OC 58.2 - Allele specific knock-out of mutant Von Willebrand factor in heterozygous Von Willebrand disease type 2A patient-derived ECFCs

Tuesday, June 25, 2024

09:45 – 10:00 ICT

Room: 211 A-D

Presenting Author(s)

Isabel Bär, MSc

PhD candidate
Erasmus Medical Center Rotterdam
Rotterdam, Zuid-Holland, Netherlands

Co-Author(s)

PB

Petra Bürgisser

Research Technician
Hematology, Erasmus MC University Medical Center, Rotterdam, The Netherlands
Rotterdam, Zuid-Holland, Netherlands
Iris van Moort

Postdoctoral researcher
Erasmus MC University Medical Center, Rotterdam, The Netherlands
Rotterdam, Zuid-Holland, Netherlands
Calvin B. van Kwawegen, MSc

PhD candidate
Erasmus MC, University Medical Centre Rotterdam, Department of Haematology, Rotterdam, The Netherlands
Rotterdam, Zuid-Holland, Netherlands
JV

Jan Voorberg

Professor
Department of Molecular Hematology, Sanquin Research and Landsteiner Laboratory, Amsterdam, the Netherlands
Amsterdam, Noord-Holland, Netherlands
Jeroen Eikenboom

MD, PhD, prof
Department of Thrombosis and Hemostasis, Leiden University Medical Center, Leiden, The Netherlands
Leiden, Zuid-Holland, Netherlands
Frank W.G Leebeek, MD PhD

Internist-hematologist, Professor
Department of Hematology, Erasmus University Medical Center, Rotterdam, The Netherlands
Rotterdam, Zuid-Holland, Netherlands
Ruben Bierings, PhD

Associate Professor
Hematology, Erasmus MC University Medical Center, Rotterdam, The Netherlands
Rotterdam, Zuid-Holland, Netherlands

Background: Von Willebrand Disease (VWD) is a heterogeneous bleeding disorder arising from a variety of mutations in the von Willebrand Factor (VWF) gene. Due to the dominant negative effects of heterozygous mutations in VWF, patients could potentially benefit from a strategy that selectively targets the expression of the mutant allele. Recent work has shown phenotypic improvement in endothelial cells of a VWD type 2A patient after allele-selective targeting of a dominant negative VWF allele using siRNAs against single nucleotide polymorphisms (SNPs) (De Jong, 2020; PMID: 32803740).

Aims: Proof of concept for durable phenotypic correction of VWD by permanently knocking out a dominant negative VWF mutation using allele-selective targeting of SNPs in VWF using CRISPR/Cas9.

Methods: Endothelial colony forming cells (ECFCs) were isolated from venous blood of a VWD type 2A patient from the Willebrand in the Netherlands (WiN) cohort, with a heterozygous p.C1190R (c.3568T>C) mutation. Long-read sequencing was used to identify nucleotides of a heterozygous SNP in VWF (rs1800378) that is on the same allele as the p.C1190R mutation. gRNAs were designed against both SNP nucleotides to knock out the healthy or mutant allele, respectively, using CRISPR/Cas9. Amplicon NGS and confocal analysis were used to monitor genotype and phenotype.

Results: p.C1190R heterozygous ECFCs were characterized for VWF processing and trafficking defects, including retention of VWF in the endoplasmic reticulum (ER) and aberrations of Weibel-Palade body morphology. NGS revealed allele-selective introduction of DSBs at the targeted sites, as evidenced by indel mutations around the predicted Cas9 cut sites. Ablation of the healthy allele led to all VWF being retained in the ER and a total lack of WPBs. Equally, the knock-out (KO) of the p.C1190R carrying allele resolved the ER retention of VWF and resulted in restoration of elongated WPBs.

Conclusion(s): These results show proof-of-concept for phenotypic correction after allele-selective KO of VWD alleles using CRISPR/Cas9.