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In vivo cytidine base editing of hepatocytes without detectable off-target mutations in RNA and DNA

Nature Biomedical Engineering volume 5, pages 179–189 (2021)Cite this article

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Abstract

Base editors are RNA-programmable deaminases that enable precise single-base conversions in genomic DNA. However, off-target activity is a concern in the potential use of base editors to treat genetic diseases. Here, we report unbiased analyses of transcriptome-wide and genome-wide off-target modifications effected by cytidine base editors in the liver of mice with phenylketonuria. The intravenous delivery of intein-split cytidine base editors by dual adeno-associated viruses led to the repair of the disease-causing mutation without generating off-target mutations in the RNA and DNA of the hepatocytes. Moreover, the transient expression of a cytidine base editor mRNA and a relevant single-guide RNA intravenously delivered by lipid nanoparticles led to ~21% on-target editing and to the reversal of the disease phenotype; there were also no detectable transcriptome-wide and genome-wide off-target edits. Our findings support the feasibility of therapeutic cytidine base editing to treat genetic liver diseases.

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Fig. 1: Transcriptome-wide C-to-U editing analysis after AAV-mediated SaKKH–CBE3 treatment.
Fig. 2: Genome-wide off-target analysis after AAV-mediated SaKKH–CBE3 treatment.
Fig. 3: Correction of the disease locus of the Pahenu2 mouse model using LNP-mediated delivery of base-editing components.
Fig. 4: Transcriptome- and genome-wide off-target analysis after LNP-mediated SaKKH–CBE3 treatment.

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Data availability

The main data supporting the results in this study are available within the paper and its Supplementary Information. The raw and analysed datasets generated during the study are too large to be publicly shared, yet they are available for research purposes from the corresponding author on reasonable request. High-throughput data are publicly available under the following accession numbers: GSE148349 (Gene Expression Omnibus datasets for HTS and RNA-seq), PRJEB38134 (Sequence Read Archive dataset for WGS) and PRJEB40008 (fastq files).

Code availability

The scripts used to quantify on-target and off-target editing are available at https://github.com/HLindsay/Villiger_deaminase.

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Acknowledgements

We thank S. Iyer and J. K. Joung for sharing technical details about variant filtering for transcriptome off-target identification; H. Vatandaslar and S. Pantasis for their support with primary hepatocyte isolations and microscopy analysis; the members of the FGCZ for WGS and RNA-seq; H. M. Grisch-Chan for her help with l-Phe analysis; M. Tanner and N. Rimann for their support with animal work; and D. Lenggenhager for the evaluation of histological samples for pathologies. This work was supported by a Swiss National Science Foundation grant (no. 310030_185293, to G.S.), Swiss National Science Foundation Sinergia grant (no. 180257, to B.T.), the Swiss National Science Foundation grant (no. 205 321_169 612, to J.H.), the Novartis Foundation for Medical-Biological Research (no. 19A004, to D.W.) and a PHRT grant (no. 528, to G.S., M.S. and B.T.).

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Author notes

  1. Femke Ringnalda

    Present address: Princess Máxima Center for Pediatric Oncology, Utrecht, the Netherlands

  2. These authors contributed equally: Lukas Villiger, Tanja Rothgangl.

Authors and Affiliations

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

    Lukas Villiger, Tanja Rothgangl, Sharan Janjuha, Femke Ringnalda, Markus Stoffel & Gerald Schwank

  2. Institute for Pharmacology and Toxicology, University of Zurich, Zurich, Switzerland

    Lukas Villiger, Tanja Rothgangl, Dominik Witzigmann, Sharan Janjuha & Gerald Schwank

  3. Division of Pharmaceutical Technology, Department of Pharmaceutical Sciences, University of Basel, Basel, Switzerland

    Dominik Witzigmann

  4. Oncode Institute, Princess Máxima Center for Pediatric Oncology, Utrecht, the Netherlands

    Rurika Oka & Ruben van Boxtel

  5. Acuitas Therapeutics, Vancouver, British Columbia, Canada

    Paulo J. C. Lin, Mitchell B. Beattie & Ying K. Tam

  6. Functional Genomics Center Zurich, ETH Zurich/University of Zurich, Zurich, Switzerland

    Weihong Qi & Hubert Rehrauer

  7. Institute for Pharmaceutical Sciences, ETH Zurich, Zurich, Switzerland

    Christian Berk & Jonathan Hall

  8. Zurich Center for Integrative Human Physiology, Zurich, Switzerland

    Beat Thöny

  9. Neuroscience Center Zurich, Zurich, Switzerland

    Beat Thöny

  10. Division of Metabolism, University Children’s Hospital Zurich and Children’s Research Centre, Zurich, Switzerland

    Beat Thöny

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Contributions

L.V. and T.R. designed the study, performed experiments, analysed data and wrote the manuscript. D.W. contributed to the design of the study and edited the manuscript. P.J.C.L. and Y.K.T. developed, prepared and characterized LNPs, contributed to the design of the study and edited the manuscript. M.B.B. assisted with LNP formulation. C.B. and J.H. assisted with the design of sgRNA modifications. F.R. conducted in vitro transfection experiments. S.J. conducted HTS data analysis. W.Q. and H.R. analysed RNA-seq data. R.O. and R.v.B. performed WGS analysis. M.S. and B.T. provided reagents and conceptual advice. G.S. designed and supervised the research and wrote the manuscript. All of the authors approved the final version.

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Correspondence to Gerald Schwank.

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P.J.C.L., M.B.B. and Y.K.T. are employees of Acuitas Therapeutics.

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Villiger, L., Rothgangl, T., Witzigmann, D. et al. In vivo cytidine base editing of hepatocytes without detectable off-target mutations in RNA and DNA. Nat Biomed Eng 5, 179–189 (2021). https://doi.org/10.1038/s41551-020-00671-z

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