PB0797 - Restoration of VWF in Von Willebrand Disease type 3 canine ECFCs through CRISPR-Cas9 mediated gene editing

Monday, July 11, 2022

6:30 PM – 7:30 PM

Location: Exhibition

Presentation Format: In-Person

Presenting Author(s)

Alastair Barraclough

PhD Candidate
Amsterdam UMC, University of Amsterdam, Emma Children’s Hospital, Amsterdam, The Netherlands
Amsterdam, Noord-Holland, Netherlands

Co-Author(s)

AD

Andera D'Amico

Post Doctoral Researcher
Sanquin Research and Landsteiner Laboratory
Amsterdam, Noord-Holland, Netherlands
RL

Rodrigo Leite de Oliveira

Professor
CRISPR Expertise Center, Cancer Center Amsterdam, Amsterdam University Medical Cente
Amsterdam, Noord-Brabant, Netherlands
Ruben Bierings, PhD

Principal investigator
Department of Hematology, Erasmus MC, University Medical Center Rotterdam, the Netherlands
Rotterdam, Zuid-Holland, Netherlands
Frank W.G. Leebeek, MD, PhD

Professor and Head of Department of Hematology
Erasmus MC, University Medical Center Rotterdam, The Netherlands
Rotterdam, Zuid-Holland, Netherlands
JE

Jeroen C.J. Eikenboom

Professor, Internist, Head of Div. T&H
Department of Internal Medicine, Division of Thrombosis and Hemostasis, Einthoven Laboratory for Vascular and Regenerative Medicine, Leiden University Medical Center, Leiden, The Netherlands
Leiden, Zuid-Holland, Netherlands
SD

Simon F. De Meyer

Prof., Dr., Eng.
Laboratory for Thrombosis Research, KU Leuven Campus Kulak Kortrijk, Kortrijk, Belgium
Kortrijk, West-Vlaanderen, Belgium
Karen Vanhoorelbeke, PhD

Prof., Dr.
Laboratory for Thrombosis Research, KU Leuven Campus Kulak Kortrijk, Kortrijk, Belgium
Kortrijk, West-Vlaanderen, Belgium
KF

Karin Fijnvandraat

Professor of Pediatric Hematology
Department of Pediatric Hematology, Amsterdam UMC, University of Amsterdam, Amsterdam, the Netherlands
Amsterdam, Noord-Holland, Netherlands
JV

Jan Voorberg

Professor, Principal investigator
Department of Molecular Hematology, Sanquin Research and Landsteiner Laboratory
Amsterdam, Noord-Holland, Netherlands

Background: Von Willebrand Disease (VWD) is a common and complex bleeding disorder due to the large variance in pathogenic mutation distributions between patients and families. The most severe form is type 3 which is characterised by the inability to produce Von Willebrand Factor (VWF). Robust gene editing technologies such as CRISPR-Cas9 can provide treatment options to patients with all forms of VWF. Presented here is a previously identified type 3 canine VWD ECFC (endothelial colony forming cell) model. This model can demonstrate gene correction for VWD type 3 in-vitro, which could be further translated to a canine in-vivo model.

Aims: To deploy CRISPR-Cas9 gene correction technologies to permanently correct the pathogenic homozygous G>A mutation present in VWD type 3 canine ECFCs and restore normal VWF.

Methods: Canine ECFCs previously identified as type 3 are subjected to CRISPR-Cas9 gene correction utilising adenine base editing through viral and non-viral delivery; and homology directed repair (HDR) via a ribonucleoprotein. Qualitative and quantitative phenotypic confirmation of VWF produced by clonal populations of transformed cells can be monitored via VWF:Ag (antigen) and immunostaining to confirm abundance and localisation of VWF in the Weibel-Palade-bodies. Glycoprotein Ib binding assays (VWF:GPlbM) will determine if the produced VWF is functional.

Results: Canine ECFCs have been isolated and their phenotypes confirmed. Non-viral delivery systems for base editing are being deployed to transfect ECFCs. For the HDR, sgRNAs are currently being tested for efficiency.

Conclusion(s): Gene editing is a powerful tool for treating patients with varying forms of VWD and can be tailored to each individual. A larger screening of the population to identify common pathogenic mutations could open gene editing as wider and more accessible treatment.

This research received funding from the Netherlands Organization for Scientific Research (NWO), Domain Applied and Engineering Sciences (TTW), ‘Connecting Innovators’ Open Technology Programme, Project#18712