New gene-editing therapy targets shared genetic markers to treat VWD
CRISPR disabled mutations and restored health to patient cells in early study
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Scientists have developed a gene-editing therapy to selectively disable the genetic mutations responsible for Von Willebrand disease (VWD) type 2, regardless of subtype, potentially offering a permanent fix for several difficult-to-treat forms of the bleeding disorder.
In a new proof-of-principle study, researchers demonstrated that using CRISPR-Cas9 gene-editing technology could restore the health of blood cells from two patients with different disease subtypes, effectively allowing healthy genes to take over and produce functional clotting proteins.
“The results presented in this study lay the groundwork for future studies aimed at refining and expanding therapeutic strategies for VWD,” researchers wrote.
The study, “Allele-selective Disruption of Pathogenic VWF Variants in Type 2 Von Willebrand Disease using CRISPR/Cas9,” was published in Blood Advances.
The challenge of dominant mutations
Most cases of VWD are caused by mutations in the VWF gene, leading to reduced von Willebrand factor (VWF) activity, a protein involved in blood clotting. Without sufficient VWF, either due to low protein levels or dysfunctional VWF, the blood clotting process is impaired, leading to easy bruising or unusual bleeding.
Current treatments for VWD aim to boost VWF levels and restore blood clotting. This includes desmopressin, a lab-made hormone that increases VWF levels, and replacement therapy, which provides patients with a functional version of VWF.
Some forms of the disease, such as subtypes of VWD type 2, are caused by heterozygous dominant-negative VWF mutations. That is, while one VWF gene is mutated, the other is normal, but mutant forms can disrupt the function of the normal version, complicating treatments that increase VWF.
CRISPR-Cas9 is a gene-editing tool that acts like molecular scissors, precisely cutting and modifying DNA at specific locations. Its therapeutic potential has been demonstrated by the approval of Casgevy (exagamglogene autotemcel), a gene-editing therapy for the blood disorder sickle cell disease.
In this report, a team in the Netherlands investigated CRISPR-Cas9 as a potential treatment for patients with VWD who carry heterozygous dominant-negative VWF mutations. The approach was to permanently inactivate the mutant form of VWF using CRISPR-Cas9, while leaving the healthy form intact, thereby producing only functional VWF protein.
“This strategy may convert [VWD type 2] patients into heterozygous null carriers [one active, one inactive VWF gene], that are typically asymptomatic or, in rare cases, mildly affected,” the researchers wrote.
The team collected endothelial colony-forming cells (ECFCs), which are immature cells that grow into mature VWF-producing cells, from two VWD patients: one with type 2A and one with type 2B. Both patients exhibited reduced VWF levels and recurrent bleeding. ECFCs were also collected from a healthy individual as a control.
The most straightforward way to selectively disrupt a disease-causing gene is to target the mutation site with CRISPR-Cas9, while leaving the healthy gene intact. However, with more than 750 VWF known mutations associated with VWD, it’s really not feasible to make a CRISPR-Cas9 treatment for each mutant.
Instead, the team targeted a common single-nucleotide polymorphism (SNP), rs1800378, a single but distinct change in the DNA code on the mutated VWF gene. Targeting this SNP has been shown to selectively silence the defective VWF gene. Both patients carried this SNP, which is also found in about half of VWD patients.
Restoring cell health across subtypes
Using a lentiviral vector to deliver CRISPR-Cas9 to cells, experiments confirmed that the treatment selectively silenced a single target VWF gene in healthy ECFCs.
The researchers then tested the system in ECFCs derived from the VWD type 2A patient. Results showed that the CRISPR-Cas9 treatment efficiently and selectively disrupted the mutated VWF gene, while maintaining the VWF activity of the non-targeted healthy gene.
Further testing confirmed selective reduction of the mutant VWF protein, accompanied by reversal of ECFC disease characteristics (phenotype). Overall protein production in ECFCs, besides VWF, was not affected by the treatment.
To assess whether the approach can be applied to other VWD subtypes caused by different VWF mutations, the team used ECFCs derived from a type 2B patient. While the treatment exhibited a similar pattern of gene editing, the overall efficiency was slightly reduced. Even so, the CRISPR-Cas9 treatment disrupted the mutant gene but not the healthy gene, thereby restoring the health of ECFCs.
“Our data show that CRISPR/Cas9 targeting of a common heterozygous SNP can be used to selectively knock out a heterozygous disease [gene copy] in a VWF variant-independent manner,” the researchers wrote. This led to the “stable phenotypic rescue of the cells responsible for VWF production regardless of the VWD subtype involved.”
