title: In vivo genome editing restores haemostasis in a mouse model of haemophilia.
abstract: Editing of the human genome to correct disease-causing mutations is a promising approach for the treatment of genetic disorders. Genome editing improves on simple gene-replacement strategies by effecting in situ correction of a mutant gene, thus restoring normal gene function under the control of endogenous regulatory elements and reducing risks associated with random insertion into the genome. Gene-specific targeting has historically been limited to mouse embryonic stem cells. The development of zinc finger nucleases (ZFNs) has permitted efficient genome editing in transformed and primary cells that were previously thought to be intractable to such genetic manipulation. In vitro, ZFNs have been shown to promote efficient genome editing via homology-directed repair by inducing a site-specific double-strand break (DSB) at a target locus, but it is unclear whether ZFNs can induce DSBs and stimulate genome editing at a clinically meaningful level in vivo. Here we show that ZFNs are able to induce DSBs efficiently when delivered directly to mouse liver and that, when co-delivered with an appropriately designed gene-targeting vector, they can stimulate gene replacement through both homology-directed and homology-independent targeted gene insertion at the ZFN-specified locus. The level of gene targeting achieved was sufficient to correct the prolonged clotting times in a mouse model of haemophilia B, and remained persistent after induced liver regeneration. Thus, ZFN-driven gene correction can be achieved in vivo, raising the possibility of genome editing as a viable strategy for the treatment of genetic disease.
authors.affiliation: Division of Hematology, CTRB 5000, Children's Hospital of Philadelphia, 3501 Civic Center Boulevard, Philadelphia, Pennsylvania 19104, USA.
authors.forename: Hojun
authors.initials: H
authors.lastname: Li
authors.forename: Virginia
authors.initials: V
authors.lastname: Haurigot
authors.forename: Yannick
authors.initials: Y
authors.lastname: Doyon
authors.forename: Tianjian
authors.initials: T
authors.lastname: Li
authors.forename: Sunnie Y
authors.initials: SY
authors.lastname: Wong
authors.forename: Anand S
authors.initials: AS
authors.lastname: Bhagwat
authors.forename: Nirav
authors.initials: N
authors.lastname: Malani
authors.forename: Xavier M
authors.initials: XM
authors.lastname: Anguela
authors.forename: Rajiv
authors.initials: R
authors.lastname: Sharma
authors.forename: Lacramiora
authors.initials: L
authors.lastname: Ivanciu
authors.forename: Samuel L
authors.initials: SL
authors.lastname: Murphy
authors.forename: Jonathan D
authors.initials: JD
authors.lastname: Finn
authors.forename: Fayaz R
authors.initials: FR
authors.lastname: Khazi
authors.forename: Shangzhen
authors.initials: S
authors.lastname: Zhou
authors.forename: David E
authors.initials: DE
authors.lastname: Paschon
authors.forename: Edward J
authors.initials: EJ
authors.lastname: Rebar
authors.forename: Frederic D
authors.initials: FD
authors.lastname: Bushman
authors.forename: Philip D
authors.initials: PD
authors.lastname: Gregory
authors.forename: Michael C
authors.initials: MC
authors.lastname: Holmes
authors.forename: Katherine A
authors.initials: KA
authors.lastname: High
mesh_terms: D000818:Animals; D001483:Base Sequence; D045744:Cell Line, Tumor; D053903:DNA Breaks, Double-Stranded; D004260:DNA Repair / Q000235:genetics*; D004195:Disease Models, Animal; D004720:Endonucleases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D005091:Exons / Q000235:genetics; D005164:Factor IX / Q000032:analysis / Q000235:genetics; D018390:Gene Targeting / Q000379:methods*; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics; D016678:Genome / Q000235:genetics*; D057809:HEK293 Cells; D002836:Hemophilia B / Q000235:genetics* / Q000503:physiopathology; D006487:Hemostasis; D006801:Humans; D007438:Introns / Q000235:genetics; D008099:Liver / Q000378:metabolism; D008115:Liver Regeneration; D051379:Mice; D008810:Mice, Inbred C57BL; D009154:Mutation / Q000235:genetics; D010641:Phenotype; D017385:Sequence Homology; D016335:Zinc Fingers
chemical_list: D005164:Factor IX; D004720:Endonucleases