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Optimization of the activity and biodegradability of ionizable lipids for mRNA delivery via directed chemical evolution

Nature Biomedical Engineering volume 8, pages 1412–1424 (2024)Cite this article

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Abstract

Ionizable lipids largely determine the biocompatibility of lipid nanoparticles (LNPs) and the efficacy for mRNA delivery. Rational design and combinatorial synthesis have led to the development of potent and biodegradable ionizable lipids, yet methodologies for the stepwise optimization of ionizable lipid structure are lacking. Here we show that iterative chemical derivatization and combinatorial chemistry, and in particular the amine–aldehyde–alkyne coupling reaction, can be leveraged to iteratively accelerate the structural optimization of propargylamine-based ionizable lipids (named A3-lipids) to improve their delivery activity and biodegradability. Through five cycles of such directed chemical evolution, we identified dozens of biodegradable and asymmetric A3-lipids with delivery activity comparable to or better than a benchmark ionizable lipid. We then derived structure−activity relationships for the headgroup, ester linkage and tail. Compared with standard ionizable lipids, the lead A3-lipid improved the hepatic delivery of an mRNA-based genome editor and the intramuscular delivery of an mRNA vaccine against SARS-CoV-2. Structural criteria for ionizable lipids discovered via directed chemical evolution may accelerate the development of LNPs for mRNA delivery.

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Fig. 1: A3-coupling reaction and activity- and degradability-driven directed chemical evolution of A3-lipids.
Fig. 2: Combinatorial synthesis and screening of A3-lipids in Library 1.
Fig. 3: Optimization of symmetric A3-lipids with biodegradable alkynes in Library 2 and asymmetric A3-lipids with long-chain aldehydes in Library 3.
Fig. 4: Optimization of biodegradable tails and headgroups for asymmetric A3-lipids in Libraries 4 and 5.
Fig. 5: Characterization of A3-LNP and its application in delivering mRNA-based gene editors and vaccines.

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Data availability

All relevant data supporting the findings of this study are available within the paper and its 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) and an American Cancer Society Research Scholar Grant (RSG-22-122-01-ET). J.M.W. acknowledges support from iECURE. We thank S. Steimle from the Beckman Center for Cryo Electron Microscopy at UPenn Perelman School of Medicine (RRID: SCR_022375) for help in characterizing the morphology of LNPs; the UPenn Gene Therapy Program NAT Core for sequencing service and K. Martins for processing the NGS data.

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Authors and Affiliations

  1. Department of Bioengineering, University of Pennsylvania, Philadelphia, PA, USA

    Xuexiang Han, Rohan Palanki, Kelsey L. Swingle, Junchao Xu, Ningqiang Gong, Lulu Xue, Qiangqiang Shi, Il-Chul Yoon & Michael J. Mitchell

  2. Key Laboratory of RNA Innovation, Science and Engineering, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Shanghai, China

    Xuexiang Han

  3. Department of Medicine, University of Pennsylvania, Philadelphia, PA, USA

    Mohamad-Gabriel Alameh, Ying Xu, Rakan El-Mayta, Garima Dwivedi & Drew Weissman

  4. Penn Institute for RNA Innovation, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA

    Mohamad-Gabriel Alameh, Drew Weissman & Michael J. Mitchell

  5. Gene Therapy Program, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA

    Claude C. Warzecha & James M. Wilson

  6. Abramson Cancer Center, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA

    Michael J. Mitchell

  7. Institute for Immunology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA

    Michael J. Mitchell

  8. Cardiovascular Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA

    Michael J. Mitchell

  9. Institute for Regenerative Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA

    Michael J. Mitchell

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Contributions

X.H. and M.J.M. conceptualized the project. X.H. and M.-G.A. developed the methodology. X.H., M.-G.A., Y.X., R.P., J.X., R.E.-M., G.D., C.C.W. and I.-C.Y. conducted investigations. X.H. and Y.X. performed visualization. M.J.M. acquired funding and supervised the project. X.H. and M.J.M. wrote the original draft. J.X., M.-G.A., Y.X., R.P., L.X., N.G., R.E.-M., K.L.S., Q.S., C.C.W., J.M.W., D.W. and M.J.M. reviewed and edited the manuscript.

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Correspondence to Michael J. Mitchell.

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Competing interests

X.H. and M.J.M. have filed a patent application based on this work. 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 and Ceva Santé Animale. He has sponsored research agreements with Alexion Pharmaceuticals, 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., Xu, Y. et al. Optimization of the activity and biodegradability of ionizable lipids for mRNA delivery via directed chemical evolution. Nat. Biomed. Eng 8, 1412–1424 (2024). https://doi.org/10.1038/s41551-024-01267-7

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