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Membrane-destabilizing ionizable phospholipids for organ-selective mRNA delivery and CRISPR–Cas gene editing

Abstract

Endosomal escape remains a fundamental barrier hindering the advancement of nucleic acid therapeutics. Taking inspiration from natural phospholipids that comprise biological membranes, we report the combinatorial synthesis of multi-tailed ionizable phospholipids (iPhos) capable of delivering messenger RNA or mRNA/single-guide RNA for gene editing in vivo. Optimized iPhos lipids are composed of one pH-switchable zwitterion and three hydrophobic tails, which adopt a cone shape in endosomal acidic environments to facilitate membrane hexagonal transformation and subsequent cargo release from endosomes. Structure–activity relationships reveal that iPhos chemical structure can control in vivo efficacy and organ selectivity. iPhos lipids synergistically function with various helper lipids to formulate multi-component lipid nanoparticles (called iPLNPs) for selective organ targeting. Zwitterionic, ionizable cationic and permanently cationic helper lipids enable tissue-selective mRNA delivery and CRISPR–Cas9 gene editing in spleen, liver and lungs (respectively) following intravenous administration. This rational design of functional phospholipids demonstrates substantial value for gene editing research and therapeutic applications.

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Fig. 1: A combinatorial library of iPhos lipids was chemically synthesized and studied, which led to the elucidation of a physical mechanism of action for enhanced endosomal escape.
Fig. 2: SAR of iPhos lipids for luciferase mRNA delivery in vitro.
Fig. 3: Model membrane studies of endosomal escape demonstrated the mechanism of iPhos lipid-mediated RNA delivery with correlation to chemical structure.
Fig. 4: Structure–activity studies revealed that iPhos lipid structure controlled in vivo efficacy and organ selectivity.
Fig. 5: iPhos outperformed traditional phospholipids, and functioned with different helper lipids for organ-selective RNA delivery.
Fig. 6: iPLNPs enabled CRISPR–Cas9 gene editing selectively in liver and lungs and possessed potential for clinical translation.

Data availability

All relevant data supporting the findings of this study are available within the paper and Supplementary Information. The raw data is available from the corresponding author upon request.

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Acknowledgements

D.J.S. acknowledges financial support from the National Institutes of Health (NIH) National Institute of Biomedical Imaging and Bioengineering (NIBIB) (grant no. R01 EB025192-01A1), the American Cancer Society (ACS) (grant no. RSG-17-012-01), the Welch Foundation (grant no. I-1855) and the Cystic Fibrosis Foundation (CFF) (grant no. SIEGWA18XX0). T.W. acknowledges financial support from the Cancer Prevention and Research Institute of Texas (CPRIT) Training grant (no. RP160157). We acknowledge the UTSW Tissue Resource, supported in part by the National Cancer Institute (grant no. 5P30CA142543) and the Moody Foundation Flow Cytometry Facility.

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S.L., Q.C. and D.J.S. designed the research. S.L., Q.C., T.W., X.Y., L.T.J. and L.F. performed the experiments. All the authors were involved in the data analyses. S.L. and D.J.S. wrote the manuscript, and all authors discussed and commented on it. D.J.S. directed the research.

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Correspondence to Daniel J. Siegwart.

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D.J.S., S.L., Q.C., T.W. and X.Y., and the Reagents of the University of Texas System have filed a patent application on this technology.

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Peer review information Nature Materials thanks Bruno Pitard, John Rossi and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Liu, S., Cheng, Q., Wei, T. et al. Membrane-destabilizing ionizable phospholipids for organ-selective mRNA delivery and CRISPR–Cas gene editing. Nat. Mater. 20, 701–710 (2021). https://doi.org/10.1038/s41563-020-00886-0

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