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Oncogene Knockdown via Active Loading of Small RNAs into Extracellular Vesicles by Sonication

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Abstract

Extracellular vesicles (EVs), including exosomes and microvesicles, have emerged as promising drug delivery vehicles for small RNAs (siRNA and miRNA) due to their natural role in intercellular RNA transport. However, the application of EVs for therapeutic RNA delivery may be limited by loading approaches that can induce cargo aggregation or degradation. Here, we report the use of sonication as a means to actively load functional small RNAs into EVs. Conditions under which EVs could be loaded with small RNAs with minimal detectable aggregation were identified, and EVs loaded with therapeutic siRNA via sonication were observed to be taken up by recipient cells and capable of target mRNA knockdown leading to reduced protein expression. This system was ultimately applied to reduce expression of HER2, an oncogenic receptor tyrosine kinase that critically mediates breast cancer development and progression, and could be extended to other therapeutic targets. These results define important parameters informing the application of sonication as a small RNA loading method for EVs and demonstrate the potential utility of this approach for versatile cancer therapy.

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References

  1. Alvarez-Erviti, L., Y. Seow, H. Yin, C. Betts, S. Lakhal, and M. J. Wood. Delivery of siRNA to the mouse brain by systemic injection of targeted exosomes. Nat. Biotechnol. 29:341–345, 2011.

    Article  Google Scholar 

  2. Andaloussi, E. L., I. Mager, X. O. Breakefield, and M. J. Wood. Extracellular vesicles: biology and emerging therapeutic opportunities. Nat. Rev. Drug Discov. 12:347–357, 2013.

    Article  Google Scholar 

  3. Arteaga, C. L., M. X. Sliwkowski, C. K. Osborne, E. A. Perez, F. Puglisi, and L. Gianni. Treatment of HER2-positive breast cancer: current status and future perspectives. Nat. Rev. Clin. Oncol. 9:16–32, 2012.

    Article  Google Scholar 

  4. Baselga, J., and S. M. Swain. Novel anticancer targets: revisiting ERBB2 and discovering ERBB3. Nat. Rev. Cancer. 9:463–475, 2009.

    Article  Google Scholar 

  5. Blenkiron, C., and E. A. Miska. miRNAs in cancer: approaches, aetiology, diagnostics and therapy. Hum. Mol. Genet. 16(Spec No 1):R106–R113, 2007.

    Article  Google Scholar 

  6. Bumcrot, D., M. Manoharan, V. Koteliansky, and D. W. Sah. RNAi therapeutics: a potential new class of pharmaceutical drugs. Nat. Chem. Biol. 2:711–719, 2006.

    Article  Google Scholar 

  7. Choudhury, A., J. Charo, S. K. Parapuram, R. C. Hunt, D. M. Hunt, B. Seliger, and R. Kiessling. Small interfering RNA (siRNA) inhibits the expression of the Her2/neu gene, upregulates HLA class I and induces apoptosis of Her2/neu positive tumor cell lines. Int. J. Cancer. 108:71–77, 2004.

    Article  Google Scholar 

  8. Coelho, T., D. Adams, A. Silva, P. Lozeron, P. N. Hawkins, T. Mant, J. Perez, J. Chiesa, S. Warrington, E. Tranter, M. Munisamy, R. Falzone, J. Harrop, J. Cehelsky, B. R. Bettencourt, M. Geissler, J. S. Butler, A. Sehgal, R. E. Meyers, Q. Chen, T. Borland, R. M. Hutabarat, V. A. Clausen, R. Alvarez, K. Fitzgerald, C. Gamba-Vitalo, S. V. Nochur, A. K. Vaishnaw, D. W. Sah, J. A. Gollob, and O. B. Suhr. Safety and efficacy of RNAi therapy for transthyretin amyloidosis. N. Engl. J. Med. 369:819–829, 2013.

    Article  Google Scholar 

  9. Cooper, J. M., P. B. Wiklander, J. Z. Nordin, R. Al-Shawi, M. J. Wood, M. Vithlani, A. H. Schapira, J. P. Simons, S. El-Andaloussi, and L. Alvarez-Erviti. Systemic exosomal siRNA delivery reduced alpha-synuclein aggregates in brains of transgenic mice. Mov. Disord. 29:1476–1485, 2014.

    Article  Google Scholar 

  10. De Jong, W. H., and P. J. Borm. Drug delivery and nanoparticles: applications and hazards. Int. J. Nanomed. 3:133–149, 2008.

    Article  Google Scholar 

  11. El-Andaloussi, S., Y. Lee, S. Lakhal-Littleton, J. Li, Y. Seow, C. Gardiner, L. Alvarez-Erviti, I. L. Sargent, and M. J. Wood. Exosome-mediated delivery of siRNA in vitro and in vivo. Nat. Protoc. 7:2112–2126, 2012.

    Article  Google Scholar 

  12. Esmaeilzadeh-Gharehdaghi, E., A. Amani, M. R. Khoshayand, M. Banan, E. Esmaeilzadeh-Gharehdaghi, M. A. Amini, and M. A. Faramarzi. Chitosan nanoparticles for siRNA delivery: optimization of processing/formulation parameters. Nucleic Acid Ther. 24:420–427, 2014.

    Article  Google Scholar 

  13. Faltus, T., J. Yuan, B. Zimmer, A. Kramer, S. Loibl, M. Kaufmann, and K. Strebhardt. Silencing of the HER2/neu gene by siRNA inhibits proliferation and induces apoptosis in HER2/neu-overexpressing breast cancer cells. Neoplasia 6:786–795, 2004.

    Article  Google Scholar 

  14. Farooqi, A. A., Z. U. Rehman, and J. Muntane. Antisense therapeutics in oncology: current status. Onco Targets Ther. 7:2035–2042, 2014.

    Article  Google Scholar 

  15. Gavrilov, K., and W. M. Saltzman. Therapeutic siRNA: principles, challenges, and strategies. Yale J. Biol. Med. 85:187–200, 2012.

    Google Scholar 

  16. Gyorgy, B., M. E. Hung, X. O. Breakefield, and J. N. Leonard. Therapeutic applications of extracellular vesicles: clinical promise and open questions. Annu. Rev. Pharmacol. Toxicol. 55:439–464, 2015.

    Article  Google Scholar 

  17. Haney, M. J., N. L. Klyachko, Y. Zhao, R. Gupta, E. G. Plotnikova, Z. He, T. Patel, A. Piroyan, M. Sokolsky, A. V. Kabanov, and E. V. Batrakova. Exosomes as drug delivery vehicles for Parkinson’s disease therapy. J. Control Release 207:18–30, 2015.

    Article  Google Scholar 

  18. Kim, M. S., M. J. Haney, Y. Zhao, V. Mahajan, I. Deygen, N. L. Klyachko, E. Inskoe, A. Piroyan, M. Sokolsky, O. Okolie, S. D. Hingtgen, A. V. Kabanov, and E. V. Batrakova. Development of exosome-encapsulated paclitaxel to overcome MDR in cancer cells. Nanomedicine 12(3):655–664, 2016.

    Google Scholar 

  19. Kooijmans, S. A., S. Stremersch, K. Braeckmans, S. C. de Smedt, A. Hendrix, M. J. Wood, R. M. Schiffelers, K. Raemdonck, and P. Vader. Electroporation-induced siRNA precipitation obscures the efficiency of siRNA loading into extracellular vesicles. J. Control Release 172:229–238, 2013.

    Article  Google Scholar 

  20. Kundu, A. K., P. K. Chandra, S. Hazari, Y. V. Pramar, S. Dash, and T. K. Mandal. Development and optimization of nanosomal formulations for siRNA delivery to the liver. Eur. J. Pharm. Biopharm. 80:257–267, 2012.

    Article  Google Scholar 

  21. Lamichhane, T. N., R. S. Raiker, and S. M. Jay. Exogenous DNA loading into extracellular vesicles via electroporation is size-dependent and enables limited gene delivery. Mol. Pharm. 12:3650–3657, 2015.

    Article  Google Scholar 

  22. Lamichhane, T. N., S. Sokic, J. S. Schardt, R. S. Raiker, J. W. Lin, and S. M. Jay. Emerging roles for extracellular vesicles in tissue engineering and regenerative medicine. Tissue Eng. Part B Rev. 21:45–54, 2015.

    Article  Google Scholar 

  23. McClorey, G., and M. J. Wood. An overview of the clinical application of antisense oligonucleotides for RNA-targeting therapies. Curr. Opin. Pharmacol. 24:52–58, 2015.

    Article  Google Scholar 

  24. Moasser, M. M. The oncogene HER2: its signaling and transforming functions and its role in human cancer pathogenesis. Oncogene 26:6469–6487, 2007.

    Article  Google Scholar 

  25. Munoz, J. L., S. A. Bliss, S. J. Greco, S. H. Ramkissoon, K. L. Ligon, and P. Rameshwar. Delivery of functional anti-miR-9 by mesenchymal stem cell-derived exosomes to glioblastoma multiforme cells conferred chemosensitivity. Mol. Ther. Nucleic Acids 2:e126, 2013.

    Article  Google Scholar 

  26. Ohno, S., M. Takanashi, K. Sudo, S. Ueda, A. Ishikawa, N. Matsuyama, K. Fujita, T. Mizutani, T. Ohgi, T. Ochiya, N. Gotoh, and M. Kuroda. Systemically injected exosomes targeted to EGFR deliver antitumor microRNA to breast cancer cells. Mol. Ther. 21:185–191, 2013.

    Article  Google Scholar 

  27. Peer, D., and J. Lieberman. Special delivery: targeted therapy with small RNAs. Gene Ther. 18:1127–1133, 2011.

    Article  Google Scholar 

  28. Smyth, T., M. Kullberg, N. Malik, P. Smith-Jones, M. W. Graner, and T. J. Anchordoquy. Biodistribution and delivery efficiency of unmodified tumor-derived exosomes. J. Control Release. 199:145–155, 2015.

    Article  Google Scholar 

  29. Tan, W. B., S. Jiang, and Y. Zhang. Quantum-dot based nanoparticles for targeted silencing of HER2/neu gene via RNA interference. Biomaterials 28:1565–1571, 2007.

    Article  Google Scholar 

  30. Thiel, K. W., L. I. Hernandez, J. P. Dassie, W. H. Thiel, X. Liu, K. R. Stockdale, A. M. Rothman, F. J. Hernandez, J. O. McNamara, 2nd, and P. H. Giangrande. Delivery of chemo-sensitizing siRNAs to HER2 + -breast cancer cells using RNA aptamers. Nucleic Acids Res. 40:6319–6337, 2012.

    Article  Google Scholar 

  31. Valadi, H., K. Ekstrom, A. Bossios, M. Sjostrand, J. J. Lee, and J. O. Lotvall. Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells. Nat. Cell Biol. 9:654–659, 2007.

    Article  Google Scholar 

  32. Whitehead, K. A., R. Langer, and D. G. Anderson. Knocking down barriers: advances in siRNA delivery. Nat. Rev. Drug Discov. 8:129–138, 2009.

    Article  Google Scholar 

  33. Wichmann, H., A. Guttler, M. Bache, H. Taubert, S. Rot, J. Kessler, A. W. Eckert, M. Kappler, and D. Vordermark. Targeting of EGFR and HER2 with therapeutic antibodies and siRNA: a comparative study in glioblastoma cells. Strahlenther. Onkol. 191:180–191, 2015.

    Article  Google Scholar 

  34. Wiklander, O. P., J. Z. Nordin, A. O’Loughlin, Y. Gustafsson, G. Corso, I. Mager, P. Vader, Y. Lee, H. Sork, Y. Seow, N. Heldring, L. Alvarez-Erviti, C. E. Smith, K. Le Blanc, P. Macchiarini, P. Jungebluth, M. J. Wood, and S. E. Andaloussi. Extracellular vesicle in vivo biodistribution is determined by cell source, route of administration and targeting. J. Extracell. Vesicles. 4:26316, 2015.

    Article  Google Scholar 

  35. Wittrup, A., and J. Lieberman. Knocking down disease: a progress report on siRNA therapeutics. Nat. Rev. Genet. 16:543–552, 2015.

    Article  Google Scholar 

  36. Zhao, D., Y. Sui, and X. Zheng. miR-331-3p inhibits proliferation and promotes apoptosis by targeting HER2 through the PI3K/Akt and ERK1/2 pathways in colorectal cancer. Oncol. Rep. 35:1075–1082, 2016.

    Google Scholar 

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Acknowledgments

This work was supported by NIH R00 Grant HL112905, by an ORAU Ralph E. Powe Junior Faculty Enhancement Award, and by two University of Maryland Tier 1 seed Grants (all to SMJ).

Author Contributions

TNL, AJ, DBP, BP, NKL, NA and JSS performed the research and analyzed data. TNL, AJ, DBP and SMJ contributed to conception and design of experiments and wrote the manuscript. All authors reviewed, edited and approved of the final manuscript.

Conflict of Interest

Authors Tek N. Lamichhane, Anjana Jeyaram, Divya B. Patel, Babita Parajuli, Natalie K. Livingston, Navein Arumugasaamy, John S. Schardt and Steven M. Jay declare that they have no conflicts of interest.

Ethics and Informed Consent

No human studies were carried out by the authors for this article. No animal studies were carried out by the authors for this article.

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Correspondence to Steven M. Jay.

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Associate Editor Tejal Desai oversaw the review of this article.

Steven M. Jay

is an Assistant Professor in the Fischell Department of Bioengineering at the University of Maryland. Through generous support from the University and the National Institutes of Health (NIH), he has established a laboratory focused on biotherapeutic development and delivery, with specific interests in cancer therapy and vascular regeneration. Originally from the great state of Kentucky, Dr. Jay received a B.S.B.E. (Biological Engineering) from the University of Georgia before completing his Ph.D. in Biomedical Engineering under Dr. Mark Saltzman at Yale University. He then received postdoctoral training jointly from Dr. Richard T. Lee at the Brigham and Women’s Hospital and Dr. Linda Griffith at the Massachusetts Institute of Technology, and has also trained with Drs. Brian Rymond and Russ Mumper (University of Kentucky), Drs. Karen Burg and Thomas Jenkins (Clemson University), Dr. William Kisaalita (University of Georgia) and Drs. Themis Kyriakides and Jordan Pober (Yale University) among others. In addition to the NIH and the University of Maryland, Dr. Jay’s work has been supported by the National Science Foundation and the Oak Ridge Associated Universities. Dr. Jay’s research is enabled by the fortitude, diligence and intelligence of his trainees and by inspiration from his colleagues, friends and family.

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Lamichhane, T.N., Jeyaram, A., Patel, D.B. et al. Oncogene Knockdown via Active Loading of Small RNAs into Extracellular Vesicles by Sonication. Cel. Mol. Bioeng. 9, 315–324 (2016). https://doi.org/10.1007/s12195-016-0457-4

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