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Exosome-mediated delivery of siRNA in vitro and in vivo

Nature Protocols volume 7, pages 2112–2126 (2012)Cite this article

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Abstract

The use of small interfering RNAs (siRNAs) to induce gene silencing has opened a new avenue in drug discovery. However, their therapeutic potential is hampered by inadequate tissue-specific delivery. Exosomes are promising tools for drug delivery across different biological barriers. Here we show how exosomes derived from cultured cells can be harnessed for delivery of siRNA in vitro and in vivo. This protocol first describes the generation of targeted exosomes through transfection of an expression vector, comprising an exosomal protein fused with a peptide ligand. Next, we explain how to purify and characterize exosomes from transfected cell supernatant. Next, we detail crucial steps for loading siRNA into exosomes. Finally, we outline how to use exosomes to efficiently deliver siRNA in vitro and in vivo in mouse brain. Examples of anticipated results in which exosome-mediated siRNA delivery is evaluated by functional assays and imaging are also provided. The entire protocol takes ∼3 weeks.

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Figure 1
Figure 2: The cloning site for introducing the targeting ligands between XhoI and BspEI at the N terminus of Lamp2b.
Figure 3: Sample NTA profiles of exosomes from BMDCs (a) and HEK cells (b).
Figure 4: Typical flow cytometry profiles of BMDC-derived exosomes, using LAMP-1 and MHC-II as exosomal markers.
Figure 5: Typical exosome treatment results in vitro.
Figure 6: RNAi response of BACE1 in the cortex of mice.

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Acknowledgements

S.E.-A. was supported by a postdoctoral research fellowship from the Swedish Society of Medical Research (SSMF). C.G. was supported by a Wellcome Trust Technology Development Grant GR087730. Y.L. is funded by the Agency of Science, Technology and Research (A*STAR), Singapore.

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Author notes

  1. Samir El-Andaloussi, Yi Lee and Samira Lakhal-Littleton: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, UK

    Samir El-Andaloussi, Yi Lee, Samira Lakhal-Littleton, Jinghuan Li & Matthew J A Wood

  2. Department of Laboratory Medicine, Karolinska Institutet, Huddinge, Sweden

    Samir El-Andaloussi

  3. Molecular Engineering Laboratory, Proteos, Agency for Science Technology and Research, Singapore, Singapore

    Yiqi Seow

  4. Nuffield Department of Obstetrics and Gynaecology, John Radcliffe Hospital, Oxford, UK

    Chris Gardiner & Ian L Sargent

  5. University Department of Clinical Neurosciences, Institute of Neurology, University College London, London, UK

    Lydia Alvarez-Erviti

Authors
  1. Samir El-Andaloussi
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Contributions

M.J.A.W., L.A.-E. and Y.S. initially developed the protocol. S.E.-A., S.L.-L., Y.L. and M.J.A.W. drafted and wrote most of the protocol. All authors contributed with data: S.E.-A. performed in vitro knockdown studies with Tat exosomes, Y.L. provided the CellMask data, S.L.-L. ran flow cytometry and provided BACE-1 in vitro RNAi data, Y.S. performed the cloning and provided the constructs, J.L. wrote the western blotting protocol, L.A.-E. provided in vivo RNAi data and western blot photographs, and C.G. and I.L.S. performed the NTA analysis.

Corresponding author

Correspondence to Matthew J A Wood.

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Competing interests

Patents filed are as follows: WO2010/119256, priority date April 2009; UK1121070.5 and UK1121069.7, filed December 2011.

Supplementary information

Supplementary Figure 1

Detection of exosomal markers by western blot. Flotillin-1 and Lamp-1 are both present in BMDC cells (C) and BMDC-derived exosomes (E) using 20μg of protein sample for loading on gels. These markers are normally membrane proteins that are present on the surface of exosomes and inside cells depending on the cell type. A list of exosomal markers found in exosomes derived from different cells and organisms can be found in the exosome database: ExoCarta (http://www.exocarta.org/). (PDF 358 kb)

Supplementary Figure 2

Knockdown of BACE-1 in N2A cells following treatment with 10μg RVG-exosomes per well and increasing amounts of siRNA in a 24 well plate. Optimal ratio is 1:1 according to the observed RNAi responses. This ratio might vary between cells wherefore it is advised to screen the ratio in initial experiments. N=3 and error bars represents the SD. (PDF 324 kb)

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El-Andaloussi, S., Lee, Y., Lakhal-Littleton, S. et al. Exosome-mediated delivery of siRNA in vitro and in vivo. Nat Protoc 7, 2112–2126 (2012). https://doi.org/10.1038/nprot.2012.131

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