Abstract

CRISPR–Cas-based genome editing holds great promise for targeting genetic disorders, including inborn errors of hepatocyte metabolism. Precise correction of disease-causing mutations in adult tissues in vivo, however, is challenging. It requires repair of Cas9-induced double-stranded DNA (dsDNA) breaks by homology-directed mechanisms, which are highly inefficient in nondividing cells. Here we corrected the disease phenotype of adult phenylalanine hydroxylase (Pah)enu2 mice, a model for the human autosomal recessive liver disease phenylketonuria (PKU)1, using recently developed CRISPR–Cas-associated base editors2,3,4. These systems enable conversion of C∙G to T∙A base pairs and vice versa, independent of dsDNA break formation and homology-directed repair (HDR). We engineered and validated an intein-split base editor, which allows splitting of the fusion protein into two parts, thereby circumventing the limited cargo capacity of adeno-associated virus (AAV) vectors. Intravenous injection of AAV-base editor systems resulted in Pahenu2 gene correction rates that restored physiological blood phenylalanine (l-Phe) levels below 120 µmol/l [5]. We observed mRNA correction rates up to 63%, restoration of phenylalanine hydroxylase (PAH) enzyme activity, and reversion of the light fur phenotype in Pahenu2 mice. Our findings suggest that targeting genetic diseases in vivo using AAV-mediated delivery of base-editing agents is feasible, demonstrating potential for therapeutic application.

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Data availability

All requests for materials and data are promptly reviewed by the ETH Zurich to verify whether the request is subject to any intellectual property or confidentiality obligations. Any materials and data that can be shared will be released via a Material Transfer Agreement. High-throughput sequencing data can be found at EMBL-EBI Array Express (accession number: E-MTAB-7154).

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Acknowledgements

We thank J.-C. Paterna from the Viral Vector Facility of the Neuroscience Center Zurich for supplying vectors, the Functional Genomics Centre Zurich for sequencing in vivo samples, M. Rodriguez, A. Apladas, N. Rimann for their support with animal work, A. Garcete and I. Kleiber from the immunohistology laboratory for surgical research of the University Hospital Zurich for immunhistochemistry of liver samples. pJL-SaKKH-BE3 and pBK-YE1-BE3 were a gift from D. Liu (Addgene plasmid no. 85170 and no. 85174). WN10151 was a gift from E. Welker (Addgene plasmid no. 80441). pX601-AAV-CMV::NLS-SaCas9-NLS-3xHA-bGHpA;U6::BsaI-sgRNA was a gift from F. Zhang (Addgene plasmid no. 61591), pLenti CMV GFP Puro (658-5) was a gift from E. Campeau and P. Kaufman (Addgene plasmid no. 17448). pCMV-VSV-G was a gift from B. Weinberg (Addgene plasmid no. 8454) and psPAX2 was a gift from D. Trono (Addgene plasmid no. 12260). This work was funded by the SNSF (31003A_160230). L.V. holds an MD/PhD scholarship from the Swiss National Science Foundation.

Author information

Affiliations

  1. Department Biology, Institute for Molecular Health Sciences, ETH Zurich, Zurich, Switzerland

    • Lukas Villiger
    • , Femke Ringnalda
    •  & Gerald Schwank
  2. Division of Metabolism, University Children’s Hospital Zurich and Children’s Research Centre, Zurich, Switzerland

    • Hiu Man Grisch-Chan
    • , Gabriella Allegri
    • , Ralph Fingerhut
    •  & Beat Thöny
  3. SIB Swiss Institute of Bioinformatics, University of Zurich, Zurich, Switzerland

    • Helen Lindsay
    •  & Mark D. Robinson
  4. Institute of Molecular Life Sciences, University of Zurich, Zurich, Switzerland

    • Helen Lindsay
    • , Johannes Häberle
    •  & Mark D. Robinson
  5. Department of Biology, Institute of Biochemistry, ETH Zurich, Zurich, Switzerland

    • Chiara B. Pogliano
    •  & Joao Matos
  6. Zurich Center for Integrative Human Physiology, Zurich, Switzerland

    • Johannes Häberle
  7. Neuroscience Center Zurich, Zurich, Switzerland

    • Johannes Häberle
    •  & Beat Thöny
  8. Swiss Newborn Screening Laboratory, University Children’s Hospital, Zurich, Switzerland

    • Ralph Fingerhut

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Contributions

L.V. designed the research, performed experiments, analyzed data, and wrote the manuscript. F.R. conducted cryosections, fluorescent imaging, and western blot experiments and analysis thereof. J.M. and C.B.P performed western blot experiments. H.M.G.-C. and G.A. assisted with in vitro PAH assays and data analysis thereof. H.L. wrote all R scripts and assisted with HTS data analysis. R.F. analyzed blood l-Phe levels. B.T., M.D.R., J.H., and H.M.G.-C. assisted with the design of experiments G.S. designed and supervised the research and wrote the manuscript. All authors approved the final version.

Competing interests

The authors declare no competing interests.

Corresponding author

Correspondence to Gerald Schwank.

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DOI

https://doi.org/10.1038/s41591-018-0209-1