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Restoration of dystrophin expression in mice by suppressing a nonsense mutation through the incorporation of unnatural amino acids

Abstract

Approximately 11% of monogenic diseases involve nonsense mutations that are caused by premature termination codons. These codons can in principle be read-through via the site-specific incorporation of unnatural amino acids to generate full-length proteins with minimal loss of function. Here we report that aminoacyl-tRNA-synthase–tRNA pairs specific for the desired unnatural amino acids can be used to read through a nonsense mutation in the dystrophin gene. We show partial restoration of dystrophin expression in differentiated primary myoblasts (from a mdx mouse model and a patient with Duchenne muscular dystrophy), and restoration of muscle function in two mouse models: mdx mice, via viral delivery of the engineered tRNA-synthase–tRNA pair intraperitoneally or intramuscularly and of the associated unnatural amino acid intraperitoneally; and mice produced by crossing mdx mice and transgenic mice with a chromosomally integrated pair, via intraperitoneal delivery of the unnatural amino acid. The incorporation of unnatural amino acids to restore endogenous protein expression could be explored for therapeutic use.

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Fig. 1: Engineered PylRS–tRNAPyl system to read-through endogenous PTCs for restoration of dystrophin expression in vitro.
Fig. 2: Dystrophin restoration in differentiated myoblasts derived from a mdx mouse and a patient with DMD after PylRS–tRNAPyl transfection.
Fig. 3: Dystrophin restoration in mdx mouse model after plasmid electroporation in vivo.
Fig. 4: Validation of the \({\rm{PylRS}}-{{\rm{tRNA}}_{{\rm{UUA}}}^{{\rm{Pyl}}}}\) system to restore endogenous dystrophin in trans/mdx mice.
Fig. 5: Evaluation of the in vivo safety of the PylRS–tRNAPyl system.
Fig. 6: In vivo restoration of dystrophin by AAV delivery of the \({\rm{PylRS}}-{{\rm{tRNA}}_{{\rm{UUA}}}^{{\rm{Pyl}}}}\) system.

Data availability

The main data supporting the findings of this study are available within this paper and its Supplementary Information. Transcriptomics data of different mouse tissues are available in the Gene Expression Omnibus (GEO) repository, with accession code GSE158961. The UAA incorporation sites were based on reported DMD cases from the Human Gene Mutation Database (http://www.hgmd.cf.ac.uk/ac/index.php). Source data are provided with this paper.

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Acknowledgements

This work was supported by the National Science and Technology Major Projects for Major New Drugs Innovation and Development (grant numbers 2018ZX09711003-001-003 and 2018ZX09J18114) of China.

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Contributions

Q.Y. and Q.X. conceived the idea and designed the experiments. Q.Y. and N.S. performed the experiments and offered the original images. H.Z. and N.S. organized the results, analysed the data, prepared the figures and wrote the manuscript. H.L. and X.Y. edited the manuscript and gave technical advice. All other authors participated in some of the experiments, results or discussion. Q.X. supervised the study.

Corresponding author

Correspondence to Qing Xia.

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Competing interests

Q.Y. and Q.X. are listed as co-inventors on patents regarding the PylRS–tRNA system for dystrophin read-through and DMD therapy (PCT/CN2017/075577 and CN 2016/10134657.6). The other authors declare no competing interests.

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Shi, N., Yang, Q., Zhang, H. et al. Restoration of dystrophin expression in mice by suppressing a nonsense mutation through the incorporation of unnatural amino acids. Nat Biomed Eng 6, 195–206 (2022). https://doi.org/10.1038/s41551-021-00774-1

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