Hemophilia A ameliorated in mice by CRISPR-based in vivo genome editing of human Factor VIII.

Hemophilia A is a monogenic disease with a blood clotting factor VIII (FVIII) deficiency caused by mutation in the factor VIII (F8) gene. Current and emerging treatments such as FVIII protein injection and gene therapies via AAV-delivered F8 transgene in an episome are costly and nonpermanent. Here, we describe a CRISPR/Cas9-based in vivo genome editing method, combined with non-homologous end joining, enabling permanent chromosomal integration of a modified human B domain deleted-F8 (BDD-F8) at the albumin (Alb) locus in liver cells. To test the approach in mice, C57BL/6 mice received tail vein injections of two vectors, AAV8-SaCas9-gRNA, targeting Alb intron 13, and AAV8-BDD-F8. This resulted in BDD-F8 insertion at the Alb locus and FVIII protein expression in the liver of vector-, but not vehicle-, treated mice. Using this approach in hemophilic mice, BDD-F8 was expressed in liver cells as functional human FVIII, leading to increased plasma levels of FVIII and restoration of blood clotting properties in a dose-dependent manor for at least 7 months, with no detectable liver toxicity or meaningful off-target effects. Based on these findings, our BDD-F8 genome editing approach may offer an efficacious, long-term and safe treatment for patients with hemophilia A.