Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Reduction of the therapeutic dose of silencing RNA by packaging it in extracellular vesicles via a pre-microRNA backbone

Nature Biomedical Engineering volume 4, pages 52–68 (2020)Cite this article

Subjects

Abstract

A small percentage of the short interfering RNA (siRNA) delivered via passive lipid nanoparticles and other delivery vehicles reaches the cytoplasm of cells. The high doses of siRNA and delivery vehicle that are thus required to achieve therapeutic outcomes can lead to toxicity. Here, we show that the integration of siRNA sequences into a Dicer-independent RNA stem–loop based on pre-miR-451 microRNA—which is highly enriched in small extracellular vesicles secreted by many cell types—reduces the expression of the genes targeted by the siRNA in the liver, intestine and kidney glomeruli of mice at siRNA doses that are at least tenfold lower than the siRNA doses typically delivered via lipid nanoparticles. Small extracellular vesicles that efficiently package siRNA can significantly reduce its therapeutic dose.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Rent or buy this article

Get just this article for as long as you need it

$39.95

Learn more

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: sEVs contain few miRNAs and are enriched in miR-451 compared with cells.
Fig. 2: Reprogramming the pre-miR-451 backbone with siRNAs causes their enrichment in sEVs.
Fig. 3: sEVs loaded with siRNA integrated in the pre-miR-451 backbone efficiently deliver siRNA to primary motor neurons.
Fig. 4: Intravenously injected sEVs loaded with siRNA integrated in the pre-miR-451 backbone knock down target expression in mouse liver and small intestine.
Fig. 5: sEVs loaded with siRNA integrated in the pre-miR-451 backbone knock down target expression with lower doses than lipid nanoparticles or electroporated sEVs.
Fig. 6: Quantitative FISH accurately measures mRNA in mouse tissues.
Fig. 7: sEVs packaged with siRNA knock down target genes in specific regions and cell types of liver, small intestine and kidney.
Fig. 8: sEVs packaged with siRNA targeting TTR reduce TTR levels in blood by more than 85%.

Data availability

The main data supporting the results of this study are available within the paper and its Supplementary Information. The raw and analysed datasets are too numerous to be readily shared publicly, but can be obtained for research purposes from the corresponding author on reasonable request.

References

  1. Ha, M. & Kim, V. N. Regulation of microRNA biogenesis. Nat. Rev. Mol. Cell Biol. 15, 509–524 (2014).

    CAS  PubMed  Google Scholar 

  2. Fitzgerald, K. et al. A highly durable RNAi therapeutic inhibitor of PCSK9. N. Engl. J. Med. 376, 41–51 (2017).

    CAS  PubMed  Google Scholar 

  3. Coelho, T. et al. Safety and efficacy of RNAi therapy for transthyretin amyloidosis. N. Engl. J. Med. 369, 819–829 (2013).

    CAS  PubMed  Google Scholar 

  4. Whitehead, Ka, Langer, R. & Anderson, D. G. Knocking down barriers: advances in siRNA delivery. Nat. Rev. Drug Discov. 8, 129–138 (2009).

    CAS  PubMed  Google Scholar 

  5. Kanasty, R., Dorkin, J. R., Vegas, A. & Anderson, D. Delivery materials for siRNA therapeutics. Nat. Mater. 12, 967–977 (2013).

    CAS  PubMed  Google Scholar 

  6. Wittrup, A. et al. Visualizing lipid-formulated siRNA release from endosomes and target gene knockdown. Nat. Biotechnol. 33, 1–9 (2015).

    Google Scholar 

  7. Pei, Y. et al. Quantitative evaluation of siRNA delivery in vivo. RNA 16, 2553–2563 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  8. Veldhoen, S., Laufer, S. D., Trampe, A. & Restle, T. Cellular delivery of small interfering RNA by a non-covalently attached cell-penetrating peptide: quantitative analysis of uptake and biological effect. Nucleic Acids Res. 34, 6561–6573 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  9. Gilleron, J. et al. Image-based analysis of lipid nanoparticle-mediated siRNA delivery, intracellular trafficking and endosomal escape. Nat. Biotechnol. 31, 638–646 (2013).

    CAS  PubMed  Google Scholar 

  10. Garber, K. Alnylam terminates revusiran program, stock plunges. Nat. Biotechnol. 34, 1213–1214 (2016).

    CAS  PubMed  Google Scholar 

  11. Fitzgerald, K. et al. A highly durable RNAi therapeutic inhibitor of PCSK9. N. Engl. J. Med. 376, 41–51 (2017).

    CAS  PubMed  Google Scholar 

  12. Colombo, M., Raposo, G. & Théry, C. Biogenesis, secretion, and intercellular interactions of exosomes and other extracellular vesicles. Annu. Rev. Cell Dev. Biol. 30, 255–289 (2014).

    CAS  PubMed  Google Scholar 

  13. Masciopinto, F. et al. Association of hepatitis C virus envelope proteins with exosomes. Eur. J. Immunol. 34, 2834–2842 (2004).

    CAS  PubMed  Google Scholar 

  14. Baj-Krzyworzeka, M. et al. Tumour-derived microvesicles carry several surface determinants and mRNA of tumour cells and transfer some of these determinants to monocytes. Cancer Immunol. Immunother. 55, 808–818 (2006).

    CAS  PubMed  Google Scholar 

  15. Ratajczak, J. et al. Embryonic stem cell-derived microvesicles reprogram hematopoietic progenitors: evidence for horizontal transfer of mRNA and protein delivery. Leukemia 20, 847–856 (2006).

    CAS  PubMed  Google Scholar 

  16. Valadi, H. et al. Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells. Nat. Cell Biol. 9, 654–659 (2007).

    CAS  PubMed  Google Scholar 

  17. Skog, J. et al. Glioblastoma microvesicles transport RNA and proteins that promote tumour growth and provide diagnostic biomarkers. Nat. Cell Biol. 10, 1470–1476 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  18. Pegtel, D. M. et al. Functional delivery of viral miRNAs via exosomes. Proc. Natl Acad. Sci. USA 107, 6328–6333 (2010).

    CAS  PubMed  Google Scholar 

  19. Alvarez-Erviti, L. et al. Delivery of siRNA to the mouse brain by systemic injection of targeted exosomes. Nat. Biotechnol. 29, 3–4 (2011).

    Google Scholar 

  20. Kamerkar, S. et al. Exosomes facilitate therapeutic targeting of oncogenic KRAS in pancreatic cancer. Nature 546, 498–503 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  21. Didiot, M.-C. et al. Exosome-mediated delivery of hydrophobically modified siRNA for Huntingtin mRNA silencing. Mol. Ther. 24, 1836–1847 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  22. Kooijmans, S. A. A. et al. Electroporation-induced siRNA precipitation obscures the efficiency of siRNA loading into extracellular vesicles. J. Control. Release 172, 229–238 (2013).

    CAS  PubMed  Google Scholar 

  23. Chevillet, J. R. et al. Quantitative and stoichiometric analysis of the microRNA content of exosomes. Proc. Natl Acad. Sci. USA 111, 14888–14893 (2014).

    CAS  PubMed  Google Scholar 

  24. Shurtleff, M., Karfilis, K. V., Temoche-Diaz, M., Ri, S. & Schekman, R. Y-box protein 1 is required to sort microRNAs into exosomes in cells and in a cell-free reaction. eLife 5, e19276 (2016).

    PubMed  PubMed Central  Google Scholar 

  25. Mukherjee, K. et al. Reversible HuR‐microRNA binding controls extracellular export of miR‐122 and augments stress response. EMBO Rep. 17, 1184–1203 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  26. Villarroya-Beltri, C. et al. Sumoylated hnRNPA2B1 controls the sorting of miRNAs into exosomes through binding to specific motifs. Nat. Commun. 4, 2980 (2013).

    PubMed  PubMed Central  Google Scholar 

  27. Yang, J. S. & Lai, E. C. Alternative miRNA biogenesis pathways and the interpretation of core miRNA pathway mutants. Mol. Cell 43, 892–903 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  28. Cifuentes, D. et al. A novel miRNA processing pathway independent of Dicer requires Argonaute2 catalytic activity. Science 328, 1694–1698 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  29. Cheloufi, S., Dos Santos, C. O., Chong, M. M. & Hannon, G. J. A dicer-independent miRNA biogenesis pathway that requires Ago catalysis. Nature 465, 584–589 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  30. Yang, J.-S. et al. Conserved vertebrate mir-451 provides a platform for Dicer-independent, Ago2-mediated microRNA biogenesis. Proc. Natl Acad. Sci. USA 107, 15163–15168 (2010).

    CAS  PubMed  Google Scholar 

  31. Stevanato, L., Thanabalasundaram, L., Vysokov, N. & Sinden, J. D. Investigation of content, stoichiometry and transfer of miRNA from human neural stem cell line derived exosomes. PLos ONE 11, e0146353 (2016).

    PubMed  PubMed Central  Google Scholar 

  32. Shelke, G. V., Lässer, C., Gho, Y. S. & Lötvall, J. Importance of exosome depletion protocols to eliminate functional and RNA-containing extracellular vesicles from fetal bovine serum. J. Extracell. Vesicles 3, 24783 (2014).

    Google Scholar 

  33. Wei, Z. & Batagov, A. O. & Carter, D. R. F. & Krichevsky, A. M. Fetal bovine serum RNA interferes with the cell culture derived extracellular RNA. Sci. Rep. 6, 31175 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  34. McKenzie, A. J. et al. KRAS–MEK signaling controls Ago2 sorting into exosomes. Cell Rep. 15, 978–987 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  35. Melo, S. A. et al. Cancer exosomes perform cell-independent microRNA biogenesis and promote tumorigenesis. Cancer Cell 26, 707–721 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  36. Eldh, M., Lötvall, J., Malmhäll, C. & Ekström, K. Importance of RNA isolation methods for analysis of exosomal RNA: evaluation of different methods. Mol. Immunol. 50, 278–286 (2012).

    CAS  PubMed  Google Scholar 

  37. Meerson, A. & Ploug, T. Assessment of six commercial plasma small RNA isolation kits using qRT-PCR and electrophoretic separation: higher recovery of microRNA following ultracentrifugation. Biol. Methods Protoc. 1, bpw003 (2016).

    Google Scholar 

  38. Gantier, M. P. et al. Analysis of microRNA turnover in mammalian cells following Dicer1 ablation. Nucleic Acids Res. 39, 5692–5703 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  39. Bartlett, D. W. & Davis, M. E. Insights into the kinetics of siRNA-mediated gene silencing from live-cell and live-animal bioluminescent imaging. Nucleic Acids Res. 34, 322–333 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  40. Furuyama, K. et al. Continuous cell supply from a Sox9-expressing progenitor zone in adult liver, exocrine pancreas and intestine. Nat. Genet. 43, 34–41 (2011).

    CAS  Google Scholar 

  41. Love, K. T. et al. Lipid-like materials for low-dose, in vivo gene silencing. Proc. Natl Acad. Sci. USA 107, 1864–1869 (2010).

    CAS  PubMed  Google Scholar 

  42. Bobrie, A., Colombo, M., Krumeich, S. & Raposo, G. Diverse subpopulations of vesicles secreted by different intracellular mechanisms are present in exosome preparations obtained by differential ultracentrifugation. J. Extracell. Vesicles 1, 18397 (2012).

    CAS  Google Scholar 

  43. Kowal, J. et al. Proteomic comparison defines novel markers to characterize heterogeneous populations of extracellular vesicle subtypes. Proc. Natl Acad.Sci. USA 113, 968–977 (2016).

    Google Scholar 

  44. Zhang, H. et al. Identification of distinct nanoparticles and subsets of extracellular vesicles by asymmetric flow field-flow fractionation. Nat. Cell Biol. 20, 332–343 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  45. Théry, C., Amigorena, S., Raposo, G. & Clayton, A. Isolation and characterization of exosomes from cell culture supernatants. Curr. Protoc. Cell Biol. Ch. 3, 1–29 (2006).

    Google Scholar 

  46. Okabe, M., Ikawa, M., Kominami, K., Nakanishi, T. & Nishimune, Y. ‘Green mice’ as a source of ubiquitous green cells. FEBS Lett. 407, 313–319 (1997).

    CAS  PubMed  Google Scholar 

  47. Gurney, M. E. et al. Motor neuron degeneration in mice that express a human Cu,Zn superoxide dismutase mutation. Science 264, 1772–1775 (1994).

    CAS  PubMed  Google Scholar 

  48. Miller, V. M., Gouvion, C. M., Davidson, B. L. & Paulson, H. L. Targeting Alzheimer’s disease genes with RNA interference: an efficient strategy for silencing mutant alleles. Nucleic Acids Res. 32, 661–668 (2004).

    CAS  PubMed  PubMed Central  Google Scholar 

  49. Foust, K. D. et al. Therapeutic AAV9-mediated suppression of mutant SOD1 slows disease progression and extends survival in models of inherited ALS. Mol. Ther. 21, 2148–2159 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  50. Lin, X. et al. A robust in vivo positive-readout system for monitoring siRNA delivery to xenograft tumors. RNA 17, 603–612 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  51. Taylor, S. C., Carbonneau, J., Shelton, D. N. & Bolvin, G. Optimization of droplet digital PCR from RNA and DNA extracts with direct comparison to RT–qPCR: clinical implications for quantification of oseltamivir-resistant subpopulations. J. Virol. Methods 224, 58–66 (2015).

    CAS  PubMed  Google Scholar 

Download references

Acknowledgements

The authors acknowledge E. Lai (Memorial Sloan-Kettering Cancer Center) for providing mouse embryonic fibroblast cell lines (WT, Ago2−/− and Ago2−/− rescued with Ago2). R.R. was funded in part by a scholarship in translational research from the Centre for Neuromuscular Disease and the University of Ottawa Brain and Mind Research Institute. This research was funded by grants from the Canadian Institutes of Health Research (proof of principle grant, PPP-141720), the National Research and Engineering Council of Canada (discovery grant no. 436104), The Quebec Consortium for Drug Discovery (CQDM Explore grant) and the ALS Association Treat Program (grant no. 15-LGCA-290) awarded to D.G.

Author information

Author notes

  1. These authors contributed equally: Ryan Reshke, James A. Taylor, Alexandre Savard.

Authors and Affiliations

  1. Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa, Canada

    Ryan Reshke, James A. Taylor, Alexandre Savard, Huishan Guo, My Tran Trung, Charles Campbell & Derrick Gibbings

  2. Ottawa Institute of Systems Biology, University of Ottawa, Ottawa, Canada

    Ryan Reshke, James A. Taylor, Alexandre Savard, Huishan Guo, My Tran Trung, Charles Campbell & Derrick Gibbings

  3. Éric Poulin Center for Neuromuscular Disease, University of Ottawa, Ottawa, Canada

    Ryan Reshke & Derrick Gibbings

  4. The Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA, USA

    Luke H. Rhym, Piotr S. Kowalski & Daniel G. Anderson

  5. Central Nervous System Disorders Drug Discovery Unit, Takeda Pharmaceuticals, Cambridge, MA, USA

    Wheaton Little

  6. Brain and Mind Research Institute, Ottawa, Canada

    Derrick Gibbings

Authors
  1. Ryan Reshke
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. James A. Taylor
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Alexandre Savard
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Huishan Guo
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Luke H. Rhym
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Piotr S. Kowalski
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. My Tran Trung
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Charles Campbell
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Wheaton Little
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Daniel G. Anderson
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Derrick Gibbings
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

J.A.T. performed cloning, lentivirus production, northern blots, analysis of RNA enrichment in sEVs and absolute quantification of RNA in sEVs. A.S. maintained mouse colonies, performed injections and tissue collections, helped analyse sEVs distribution, performed western blots of sEVs, generated cultures of primary mixed motor neurons, performed some RT–qPCR and performed and analysed microscopy. R.R. produced sEVs, performed nanoparticle tracking analysis, performed tissue collections, labelled sEVs and analysed their distribution, and analysed RNA enrichment in sEVs and mRNA target knockdown by RT–qPCR. M.T.T. maintained mouse colonies, generated mouse protocols, genotyped mice and performed injections and tissue collections. C.C. helped establish protocol for primary mixed motor neuron culture and performed some analyses of miRNA levels in sEVs. H.G. performed western blots of sEVs and density gradient analyses of sEVs. L.H.R. and P.S.K. helped design lipid nanoparticle experiments and produced C12–200 lipid nanoparticles. D.G.A. helped design lipid nanoparticle experiments and supervised L.H.R. and P.S.K. W.L. helped design experiments. J.A.T., A.S. and R.R. analysed experiments and helped design experiments. D.G. conceived the project, designed experiments and wrote the manuscript.

Corresponding author

Correspondence to Derrick Gibbings.

Ethics declarations

Competing interests

J.A.T. and D.G. are inventors on a filed patent that claims the use of the pre-miR-451 backbone for enrichment of small RNAs in sEVs. The remaining authors declare no competing interests.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Information

Supplementary Tables 1–3, Supplementary Figs. 1–5 and unprocessed western blots.

Reporting Summary

Rights and permissions

Reprints and Permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Reshke, R., Taylor, J.A., Savard, A. et al. Reduction of the therapeutic dose of silencing RNA by packaging it in extracellular vesicles via a pre-microRNA backbone. Nat Biomed Eng 4, 52–68 (2020). https://doi.org/10.1038/s41551-019-0502-4

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41551-019-0502-4

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing