Skip to main content
SpringerLink
Log in

Analysis of Oligonucleotide Biodistribution and Metabolization in Experimental Animals

  • Protocol
  • First Online:
Alternative Splicing

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2537))

Abstract

We describe methods to follow the fate of oligonucleotides after their injection into experimental animals. The quantitation in various tissues, blood or bone marrow cells is possible by chemical ligation PCR. This method works independently of chemical modifications of the oligonucleotide and/or its conjugations to lipid or peptide moieties. Moreover, metabolization intermediates can be detected by mass spectrometry. Together with a readout assay for the biochemical or physiological effects, which will differ, depending on the particular purpose of the oligonucleotide, these methods allow for a comprehensive understanding of oligonucleotide behavior in a living organism.

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 129.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 169.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 249.99
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. Stein CA, Castanotto D (2017) FDA-approved oligonucleotide therapies in 2017. Mol Ther 25(5):1069–1075. https://doi.org/10.1016/j.ymthe.2017.03.023

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Levin AA (2019) Treating disease at the RNA level with oligonucleotides. N Engl J Med 380(1):57–70. https://doi.org/10.1056/NEJMra1705346

    Article  PubMed  Google Scholar 

  3. Crooke ST, Wang S, Vickers TA, Shen W, Liang X-h (2017) Cellular uptake and trafficking of antisense oligonucleotides. Nat Biotechnol 35(3):230–237. https://doi.org/10.1038/nbt.3779

    Article  CAS  PubMed  Google Scholar 

  4. Crooke ST, Witztum JL, Bennett CF, Baker BF (2018) RNA-targeted therapeutics. Cell Metab 27(4):714–739. https://doi.org/10.1016/j.cmet.2018.03.004

    Article  CAS  PubMed  Google Scholar 

  5. Boos JA, Kirk DW, Piccolotto M-L, Zuercher W, Gfeller S, Neuner P, Dattler A, Wishart WL, Von Arx F, Beverly M, Christensen J, Litherland K, van de Kerkhof E, Swart PJ, Faller T, Beyerbach A, Morrissey D, Hunziker J, Beuvink I (2013) Whole-body scanning PCR; a highly sensitive method to study the biodistribution of mRNAs, noncoding RNAs and therapeutic oligonucleotides. Nucleic Acids Res 41(15):e145. https://doi.org/10.1093/nar/gkt515

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Brunschweiger A, Gebert LFR, Lucic M, Pradere U, Jahns H, Berk C, Hunziker J, Hall J (2016) Site-specific conjugation of drug-like fragments to an antimiR scaffold as a strategy to target miRNAs inside RISC. Chem Commun 52(1):156–159. https://doi.org/10.1039/C5CC07478A

    Article  CAS  Google Scholar 

  7. Halloy F, Iyer PS, Cwiek P, Ghidini A, Barman-Aksozen J, Wildner-Verhey van Wijk N, Theocharides APA, Minder EI, Schneider-Yin X, Schumperli D, Hall J (2020) Delivery of oligonucleotides to bone marrow to modulate ferrochelatase splicing in a mouse model of erythropoietic protoporphyria. Nucleic Acids Res 48(9):4658–4671. https://doi.org/10.1093/nar/gkaa229

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Kim J, Basiri B, Hassan C, Punt C, van der Hage E, den Besten C, Bartlett MG (2019) Metabolite profiling of the antisense oligonucleotide Eluforsen using liquid chromatography-mass spectrometry. Mol Ther Nucleic Acids 17:714–725. https://doi.org/10.1016/j.omtn.2019.07.006

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Sips L, Ediage EN, Ingelse B, Verhaeghe T, Dillen L (2019) LC–MS quantification of oligonucleotides in biological matrices with SPE or hybridization extraction. Bioanalysis 11(21):1941–1954. https://doi.org/10.4155/bio-2019-0117

    Article  CAS  PubMed  Google Scholar 

  10. Lecha M, Puy H, Deybach J-C (2009) Erythropoietic protoporphyria. Orphanet J Rare Dis 4(1):1–10. https://doi.org/10.1186/1750-1172-4-19

    Article  Google Scholar 

  11. Gouya L, Puy H, Robreau A-M, Bourgeois M, Lamoril J, Da Silva V, Grandchamp B, Deybach J-C (2002) The penetrance of dominant erythropoietic protoporphyria is modulated by expression of wildtype FECH. Nat Genet 30(1):27–28. http://www.nature.com/ng/journal/v30/n1/suppinfo/ng809_S1.html

    Article  CAS  Google Scholar 

  12. Amend SR, Valkenburg KC, Pienta KJ (2016) Murine hind limb long bone dissection and bone marrow isolation. J Vis Exp 110:53936. https://doi.org/10.3791/53936

    Article  CAS  Google Scholar 

  13. Turnpenny P, Rawal J, Schardt T, Lamoratta S, Mueller H, Weber M, Brady K (2011) Quantitation of locked nucleic acid antisense oligonucleotides in mouse tissue using a liquid-liquid extraction LC-MS/MS analytical approach. Bioanalysis 3(17):1911–1921. https://doi.org/10.4155/bio.11.100

    Article  CAS  PubMed  Google Scholar 

  14. Boos JA, Beuvink I (2016) Whole-body scanning PCR, a tool for the visualization of the in vivo biodistribution pattern of endogenous and exogenous oligonucleotides in rodents. In: Medarova Z (ed) RNA imaging: methods and protocols. Springer New York, New York, NY, pp 99–111. https://doi.org/10.1007/978-1-4939-3148-4_8

    Chapter  Google Scholar 

  15. Yu RZ, Geary RS, Monteith DK, Matson J, Truong L, Fitchett J, Levin AA (2004) Tissue disposition of 2′-O-(2-methoxy) ethyl modified antisense oligonucleotides in monkeys. J Pharm Sci 93(1):48–59. https://doi.org/10.1002/jps.10473

    Article  CAS  PubMed  Google Scholar 

  16. Nair JK, Attarwala H, Sehgal A, Wang Q, Aluri K, Zhang X, Gao M, Liu J, Indrakanti R, Schofield S, Kretschmer P, Brown CR, Gupta S, Willoughby JLS, Boshar JA, Jadhav V, Charisse K, Zimmermann T, Fitzgerald K, Manoharan M, Rajeev KG, Akinc A, Hutabarat R, Maier MA (2017) Impact of enhanced metabolic stability on pharmacokinetics and pharmacodynamics of GalNAc–siRNA conjugates. Nucleic Acids Res 45(19):10969–10977. https://doi.org/10.1093/nar/gkx818

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Baek M-S, Yu RZ, Gaus H, Grundy JS, Geary RS (2010) In vitro metabolic stabilities and metabolism of 2′-O-(methoxyethyl) partially modified Phosphorothioate antisense oligonucleotides in preincubated rat or human whole liver homogenates. Oligonucleotides 20(6):309–316. https://doi.org/10.1089/oli.2010.0252

    Article  CAS  PubMed  Google Scholar 

  18. Fontaine SD, Reid R, Robinson L, Ashley GW, Santi DV (2015) Long-term stabilization of maleimide–thiol conjugates. Bioconjug Chem 26(1):145–152. https://doi.org/10.1021/bc5005262

    Article  CAS  PubMed  Google Scholar 

  19. Wei C, Zhang G, Clark T, Barletta F, Tumey LN, Rago B, Hansel S, Han X (2016) Where did the linker-payload go? A quantitative investigation on the destination of the released linker-payload from an antibody-drug conjugate with a maleimide linker in plasma. Anal Chem 88(9):4979–4986. https://doi.org/10.1021/acs.analchem.6b00976

    Article  CAS  PubMed  Google Scholar 

  20. Brinckerhoff LH, Kalashnikov VV, Thompson LW, Yamshchikov GV, Pierce RA, Galavotti HS, Engelhard VH, Slingluff CL Jr (1999) Terminal modifications inhibit proteolytic degradation of an immunogenic mart-127–35 peptide: implications for peptide vaccines. Int J Cancer 83(3):326–334. https://doi.org/10.1002/(sici)1097-0215(19991029)83:3<326::Aid-ijc7>3.0.Co;2-x

    Article  CAS  PubMed  Google Scholar 

  21. Wolfrum C, Shi S, Jayaprakash KN, Jayaraman M, Wang G, Pandey RK, Rajeev KG, Nakayama T, Charrise K, Ndungo EM, Zimmermann T, Koteliansky V, Manoharan M, Stoffel M (2007) Mechanisms and optimization of in vivo delivery of lipophilic siRNAs. Nat Biotechnol 25(10):1149–1157. http://www.nature.com/nbt/journal/v25/n10/suppinfo/nbt1339_S1.html

    Article  CAS  Google Scholar 

  22. Biscans A, Coles A, Haraszti R, Echeverria D, Hassler M, Osborn M, Khvorova A (2019) Diverse lipid conjugates for functional extra-hepatic siRNA delivery in vivo. Nucleic Acids Res 47(3):1082–1096. https://doi.org/10.1093/nar/gky1239

    Article  CAS  PubMed  Google Scholar 

  23. Nowakowski GS, Dooner MS, Valinski HM, Mihaliak AM, Quesenberry PJ, Becker PS (2004) A specific heptapeptide from a phage display peptide library homes to bone marrow and binds to primitive hematopoietic stem cells. Stem Cells 22(6):1030–1038. https://doi.org/10.1634/stemcells.22-6-1030

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

We acknowledge financial support by the NCCR RNA and Disease of the Swiss National Science Foundation and by ETH Zürich to J.H.

Author information

Author notes

  1. François Halloy

    Present address: Department of Paediatrics, Medical Sciences Division, University of Oxford, Oxford, UK

  2. Paulina Brönnimann

    Present address: Translational Research Unit, Institute of Pathology, University of Bern, Bern, Switzerland

Authors and Affiliations

  1. Department of Chemistry and Applied Biosciences, Institute of Pharmaceutical Sciences, ETH Zürich, Zürich, Switzerland

    François Halloy, Paulina Brönnimann, Jonathan Hall & Daniel Schümperli

Authors
  1. François Halloy
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Paulina Brönnimann
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Jonathan Hall
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Daniel Schümperli
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Daniel Schümperli .

Editor information

Editors and Affiliations

  1. Biozentrum, University of Basel, Basel, Switzerland

    Peter Scheiffele

  2. Biozentrum, University of Basel, Basel, Switzerland

    Oriane Mauger

Rights and permissions

Reprints and permissions

Copyright information

© 2022 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Halloy, F., Brönnimann, P., Hall, J., Schümperli, D. (2022). Analysis of Oligonucleotide Biodistribution and Metabolization in Experimental Animals. In: Scheiffele, P., Mauger, O. (eds) Alternative Splicing. Methods in Molecular Biology, vol 2537. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2521-7_19

Download citation

  • DOI: https://doi.org/10.1007/978-1-0716-2521-7_19

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-2520-0

  • Online ISBN: 978-1-0716-2521-7

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation