Experimental cotransfection studies show that increasing the proportion of mutant VWF progressively worsens multimer formation. Conversely, reducing expression of the mutant allele shifts the multimer pattern toward normal. Dr. Eikenboom therefore proposes selectively silencing the mutant allele while preserving expression from the normal allele. Although total VWF antigen may decrease because only one allele remains active, the VWF produced should be more functional because it is composed primarily of normal subunits.
Following successful allele silencing, desmopressin could potentially release a pool of VWF with a much more normal composition and activity.
Allele-Selective siRNA
Small interfering RNA, or siRNA, can be designed to bind a specific messenger RNA sequence. Once incorporated into the RNA-induced silencing complex, the siRNA guides degradation of a complementary messenger RNA while leaving transcripts with a sequence mismatch relatively unaffected.
One possible strategy would be to design a separate siRNA for each pathogenic VWF mutation. However, because hundreds of different VWF variants have been identified, this would be impractical.
Instead, Dr. Eikenboom describes targeting common single nucleotide polymorphisms that are inherited on the same chromosome as the disease-causing variant. When the selected polymorphism is linked to the mutant allele, the siRNA can target that allele without directly targeting the pathogenic mutation itself. The normal allele, carrying a different nucleotide at the polymorphic site, remains expressed.
Evidence from Patient-Derived Endothelial Cells
This approach has been tested in endothelial colony-forming cells derived from a patient with type 2A VWD.
Untreated cells showed retention of mutant VWF within the endoplasmic reticulum. Selective silencing of the mutant allele reduced this retention and restored localization of VWF within normal-appearing Weibel-Palade bodies.
In contrast, silencing the healthy allele worsened the phenotype. VWF accumulated within the endoplasmic reticulum, and the remaining Weibel-Palade bodies appeared abnormal. These findings demonstrated that allele-selective siRNA could improve the cellular phenotype when directed at the pathogenic allele.
Evidence from Mouse Models
Dr. Eikenboom next reviews the challenges of testing allele-selective treatment in mice. Laboratory mice are genetically uniform and lack the heterozygous single nucleotide polymorphisms needed to distinguish between two alleles.
Investigators addressed this by crossing two mouse strains with naturally occurring sequence differences in their VWF genes. These differences served as proxies for human polymorphisms and allowed an siRNA to selectively suppress one allele.
In normal crossed mice, targeting one strain-specific allele reduced VWF antigen to approximately half of baseline while leaving the other allele intact. The strategy was then tested in a type 2B VWD model in which the pathogenic mutation was carried on the targeted allele.
Selective suppression of the mutant allele increased high-molecular-weight multimers and improved the large multimer index. In several treated mice, the prolonged bleeding time was also corrected. These studies support the feasibility of using sequence variation linked to a mutation to selectively silence a dominant-negative allele.
CRISPR-Cas Allele Knockout
Unlike siRNA, which produces temporary suppression, CRISPR-Cas gene editing offers the possibility of permanently disrupting a pathogenic allele.
Dr. Eikenboom describes a similar polymorphism-based strategy in which the guide RNA recognizes a sequence linked to the mutant allele but not the healthy allele. The CRISPR-Cas complex therefore cuts and disrupts the pathogenic allele while preserving the normal copy.
This approach was tested in patient-derived endothelial colony-forming cells carrying a type 2A mutation. Untreated cells showed substantial VWF retention within the endoplasmic reticulum. Disruption of the healthy allele worsened this retention. In contrast, selective knockout of the mutant allele restored VWF localization to well-formed Weibel-Palade bodies.
These experiments demonstrate that allele-selective CRISPR-Cas editing can correct a cellular VWD phenotype.
Which Patients Could Benefit?
Allele-selective silencing is most relevant when a patient has one mutant allele that interferes with the normal allele. It would not be useful for patients whose type 1 VWD results from a null allele that is already not expressed. It would also not address most type 3 VWD, where both alleles fail to produce functional VWF.
Potential candidates include many patients with heterozygous missense variants in type 1 VWD and patients with dominant type 2A, 2B, and 2M disease. The approach may therefore apply to a substantial proportion of patients, but not to all VWD subtypes.
Developmental Challenges
The siRNA platform remains preclinical. Investigators are optimizing the molecules to improve selectivity and affinity while reducing unintended effects. Endothelial-targeted lipid nanoparticles have enabled delivery in mice, but toxicity and efficacy must still be evaluated in vitro, in animal models, and eventually in humanized models.
CRISPR-Cas approaches are also at an early stage. Considerable work remains before permanent allele knockout could be evaluated safely in patients.
Gene Transfer and Gene Correction
Dr. Eikenboom also reviews approaches for patients who cannot benefit from allele-selective silencing. Earlier liver-directed gene transfer produced high levels of VWF expression in mice but failed to generate a normal multimer pattern. The expressed protein contained predominantly low-molecular-weight multimers and did not adequately correct the bleeding phenotype. These findings suggest that the cellular site of VWF production is critical and that endothelial expression may be necessary.
More recent CRISPR-Cas work has focused on correcting a frameshift mutation in a canine model of type 3 VWD. By creating a compensatory nucleotide insertion or deletion, investigators restored the reading frame. Treated cells resumed producing VWF and assembling multimers, demonstrating that direct correction of a type 3 defect may be feasible.
Comparing Genomic Strategies
Gene addition could introduce a normal VWF gene, but patients with dominant-negative disease would continue producing mutant protein unless that allele were also addressed. In type 3 VWD, where no endogenous VWF is produced, gene addition may be more straightforward.
Allele-selective silencing removes mutant expression and leaves only the normal allele active, producing lower quantities of more functional VWF. Direct correction of the mutation could theoretically restore normal expression from both alleles, but this is technically more complex.
Dr. Eikenboom presents these approaches as complementary rather than interchangeable. The most appropriate strategy will depend on the VWD subtype and the underlying molecular defect.
Conclusion
Dr. Eikenboom concludes that genomic treatment of VWD will likely require subtype-specific and mutation-informed approaches. Allele-selective siRNA and CRISPR-Cas strategies have improved VWF processing, multimer composition, and bleeding phenotypes in cellular and animal models of dominant-negative VWD.
Gene transfer and direct gene correction may offer alternatives for type 3 disease and other defects that cannot be treated through allele silencing. Although these approaches remain preclinical, they provide a foundation for therapies that address the molecular cause of VWD rather than temporarily replacing or releasing VWF.