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Lipid nanoparticle-mediated mRNA delivery to CD34+ cells in rhesus monkeys

Nature Biotechnology (2024)Cite this article

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Abstract

Transplantation of ex vivo engineered hematopoietic stem cells (HSCs) can lead to robust clinical responses but carries risks of adverse events from bone marrow mobilization, chemotherapy conditioning and other factors. HSCs have been modified in vivo using lipid nanoparticles (LNPs) decorated with targeting moieties, which increases manufacturing complexity. Here we screen 105 LNPs without targeting ligands for effective homing to the bone marrow in mouse. We report an LNP named LNP67 that delivers mRNA to murine HSCs in vivo, primary human HSCs ex vivo and CD34+ cells in rhesus monkeys (Macaca mulatta) in vivo at doses of 0.25 and 0.4 mg kg−1. Without mobilization and conditioning, LNP67 can mediate delivery of mRNA to HSCs and their progenitor cells (HSPCs), as well as to the liver in rhesus monkeys, without serum cytokine activation. These data support the hypothesis that in vivo delivery to HSCs and HSPCs in nonhuman primates is feasible without targeting ligands.

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Fig. 1: Barcoded LNPs deliver mRNA to transcriptionally defined HSCs in vivo.
Fig. 2: LNP67 delivers mRNA to cells in mouse bone marrow.
Fig. 3: LNP67 delivers mRNA to transcriptionally defined HSCs in vivo.
Fig. 4: LNP67 delivers mRNA to CD34+ cells in rhesus monkeys.
Fig. 5: LNP67 delivers mRNA to primary human HSCs ex vivo.

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Data availability

scRNA-seq raw data were deposited to the National Center for Biotechnology Information Sequence Read Archive database (SRR28416440, SRR28416441, SRR28416442, SRR28417638 and SRR28417639) under project identifier PRJNA1090779. The mouse genome GRCm39 can be accessed at https://www.ncbi.nlm.nih.gov/datasets/genome/GCF_000001635.27/. All other data are shown.

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Acknowledgements

We thank K. Tiegren at Emory University for copyediting the manuscript. We thank R. C. Guerrero-Ferreira at the Emory University Robert P. Apkarian Integrated Electron Microscopy Core Facility (RRID: SCR_023537) for cryo-TEM imaging. The cryo-TEM data described here were collected on the JEOL JEM-2200FS 200-kV TEM instrument supported by the National Science Foundation Major Research Instrumentation (grant 0923395). We thank P. Bagchi at the Emory Integrated Proteomics Core (EIPC) subsidized by the Emory University School of Medicine for the proteomics assay. For the proteomics assay at EIPC, additional support was provided by the Georgia Clinical and Translational Science Alliance of the National Institutes of Health (NIH; award number UL1TR002378). These studies were supported through the NIH Somatic Cell Genome Editing (SCGE) consortium. This was a collaborative effort between UH3-TR002855 (J.E.D. and P.J.S.) and the Nonhuman Primate Testing Center for Evaluation of Somatic Cell Genome-Editing Tools (U42 OD027094, to A.T.). Information about these studies is also provided in the SCGE toolkit, supported through the SCGE Dissemination and Coordinating Center (https://commonfund.nih.gov/editing)63. Studies were also supported through the base operating grant for the California National Primate Research Center (P51-OD011107). This work was funded in part through ATLANTIS NIH training grant in kidney, urology and hematology (TL1DK136047, to R.Z.).

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  1. These authors contributed equally: Hyejin Kim, Ryan Zenhausern.

Authors and Affiliations

  1. Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University School of Medicine, Atlanta, GA, USA

    Hyejin Kim, Ryan Zenhausern, Kara Gentry, Liming Lian, Sebastian G. Huayamares, David Loughrey, Ananda R. Podilapu, Marine Z. C. Hatit, Huanzhen Ni, Andrea Li, Aram Shajii, Hannah E. Peck, Keyi Han, Xuanwen Hua, Shu Jia, Philip J. Santangelo & James E. Dahlman

  2. Petit Institute for Bioengineering and Biosciences, Georgia Institute of Technology, Atlanta, GA, USA

    Afsane Radmand

  3. Department of Chemical Engineering, Georgia Institute of Technology, Atlanta, GA, USA

    Afsane Radmand

  4. California National Primate Research Center, University of California, Davis, Davis, CA, USA

    Michele Martinez, Charles Lee & Alice Tarantal

  5. Department of Pediatrics, University of California, Davis, Davis, CA, USA

    Charles Lee & Alice Tarantal

  6. Department of Cell Biology and Human Anatomy, University of California, Davis, Davis, CA, USA

    Alice Tarantal

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Contributions

H.K., R.Z., A.T. and J.E.D. designed the experiments and analyzed data. H.K. and R.Z. led all experiments. A.T. and J.E.D. supervised the project. K.G., L.L., S.G.H., A.R., A.L. and A.S. assisted with mouse experiments. K.G. and L.L. assisted with LNP characterization. D.L. performed next-generation sequencing. A.R.P., M.Z.C.H. and H.N. designed and synthesized the ionizable lipid. H.E.P. and P.J.S. designed and synthesized the mRNA. K.H., X.H. and S.J. performed Fourier light-field fluorescence microscopy imaging of rhesus cells. M.M., C.L. and A.T. performed rhesus macaque experiments. H.K., R.Z., A.F.T. and J.E.D. wrote the initial draft, which was sent to all authors.

Corresponding author

Correspondence to James E. Dahlman.

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Competing interests

M.Z.C.H., H.N. and J.E.D. have filed a provisional patent related to this manuscript (US patent application number 63/632,354). J.E.D. is an advisor to GV, Readout, Edge Animal Health and Nava Therapeutics and P.J.S. is a cofounder of Tether Therapeutics. None of these companies provided any financial support for this work. The other authors declare no competing interests.

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Supplementary Figs. 1–19 and references.

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Supplementary Table 1

Sequences for LNP barcodes and templates for mRNA.

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Kim, H., Zenhausern, R., Gentry, K. et al. Lipid nanoparticle-mediated mRNA delivery to CD34+ cells in rhesus monkeys. Nat Biotechnol (2024). https://doi.org/10.1038/s41587-024-02470-2

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