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


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 manipulation1. 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 locus2,3,4, 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 viable strategy for the treatment of genetic disease.

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Figure 1: F9 ZFNs cleave human F9 intron 1 and induce homology-directed repair in vitro.
Figure 2: AAV8-mediated delivery of F9 ZFNs to h F9 mut mouse liver results in cleavage of h F9 mut intron 1 in vivo.
Figure 3: F9 ZFNs promote AAV-mediated targeting of wild-type F9 exons 2–8 to h F9 mut intron 1 in vivo.
Figure 4: In vivo h F9 mut gene correction results in stable circulating factor IX.
Figure 5: Hepatic h F9 mut gene correction results in phenotypic correction of haemophilia B.

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  1. Urnov, F. D., Rebar, E. J., Holmes, M. C., Zhang, H. S. & Gregory, P. D. Genome editing with engineered zinc finger nucleases. Nature Rev. Genet. 11, 636–646 (2010)

    Article  CAS  Google Scholar 

  2. Urnov, F. D. et al. Highly efficient endogenous human gene correction using designed zinc-finger nucleases. Nature 435, 646–651 (2005)

    Article  ADS  CAS  Google Scholar 

  3. Porteus, M. H. & Baltimore, D. Chimeric nucleases stimulate gene targeting in human cells. Science 300, 763 (2003)

    Article  Google Scholar 

  4. Bibikova, M. et al. Stimulation of homologous recombination through targeted cleavage by chimeric nucleases. Mol. Cell. Biol. 21, 289–297 (2001)

    Article  CAS  Google Scholar 

  5. Cartier, N. et al. Hematopoietic stem cell gene therapy with a lentiviral vector in X-linked adrenoleukodystrophy. Science 326, 818–823 (2009)

    Article  ADS  CAS  Google Scholar 

  6. Aiuti, A. et al. Gene therapy for immunodeficiency due to adenosine deaminase deficiency. N. Engl. J. Med. 360, 447–458 (2009)

    Article  CAS  Google Scholar 

  7. Cideciyan, A. V. et al. Human gene therapy for RPE65 isomerase deficiency activates the retinoid cycle of vision but with slow rod kinetics. Proc. Natl Acad. Sci. USA 105, 15112–15117 (2008)

    Article  ADS  CAS  Google Scholar 

  8. Maguire, A. M. et al. Safety and efficacy of gene transfer for Leber’s congenital amaurosis. N. Engl. J. Med. 358, 2240–2248 (2008)

    Article  CAS  Google Scholar 

  9. Bainbridge, J. W. et al. Effect of gene therapy on visual function in Leber’s congenital amaurosis. N. Engl. J. Med. 358, 2231–2239 (2008)

    Article  CAS  Google Scholar 

  10. Cavazzana-Calvo, M. et al. Transfusion independence and HMGA2 activation after gene therapy of human β-thalassaemia. Nature 467, 318–322 (2010)

    Article  ADS  CAS  Google Scholar 

  11. Howe, S. J. et al. Insertional mutagenesis combined with acquired somatic mutations causes leukemogenesis following gene therapy of SCID-X1 patients. J. Clin. Invest. 118, 3143–3150 (2008)

    Article  CAS  Google Scholar 

  12. Hacein-Bey-Abina, S. et al. LMO2-associated clonal T cell proliferation in two patients after gene therapy for SCID-X1. Science 302, 415–419 (2003)

    Article  ADS  CAS  Google Scholar 

  13. Hanna, J. et al. Treatment of sickle cell anemia mouse model with iPS cells generated from autologous skin. Science 318, 1920–1923 (2007)

    Article  ADS  CAS  Google Scholar 

  14. Pollak, E. S. & High, K. A. in The Metabolic and Molecular Bases of Inherited Disease (eds Scriver, C. R., Beaudet, A. L., Valle, D. & Sly, W. S. ) 4393–4413 (McGraw-Hill, 2001)

  15. Green, P. Haemophilia B Mutation Database (2004)

    Google Scholar 

  16. Moehle, E. A. et al. Targeted gene addition into a specified location in the human genome using designed zinc finger nucleases. Proc. Natl Acad. Sci. USA 104, 3055–3060 (2007)

    Article  ADS  CAS  Google Scholar 

  17. Manno, C. S. et al. Successful transduction of liver in hemophilia by AAV-Factor IX and limitations imposed by the host immune response. Nature Med. 12, 342–347 (2006)

    Article  CAS  Google Scholar 

  18. Miao, C. H. et al. Inclusion of the hepatic locus control region, an intron, and untranslated region increases and stabilizes hepatic factor IX gene expression in vivo but not in vitro . Mol. Ther. 1, 522–532 (2000)

    Article  CAS  Google Scholar 

  19. Thompson, A. R. & Chen, S. H. Germ line origins of de novo mutations in hemophilia B families. Hum. Genet. 94, 299–302 (1994)

    Article  CAS  Google Scholar 

  20. Zambrowicz, B. P. et al. Disruption of overlapping transcripts in the ROSA βgeo 26 gene trap strain leads to widespread expression of β-galactosidase in mouse embryos and hematopoietic cells. Proc. Natl Acad. Sci. USA 94, 3789–3794 (1997)

    Article  ADS  CAS  Google Scholar 

  21. Lin, H. F., Maeda, N., Smithies, O., Straight, D. L. & Stafford, D. W. A coagulation factor IX-deficient mouse model for human hemophilia B. Blood 90, 3962–3966 (1997)

    CAS  PubMed  Google Scholar 

  22. Perez, E. E. et al. Establishment of HIV-1 resistance in CD4+ T cells by genome editing using zinc-finger nucleases. Nature Biotechnol. 26, 808–816 (2008)

    Article  CAS  Google Scholar 

  23. Nakai, H. et al. Extrachromosomal recombinant adeno-associated virus vector genomes are primarily responsible for stable liver transduction in vivo . J. Virol. 75, 6969–6976 (2001)

    Article  CAS  Google Scholar 

  24. Li, H. et al. Assessing the potential for AAV vector genotoxicity in a murine model. Blood 117, 3311–3319 (2011)

    Article  CAS  Google Scholar 

  25. Nakai, H. et al. AAV serotype 2 vectors preferentially integrate into active genes in mice. Nature Genet. 34, 297–302 (2003)

    Article  CAS  Google Scholar 

  26. Miller, D. G., Petek, L. M. & Russell, D. W. Adeno-associated virus vectors integrate at chromosome breakage sites. Nature Genet. 36, 767–773 (2004)

    Article  CAS  Google Scholar 

  27. Doyon, Y. et al. Heritable targeted gene disruption in zebrafish using designed zinc-finger nucleases. Nature Biotechnol. 26, 702–708 (2008)

    Article  CAS  Google Scholar 

  28. Ayuso, E. et al. High AAV vector purity results in serotype- and tissue-independent enhancement of transduction efficiency. Gene Ther. 17, 503–510 (2010)

    Article  CAS  Google Scholar 

  29. Mitchell, C. & Willenbring, H. A reproducible and well-tolerated method for 2/3 partial hepatectomy in mice. Nature Protocols 3, 1167–1170 (2008)

    Article  CAS  Google Scholar 

  30. Berry, C., Hannenhalli, S., Leipzig, J. & Bushman, F. D. Selection of target sites for mobile DNA integration in the human genome. PLoS Comput. Biol. 2, e157 (2006)

    Article  ADS  Google Scholar 

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This work was funded by the National Institutes of Health and the Howard Hughes Medical Institute.

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Authors and Affiliations



H.L., V.H., Y.D., T.L., S.L.M., P.D.G., M.C.H. and K.A.H. designed the experiments. H.L., V.H., Y.D., T.L., S.Y.W., A.S.B., N.M., X.M.A., R.S., L.I., S.L.M., J.D.F., F.R.K., S.Z., D.E.P. and E.J.R. generated reagents and performed the experiments. H.L., Y.D., F.D.B., P.D.G., M.C.H. and K.A.H. wrote and edited the manuscript.

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Correspondence to Michael C. Holmes or Katherine A. High.

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Competing interests

Y.D., T.L., S.Y.W., D.E.P., E.J.R., P.D.G. and M.C.H. were all employees of Sangamo Biosciences when involved in this work. K.A.H. holds patents related to AAV vectors in the treatment of haemophilia.

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Li, H., Haurigot, V., Doyon, Y. et al. In vivo genome editing restores haemostasis in a mouse model of haemophilia. Nature 475, 217–221 (2011).

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