Genome Biology, 2019-12, Vol.20 (1), p.276-276, Article 276
2019
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- Autor(en) / Beteiligte
- Titel
- Curing hemophilia A by NHEJ-mediated ectopic F8 insertion in the mouse
- Ist Teil von
- Genome Biology, 2019-12, Vol.20 (1), p.276-276, Article 276
- Ort / Verlag
- England: BioMed Central
- Erscheinungsjahr
- 2019
- Quelle
- SpringerLink (Online service)
- Beschreibungen/Notizen
- 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%). 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. 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.
- Sprache
- Englisch
- Identifikatoren
- ISSN: 1474-760X, 1474-7596eISSN: 1474-760XDOI: 10.1186/s13059-019-1907-9Titel-ID: cdi_doaj_primary_oai_doaj_org_article_0175546634cc4b5da1856d314090ad7d
- Format
- –
- Schlagworte
- Albumins - genetics, Amino acids, Animals, Codon, Terminator, CRISPR, CRISPR-Cas9, Design, DNA End-Joining Repair, Efficiency, Factor VIII - genetics, Gene Knock-In Techniques, Gene therapy, Genetic Therapy - methods, Genome editing, Genomes, Hemophilia, Hemophilia A, Hemophilia A - therapy, Hemostasis, Hepatocytes, Integration, Introns, Knock-in, Liver, Mice, Mutation, NHEJ, Non-homologous end joining, Plasmids, Proteins, Vectors (Biology)