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Fast and facile synthesis of amidine-incorporated degradable lipids for versatile mRNA delivery in vivo

Abstract

Lipid nanoparticles (LNPs) are widely used for mRNA delivery, with cationic lipids greatly affecting biodistribution, cellular uptake, endosomal escape and transfection efficiency. However, the laborious synthesis of cationic lipids limits the discovery of efficacious candidates and slows down scale-up manufacturing. Here we develop a one-pot, tandem multi-component reaction based on the rationally designed amine–thiol–acrylate conjugation, which enables fast (1 h) and facile room-temperature synthesis of amidine-incorporated degradable (AID) lipids. Structure–activity relationship analysis of a combinatorial library of 100 chemically diverse AID-lipids leads to the identification of a tail-like amine–ring-alkyl aniline that generally affords efficacious lipids. Experimental and theoretical studies show that the embedded bulky benzene ring can enhance endosomal escape and mRNA delivery by enabling the lipid to adopt a more conical shape. The lead AID-lipid can not only mediate local delivery of mRNA vaccines and systemic delivery of mRNA therapeutics, but can also alter the tropism of liver-tropic LNPs to selectively deliver gene editors to the lung and mRNA vaccines to the spleen.

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Fig. 1: Fast and facile synthesis of AID-lipids via T-MCR for mRNA delivery.
Fig. 2: High-throughput synthesis and screening of AID-lipids.
Fig. 3: Characterization and in vitro evaluation of 12T-O14 LNP.
Fig. 4: 12T-O14 enables local mRNA vaccine and systemic mRNA drug delivery.
Fig. 5: 12T-O14 redirects liver-tropic LNPs to selectively target lung or spleen.
Fig. 6: 12T-O14 redirects liver-tropic LNPs to deliver functional mRNAs to non-liver cells.

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Data availability

All relevant data supporting the findings of this study are available within the paper and the Supplementary Information. Source data are provided with this paper.

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Acknowledgements

M.J.M. acknowledges support from a US National Institutes of Health (NIH) Director’s New Innovator Award (DP2 TR002776), a Burroughs Wellcome Fund Career Award at the Scientific Interface (CASI), a US National Science Foundation CAREER Award (CBET-2145491) and an American Cancer Society Research Scholar Grant (RSG-22-122-01-ET). M.J.M. and J.M.W. acknowledge support from a sponsored research agreement with iECURE. We acknowledge S. Steimle from Beckman Center for Cryo Electron Microscopy at the University of Pennsylvania for help with characterizing the morphology of LNPs. We acknowledge UPenn Gene Therapy Program NAT Core for sequencing service and thank K. Martins from UPenn GTP for processing the NGS data. We thank Y. Zhong from State Key Laboratory of Natural and Biomimetic Drugs, Peking University for providing access to computing resources in molecular dynamic simulation studies. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.

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Authors

Contributions

X.H., M.-G.A., N.G., L.X. and M.J.M. designed experiments. X.H., M.-G.A., N.G., L.X., G.Z., C.C.W., G.B., R.E.-M. and G.D. performed experiments. X.H., M.-G.A., N.G. and L.X. analysed data. X.H., M.-G.A. and M.J.M. wrote the paper. All authors discussed and edited the paper content.

Corresponding authors

Correspondence to Drew Weissman or Michael J. Mitchell.

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Competing interests

X.H. and M.J.M. are inventors on a patent filed by the Trustees of the University of Pennsylvania (US provisional patent application no. 63/589,051, filed 10 October 2023) describing the amidine-incorporated degradable lipid nanoparticle technology in this paper. In accordance with the University of Pennsylvania policies and procedures and our ethical obligations as researchers, we report that D.W. is named on patents that describe the use of nucleoside-modified mRNA as a platform to deliver therapeutic proteins and vaccines. M.J.M., X.H., D.W. and M.-G.A. are also named on patents describing the use of lipid nanoparticles, and lipid compositions for nucleic acid delivery and vaccination. We have disclosed those interests fully to the University of Pennsylvania, and we have in place an approved plan for managing any potential conflicts arising from licensing of our patents. J.M.W. is a paid advisor to and holds equity in iECURE, Passage Bio and the Center for Breakthrough Medicines (CBM). He also holds equity in the former G2 Bio asset companies. He has sponsored research agreements with Amicus Therapeutics, CBM, Ceva Santé Animale, Elaaj Bio, FA212, Foundation for Angelman Syndrome Therapeutics, former G2 Bio asset companies, iECURE and Passage Bio, which are licensees of Penn technology. J.M.W. and C.C.W. are inventors on patents that have been licensed to various biopharmaceutical companies and for which they may receive payments. The other authors declare no competing interests.

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Han, X., Alameh, MG., Gong, N. et al. Fast and facile synthesis of amidine-incorporated degradable lipids for versatile mRNA delivery in vivo. Nat. Chem. (2024). https://doi.org/10.1038/s41557-024-01557-2

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