Article | Published:

Meganuclease targeting of PCSK9 in macaque liver leads to stable reduction in serum cholesterol

Nature Biotechnology volume 36, pages 717725 (2018) | Download Citation

Abstract

Clinical translation of in vivo genome editing to treat human genetic diseases requires thorough preclinical studies in relevant animal models to assess safety and efficacy. A promising approach to treat hypercholesterolemia is inactivating the secreted protein PCSK9, an antagonist of the LDL receptor. Here we show that single infusions in six non-human primates of adeno-associated virus vector expressing an engineered meganuclease targeting PCSK9 results in dose-dependent disruption of PCSK9 in liver, as well as a stable reduction in circulating PCSK9 and serum cholesterol. Animals experienced transient, asymptomatic elevations of serum transaminases owing to the formation of T cells against the transgene product. Vector DNA and meganuclease expression declined rapidly, leaving stable populations of genome-edited hepatocytes. A second-generation PCSK9-specific meganuclease showed reduced off-target cleavage. These studies demonstrate efficient, physiologically relevant in vivo editing in non-human primates, and highlight safety considerations for clinical translation.

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Acknowledgements

We thank the Penn Vector Core for supplying AAV vectors, the Program in Comparative Medicine for animal care and procedures, the Nucleic Acid Technologies Core for assistance with deep sequencing, the Immunology Core for assistance with immunology assays, the CHOP Human Pluripotent Stem Cell Core for providing CHOPWT4, H. Zhang for technical assistance, Y. Zhu for assistance on histology analyses, and C. Lee for WebLogo generation. We also thank J. Stewart for editorial assistance with the manuscript. This work was supported by Penn Medicine and Precision Biosciences.

Author information

Affiliations

  1. Gene Therapy Program, Department of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.

    • Lili Wang
    • , Camilo Breton
    • , Peter Clark
    • , Jia Zhang
    • , Lei Ying
    • , Yan Che
    • , Peter Bell
    • , Roberto Calcedo
    • , Elizabeth L Buza
    • , Alexei Saveliev
    • , Zhenning He
    • , John White
    •  & James M Wilson
  2. Precision BioSciences, Durham, North Carolina, USA.

    • Jeff Smith
    • , Janel Lape
    • , Victor V Bartsevich
    •  & Derek Jantz
  3. Department of Biostatistics and Epidemiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.

    • Mingyao Li

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Contributions

L.W., D.J., and J.M.W. conceived this study. L.W., J.S., C.B., J.Z., L.Y., R.C., A.S., and J.M.W. designed the experiments. C.B., L.Y., J. L., P.B., E.L.B., A.S., V.V.B., Z.H., and J.W. performed the experiments. P.C., Y.C., J.L., A.S., and M.L. conducted the bioinformatics analysis of the deep sequencing data. M.L. conducted statistical analyses. J.M.W., L.W., C.B., P.C., J.Z., L.Y., M.L., P.B., R.C., and D.J. wrote and edited the manuscript.

Competing interests

J.M.W. is an advisor to, holds equity in, and has a sponsored research agreement with REGENXBIO; he also has a sponsored research agreement with Ultragenyx, Biogen, and Janssen, which are licensees of Penn technology. In addition, he has sponsored research agreements with Precision Biosciences and Moderna Therapeutics. J.M.W. holds equity in Solid Bio. J.M.W. and L.W. are inventors on patents that have been licensed to various biopharmaceutical companies. J.S., J.L., V.V.B., and D.J. are employees of, and hold equity in, Precision Biosciences.

Corresponding author

Correspondence to James M Wilson.

Integrated supplementary information

Supplementary information

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  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–9

  2. 2.

    Life Sciences Reporting Summary

  3. 3.

    Supplemental Tables

    Supplementary Tables 1–4

Excel files

  1. 1.

    Supplementary Dataset 1

    Potential M1PCSK9- or M2PCSK9-mediated off-target sites in the rhesus macaque genome identified by GUIDE-seq in LLCMK2 cells. N = 5 experiments for each nuclease.

  2. 2.

    Supplementary Dataset 2

    Potential M2PCSK9-mediated off-target sites in the human genome identified by GUIDE-seq in iPSC-derived hepatocytes. N = 3 experiment conditions.

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DOI

https://doi.org/10.1038/nbt.4182