| First Author | Zhang JP | Year | 2019 |
| Journal | Genome Biol | Volume | 20 |
| Issue | 1 | Pages | 276 |
| PubMed ID | 31843008 | Mgi Jnum | J:285277 |
| Mgi Id | MGI:6393218 | Doi | 10.1186/s13059-019-1907-9 |
| Citation | Zhang JP, et al. (2019) Curing hemophilia A by NHEJ-mediated ectopic F8 insertion in the mouse. Genome Biol 20(1):276 |
| abstractText | BACKGROUND: 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 (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 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. |
