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.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2022 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
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