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Cellular uptake and trafficking of antisense oligonucleotides

Abstract

Antisense oligonucleotides (ASOs) modified with phosphorothioate (PS) linkages and different 2′ modifications can be used either as drugs (e.g., to treat homozygous familial hypercholesterolemia and spinal muscular atrophy) or as research tools to alter gene expression. PS-ASOs can enter cells without additional modification or formulation and can be designed to mediate sequence-specific cleavage of different types of RNA (including mRNA and non-coding RNA) targeted by endogenous RNase H1. Although PS-ASOs function in both the cytoplasm and nucleus, localization to different subcellular regions can affect their therapeutic potency. Cellular uptake and intracellular distribution of PS ASOs are mediated by protein interactions. The main proteins involved in these processes have been identified, and intracellular sites in which PS ASOs are active, or inactive, cataloged.

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Figure 1: Pathways of PS-ASO uptake and intracellular trafficking.
Figure 2: PS-ASOs interact with intracellular proteins.
Figure 3: PS-ASO localization in nuclear and cytoplasmic foci.
Figure 4: Kinetics of PS-ASO localization and antisense activity.

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References

  1. Crooke, S.T., Vickers, T., Lima, W. & Wu, H.-J. in Antisense Drug Technology—Principles, Strategies, and Application 2nd edn. (ed. Crooke, S.T.) 3–46 (CRC Press, Boca Raton, FL, 2008).

  2. Evers, M.M. & Toonen, L.J.A. & van Roon-Mom, W.M.C. Antisense oligonucleotides in therapy for neurodegenerative disorders. Adv. Drug Deliv. Rev. 87, 90–103 (2015).

    Article  CAS  Google Scholar 

  3. Brown, D.A. et al. Effect of phosphorothioate modification of oligodeoxynucleotides on specific protein binding. J. Biol. Chem. 269, 26801–26805 (1994).

    CAS  PubMed  Google Scholar 

  4. Liang, X.H., Sun, H., Shen, W. & Crooke, S.T. Identification and characterization of intracellular proteins that bind oligonucleotides with phosphorothioate linkages. Nucleic Acids Res. 43, 2927–2945 (2015).

    Article  CAS  Google Scholar 

  5. Swayze, E.E. & Bhat, B. in Antisense Drug Technology—Principles, Strategies, and Application 2nd edn. (ed. Crooke, S.T.) 143–182 (CRC Press, Boca Raton, FL, 2008).

  6. Geary, R.S., Yu, R.Z., Siwkoswki, A. & Leivin, A.A. in Antisense Drug Technology—Principles, Strategies, and Application 2nd edn. (ed. Crooke, S.T.) 305–326 (CRC Press, Boca Raton, FL. 2008).

  7. Dias, N. & Stein, C.A. Antisense oligonucleotides: basic concepts and mechanisms. Mol. Cancer Ther. 1, 347–355 (2002).

    Article  CAS  Google Scholar 

  8. Kurreck, J. Antisense technologies. Improvement through novel chemical modifications. Eur. J. Biochem. 270, 1628–1644 (2003).

    Article  CAS  Google Scholar 

  9. Lee, R.G., Crosby, J., Baker, B.F., Graham, M.J. & Crooke, R.M. Antisense technology: an emerging platform for cardiovascular disease therapeutics. J. Cardiovasc. Transl. Res. 6, 969–980 (2013).

    Article  Google Scholar 

  10. Bennett, C.F. & Swayze, E.E. RNA targeting therapeutics: molecular mechanisms of antisense oligonucleotides as a therapeutic platform. Annu. Rev. Pharmacol. Toxicol. 50, 259–293 (2010).

    Article  CAS  Google Scholar 

  11. Geary, R.S., Norris, D., Yu, R. & Bennett, C.F. Pharmacokinetics, biodistribution and cell uptake of antisense oligonucleotides. Adv. Drug Deliv. Rev. 87, 46–51 (2015).

    Article  CAS  Google Scholar 

  12. Trülzsch, B. & Wood, M. Applications of nucleic acid technology in the CNS. J. Neurochem. 88, 257–265 (2004).

    Article  Google Scholar 

  13. Juliano, R.L. The delivery of therapeutic oligonucleotides. Nucleic Acids Res. 44, 6518–6548 (2016).

    Article  Google Scholar 

  14. Vlassov, V.V., Vlassova, I.E. & Pautova, L.V. Oligonucleotides and polynucleotides as biologically active compounds. Prog. Nucleic Acid Res. Mol. Biol. 57, 95–143 (1997).

    Article  CAS  Google Scholar 

  15. Iversen, P.L., Zhu, S., Meyer, A. & Zon, G. Cellular uptake and subcellular distribution of phosphorothioate oligonucleotides into cultured cells. Antisense Res. Dev. 2, 211–222 (1992).

    Article  CAS  Google Scholar 

  16. Ugarte-Uribe, B. et al. Synthesis, cell-surface binding, and cellular uptake of fluorescently labeled glucose-DNA conjugates with different carbohydrate presentation. Bioconjug. Chem. 21, 1280–1287 (2010).

    Article  CAS  Google Scholar 

  17. Cheng, C.J. & Saltzman, W.M. Enhanced siRNA delivery into cells by exploiting the synergy between targeting ligands and cell-penetrating peptides. Biomaterials 32, 6194–6203 (2011).

    Article  CAS  Google Scholar 

  18. Yu, C., Brussaard, A.B., Yang, X., Listerud, M. & Role, L.W. Uptake of antisense oligonucleotides and functional block of acetylcholine receptor subunit gene expression in primary embryonic neurons. Dev. Genet. 14, 296–304 (1993).

    Article  CAS  Google Scholar 

  19. Koller, E. et al. Mechanisms of single-stranded phosphorothioate modified antisense oligonucleotide accumulation in hepatocytes. Nucleic Acids Res. 39, 4795–4807 (2011).

    Article  CAS  Google Scholar 

  20. Juliano, R.L., Ming, X., Carver, K. & Laing, B. Cellular uptake and intracellular trafficking of oligonucleotides: implications for oligonucleotide pharmacology. Nucleic Acid Ther. 24, 101–113 (2014).

    Article  CAS  Google Scholar 

  21. Juliano, R.L., Carver, K., Cao, C. & Ming, X. Receptors, endocytosis, and trafficking: the biological basis of targeted delivery of antisense and siRNA oligonucleotides. J. Drug Target. 21, 27–43 (2013).

    Article  CAS  Google Scholar 

  22. Goldsack, N.C. et al. Integrin mediated uptake of antisense oligonucleotides to the PAR-1 thrombin receptor inhibits thrombin induced fibroblast proliferation. FASEB J. 12, A434 (1998).

  23. Alam, M.R. et al. Intracellular delivery of an anionic antisense oligonucleotide via receptor-mediated endocytosis. Nucleic Acids Res. 36, 2764–2776 (2008).

    Article  CAS  Google Scholar 

  24. Ming, X. et al. Intracellular delivery of an antisense oligonucleotide via endocytosis of a G-protein-coupled receptor. Nucleic Acids Res. 38, 6567–6576 (2010).

    Article  CAS  Google Scholar 

  25. Miller, C.M. et al. Stabilin-1 and Stabilin-2 are specific receptors for the cellular internalization of phosphorothioate-modified antisense oligonucleotides (ASOs) in the liver. Nucleic Acids Res. 44, 2782–2794 (2016).

    Article  Google Scholar 

  26. Ezzat, K. et al. Self-Assembly into Nanoparticles Is Essential for Receptor Mediated Uptake of Therapeutic Antisense Oligonucleotides. Nano Lett. 15, 4364–4373 (2015).

    Article  CAS  Google Scholar 

  27. Kortylewski, M. et al. In vivo delivery of siRNA to immune cells by conjugation to a TLR9 agonist enhances antitumor immune responses. Nat. Biotechnol. 27, 925–932 (2009).

    Article  CAS  Google Scholar 

  28. Kandimalla, E.R. et al. Design, synthesis and biological evaluation of novel antagonist compounds of Toll-like receptors 7, 8 and 9. Nucleic Acids Res. 41, 3947–3961 (2013).

    Article  CAS  Google Scholar 

  29. Weidner, D.A., Valdez, B.C., Henning, D., Greenberg, S. & Busch, H. Phosphorothioate oligonucleotides bind in a non-sequence-specific manner to the nucleolar protein C23/nucleolin. FEBS Lett. 366, 146–150 (1995).

    Article  CAS  Google Scholar 

  30. Kotula, J.W. et al. Aptamer-mediated delivery of splice-switching oligonucleotides to the nuclei of cancer cells. Nucleic Acid Ther. 22, 187–195 (2012).

    Article  CAS  Google Scholar 

  31. Sahay, G. et al. Efficiency of siRNA delivery by lipid nanoparticles is limited by endocytic recycling. Nat. Biotechnol. 31, 653–658 (2013).

    Article  CAS  Google Scholar 

  32. Gilleron, J. et al. Image-based analysis of lipid nanoparticle-mediated siRNA delivery, intracellular trafficking and endosomal escape. Nat. Biotechnol. 31, 638–646 (2013).

    Article  CAS  Google Scholar 

  33. Beltinger, C. et al. Binding, uptake, and intracellular trafficking of phosphorothioate-modified oligodeoxynucleotides. J. Clin. Invest. 95, 1814–1823 (1995).

    Article  CAS  Google Scholar 

  34. Bennett, C.F. in Antisense Drug Technology—Principles, Strategies, and Application 2nd edn. (ed. Crooke, S.T.) 273–304 (CRC Press, Boca Raton, FL, 2006).

  35. Mou, T.C. & Gray, D.M. The high binding affinity of phosphorothioate-modified oligomers for Ff gene 5 protein is moderated by the addition of C-5 propyne or 2′-O-methyl modifications. Nucleic Acids Res. 30, 749–758 (2002).

    Article  CAS  Google Scholar 

  36. Liang, X.H. et al. Hsp90 protein interacts with phosphorothioate oligonucleotides containing hydrophobic 2′-modifications and enhances antisense activity. Nucleic Acids Res. 44, 3892–3907 (2016).

    Article  CAS  Google Scholar 

  37. Juliano, R.L., Ming, X. & Nakagawa, O. Cellular uptake and intracellular trafficking of antisense and siRNA oligonucleotides. Bioconjug. Chem. 23, 147–157 (2012).

    Article  CAS  Google Scholar 

  38. Juliano, R.L. & Carver, K. Cellular uptake and intracellular trafficking of oligonucleotides. Adv. Drug Deliv. Rev. 87, 35–45 (2015).

    Article  CAS  Google Scholar 

  39. Reyes-Reyes, E.M., Teng, Y. & Bates, P.J. A new paradigm for aptamer therapeutic AS1411 action: uptake by macropinocytosis and its stimulation by a nucleolin-dependent mechanism. Cancer Res. 70, 8617–8629 (2010).

    Article  CAS  Google Scholar 

  40. Alam, M.R. et al. The biological effect of an antisense oligonucleotide depends on its route of endocytosis and trafficking. Oligonucleotides 20, 103–109 (2010).

    Article  CAS  Google Scholar 

  41. Stein, C.A. et al. Efficient gene silencing by delivery of locked nucleic acid antisense oligonucleotides, unassisted by transfection reagents. Nucleic Acids Res. 38 e3 (2010).

    Article  Google Scholar 

  42. Ming, X., Sato, K. & Juliano, R.L. Unconventional internalization mechanisms underlying functional delivery of antisense oligonucleotides via cationic lipoplexes and polyplexes. J. Control. Release 153, 83–92 (2011).

    Article  CAS  Google Scholar 

  43. Biessen, E.A., Vietsch, H., Kuiper, J., Bijsterbosch, M.K. & Berkel, T.J. Liver uptake of phosphodiester oligodeoxynucleotides is mediated by scavenger receptors. Mol. Pharmacol. 53, 262–269 (1998).

    Article  CAS  Google Scholar 

  44. El-Sayed, A. & Harashima, H. Endocytosis of gene delivery vectors: from clathrin-dependent to lipid raft-mediated endocytosis. Mol. Ther. 21, 1118–1130 (2013).

    Article  CAS  Google Scholar 

  45. Prakash, T.P. et al. Targeted delivery of antisense oligonucleotides to hepatocytes using triantennary N-acetyl galactosamine improves potency 10-fold in mice. Nucleic Acids Res. 42, 8796–8807 (2014).

    Article  CAS  Google Scholar 

  46. Wada, S. et al. Evaluation of the effects of chemically different linkers on hepatic accumulations, cell tropism and gene silencing ability of cholesterol-conjugated antisense oligonucleotides. J. Control. Release 226, 57–65 (2016).

    Article  CAS  Google Scholar 

  47. Vickers, T.A. & Crooke, S.T. Development of a quantitative BRET affinity assay for nucleic acid-protein interactions. PLoS One 11, e0161930 (2016).

    Article  Google Scholar 

  48. Wang, S., Sun, H., Tanowitz, M., Liang, X.H. & Crooke, S.T. Annexin A2 facilitates endocytic trafficking of antisense oligonucleotides. Nucleic Acids Res. 44, 7314–7330 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  49. Shen, W., Liang, X.H. & Crooke, S.T. Phosphorothioate oligonucleotides can displace NEAT1 RNA and form nuclear paraspeckle-like structures. Nucleic Acids Res. 42, 8648–8662 (2014).

    Article  CAS  Google Scholar 

  50. Machleidt, T. et al. NanoBRET—a novel BRET platform for the analysis of protein-protein interactions. ACS Chem. Biol. 10, 1797–1804 (2015).

    Article  CAS  Google Scholar 

  51. Chin, D.J., Green, G.A., Zon, G., Szoka, F.C. Jr. & Straubinger, R.M. Rapid nuclear accumulation of injected oligodeoxyribonucleotides. New Biol. 2, 1091–1100 (1990).

    CAS  PubMed  Google Scholar 

  52. Bennett, C.F., Chiang, M.Y., Chan, H., Shoemaker, J.E. & Mirabelli, C.K. Cationic lipids enhance cellular uptake and activity of phosphorothioate antisense oligonucleotides. Mol. Pharmacol. 41, 1023–1033 (1992).

    CAS  PubMed  Google Scholar 

  53. Leonetti, J.P., Mechti, N., Degols, G., Gagnor, C. & Lebleu, B. Intracellular distribution of microinjected antisense oligonucleotides. Proc. Natl. Acad. Sci. USA 88, 2702–2706 (1991).

    Article  CAS  Google Scholar 

  54. Lorenz, P., Baker, B.F., Bennett, C.F. & Spector, D.L. Phosphorothioate antisense oligonucleotides induce the formation of nuclear bodies. Mol. Biol. Cell 9, 1007–1023 (1998).

    Article  CAS  Google Scholar 

  55. Lorenz, P., Misteli, T., Baker, B.F., Bennett, C.F. & Spector, D.L. Nucleocytoplasmic shuttling: a novel in vivo property of antisense phosphorothioate oligodeoxynucleotides. Nucleic Acids Res. 28, 582–592 (2000).

    Article  CAS  Google Scholar 

  56. Liang, X.H., Shen, W., Sun, H., Prakash, T.P. & Crooke, S.T. TCP1 complex proteins interact with phosphorothioate oligonucleotides and can co-localize in oligonucleotide-induced nuclear bodies in mammalian cells. Nucleic Acids Res. 42, 7819–7832 (2014).

    Article  CAS  Google Scholar 

  57. Marcusson, E.G., Bhat, B., Manoharan, M., Bennett, C.F. & Dean, N.M. Phosphorothioate oligodeoxyribonucleotides dissociate from cationic lipids before entering the nucleus. Nucleic Acids Res. 26, 2016–2023 (1998).

    Article  CAS  Google Scholar 

  58. Zelphati, O. & Szoka, F.C. Jr. Mechanism of oligonucleotide release from cationic liposomes. Proc. Natl. Acad. Sci. USA 93, 11493–11498 (1996).

    Article  CAS  Google Scholar 

  59. Tarkanyi, I. et al. Inhibition of human telomerase by oligonucleotide chimeras, composed of an antisense moiety and a chemically modified homo-oligonucleotide. FEBS Lett. 579, 1411–1416 (2005).

    Article  CAS  Google Scholar 

  60. Castanotto, D. et al. A cytoplasmic pathway for gapmer antisense oligonucleotide-mediated gene silencing in mammalian cells. Nucleic Acids Res. 43, 9350–9361 (2015).

    Article  CAS  Google Scholar 

  61. Kubo, T. et al. Controlled intracellular localization and enhanced antisense effect of oligonucleotides by chemical conjugation. Org. Biomol. Chem. 3, 3257–3259 (2005).

    Article  CAS  Google Scholar 

  62. Juliano, R., Bauman, J., Kang, H. & Ming, X. Biological barriers to therapy with antisense and siRNA oligonucleotides. Mol. Pharm. 6, 686–695 (2009).

    Article  CAS  Google Scholar 

  63. Yang, B. et al. High-throughput screening identifies small molecules that enhance the pharmacological effects of oligonucleotides. Nucleic Acids Res. 43, 1987–1996 (2015).

    Article  CAS  Google Scholar 

  64. Vickers, T.A. & Crooke, S.T. Antisense oligonucleotides capable of promoting specific target mRNA reduction via competing RNase H1-dependent and independent mechanisms. PLoS One 9, e108625 (2014).

    Article  Google Scholar 

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Acknowledgements

This work is supported by internal funding from Ionis Pharmaceuticals. The authors wish to thank R. Crooke for helpful comments, T. Kniss and D. Parrett for editing and administrative assistance, and T. Reigle for help in figure preparation.

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Correspondence to Stanley T Crooke.

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This work was funded by Ionis Pharmaceuticals. All authors are employees of Ionis Pharmaceuticals.

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Crooke, S., Wang, S., Vickers, T. et al. Cellular uptake and trafficking of antisense oligonucleotides. Nat Biotechnol 35, 230–237 (2017). https://doi.org/10.1038/nbt.3779

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