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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

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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.

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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%.

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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.

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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.

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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

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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.

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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.

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

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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

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