Abstract
Here we describe formulations comprising individual, polymer-complexed self-amplifying RNA (saRNA) molecules, designed for vaccination against infectious diseases and other prophylactic and therapeutic applications. When exposed to a large excess of the cationic polymer polyethylenimine (PEI), the single saRNA molecules in solution reorganize from an extended to a globular organization, characterized by a high packing density, low polymer mass fraction and, consequently, a very small size of the polyplex nanoparticles of about 30 nm. This format of PEI-complexed saRNA exhibits enhanced biological activity in comparison with previously described saRNA/PEI formulations, both in vitro and in vivo. In vaccination models, relevant immune responses at lower doses are achieved, offering potential advantages for practical use. We found that the single PEI-complexed RNA molecules are also present in conventional formulations to some degree. The direct correlation between the single-molecule fraction with activity suggests that it is this format that predominantly contributes to activity in the different formulation types. Complexation is driven by mechanisms of self-assembly between oppositely charged polyelectrolytes, making this protocol broadly applicable to various cationic polymers and RNA constructs. With their small size and good stability in biofluids, these compacted RNA molecules are also promising for the systemic delivery of genetic material to compartments that are difficult to reach with larger particles.
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Data availability
The data that support the findings of this study are available from the corresponding author, upon reasonable request. For the SANS measurements, the raw images are public via ISIS Neutron Source at https://data.isis.stfc.ac.uk/doi/INVESTIGATION/111243135/ and https://doi.org/10.5286/ISIS.E.RB2000156. Source data are provided with this paper.
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Acknowledgements
Figures 1, 3 and 4 were illustrated and designed by Elvire Thouvenot and are reproduced with permission, copyright 2022 Elvire Thouvenot. We thank JASCO and S. Suzuki for making available the HTS Module for early CD measurements and helping throughout the establishment of the HTS protocol used in this Article. We acknowledge A. Westenberger for excellent technical assistance. For SANS data analysis, this work benefitted from the use of the SasView application, originally developed under NSF award DMR-0520547. For SANS data analysis, the software package SasView was used. SasView contains code developed with funding from the European Union’s Horizon 2020 research and innovation programme under the SINE2020 project, grant agreement no. 654000.
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Conceptualization by J.M.H., S.E., A.S. and H.H. Methodology by J.M.H., S.E., J.S., A.S. and H.H. Investigation by J.M.H., T.B.S., J.S., K.M., T.B., L.P.C., M.A.S. and D.I.S. Visualization by J.M.H., S.E. and H.H. Project administration by S.E., A.S., H.H. and U.S. Supervision by S.E., A.S., H.H. and U.S. Writing—original draft by J.M.H., S.E. and H.H. Writing— review and editing by J.M.H., T.B.S., S.E., K.M., A.S., J.S., T.B., L.P.C., M.A.S., D.I.S. and H.H.
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U.S. is a management board member at BioNTech SE. T.B.S., J.S. and T.B. are employees at BioNTech SE. J.M.H., T.B.S., S.E., A.S., T.B., H.H. and U.S. are inventors on patents and patent applications related to RNA technology and/or have securities from BioNTech SE. All other authors declare no competing interests.
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Moreno Herrero, J., Stahl, T.B., Erbar, S. et al. Compact polyethylenimine-complexed mRNA vaccines. Nat. Nanotechnol. 20, 1323–1331 (2025). https://doi.org/10.1038/s41565-025-01961-w
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DOI: https://doi.org/10.1038/s41565-025-01961-w
