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

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

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Abstract Background Hemophilia A, a bleeding disorder resulting from F8 mutations, can only be cured by gene therapy.A promising strategy u11-200ps is CRISPR-Cas9-mediated precise insertion of F8 in hepatocytes at highly expressed gene loci, such as albumin (Alb).Unfortunately, the precise in vivo integration efficiency of a long insert is very low (~ 0.1%).

Results 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 find that the integration of a double-cut donor at the Alb voyage et cie discount code locus in mouse liver is mainly through non-homologous end joining (NHEJ)-mediated knock-in.We then target BDDF8 to multiple sites on introns 11 and 13 and find that NHEJ-mediated insertion of BDDF8 restores hemostasis.Finally, using 3 AAV8 vectors to deliver genome editing components, including Cas9, sgRNA, and BDDF8 donor, we observe the same therapeutic effects.

A follow-up of 100 mice over 1 year shows no adverse effects.Conclusions 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.