Allele-selective disruption of pathogenic VWF variants in type 2 von Willebrand disease using CRISPR/Cas9.

In contrast to major innovations in treating severe hemophilia, the 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 the treatment of VWD have been developed, largely owing 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 with VWD caused by heterozygous dominant-negative VWF variants. Our strategy permanently inactivates VWF variants by selectively disrupting the pathogenic allele's open reading frame via the introduction of indels by Cas9. To circumvent the challenge of designing variant-specific strategies, we targeted the common single nucleotide polymorphism (SNP) rs1800378 in VWF. We used endothelial colony-forming cells (ECFCs) from patients with VWD2A and VWD2B with heterozygous p.C1190R and p.R1306W variants, respectively, to demonstrate ex vivo proof of principle. Using next-generation sequencing analysis, we show efficient and allele-selective knockout of VWF, while maintaining VWF expression of the nontargeted 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.