Blood Outgrowth Endothelial Cells (BOECs) to study the pathophysiology of von Willebrand disease and the feasibility of in vitro correction of von Willebrand factor defects

Von Willebrand factor (VWF) plays an essential role in primary hemostasis. It
is synthesized in endothelial cells and megakaryocytes and is stored in
Weibel-Palade bodies and *-granules, respectively. Qualitative and quantitative
defects of VWF lead to the most common, inherited bleeding disorder von
Willebrand disease (VWD). Patients with types 1 and 2 VWD are usually
heterozygous for missense mutations in the VWF gene that exert a
dominant-negative effect of the mutant allele on the wild type allele resulting
in additional quantitative and qualitative defects of VWF. Current therapy for
VWD is based on inducing the release of endogenously synthesized VWF or
infusion of exogenous VWF concentrates. However, factor concentrates have a
short half-life, are very expensive and will not correct the negative effects
of the still circulating mutant VWF. A new therapeutic strategy could be to
silence the expression of the mutant VWF allele by RNA-targeted therapy or
CRISPR/Cas9-editing to decrease the synthesis of the mutant VWF and as a
consequence increase the synthesis of normal VWF. For the development of new
therapeutic strategies a good understanding of the pathophysiology of VWF
mutations is necessary. The most optimal model for studying the VWF biology and
RNA-targeted silencing are the blood outgrowth endothelial cells (BOECs). An
alternative approach to obtain patient specific VWF producing cells is to
reprogram peripheral blood mononuclear cells (PBMCs) into induced pluripotent
stem cells (iPSCs) that can subsequently be differentiated into endothelial
cells (iPSC-ECs) and megakaryocytes (iPSC-MKs).

1. To establish optimized approaches for the isolation and characterization of
BOECs and iPSC-ECs
2. To further explore the biology of VWF, the pathophysiology of different
subtypes of VWD and the role of VWF as a signalling protein in for example
angiogenesis using BOECs and iPSC derived endothelial cells and
megakaryocytes/platelets.
3. To develop in vitro a therapeutic strategy for VWD by silencing of
dominant-negative mutant VWF alleles using RNA-targeted silencing or
CRISPR/Cas9-editing in BOECs and iPSC-ECs

The study is a multicenter, basic science, experimental in vitro,
non-therapeutic study.

The burden for the participants is minimal as the research is restricted to a
single 30 minutes visit to the hospital including a short interview on medical
history and medication use and a venepuncture with a total maximum blood draw
of 70 mL.

Participants may be requested to participate in repeated sampling, however this
will be maximised to three times and the repeated sampling will be a maximum of
50 mL of blood. For repeated sampling the participants will be asked for a new
informed consent.

The risk of venepuncture is negligible. Proper manual compression of the
puncture site for a few minutes will prevent any bruises.

Patients do not directly benefit from this research. Their blood samples will
be used in a fundamental research project only.

The study is focused on pathophysiologic aspects and potential new therapeutic
approaches of VWD and can only be performed in this study population.