Skip to main content

Continuous Delivery of Oligonucleotides into the Brain

  • Protocol
  • First Online:
MicroRNA Technologies

Part of the book series: Neuromethods ((NM,volume 128))

Abstract

The growing field of RNA neurobiology dictates development and improvement of effective and reliable in vivo techniques to address the function of particular microRNA molecules within the brain. Here we describe a novel method involving continuous delivery of oligonucleotides into a brain region of interest by osmotic pump infusion. The approach implements application of double-stranded microRNA-mimics with only two LNA moieties at the 3′-end and additionally one at the 5′-end of the sense strand. This method holds promise for long-lasting and specific siRNA upregulation in vivo, especially in the Dicer-depleted systems, where other approaches are limited or not applicable. Being robust and effective, various techniques described in this chapter can be easily modified in order to achieve up- or downregulation of expression of specific RNA molecules, bi- or unilateral infusions or injections, and in vivo “screening” strategy allowing to start from a bigger group of RNA molecules and end up with identification of single RNA species critical for a phenotype.

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

Access this chapter

Subscribe and save

Springer+ Basic
EUR 32.99 /Month
  • Get 10 units per month
  • Download Article/Chapter or eBook
  • 1 Unit = 1 Article or 1 Chapter
  • Cancel anytime
Subscribe now

Buy Now

Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 84.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 159.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 109.99
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

Similar content being viewed by others

References

  1. Bartel DP (2009) MicroRNAs: target recognition and regulatory functions. Cell 136(2):215–233. doi:10.1016/j.cell.2009.01.002, S0092-8674(09)00008-7 [pii]

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Mikl M, Vendra G, Doyle M, Kiebler MA (2010) RNA localization in neurite morphogenesis and synaptic regulation: current evidence and novel approaches. J Comp Physiol A Neuroethol Sens Neural Behav Physiol 196(5):321–334. doi:10.1007/s00359-010-0520-x

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Schratt G (2009) microRNAs at the synapse. Nat Rev Neurosci 10(12):842–849. doi:10.1038/nrn2763, nrn2763 [pii]

    Article  CAS  PubMed  Google Scholar 

  4. Konopka W, Schutz G, Kaczmarek L (2011) The microRNA contribution to learning and memory. Neuroscientist 17(5):468–474. doi:10.1177/1073858411411721

    Article  CAS  PubMed  Google Scholar 

  5. Konopka W, Kiryk A, Novak M, Herwerth M, Parkitna JR, Wawrzyniak M, Kowarsch A, Michaluk P, Dzwonek J, Arnsperger T, Wilczynski G, Merkenschlager M, Theis FJ, Kohr G, Kaczmarek L, Schutz G (2010) MicroRNA loss enhances learning and memory in mice. J Neurosci 30(44):14835–14842. doi:10.1523/JNEUROSCI.3030-10.2010, 30/44/14835 [pii]

    Article  CAS  PubMed  Google Scholar 

  6. Vinnikov IA, Hajdukiewicz K, Reymann J, Beneke J, Czajkowski R, Roth LC, Novak M, Roller A, Dörner N, Starkuviene V, Theis FJ, Erfle H, Schütz G, Grinevich V, Konopka W (2014) Hypothalamic miR-103 protects from hyperphagic obesity in mice. J Neurosci 34(32):10659–10674. doi:10.1523/jneurosci.4251-13.2014

    Article  PubMed  Google Scholar 

  7. Verma P, Augustine GJ, Ammar MR, Tashiro A, Cohen SM (2015) A neuroprotective role for microRNA miR-1000 mediated by limiting glutamate excitotoxicity. Nat Neurosci 18(3):379–385. doi:10.1038/nn.3935

    CAS  PubMed  Google Scholar 

  8. Gao J, Wang WY, Mao YW, Graff J, Guan JS, Pan L, Mak G, Kim D, Su SC, Tsai LH (2010) A novel pathway regulates memory and plasticity via SIRT1 and miR-134. Nature 466(7310):1105–1109. doi:10.1038/nature09271, nature09271 [pii]

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Tan CL, Plotkin JL, Veno MT, von Schimmelmann M, Feinberg P, Mann S, Handler A, Kjems J, Surmeier DJ, O’Carroll D, Greengard P, Schaefer A (2013) MicroRNA-128 governs neuronal excitability and motor behavior in mice. Science 342(6163):1254–1258. doi:10.1126/science.1244193

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Xie J, Ameres SL, Friedline R, Hung JH, Zhang Y, Xie Q, Zhong L, Su Q, He R, Li M, Li H, Mu X, Zhang H, Broderick JA, Kim JK, Weng Z, Flotte TR, Zamore PD, Gao G (2012) Long-term, efficient inhibition of microRNA function in mice using rAAV vectors. Nat Methods 9(4):403–409. doi:10.1038/nmeth.1903

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Ran FA, Cong L, Yan WX, Scott DA, Gootenberg JS, Kriz AJ, Zetsche B, Shalem O, Wu X, Makarova KS, Koonin EV, Sharp PA, Zhang F (2015) In vivo genome editing using Staphylococcus aureus Cas9. Nature. doi:10.1038/nature14299

    Google Scholar 

  12. Platt RJ, Chen S, Zhou Y, Yim MJ, Swiech L, Kempton HR, Dahlman JE, Parnas O, Eisenhaure TM, Jovanovic M, Graham DB, Jhunjhunwala S, Heidenreich M, Xavier RJ, Langer R, Anderson DG, Hacohen N, Regev A, Feng G, Sharp PA, Zhang F (2014) CRISPR-Cas9 knockin mice for genome editing and cancer modeling. Cell 159(2):440–455. doi:10.1016/j.cell.2014.09.014

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Christensen M, Larsen LA, Kauppinen S, Schratt G (2010) Recombinant adeno-associated virus-mediated microRNA delivery into the postnatal mouse brain reveals a role for miR-134 in dendritogenesis in vivo. Front Neural Circuits 3:16. doi:10.3389/neuro.04.016.2009

    PubMed  PubMed Central  Google Scholar 

  14. Zovoilis A, Agbemenyah HY, Agis-Balboa RC, Stilling RM, Edbauer D, Rao P, Farinelli L, Delalle I, Schmitt A, Falkai P, Bahari-Javan S, Burkhardt S, Sananbenesi F, Fischer A (2011) microRNA-34c is a novel target to treat dementias. EMBO J 30(20):4299–4308. doi:10.1038/emboj.2011.327

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Krützfeldt J, Rajewsky N, Braich R, Rajeev KG, Tuschl T, Manoharan M, Stoffel M (2005) Silencing of microRNAs in vivo with ‘antagomirs’. Nature 438(7068):685–689. doi:10.1038/nature04303

    Article  PubMed  Google Scholar 

  16. Krutzfeldt J, Kuwajima S, Braich R, Rajeev KG, Pena J, Tuschl T, Manoharan M, Stoffel M (2007) Specificity, duplex degradation and subcellular localization of antagomirs. Nucleic Acids Res 35(9):2885–2892. doi:10.1093/nar/gkm024, gkm024 [pii]

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Esau C, Davis S, Murray SF, Yu XX, Pandey SK, Pear M, Watts L, Booten SL, Graham M, McKay R, Subramaniam A, Propp S, Lollo BA, Freier S, Bennett CF, Bhanot S, Monia BP (2006) miR-122 regulation of lipid metabolism revealed by in vivo antisense targeting. Cell Metab 3(2):87–98. doi:10.1016/j.cmet.2006.01.005

    Article  CAS  PubMed  Google Scholar 

  18. Liu J, Yu D, Aiba Y, Pendergraff H, Swayze EE, Lima WF, Hu J, Prakash TP, Corey DR (2013) ss-siRNAs allele selectively inhibit ataxin-3 expression: multiple mechanisms for an alternative gene silencing strategy. Nucleic Acids Res 41(20):9570–9583. doi:10.1093/nar/gkt693

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Gatfield D, Le Martelot G, Vejnar CE, Gerlach D, Schaad O, Fleury-Olela F, Ruskeepaa AL, Oresic M, Esau CC, Zdobnov EM, Schibler U (2009) Integration of microRNA miR-122 in hepatic circadian gene expression. Genes Dev 23(11):1313–1326. doi:10.1101/gad.1781009

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Rayner KJ, Esau CC, Hussain FN, McDaniel AL, Marshall SM, van Gils JM, Ray TD, Sheedy FJ, Goedeke L, Liu X, Khatsenko OG, Kaimal V, Lees CJ, Fernandez-Hernando C, Fisher EA, Temel RE, Moore KJ (2011) Inhibition of miR-33a/b in non-human primates raises plasma HDL and lowers VLDL triglycerides. Nature 478(7369):404–407. doi:10.1038/nature10486

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Elmen J, Lindow M, Silahtaroglu A, Bak M, Christensen M, Lind-Thomsen A, Hedtjarn M, Hansen JB, Hansen HF, Straarup EM, McCullagh K, Kearney P, Kauppinen S (2007) Antagonism of microRNA-122 in mice by systemically administered LNA-antimiR leads to up-regulation of a large set of predicted target mRNAs in the liver. Nucleic Acids Res 36(4):1153–1162. doi:10.1093/nar/gkm1113

    Article  PubMed  PubMed Central  Google Scholar 

  22. Elmén J, Lindow M, Schütz S, Lawrence M, Petri A, Obad S, Lindholm M, Hedtjärn M, Hansen HF, Berger U, Gullans S, Kearney P, Sarnow P, Straarup EM, Kauppinen S (2008) LNA-mediated microRNA silencing in non-human primates. Nature 452(7189):896–899. doi:10.1038/nature06783

    Article  PubMed  Google Scholar 

  23. Lanford RE, Hildebrandt-Eriksen ES, Petri A, Persson R, Lindow M, Munk ME, Kauppinen S, Orum H (2009) Therapeutic silencing of microRNA-122 in primates with chronic hepatitis C virus infection. Science 327(5962):198–201. doi:10.1126/science.1178178

    Article  PubMed  PubMed Central  Google Scholar 

  24. Zhang Y, Wang Z, Gemeinhart RA (2013) Progress in microRNA delivery. J Control Release 172(3):962–974. doi:10.1016/j.jconrel.2013.09.015

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Elmen J, Thonberg H, Ljungberg K, Frieden M, Westergaard M, Xu Y, Wahren B, Liang Z, Orum H, Koch T, Wahlestedt C (2005) Locked nucleic acid (LNA) mediated improvements in siRNA stability and functionality. Nucleic Acids Res 33(1):439–447. doi:10.1093/nar/gki193, 33/1/439 [pii]

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Yu D, Pendergraff H, Liu J, Kordasiewicz HB, Cleveland DW, Swayze EE, Lima WF, Crooke ST, Prakash TP, Corey DR (2012) Single-stranded RNAs use RNAi to potently and allele-selectively inhibit mutant huntingtin expression. Cell 150(5):895–908. doi:10.1016/j.cell.2012.08.002

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Mook OR, Baas F, de Wissel MB, Fluiter K (2007) Evaluation of locked nucleic acid-modified small interfering RNA in vitro and in vivo. Mol Cancer Ther 6(3):833–843. doi:10.1158/1535-7163.mct-06-0195

    Article  CAS  PubMed  Google Scholar 

  28. Mong JA, Devidze N, Goodwillie A, Pfaff DW (2003) Reduction of lipocalin-type prostaglandin D synthase in the preoptic area of female mice mimics estradiol effects on arousal and sex behavior. Proc Natl Acad Sci U S A 100(25):15206–15211. doi:10.1073/pnas.2436540100

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Bramsen JB, Laursen MB, Damgaard CK, Lena SW, Ravindra Babu B, Wengel J, Kjems J (2007) Improved silencing properties using small internally segmented interfering RNAs. Nucleic Acids Res 35(17):5886–5897. doi:10.1093/nar/gkm548

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Su J, Baigude H, McCarroll J, Rana TM (2011) Silencing microRNA by interfering nanoparticles in mice. Nucleic Acids Res 39(6):e38. doi:10.1093/nar/gkq1307

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. John M, Constien R, Akinc A, Goldberg M, Moon Y-A, Spranger M, Hadwiger P, Soutschek J, Vornlocher H-P, Manoharan M, Stoffel M, Langer R, Anderson DG, Horton JD, Koteliansky V, Bumcrot D (2007) Effective RNAi-mediated gene silencing without interruption of the endogenous microRNA pathway. Nature 449(7163):745–747. doi:10.1038/nature06179

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Trajkovski M, Hausser J, Soutschek J, Bhat B, Akin A, Zavolan M, Heim MH, Stoffel M (2011) MicroRNAs 103 and 107 regulate insulin sensitivity. Nature 474(7353):649–653. doi:10.1038/nature10112, nature10112 [pii]

    Article  CAS  PubMed  Google Scholar 

  33. Lee ST, Chu K, Jung KH, Kim JH, Huh JY, Yoon H, Park DK, Lim JY, Kim JM, Jeon D, Ryu H, Lee SK, Kim M, Roh JK (2012) miR-206 regulates brain-derived neurotrophic factor in Alzheimer disease model. Ann Neurol 72(2):269–277. doi:10.1002/ana.23588

    Article  CAS  PubMed  Google Scholar 

  34. Cunningham MG, O’Connor RP, Wong SE (2008) Construction and implantation of a microinfusion system for sustained delivery of neuroactive agents. J Vis Exp. doi:10.3791/716, 716 [pii]

    PubMed  PubMed Central  Google Scholar 

  35. Zovoilis A, Agbemenyah HY, Agis-Balboa RC, Stilling RM, Edbauer D, Rao P, Farinelli L, Delalle I, Schmitt A, Falkai P, Bahari-Javan S, Burkhardt S, Sananbenesi F, Fischer A (2011) microRNA-34c is a novel target to treat dementias. EMBO J 30(20):4299–4308. doi:10.1038/emboj.2011.327, emboj2011327 [pii]

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Paxinos G, Franklin KBJ (2001) The mouse brain in stereotaxic coordinates, 2nd edn. Academic Press, San Diego

    Google Scholar 

Download references

Acknowledgments

The authors declare no competing financial interests. This work has been supported by the DFG through SFB488, the EU through grant LSHM-CT-2005-018652 (CRESCENDO), the BMBF through NGFNplus grants FZK-01GS08153 and 01GS08142, and the HGF through Initiative CoReNe (Network II, E2) the National Science Centre (Poland) grant (SONATA) 2011/01/D/NZ4/03744, grant (HARMONIA) 2013/08/M/NZ3/01045, and the Academy of Finland. We thank Jörg Krummheuer for the protocol for preparation of the LNA-oligonucleotides for injection as well as assistance in developing the oligonucleotide synthesis strategy, Günther Schütz for support, Lena Roth for assistance with the infusion technique. Special thanks goes to Adair Oesterle for assistance with techniques to fabricate micropipettes.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Ilya A. Vinnikov .

Editor information

Editors and Affiliations

Rights and permissions

Reprints and permissions

Copyright information

© 2016 Springer Science+Business Media New York

About this protocol

Cite this protocol

Vinnikov, I.A., Domanskyi, A., Konopka, W. (2016). Continuous Delivery of Oligonucleotides into the Brain. In: Kye, M. (eds) MicroRNA Technologies. Neuromethods, vol 128. Humana Press, New York, NY. https://doi.org/10.1007/7657_2016_3

Download citation

  • DOI: https://doi.org/10.1007/7657_2016_3

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7173-2

  • Online ISBN: 978-1-4939-7175-6

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics