Abstract
The blood–brain barrier (BBB) restricts the systemic delivery of messenger RNAs (mRNAs) into diseased neurons. Although leucocyte-derived extracellular vesicles (EVs) can cross the BBB at inflammatory sites, it is difficult to efficiently load long mRNAs into the EVs and to enhance their neuronal uptake. Here we show that the packaging of mRNA into leucocyte-derived EVs and the endocytosis of the EVs by neurons can be enhanced by engineering leucocytes to produce EVs that incorporate retrovirus-like mRNA-packaging capsids. We transfected immortalized and primary bone-marrow-derived leucocytes with DNA or RNA encoding the capsid-forming activity-regulated cytoskeleton-associated (Arc) protein as well as capsid-stabilizing Arc 5’-untranslated-region RNA elements. These engineered EVs inherit endothelial adhesion molecules from donor leukocytes, recruit endogenous enveloping proteins to their surface, cross the BBB, and enter the neurons in neuro-inflammatory sites. Produced from self-derived donor leukocytes, the EVs are immunologically inert, and enhanced the neuronal uptake of the packaged mRNA in a mouse model of low-grade chronic neuro-inflammation.
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Data availability
The data supporting the results in this study are available within the paper and its Supplementary Information. The raw patient data are available from the authors, subject to Institutional Review Board approval. Source data for the figures are provided with this paper. The raw and analysed datasets generated during the study are available for research purposes from the corresponding authors on reasonable request. Source data are provided with this paper.
Code availability
The code for the custom macro generated for this study is provided in Supplementary Information.
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Acknowledgements
We thank S. Wilkens from SUNY Upstate Medical University for assistance with TEM imaging; R. Williams, T. Abratte and the BRC Imaging Facility at the Cornell Institute of Biotechnology (RRID:SCR_021741) for imaging experiments, with NIH S10OD025049 for the IVIS-spectrum optical imager in Cornell’s BRC Imaging Facility; P. Schweitzer and the BRC Genomics Facility (RRID:SCR_021727) at the Cornell Institute of Biotechnology for the sequencing experiments; L. Tesfa, J. Mahoney and the BRC Flow Cytometry Facility (RRID:SCR_021740) for flow cytometry data; T. Totman, E. Feldman, F. Burgus and the CARE at Cornell for service and advice on care of animals; R. Felt and the IACUC, Cornell for help composing and managing animal protocols; A. Recknagel for advice on imaging; Herbert Fountain for inspiring discussions about anti-aging therapies; and P. Miller for help in maintaining high-standard laboratory practices. S.J. acknowledges start-up support from Cornell University, including the Robert Langer ’70 Family and Friends Professorship, Cornell NEXT Nano Initiative and Cornell Engineering’s inaugural Sprout Awards.
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Contributions
W. Gu, R.L. and S.J. conceptualized the work. W. Gu, S.L., K.L., S.C., N.E., Y.Y., M.C., Y.-W.C., W. Gao and T.S. acquired the data. W. Gu, S.L., S.C., Y.Y., Y.-W.C. and W. Gao contributed to data analyses and to the interpretation of the results. W. Gu, Z.Y., K.S.-G., R.H., P.M., C.W., C. Seo, A.G., C. Schaffer, N.N., R.C., Q.Y., M.W., R.L. and S.J. provided advice, support and supervision. W. Gu wrote the manuscript. W. Gu, C. Schaffer, N.N., R.C., M.W., R.L. and S.J. edited the manuscript.
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S.J., W. Gu, S.L. and Z.Y. are authors of a patent application related to this work (PCT/US2022/027568) filed by Cornell University. All other authors declare no competing interests.
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Nature Biomedical Engineering thanks Koen Breyne, Takahiro Ochiya and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.
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Extended data
Extended Data Fig. 1 Optimization of CMDR dye staining for the quantification of total EV uptake.
To aid in quantifying EVs both in solution and within recipient cells, we employed plasma membrane staining using CMDR. We further refined the number of EVs introduced to recipient cells. The final parameters settled on a 1:5000 CMDR dye concentration and a 2X EV concentration, indicating EVs derived from 2 donor cells (over a span of 24-40 hours) were designated for a single recipient cell.
Extended Data Fig. 2 Density-related expression of the EV markers CD63 and CD81 in capsid+/ stabilizer+ eraEVs.
(a) Western blot analysis illustrating CD63 and Arc expression across various EV subpopulations, differentiated by density through density gradient centrifugation. The 70% sucrose cushion layer—representing the highest density—displayed a marked increase in both CD63 and Arc. Equal amounts of total proteins were loaded across all lanes. Displayed is one representative trial out of three independent experiments. (b) Immunomagnetic positive selection was conducted to isolate CD81+ EVs from filtered supernatant cell culture media laden with both control and engineered EVs. Following isolation, the CD81+ EVs were lysed to facilitate protein quantification and subsequent western blot analysis. (c) Utilizing the Pierce 660 nm Protein Assay, total proteins harvested from CD81+ EVs were quantified. Notably, there was a 2.3-fold upsurge in the protein count extracted from CD81 + /Arc+ engineered EVs in contrast to the capsid‒ control. (d) Western blotting of the CD81 + EV lysates affirmed the presence of Arc proteins. The absence of reducing agents in the sample buffer was deliberate since detection antibodies for CD9/CD63/CD81 often identify the disulfide bond associated with antigen epitopes. Under non-reducing conditions, substantial Arc oligomers are evident. Notably, despite comparable levels of monomer Arc, the quantity of Arc oligomers was significantly amplified in stabilizer+ EVs, underscoring the stabilization effect imparted by the A5U motif. Owing to our exclusion of an Arc knockout cell line, endogenous Arc was discernible in the no-transfection control set. Abbreviations: NT - no transfection control; AA - Arc/A5U-GFP; AG - Arc/GFP. (e-f) Quantitative breakdown depicting the distribution of Arc protein monomers and oligomers in each sample set, as determined through Western blot analysis. The figure presents one representative trial from three independent repeats. For uncropped western gels, refer to SD_ED_FIG2.
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Source data
Source Data for Extended Data Fig. 2
Unprocessed western blots.
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Gu, W., Luozhong, S., Cai, S. et al. Extracellular vesicles incorporating retrovirus-like capsids for the enhanced packaging and systemic delivery of mRNA into neurons. Nat. Biomed. Eng (2024). https://doi.org/10.1038/s41551-023-01150-x
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DOI: https://doi.org/10.1038/s41551-023-01150-x
