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Combinatorial development of nebulized mRNA delivery formulations for the lungs

Nature Nanotechnology (2023)Cite this article

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Abstract

Inhaled delivery of mRNA has the potential to treat a wide variety of diseases. However, nebulized mRNA lipid nanoparticles (LNPs) face several unique challenges including stability during nebulization and penetration through both cellular and extracellular barriers. Here we develop a combinatorial approach addressing these barriers. First, we observe that LNP formulations can be stabilized to resist nebulization-induced aggregation by altering the nebulization buffer to increase the LNP charge during nebulization, and by the addition of a branched polymeric excipient. Next, we synthesize a combinatorial library of ionizable, degradable lipids using reductive amination, and evaluate their delivery potential using fully differentiated air–liquid interface cultured primary lung epithelial cells. The final combination of ionizable lipid, charge-stabilized formulation and stability-enhancing excipient yields a significant improvement in lung mRNA delivery over current state-of-the-art LNPs and polymeric nanoparticles.

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Fig. 1: Formulation optimization of LNPs for nebulized delivery using DOE.
Fig. 2: Excipients and buffer modifications for improved LNP stability and in vivo delivery.
Fig. 3: Synthesis and screening of a biodegradable lipid library.
Fig. 4: In vivo testing and in vitro–in vivo comparison of top lipids.
Fig. 5: Pharmacokinetics and pharmacodynamics of top-performing LNPs.
Fig. 6: Evaluating functional mRNA delivery to lung epithelium with IR-117-17 LNPs in the Ai14 mouse model.

Data availability

All data that support the findings of this study are provided within the paper and its Supplementary Information. Source data are provided with this paper.

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Acknowledgements

This work was supported by the NIH (grant numbers UG3HL147367 and R01 HL162564-02 to A.Y.J., F.E., C.M. and D.G.A.) and Sanofi (formerly Translate Bio, to I.O.R., Y.H., R.S.M. and D.G.A.). J.W. was supported by the Cystic Fibrosis Foundation under award WITTEN19XX0. S.M. and F.A.O. were supported by the MIT Undergraduate Research Opportunities Program. We thank the Koch Institute Swanson Biotechnology Center for technical support, specifically the Animal Imaging & Preclinical Testing, Histology, Nanotechnology Materials, BioMicro Center and Microscopy core facilities. This work was also supported in part by the Koch Institute Support (core) Grant P30-CA14051 from the National Cancer Institute.

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  1. These authors contributed equally: Allen Y. Jiang, Jacob Witten, Idris O. Raji.

Authors and Affiliations

  1. Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA

    Allen Y. Jiang, Jacob Witten, Idris O. Raji, Rajith S. Manan, Robert Langer & Daniel G. Anderson

  2. David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA, USA

    Allen Y. Jiang, Jacob Witten, Idris O. Raji, Corina MacIsaac, Yizong Hu, Rajith S. Manan, Robert Langer & Daniel G. Anderson

  3. Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA

    Jacob Witten, Sabrina Meng, Favour A. Oladimeji, Robert Langer & Daniel G. Anderson

  4. Department of Anesthesiology, Boston Children’s Hospital, Boston, MA, USA

    Idris O. Raji, Robert Langer & Daniel G. Anderson

  5. Harvard and MIT Division of Health Science and Technology, Massachusetts Institute of Technology, Cambridge, MA, USA

    Feyisayo Eweje, Corina MacIsaac, Robert Langer & Daniel G. Anderson

  6. Harvard/MIT MD–PhD Program, Boston, MA, USA

    Feyisayo Eweje

  7. Institute for Medical Engineering and Science, Massachusetts Institute of Technology, Cambridge, MA, USA

    Robert Langer & Daniel G. Anderson

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Contributions

A.Y.J., J.W. and I.O.R. conceived the project, contributed equally and all reserve the right to list themselves first on their CVs. A.Y.J., J.W., I.O.R., F.E., C.M., S.M., F.A.O., Y.H. and R.S.M. performed the experiments and analysed data. A.Y.J., J.W., R.L. and D.G.A. discussed the results and wrote the paper with input from all authors. R.L. and D.G.A. acquired funding and supervised the project.

Corresponding author

Correspondence to Daniel G. Anderson.

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Competing interests

A.Y.J., J.W., I.O.R. and D.G.A. have filed a patent for the biodegradable lipid library described herein (US Patent Application No. 18080299). D.G.A. receives research funding from Sanofi/Translate Bio, and is a founder of oRNA Tx. R.L. is co-founder and a director of Moderna. He also serves on the board and has equity in Particles For Humanity. For a list of entities with which R.L. is, or has been recently involved, compensated or uncompensated, see https://www.dropbox.com/s/yc3xqb5s8s94v7x/Rev%20Langer%20COI.pdf?dl=0. The other authors declare no competing interests.

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Nature Nanotechnology thanks Dan Peer, Bruno Pitard and the other, anonymous, reviewers for their contribution to the peer review of this work.

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Jiang, A.Y., Witten, J., Raji, I.O. et al. Combinatorial development of nebulized mRNA delivery formulations for the lungs. Nat. Nanotechnol. (2023). https://doi.org/10.1038/s41565-023-01548-3

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