In vivo genome editing restores haemostasis in a mouse model of haemophilia

• Published in: Nature (London) 2011-07, Vol.475 (7355), p.217-221
• Main Authors: Li, Hojun, Haurigot, Virginia, Doyon, Yannick, Li, Tianjian, Wong, Sunnie Y., Bhagwat, Anand S., Malani, Nirav, Anguela, Xavier M., Sharma, Rajiv, Ivanciu, Lacramiora, Murphy, Samuel L., Finn, Jonathan D., Khazi, Fayaz R., Zhou, Shangzhen, Paschon, David E., Rebar, Edward J., Bushman, Frederic D., Gregory, Philip D., Holmes, Michael C., High, Katherine A.
• Format: Article
• Language: English
• Subjects: 631/326/596/2561; 631/61/51/2315; 692/308; 692/699/1541; Animals; Base Sequence; Biological and medical sciences; Cell Line, Tumor; Disease Models, Animal; DNA Breaks, Double-Stranded; DNA Repair - genetics; Editing; Endonucleases - chemistry; Endonucleases - genetics; Endonucleases - metabolism; Exons - genetics; Factor IX - analysis; Factor IX - genetics; Gene expression; Gene Targeting - methods; Gene therapy; Genetic aspects; Genetic disorders; Genetic engineering; Genetic Therapy - methods; Genetic Vectors - genetics; Genome - genetics; Genomes; Health aspects; HEK293 Cells; Hematologic and hematopoietic diseases; Hemophilia; Hemophilia B - genetics; Hemophilia B - physiopathology; Hemostasis; Humanities and Social Sciences; Humans; Introns - genetics; letter; Liver - metabolism; Liver Regeneration; Medical sciences; Mice; Mice, Inbred C57BL; multidisciplinary; Mutation; Mutation - genetics; Phenotype; Physiological aspects; Platelet diseases and coagulopathies; Rodents; Science; Science (multidisciplinary); Sequence Homology; Stem cells; Zinc Fingers
• ISSN: 0028-0836; 1476-4687
• DOI: 10.1038/nature10177

Gene correction in a blood disorder Direct editing of disease-causing mutations has obvious attractions for the treatment of genetic disorders if the many practical obstacles to the technique can be overcome. One promising line of research centres on the development of zinc finger nucleases (ZFNs) produced by fusing an engineered zinc finger DNA-binding domain to an endonuclease. These artificial enzymes induce efficient gene correction in cultured cells. Li et al . now report that zinc finger nucleases induce double-strand breaks in specifically selected locations on the genome and stimulate genome editing at a clinically meaningful level in vivo . In a proof-of-principle experiment, ZFNs delivered to the liver in a mouse model of haemophilia B achieved a level of gene replacement that was sufficient to correct the clotting defect, and the effect persisted following liver regeneration. 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 1 . 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 2 , 3 , 4 , 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 vi