000640497 001__ 640497 000640497 005__ 20251120212636.0 000640497 0247_ $$2doi$$a10.1038/s41565-025-01961-w 000640497 0247_ $$2ISSN$$a1748-3387 000640497 0247_ $$2ISSN$$a1748-3395 000640497 037__ $$aPUBDB-2025-04819 000640497 041__ $$aEnglish 000640497 082__ $$a600 000640497 1001_ $$aMoreno Herrero, Jorge$$b0 000640497 245__ $$aCompact polyethylenimine-complexed mRNA vaccines 000640497 260__ $$aLondon [u.a.]$$bNature Publishing Group$$c2025 000640497 3367_ $$2DRIVER$$aarticle 000640497 3367_ $$2DataCite$$aOutput Types/Journal article 000640497 3367_ $$0PUB:(DE-HGF)16$$2PUB:(DE-HGF)$$aJournal Article$$bjournal$$mjournal$$s1763638650_1728937 000640497 3367_ $$2BibTeX$$aARTICLE 000640497 3367_ $$2ORCID$$aJOURNAL_ARTICLE 000640497 3367_ $$00$$2EndNote$$aJournal Article 000640497 500__ $$aWaiting for fulltext 000640497 520__ $$aHere 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. 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