Allele-selective Disruption of Pathogenic VWF Variants in Type 2 Von Willebrand Disease using CRISPR/Cas9 Open Access

• Targeting CRISPR/Cas9 to the common SNP rs1800378 allele-selectively disrupts VWF alleles.

• Disruption of pathogenic VWF alleles in heterozygous VWD patient-ECFCs leads to a healthy VWF phenotype in vitro.

In contrast to major innovations in treating severe hemophilia, treatment of severe von Willebrand disease (VWD) remains limited to intravenous infusion of von Willebrand factor (VWF) concentrates. To date, no gene therapy-based approaches for treatment of VWD have been developed, largely due to the disease's heterogeneous mutational landscape and the challenge of specifically targeting VWF production in endothelial cells. In this study we developed a novel gene therapy strategy for patients suffering from VWD caused by heterozygous dominant-negative VWF variants. Our strategy permanently inactivates VWF variants by selectively disrupting the pathogenic allele's open reading frame (ORF) via introduction of indels by Cas9. To circumvent the challenge of designing variant-specific strategies, we targeted the common SNP rs1800378 in VWF. We used endothelial colony forming cells (ECFCs) from VWD2A and VWD2B patients with heterozygous p.C1190R and p.R1306W variants, respectively, to demonstrate ex vivo proof-of-principle. Using NGS analysis we show efficient and allele-selective knock-out of VWF, while maintaining VWF expression of the non-targeted allele. Variant mapping mass spectrometry that discriminates between wild type and variant VWF proteoforms confirmed selective reduction of variant allele expression, which was accompanied by reversal of cellular disease phenotypes in ECFCs. This study shows the feasibility of a novel gene editing strategy for VWD that, by virtue of its targeting of a common SNP, can be broadly applicable and can be used to design treatments for VWD without being constrained by the disease-causing variant, pathogenic mechanism, or VWD subtype.