Self-assembled peptide–poloxamine nanoparticles enable in vitro and in vivo genome restoration for cystic fibrosis

Abstract

Developing safe and efficient non-viral delivery systems remains a major challenge for in vivo applications of gene therapy, especially in cystic fibrosis. Unlike conventional cationic polymers or lipids, the emerging poloxamine-based copolymers display promising in vivo gene delivery capabilities. However, poloxamines are invalid for in vitro applications and their in vivo transfection efficiency is still low compared with viral vectors. Here, we show that peptides developed by modular design approaches can spontaneously form compact and monodisperse nanoparticles with poloxamines and nucleic acids via self-assembly. Both messenger RNA and plasmid DNA expression mediated by peptide-poloxamine nanoparticles are greatly boosted in vitro and in the lungs of cystic fibrosis mice with negligible toxicity. Peptide–poloxamine nanoparticles containing integrating vectors enable successful in vitro and in vivo long-term restoration of cystic fibrosis transmembrane conductance regulator deficiency with a safe integration profile. Our dataset provides a new framework for designing non-viral gene delivery systems qualified for in vivo genetic modifications.

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Fig. 1: Schematic of the multi-modular peptide-based gene transfection platform used in this study.
Fig. 2: Characterization and mechanism studies of the ternary complex and binary counterpart.
Fig. 3: In vitro evaluation of the ternary complex.
Fig. 4: In vitro long-term exogenous gene expression mediated by ternary complexes.
Fig. 5: In vivo evaluation of the ternary complex.
Fig. 6: In vivo long-term transgene expression mediated by ternary complexes.

Data availability

All of the primary data that support the findings of this study are available from the corresponding author on request.

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Acknowledgements

The authors are grateful to G. Vassaux (University of Nice Sophia Antipolis) and B. Pitard (University of Nantes) for introducing and providing poloxamine 704, and to S. Hyde (University of Oxford) for providing a CpG-depleted CFTR expression cassette. Special thanks go to S. L. Hart (University College London) for developing targeting elements showing high affinity to human airway epithelial cells. We also thank J. Geiger (Ethris) for providing SNIM-mRNA, M. K. Aneja (Ethris) for careful proofreading of the manuscript, Y. Liu, J. Rejman and J. Zhang (Ludwig Maximilian University of Munich) for technical support, and J. Tang (University of Queensland) for assistance with the Prism software. This work was supported by the Bundesministerium für Bildung und Forschung under project number 01GM1106A.

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Contributions

S. Guan and J.R. conceived and developed the delivery system. C.R. and Z.I. generated the SB transposon system. A.M., G.H., S.H., W.G. and S. Guan designed and performed the animal experiments. S. Guan, L.Z. and A.S. characterized the nanoparticles. S. Guan, A.S. and S.M.J. performed the in vitro experiments. A.M., S.H., P.B., S. Glage, S.L., F.S. and C.M. performed the in vivo endpoint studies. Y.K. was responsible for the bioinformatics and integration sites analysis. B.T., C.R., Z.I. and J.R. designed and supervised the research. S. Guan and J.R. wrote the manuscript with comments from all authors.

Corresponding author

Correspondence to Joseph Rosenecker.

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Competing interests

S. Guan and J.R. have filed patent applications on the subject matter related to this study. C.R. is a scientific co-founder and chief executive officer of the biotechnology company Ethris, which focuses on the development of mRNA therapies. G.H. is an employee of Ethris. Y.K. is an employee of Eurofins GATC Biotech. The other authors declare no competing interests.

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Guan, S., Munder, A., Hedtfeld, S. et al. Self-assembled peptide–poloxamine nanoparticles enable in vitro and in vivo genome restoration for cystic fibrosis. Nat. Nanotechnol. 14, 287–297 (2019). https://doi.org/10.1038/s41565-018-0358-x

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