Patients with von Willebrand disease (VWD) have a defect in the process of blood coagulation. Therefore, they develop bruises easily and it takes longer for a bleeding to stop. Patients develop VWD because of a defect in von Willebrand factor (VWF), a protein with an important role in blood coagulation. VWF is mainly important in attracting platelets to sites of vascular damage. VWF also circulates in the blood. In the blood VWF binds another coagulation protein, coagulation factor VIII (FVIII). Because of binding of FVIII to VWF, FVIII can survive longer in the bloodstream. This results in a better function of FVIII. Patients with VWD usually have less VWF in the blood, or they have a functional defect in VWF.

VWF functions better when multiple VWF molecules join forces. Together they form a large VWF multimer. Separate VWF molecules are produced by two different alleles, the mother- and the father allele. In case of VWD, only one of the two alleles is defect. Therefore half of the molecules in the VWF multimer is defect. This has however consequences for the whole VWF multimer. We hypothesized that if we are able to inhibit the production of the defective VWF molecule, this would lead to a fully functional VWF protein. Using the LSBR grant we have been able to test this hypothesis in the past three years.

The VWF gene on the DNA is transcribed into the VWF mRNA. The mRNA is then translated to the VWF protein. The conversion of mRNA to protein could be inhibited by small RNA molecules, so-called small interfering RNAs or siRNAs. We used these siRNAs to inhibit the production of the defective VWF molecules. However, between the healthy and defective VWF allele there is only one nucleotide variation. This complicates the design of siRNAs that only inhibit the defective allele, with minor inhibition of the healthy allele. We therefore tested several different siRNAs for their function.

We made a selection of very effective siRNAs by using a cell model in which both healthy and defective VWF was inserted. Together with VWF, we also inserted the siRNA. Then, we tested on a protein level whether there was less production of the defective VWF, but still normal production of the healthy VWF. By doing this, we were able to select siRNAs that were efficient and specific for inhibition of the defective VWF molecule.
In the body, VWF is produced by endothelial cells. These cells line the inner wall of the blood vessels. We have been able to isolate endothelial cells from a VWD patient. This patient has a defect in the process of multimerization, which we could also observe in the cells of the patient. We tested an siRNA that should inhibit solely production of the defective VWF molecule. We saw that after siRNA treatment, the cells indeed started to produce fully functional VWF multimers. We also tried to inhibit the healthy VWF allele, leaving a more defective protein. We indeed observed a worsening in VWF multimerization thereafter. This proves that our technique has the expected effects.

Altogether we could conclude that the siRNAs are effective and very specifically inhibit the production of the defective VWF protein. We showed this in two different cell models for VWD. We extent this research in a VWD mouse model. The use of mouse models is important to show that our approach is also effective in a physiological system. Altogether, the results in the cell models show that allele-specific siRNAs hold promise as a potential treatment for VWD patients.

Thesis 2020: Personalized treatment for Von Willebrand Disease, Annika de Jong