Curing hemophilia A by NHEJ-mediated ectopic F8 insertion in mouse

Hemophilia A, a bleeding disorder resulting from F8 mutations, can only be cured by gene therapy. A promising strategy is CRISPR-Cas9-mediated precise insertion of F8 in hepatocytes at highly expressed gene loci, such as albumin. Unfortunately, the precise in vivo integration efficiency of a long insert is very low (~0.1%). Here we report that the use of a double-cut donor leads to a 10- to 20-fold increase in liver editing efficiency, thereby completely reconstituting serum F8 activity in a mouse model of hemophilia A after hydrodynamic injection of Cas9-sgAlb and B-domain-deleted (BDD) F8 donor plasmids. We found that the integration of double cut donor at Alb locus in mouse liver is mainly through nonhomologous end joining (NHEJ)-mediated knockin. We then targeted BDDF8 to multiple sites on introns 11 and 13 and found that NHEJ-mediated insertion of BDDF8 restores hemostasis. Finally, using three AAV8 vectors to deliver genome editing components, including Cas9, sgRNA, and BDDF8 donor, we observed the same therapeutic effects. A follow-up of 100 mice over one year showed no adverse effects. These findings lay the foundation for curing hemophilia A by NHEJ knock-in of BDDF8 at Alb introns after AAV-mediated delivery of editing components.