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

Lukas Villiger; Tanja Rothgangl; Dominik Witzigmann; Rurika Oka; Paulo J C Lin; Weihong Qi; Sharan Janjuha; Christian Berk; Femke Ringnalda; Mitchell B Beattie; Markus Stoffel; Beat Thöny; Jonathan Hall; Hubert Rehrauer; Ruben van Boxtel; Ying K Tam; Gerald Schwank

doi:10.1038/s41551-020-00671-z

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

Lukas Villiger, Tanja Rothgangl, Dominik Witzigmann, Rurika Oka, Paulo J C Lin, Weihong Qi, Sharan Janjuha, Christian Berk, Femke Ringnalda, Mitchell B Beattie, Markus Stoffel, Beat Thöny, Jonathan Hall, Hubert Rehrauer, Ruben van Boxtel, Ying K Tam, Gerald Schwank

Group van Boxtel

Research output: Contribution to journal › Article › peer-review

24 Citations (Scopus)

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.

Original language	English
Pages (from-to)	179-189
Number of pages	11
Journal	Nature biomedical engineering
Volume	5
Issue number	2
DOIs	https://doi.org/10.1038/s41551-020-00671-z
Publication status	Published - Feb 2021

Keywords

Adenoviridae/physiology
Animals
Cytidine/genetics
DNA/genetics
Gene Editing/methods
Genetic Vectors/physiology
HEK293 Cells
Hepatocytes/metabolism
Humans
Mice, Inbred C57BL
RNA/genetics

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Villiger, L., Rothgangl, T., Witzigmann, D., Oka, R., Lin, P. J. C., Qi, W., Janjuha, S., Berk, C., Ringnalda, F., Beattie, M. B., Stoffel, M., Thöny, B., Hall, J., Rehrauer, H., van Boxtel, R., Tam, Y. K., & Schwank, G. (2021). In vivo cytidine base editing of hepatocytes without detectable off-target mutations in RNA and DNA. Nature biomedical engineering, 5(2), 179-189. https://doi.org/10.1038/s41551-020-00671-z

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

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.",

keywords = "Adenoviridae/physiology, Animals, Cytidine/genetics, DNA/genetics, Gene Editing/methods, Genetic Vectors/physiology, HEK293 Cells, Hepatocytes/metabolism, Humans, Mice, Inbred C57BL, RNA/genetics",

author = "Lukas Villiger and Tanja Rothgangl and Dominik Witzigmann and Rurika Oka and Lin, {Paulo J C} and Weihong Qi and Sharan Janjuha and Christian Berk and Femke Ringnalda and Beattie, {Mitchell B} and Markus Stoffel and Beat Th{\"o}ny and Jonathan Hall and Hubert Rehrauer and {van Boxtel}, Ruben and Tam, {Ying K} and Gerald Schwank",

note = "Publisher Copyright: {\textcopyright} 2021, The Author(s), under exclusive licence to Springer Nature Limited.",

year = "2021",

month = feb,

doi = "10.1038/s41551-020-00671-z",

language = "English",

volume = "5",

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Villiger, L, Rothgangl, T, Witzigmann, D, Oka, R, Lin, PJC, Qi, W, Janjuha, S, Berk, C, Ringnalda, F, Beattie, MB, Stoffel, M, Thöny, B, Hall, J, Rehrauer, H, van Boxtel, R, Tam, YK & Schwank, G 2021, 'In vivo cytidine base editing of hepatocytes without detectable off-target mutations in RNA and DNA', Nature biomedical engineering, vol. 5, no. 2, pp. 179-189. https://doi.org/10.1038/s41551-020-00671-z

TY - JOUR

T1 - In vivo cytidine base editing of hepatocytes without detectable off-target mutations in RNA and DNA

AU - Villiger, Lukas

AU - Rothgangl, Tanja

AU - Witzigmann, Dominik

AU - Oka, Rurika

AU - Lin, Paulo J C

AU - Qi, Weihong

AU - Janjuha, Sharan

AU - Berk, Christian

AU - Ringnalda, Femke

AU - Beattie, Mitchell B

AU - Stoffel, Markus

AU - Thöny, Beat

AU - Hall, Jonathan

AU - Rehrauer, Hubert

AU - van Boxtel, Ruben

AU - Tam, Ying K

AU - Schwank, Gerald

PY - 2021/2

Y1 - 2021/2

N2 - 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.

AB - 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.

KW - Adenoviridae/physiology

KW - Animals

KW - Cytidine/genetics

KW - DNA/genetics

KW - Gene Editing/methods

KW - Genetic Vectors/physiology

KW - HEK293 Cells

KW - Hepatocytes/metabolism

KW - Humans

KW - Mice, Inbred C57BL

KW - RNA/genetics

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U2 - 10.1038/s41551-020-00671-z

DO - 10.1038/s41551-020-00671-z

M3 - Article

C2 - 33495639

VL - 5

SP - 179

EP - 189

JO - Nature biomedical engineering

JF - Nature biomedical engineering

SN - 2157-846X

IS - 2

ER -

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

Abstract

Keywords

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