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

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

Author information

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.

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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