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  • Brief Communication
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Complete substitution with modified nucleotides in self-amplifying RNA suppresses the interferon response and increases potency

Abstract

The use of modified nucleotides to suppress the interferon response and maintain translation of self-amplifying RNA (saRNA), which has been achieved for mRNA, has not yet succeeded. We identify modified nucleotides that, when substituted at 100% in saRNA, confer innate immune evasion and robust long-term protein expression, and when formulated as a vaccine, protect against lethal SARS-CoV-2 challenge in mice. This discovery advances saRNA therapeutics by enabling prolonged protein expression at low doses.

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Fig. 1: Identification of modified nucleotides compatible with saRNA and their in vitro bioactivity.
Fig. 2: Development and characterization of a fully modified saRNA vaccine against SARS-CoV-2.

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Data availability

Antigen sequences used in the current study are accessible from NCBI. The NCBI reference sequence for the influenza A hemagglutinin (Influenza A virus (A/California/07/2009(H1N1))) is available at YP_009118626.1, and the NCBI reference sequence for the SARS-CoV-2 spike protein with K986P-stabilizing and V987P-stabilizing mutations (Wuhan-Hu-1) is available at YP_009724390.1. The source data supporting the findings of the study are available with the manuscript as a supplementary file.

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Acknowledgements

W.W.W. acknowledges support from the National Institutes of Health (NIH) (U01CA265713, R01EB029483, R01DK132576 and R01EB031904). M.W.G. acknowledges support from the NIH (U01CA265713) and the William Fairfield Warren Professorship. F.D. acknowledges support from a Boston University startup fund, a Peter Paul Career Development Professorship and a National Institute of Allergy and Infectious Diseases K22 transition award (K22AI144050). D.K. acknowledges support from the NIH T32 Immunology Training Program (T32AI007309). We thank the IVIS Imaging Core Facility (1S10RR024523-01), the NEIDL animal core and the NEIDL operations staff for their outstanding support. T.S. acknowledges support from a Lung Cancer Research Fellowship. J.E.M. acknowledges support from the National Science Foundation (NSF) Graduate Research Fellowship Program (GRFP). J.E.M. and J.R.K. were supported by the NIH T32 Translational Research in Biomaterials Training Program (T32EB006359). This material is based upon work supported by the NSF GRFP (2234657). Any opinions, findings and conclusions or recommendations expressed in this material are those of the authors and do not necessarily reflect the views of the NSF. This research was supported in part by an NIH training grant at Boston University (T32EB006359). The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH. Fig. 2 was partially generated with BioRender.com. Source data are provided with this paper.

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J.E.M., J.R.K., M.W.G. and W.W.W. conceptualized the study. J.E.M. and J.R.K. designed and assembled RNA constructs and conducted in vitro experiments. D.K. and E.C.C. performed in vivo experiments involving the SARS-CoV-2 vaccine. F.C. performed studies characterizing vaccine immunogenicity. T.-Y.S. and J.E.M. performed in vivo bioluminescent imaging. J.E.M., J.R.K., M.W.G., W.W.W. and F.D. were involved in study design. All authors provided feedback and contributed to manuscript preparation.

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Correspondence to Florian Douam, Wilson W. Wong or Mark W. Grinstaff.

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W.W.W. holds equity in Senti Biosciences, 4Immune Therapeutics and Keylicon Biosciences. A patent has been filed based on the findings of this work (J.E.M., W.W.W., M.W.G. and J.R.K.). J.E.M., M.W.G. and J.R.K. hold equity in Keylicon Biosciences. All other authors have no competing interests.

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McGee, J.E., Kirsch, J.R., Kenney, D. et al. Complete substitution with modified nucleotides in self-amplifying RNA suppresses the interferon response and increases potency. Nat Biotechnol (2024). https://doi.org/10.1038/s41587-024-02306-z

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