Skip to main content
SpringerLink
Log in

Loading of Extracellular Vesicles with Hydrophobically Modified siRNAs

  • Protocol
Extracellular RNA

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1740))

Abstract

Delivery represents a significant barrier to the clinical advancement of oligonucleotide therapeutics. Small, endogenous extracellular vesicles (EVs) have the potential to act as oligonucleotide delivery vehicles, but robust and scalable methods for loading RNA therapeutic cargo into vesicles are lacking. Here we describe the efficient loading of hydrophobically modified siRNAs (hsiRNAs) into EVs upon co-incubation, without altering vesicle size distribution or integrity. This method is expected to advance the development of EV-based therapies for the treatment of a broad range of disorders.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 69.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 89.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 119.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Khvorova A, Watts JK (2017) The chemical evolution of oligonucleotide therapies of clinical utility. Nat Biotechnol 35(3):238–248. https://doi.org/10.1038/nbt.3765

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Byrne M, Tzekov R, Wang Y, Rodgers A, Cardia J, Ford G, Holton K, Pandarinathan L, Lapierre J, Stanney W, Bulock K, Shaw S, Libertine L, Fettes K, Khvorova A, Kaushal S, Pavco P (2013) Novel hydrophobically modified asymmetric RNAi compounds (sd-rxRNA) demonstrate robust efficacy in the eye. J Ocul Pharmacol Ther 29(10):855–864. https://doi.org/10.1089/jop.2013.0148

    Article  CAS  PubMed  Google Scholar 

  3. Geary RS, Norris D, Yu R, Bennett CF (2015) Pharmacokinetics, biodistribution and cell uptake of antisense oligonucleotides. Adv Drug Deliv Rev 87:46–51. https://doi.org/10.1016/j.addr.2015.01.008

    Article  CAS  PubMed  Google Scholar 

  4. Alterman JF, Hall LM, Coles AH, Hassler MR, Didiot MC, Chase K, Abraham J, Sottosanti E, Johnson E, Sapp E, Osborn MF, Difiglia M, Aronin N, Khvorova A (2015) Hydrophobically modified siRNAs silence huntingtin mRNA in primary neurons and mouse brain. Mol Ther Nucleic Acids 4:e266. https://doi.org/10.1038/mtna.2015.38

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Ly S, Navaroli D, Didiot M, Cardia J, Pandarinathan L, Alterman J, Fogarty K, Standley C, Lifshitz L, Bellve K, Prot M, Echeverria D, Corvera S, Khvorova A (2016) Visualization of self-delivering hydrophobically modified siRNA cellular internalization. Nucleic Acids Res. https://doi.org/10.1093/nar/gkw1005

  6. Distler JH, Huber LC, Hueber AJ, Reich CF 3rd, Gay S, Distler O, Pisetsky DS (2005) The release of microparticles by apoptotic cells and their effects on macrophages. Apoptosis 10(4):731–741. https://doi.org/10.1007/s10495-005-2941-5

    Article  CAS  PubMed  Google Scholar 

  7. Muralidharan-Chari V, Clancy JW, Sedgwick A, D'Souza-Schorey C (2010) Microvesicles: mediators of extracellular communication during cancer progression. J Cell Sci 123(Pt 10):1603–1611. https://doi.org/10.1242/jcs.064386

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Tetta C, Ghigo E, Silengo L, Deregibus MC, Camussi G (2013) Extracellular vesicles as an emerging mechanism of cell-to-cell communication. Endocrine 44(1):11–19. https://doi.org/10.1007/s12020-012-9839-0

    Article  CAS  PubMed  Google Scholar 

  9. Alvarez-Erviti L, Seow Y, Yin H, Betts C, Lakhal S, Wood MJ (2011) Delivery of siRNA to the mouse brain by systemic injection of targeted exosomes. Nat Biotechnol 29(4):341–345. https://doi.org/10.1038/nbt.1807

    Article  CAS  PubMed  Google Scholar 

  10. Cooper JM, Wiklander PB, Nordin JZ, Al-Shawi R, Wood MJ, Vithlani M, Schapira AH, Simons JP, El-Andaloussi S, Alvarez-Erviti L (2014) Systemic exosomal siRNA delivery reduced alpha-synuclein aggregates in brains of transgenic mice. Mov Disord 29(12):1476–1485. https://doi.org/10.1002/mds.25978

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Ohno S, Takanashi M, Sudo K, Ueda S, Ishikawa A, Matsuyama N, Fujita K, Mizutani T, Ohgi T, Ochiya T, Gotoh N, Kuroda M (2013) Systemically injected exosomes targeted to EGFR deliver antitumor microRNA to breast cancer cells. Mol Ther 21(1):185–191. https://doi.org/10.1038/mt.2012.180

    Article  CAS  PubMed  Google Scholar 

  12. Mizrak A, Bolukbasi MF, Ozdener GB, Brenner GJ, Madlener S, Erkan EP, Strobel T, Breakefield XO, Saydam O (2013) Genetically engineered microvesicles carrying suicide mRNA/protein inhibit schwannoma tumor growth. Mol Ther 21(1):101–108. https://doi.org/10.1038/mt.2012.161

    Article  CAS  PubMed  Google Scholar 

  13. Kosaka N, Iguchi H, Yoshioka Y, Hagiwara K, Takeshita F, Ochiya T (2012) Competitive interactions of cancer cells and normal cells via secretory microRNAs. J Biol Chem 287(2):1397–1405. https://doi.org/10.1074/jbc.M111.288662

    Article  CAS  PubMed  Google Scholar 

  14. Kooijmans SA, Stremersch S, Braeckmans K, de Smedt SC, Hendrix A, Wood MJ, Schiffelers RM, Raemdonck K, Vader P (2013) Electroporation-induced siRNA precipitation obscures the efficiency of siRNA loading into extracellular vesicles. J Control Release 172(1):229–238. https://doi.org/10.1016/j.jconrel.2013.08.014

    Article  CAS  PubMed  Google Scholar 

  15. Didiot MC, Hall LM, Coles AH, Haraszti RA, Godinho BM, Chase K, Sapp E, Ly S, Alterman JF, Hassler MR, Echeverria D, Raj L, Morrissey DV, DiFiglia M, Aronin N, Khvorova A (2016) Exosome-mediated delivery of hydrophobically modified siRNA for huntingtin mRNA silencing. Mol Ther. https://doi.org/10.1038/mt.2016.126

  16. Wang K, Zhang S, Weber J, Baxter D, Galas DJ (2010) Export of microRNAs and microRNA-protective protein by mammalian cells. Nucleic Acids Res 38(20):7248–7259. https://doi.org/10.1093/nar/gkq601

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Pegtel DM, Cosmopoulos K, Thorley-Lawson DA, van Eijndhoven MA, Hopmans ES, Lindenberg JL, de Gruijl TD, Wurdinger T, Middeldorp JM (2010) Functional delivery of viral miRNAs via exosomes. Proc Natl Acad Sci U S A 107(14):6328–6333. https://doi.org/10.1073/pnas.0914843107

    Article  PubMed  PubMed Central  Google Scholar 

  18. Valadi H, Ekstrom K, Bossios A, Sjostrand M, Lee JJ, Lotvall JO (2007) Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells. Nat Cell Biol 9(6):654–659. https://doi.org/10.1038/ncb1596

    Article  CAS  PubMed  Google Scholar 

  19. El Andaloussi S, Lakhal S, Mager I, Wood MJ (2013) Exosomes for targeted siRNA delivery across biological barriers. Adv Drug Deliv Rev 65(3):391–397. https://doi.org/10.1016/j.addr.2012.08.008

    Article  CAS  PubMed  Google Scholar 

  20. Kooijmans SA, Vader P, van Dommelen SM, van Solinge WW, Schiffelers RM (2012) Exosome mimetics: a novel class of drug delivery systems. Int J Nanomedicine 7:1525–1541. https://doi.org/10.2147/IJN.S29661

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Pan Q, Ramakrishnaiah V, Henry S, Fouraschen S, de Ruiter PE, Kwekkeboom J, Tilanus HW, Janssen HL, van der Laan LJ (2012) Hepatic cell-to-cell transmission of small silencing RNA can extend the therapeutic reach of RNA interference (RNAi). Gut 61(9):1330–1339. https://doi.org/10.1136/gutjnl-2011-300449

    Article  CAS  PubMed  Google Scholar 

  22. Lasser C (2012) Exosomal RNA as biomarkers and the therapeutic potential of exosome vectors. Expert Opin Biol Ther 12(Suppl 1):S189–S197. https://doi.org/10.1517/14712598.2012.680018

    Article  CAS  PubMed  Google Scholar 

  23. Lee Y, El Andaloussi S, Wood MJ (2012) Exosomes and microvesicles: extracellular vesicles for genetic information transfer and gene therapy. Hum Mol Genet 21(R1):R125–R134. https://doi.org/10.1093/hmg/dds317

    Article  CAS  PubMed  Google Scholar 

  24. Marcus ME, Leonard JN (2013) FedExosomes: engineering therapeutic biological nanoparticles that truly deliver. Pharmaceuticals (Basel) 6(5):659–680. https://doi.org/10.3390/ph6050659

    Article  CAS  Google Scholar 

  25. Nazarenko I, Rupp AK, Altevogt P (2013) Exosomes as a potential tool for a specific delivery of functional molecules. Methods Mol Biol 1049:495–511. https://doi.org/10.1007/978-1-62703-547-7_37

    Article  CAS  PubMed  Google Scholar 

  26. Zomer A, Vendrig T, Hopmans ES, van Eijndhoven M, Middeldorp JM, Pegtel DM (2010) Exosomes: fit to deliver small RNA. Commun Integr Biol 3(5):447–450. https://doi.org/10.4161/cib.3.5.12339

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Thery C, Amigorena S, Raposo G, Clayton A (2006) Isolation and characterization of exosomes from cell culture supernatants and biological fluids. Curr Protoc Cell Biol. Chapter 3:Unit 3.22. https://doi.org/10.1002/0471143030.cb0322s30

  28. Lotvall J, Hill AF, Hochberg F, Buzas EI, Di Vizio D, Gardiner C, Gho YS, Kurochkin IV, Mathivanan S, Quesenberry P, Sahoo S, Tahara H, Wauben MH, Witwer KW, Thery C (2014) Minimal experimental requirements for definition of extracellular vesicles and their functions: a position statement from the International Society for Extracellular Vesicles. J Extracell Vesicles 3:26913. https://doi.org/10.3402/jev.v3.26913

    Article  PubMed  Google Scholar 

  29. Roehl I, Schuster M, Seiffert S (2011) Oligonucleotide detection method. US Patent Application US20110201006A1

    Google Scholar 

Download references

Acknowledgments

We thank the members of the Khvorova and Aronin Laboratories, NIH Extracellular RNA Consortium and CHDI Foundation Inc. for helpful discussions. This work is supported in part NIH UH2-UH3 grant TR 000888 05 to N.A. and A.K., NIH grants RO1GM10880304, RO1NS10402201, and S10 OD020012 to A.K. and CHDI Foundation (Research Agreement A-6119, JSC A6367) to N.A. Marie-Cecile Didiot was supported by Huntington’s Disease Society of America Postdoctoral Fellowship. The authors declare no conflict of interest.

Author information

Authors and Affiliations

  1. RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, MA, USA

    Marie-Cecile Didiot, Reka A. Haraszti, Neil Aronin & Anastasia Khvorova

  2. Department of Molecular Medicine, University of Massachusetts Medical School, Worcester, MA, USA

    Marie-Cecile Didiot, Reka A. Haraszti & Anastasia Khvorova

  3. Department of Medicine, University of Massachusetts Medical School, Worcester, MA, USA

    Neil Aronin

Authors
  1. Marie-Cecile Didiot
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Reka A. Haraszti
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Neil Aronin
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Anastasia Khvorova
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Anastasia Khvorova .

Editor information

Editors and Affiliations

  1. Department of Transplantation, Mayo Clinic, Jacksonville, Florida, USA

    Tushar Patel

Rights and permissions

Reprints and permissions

Copyright information

© 2018 Springer Science+Business Media, LLC

About this protocol

Cite this protocol

Didiot, MC., Haraszti, R.A., Aronin, N., Khvorova, A. (2018). Loading of Extracellular Vesicles with Hydrophobically Modified siRNAs. In: Patel, T. (eds) Extracellular RNA. Methods in Molecular Biology, vol 1740. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7652-2_16

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-7652-2_16

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7651-5

  • Online ISBN: 978-1-4939-7652-2

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation