PB0274 - Re-establishing von Willebrand Factor production in endothelial cells derived from von Willebrand Disease type 3 canine models through CRISPR-Cas9 mediated gene editing

Sunday, June 23, 2024

13:45 – 14:45 ICT

Room: Exhibition Hall

Presenting Author(s)

Alastair Barraclough, MSc

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

Co-Author(s)

TN

Timothy Nichols

Director of the Francis Owen Blood Research Laboratory
Department of Pathology and Laboratory Medicine and Blood Research Center, University of North Carolina School of Medicine, Chapel Hill, NC USA.
Chapel Hill, North Carolina, United States
EM

Elizabeth Merricks

Research Specialist
Department of Pathology and Laboratory Medicine and Blood Research Center, University of North Carolina School of Medicine, Chapel Hill, NC USA.
Chapel Hill, North Carolina, United States
Karen Vanhoorelbeke

Full professor
Laboratory for Thrombosis Research, IRF Life Sciences, KU Leuven Campus Kulak Kortrijk, Kortrijk, Belgium
Kortrijk, West-Vlaanderen, Belgium
SD

Simon F. De Meyer

Full professor
Laboratory for Thrombosis Research, IRF Life Sciences, KU Leuven Campus Kulak Kortrijk, Kortrijk, Belgium
Kortrijk, West-Vlaanderen, Belgium
Frank W.G Leebeek, MD PhD

Internist-hematologist, Professor
Department of Hematology, Erasmus University Medical Center, Rotterdam, The Netherlands
Rotterdam, Zuid-Holland, Netherlands
Jeroen Eikenboom

MD, PhD, prof
Department of Thrombosis and Hemostasis, Leiden University Medical Center, Leiden, The Netherlands
Leiden, Zuid-Holland, Netherlands
KF

Karin Fijnvandraat

Professor
Emma Kinderziekenhuis/AMC, University of Amsterdam, Netherlands
Amsterdam, Noord-Holland, Netherlands
Ruben Bierings, PhD

Associate Professor
Hematology, Erasmus MC University Medical Center, 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

Background: Von Willebrand Disease (VWD) is the most commonly inherited bleeding disorder. Type 3 VWD is the most severe, as patients do not produce VWF; subsequently affecting their haemostatic capabilities to prevent bleeding. Gene therapies have been emerging as powerful alternative treatments for numerous diseases, and due to the robust nature of CRISPR-Cas9 based therapies; these can be deployed to treat various forms of VWD, including type 3. Presented here are two VWD type 3 canine derived endothelial cell (EC) models which can have healthy VWF restored via various CRISPR-Cas9 mediated gene correction technologies.

Aims: To restore the healthy VWF genotype in different VWD type 3 canine ECs and determine if functional VWF can be produced.

Methods: For a donor splice model which harbours a premature termination codon (PTC), adenine base editing is being utilised to repair the canonical splice site. For a model with a single bp deletion resulting in a frameshift and PTC, a WT-Cas9 is being delivered to cells in a Ribonucleoprotein (RNP) format in combination with an exogenous single-stranded oligodeoxynucleotide (ssODN) template encoding the desired corrections as a means of homology directed repair (HDR).

Results: Adenine base editing has been optimised in human ECFCs through a lentiviral based system; with editing efficiencies ranging from 40-90%. Initial experiments have transitioned this system into canine cells where multiple gRNAs are being trialled to determine A>G conversion efficacy.
Initial HDR experiments yielded approximately 50% of cells producing VWF (NGS and immunostaining), but this cannot be entirely attributed to HDR. Current efforts are underway to distinguish between non-homologous-end-joining (NHEJ) DNA repair and HDR through a re-designed donor with additional silent modifications.

Conclusion(s): CRISPR-Cas9 technologies can be efficiently delivered to ECs and tailored to specific mutations; further screenings are required to determine feasibility across a larger panel of mutations; and if common mutations can be identified.