Letter

Therapeutic genome editing by combined viral and non-viral delivery of CRISPR system components in vivo

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Accepted:
Published online:

Abstract

The combination of Cas9, guide RNA and repair template DNA can induce precise gene editing and the correction of genetic diseases in adult mammals. However, clinical implementation of this technology requires safe and effective delivery of all of these components into the nuclei of the target tissue. Here, we combine lipid nanoparticle–mediated delivery of Cas9 mRNA with adeno-associated viruses encoding a sgRNA and a repair template to induce repair of a disease gene in adult animals. We applied our delivery strategy to a mouse model of human hereditary tyrosinemia and show that the treatment generated fumarylacetoacetate hydrolase (Fah)-positive hepatocytes by correcting the causative Fah-splicing mutation. Treatment rescued disease symptoms such as weight loss and liver damage. The efficiency of correction was >6% of hepatocytes after a single application, suggesting potential utility of Cas9-based therapeutic genome editing for a range of diseases.

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Acknowledgements

We thank M. Grompe, S. Levine, T. Jacks, P. Sharp, E. Sontheimer, C. Mello, P. Zamore, M. Moore, T. Flotte, T. Tammela, F. Sanchez-Rivera, T. Papagiannakopoulos, D. Wang, J. Moore and A. Vegas for discussions and for sharing reagents, S. Hough for technical assistance and K. Cormier for histology. This work is supported by grants from the National Institutes of Health (NIH), 5R00CA169512 and Worcester Foundation (to W.X.). H.Y. is supported by Skoltech Center and 5-U54-CA151884-04 (NIH Centers for Cancer Nanotechnology Excellence and the Harvard-MIT Center of Cancer Nanotechnology Excellence). Y.D. acknowledges support from the National Institute of Biomedical Imaging and Bioengineering for his postdoctoral fellowship 1F32EB017625. V.K. acknowledges support from the Russian scientific fund, grant number 14-34–00017. This work is supported in part by Cancer Center Support (core) grant P30-CA14051 from the NIH. We thank the Swanson Biotechnology Center for technical support. We thank C. Wang at Boston Children's Hospital Viral Core for AAV prep (supported by core grant 5P30EY012196-17). The authors acknowledge the service to the MIT community of the late S. Collier.

Author information

Affiliations

  1. David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.

    • Hao Yin
    • , Joseph R Dorkin
    • , Qiongqiong Wu
    • , Junghoon Yang
    • , Sneha Suresh
    • , Stephen Walsh
    • , Roman L Bogorad
    • , Robert Langer
    •  & Daniel G Anderson
  2. RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, Massachusetts, USA.

    • Chun-Qing Song
    • , Angela Park
    • , Aizhan Bizhanova
    •  & Wen Xue
  3. Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, Massachusetts, USA.

    • Chun-Qing Song
    • , Lihua J Zhu
    •  & Wen Xue
  4. Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.

    • Joseph R Dorkin
  5. Department of Molecular, Cell and Cancer Biology, University of Massachusetts Medical School, Worcester, Massachusetts, USA.

    • Lihua J Zhu
    • , Ankit Gupta
    • , Mehmet F Bolukbasi
    • , Scot A Wolfe
    •  & Wen Xue
  6. Program in Bioinformatics and Integrative Biology, University of Massachusetts Medical School, Worcester, Massachusetts, USA.

    • Lihua J Zhu
    •  & Zhiping Weng
  7. Department of Bioinformatics, School of Life Science and Technology, Tongji University, Shanghai, P.R. China.

    • Yingxiang Li
  8. Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, Massachusetts, USA.

    • Mehmet F Bolukbasi
    •  & Scot A Wolfe
  9. Gene Therapy Center, University of Massachusetts Medical School, Worcester, Massachusetts, USA.

    • Guangping Gao
  10. College of Pharmacy, the Ohio State University, Columbus, Ohio, USA.

    • Yizhou Dong
  11. Skolkovo Institute of Science and Technology, Skolkovo, Russia.

    • Victor Koteliansky
  12. Department of Chemistry, M.V. Lomonosov Moscow State University, Leninskie Gory, Russia.

    • Victor Koteliansky
  13. Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.

    • Robert Langer
    •  & Daniel G Anderson
  14. Harvard-MIT Division of Health Sciences & Technology, Cambridge, Massachusetts, USA.

    • Robert Langer
    •  & Daniel G Anderson
  15. Institute of Medical Engineering and Science, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.

    • Robert Langer
    •  & Daniel G Anderson

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Contributions

H.Y., W.X. and D.G.A. designed the study. H.Y. and W.X. directed the project. H.Y., C.-Q.S., J.R.D., L.J.Z., Y.L., Q.W., J.Y., S.S., A.B., A.G., M.F.B., A.P., S.W. and R.L.B. performed experiments and analyzed data. G.G., Z.W., Y.D., V.K., S.A.W. and R.L. provided reagents and conceptual advice. H.Y., W.X. and D.G.A. wrote the manuscript with comments from all authors.

Competing interests

D.G.A., H.Y., J.R.K. and W.X. have applied for patents on the subject matter of this paper. D.G.A. is a scientific co-founder of CRISPR Therapeutics.

Corresponding authors

Correspondence to Wen Xue or Daniel G Anderson.

Integrated supplementary information

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–8, Supplementary Tables 1–6 and Supplementary Sequences

  2. 2.

    Supplementary Table 7

    sgRNA2 GUIDE-seq +&- strand peaks

  3. 3.

    Supplementary Table 8

    sgRNA2 GUIDE-seq merged peaks

  4. 4.

    Supplementary Table 9

    Deep sequencing of off-target sites