DOI QR코드

DOI QR Code

Drug Discovery Perspectives of Antisense Oligonucleotides

  • Received : 2023.01.03
  • Accepted : 2023.02.13
  • Published : 2023.05.01

Abstract

The era of innovative RNA therapies using antisense oligonucleotides (ASOs), siRNAs, and mRNAs is beginning. Since the emergence of the concept of ASOs in 1978, it took more than 20 years before they were developed into drugs for commercial use. Nine ASO drugs have been approved to date. However, they target only rare genetic diseases, and the number of chemistries and mechanisms of action of ASOs are limited. Nevertheless, ASOs are accepted as a powerful modality for next-generation medicines as they can theoretically target all disease-related RNAs, including (undruggable) protein-coding RNAs and non-coding RNAs. In addition, ASOs can not only downregulate but also upregulate gene expression through diverse mechanisms of action. This review summarizes the achievements in medicinal chemistry that enabled the translation of the ASO concept into real drugs, the molecular mechanisms of action of ASOs, the structure-activity relationship of ASO-protein binding, and the pharmacology, pharmacokinetics, and toxicology of ASOs. In addition, it discusses recent advances in medicinal chemistry in improving the therapeutic potential of ASOs by reducing their toxicity and enhancing their cellular uptake.

Keywords

References

  1. Ammala, C., Drury, W. J., 3rd, Knerr, L., Ahlstedt, I., Stillemark-Billton, P., Wennberg-Huldt, C., Andersson, E. M., Valeur, E., Jansson-Lofmark, R., Janzen, D., Sundstrom, L., Meuller, J., Claesson, J., Andersson, P., Johansson, C., Lee, R. G., Prakash, T. P., Seth, P. P., Monia, B. P. and Andersson, S. (2018) Targeted delivery of antisense oligonucleotides to pancreatic β-cells. Sci. Adv. 4, eaat3386.
  2. Anderson, B. A., Freestone, G. C., Low, A., De-Hoyos, C. L., Iii, W. J. D., Ostergaard, M. E., Migawa, M. T., Fazio, M., Wan, W. B., Berdeja, A., Scandalis, E., Burel, S. A., Vickers, T. A., Crooke, S. T., Swayze, E. E., Liang, X. and Seth, P. P. (2021) Towards next generation antisense oligonucleotides: mesylphosphoramidate modification improves therapeutic index and duration of effect of gapmer antisense oligonucleotides. Nucleic Acids Res. 49, 9026-9041. https://doi.org/10.1093/nar/gkab718
  3. Baker, B. F., Lot, S. S., Kringel, J., Cheng-Flournoy, S., Villiet, P., Sasmor, H. M., Siwkowski, A. M., Chappell, L. L. and Morrow, J. R. (1999) Oligonucleotide-europium complex conjugate designed to cleave the 5' cap structure of the ICAM-1 transcript potentiates antisense activity in cells. Nucleic Acids Res. 27, 1547-1551. https://doi.org/10.1093/nar/27.6.1547
  4. Baker, Y. R., Thorpe, C., Chen, J., Poller, L. M., Cox, L., Kumar, P., Lim, W. F., Lie, L., McClorey, G., Epple, S., Singleton, D., McDonough, M. A., Hardwick, J. S., Christensen, K. E., Wood, M. J. A., Hall, J. P., El-Sagheer, A. H. and Brown, T. (2022) An LNA-amide modification that enhances the cell uptake and activity of phosphorothioate exon-skipping oligonucleotides. Nat. Commun. 13, 4036.
  5. Bhingardeve, P., Madhanagopal, B. R., Naick, H., Jain, P., Manoharan, M. and Ganesh, K. (2020) Receptor-specific delivery of peptide nucleic acids conjugated to three sequentially linked N-acetyl galactosamine moieties into hepatocytes. J. Org. Chem. 85, 8812-8824. https://doi.org/10.1021/acs.joc.0c00601
  6. Burdick, A. D., Sciabola, S., Mantena, S. R., Hollingshead, B. D., Stanton, R., Warneke, J. A., Zeng, M., Martsen, E., Medvedev, A., Makarov, S. S., Reed, L. A., Davis, J. W., 2nd and Whiteley, L. O. (2014) Sequence motifs associated with hepatotoxicity of locked nucleic acid--modified antisense oligonucleotides. Nucleic Acids Res. 42, 4882-4891. https://doi.org/10.1093/nar/gku142
  7. Burel, S. A., Hart, C. E., Cauntay, P., Hsiao, J., Machemer, T., Katz, M., Watt, A., Bui, H. H., Younis, H., Sabripour, M., Freier, S. M., Hung, G., Dan, A., Prakash, T. P., Seth, P. P., Swayze, E. E., Bennett, C. F., Crooke, S. T. and Henry, S. P. (2016) Hepatotoxicity of high affinity gapmer antisense oligonucleotides is mediated by RNase H1 dependent promiscuous reduction of very long pre-mRNA transcripts. Nucleic Acids Res. 44, 2093-2109. https://doi.org/10.1093/nar/gkv1210
  8. Burel, S. A., Machemer, T., Ragone, F. L., Kato, H., Cauntay, P., Greenlee, S., Salim, A., Gaarde, W. A., Hung, G., Peralta, R., Freier, S. M. and Henry, S. P. (2012) Unique O-methoxyethyl ribose-DNA chimeric oligonucleotide induces an atypical melanoma differentiation-associated gene 5-dependent induction of type I interferon response. J. Pharmacol. Exp. Ther. 342, 150-162. https://doi.org/10.1124/jpet.112.193789
  9. Calvo, S. E., Pagliarini, D. J. and Mootha, V. K. (2009) Upstream open reading frames cause widespread reduction of protein expression and are polymorphic among humans. Proc. Natl. Acad. Sci. U. S. A. 106, 7507-7512. https://doi.org/10.1073/pnas.0810916106
  10. Cerritelli, S. M. and Crouch, R. J. (2009) Ribonuclease H: the enzymes in eukaryotes. FEBS J. 276, 1494-1505. https://doi.org/10.1111/j.1742-4658.2009.06908.x
  11. Chelobanov, B. P., Burakova, E. A., Prokhorova, D. V., Fokina, A. A. and Stetsenko, D. A. (2017) New oligodeoxynucleotide derivatives containing N-(methanesulfonyl)-phosphoramidate (mesyl phosphoramidate) internucleotide group. Russ. J. Bioorg. Chem. 43, 664-668. https://doi.org/10.1134/S1068162017060024
  12. Clemens, P. R., Rao, V. K., Connolly, A. M., Harper, A. D., Mah, J. K., Smith, E. C., McDonald, C. M., Zaidman, C. M., Morgenroth, L. P., Osaki, H., Satou, Y., Yamashita, T. and Hoffman, E. P. (2020) Safety, tolerability, and efficacy of viltolarsen in boys with Duchenne muscular dystrophy amenable to exon 53 skipping: a phase 2 randomized clinical trial. JAMA Neurol. 77, 982-991. https://doi.org/10.1001/jamaneurol.2020.1264
  13. Crooke, S. T., Baker, B. F., Crooke, R. M. and Liang, X. H. (2021) Antisense technology: an overview and prospectus. Nat. Rev. Drug Discov. 20, 427-453.
  14. Crooke, S. T., Baker, B. F., Kwoh, T. J., Cheng, W., Schulz, D. J., Xia, S., Salgado, N., Bui, H. H., Hart, C. E., Burel, S. A., Younis, H. S., Geary, R. S., Henry, S. P. and Bhanot, S. (2016) Integrated safety assessment of 2'-O-methoxyethyl chimeric antisense oligonucleotides in nonhuman primates and healthy human volunteers. Mol. Ther. 24, 1771-1782. https://doi.org/10.1038/mt.2016.136
  15. Crooke, S. T., Baker, B. F., Pham, N. C., Hughes, S. G., Kwoh, T. J., Cai, D., Tsimikas, S., Geary, R. S. and Bhanot, S. (2018) The effects of 2'-O-methoxyethyl oligonucleotides on renal function in humans. Nucleic Acid Ther. 28, 10-22. https://doi.org/10.1089/nat.2017.0693
  16. Crooke, S. T., Baker, B. F., Witztum, J. L., Kwoh, T. J., Pham, N. C., Salgado, N., McEvoy, B. W., Cheng, W., Hughes, S. G., Bhanot, S. and Geary, R. S. (2017a) The effects of 2'-O-methoxyethyl containing antisense oligonucleotides on platelets in human clinical trials. Nucleic Acid Ther. 27, 121-129. https://doi.org/10.1089/nat.2016.0650
  17. Crooke, S. T., Seth, P. P., Vickers, T. A. and Liang, X. H. (2020a) The interaction of phosphorothioate-containing RNA targeted drugs with proteins is a critical determinant of the therapeutic effects of these agents. J. Am. Chem. Soc. 142, 14754-14771. https://doi.org/10.1021/jacs.0c04928
  18. Crooke, S. T., Vickers, T. A. and Liang, X. H. (2020b) Phosphorothioate modified oligonucleotide-protein interactions. Nucleic Acids Res. 48, 5235-5253. https://doi.org/10.1093/nar/gkaa299
  19. Crooke, S. T., Wang, S., Vickers, T. A., Shen, W. and Liang, X. H. (2017b) Cellular uptake and trafficking of antisense oligonucleotides. Nat. Biotechnol. 35, 230-237. https://doi.org/10.1038/nbt.3779
  20. De Mesmaeker, A., Waldner, A., Lebreton, J., Hoffmann, P., Fritsch, V., Wolf, R. M. and Freier, S. M. (1994) Amides as a new type of backbone modification in oligonucleotides. Angew. Chem. Int. Ed. Engl. 33, 226-229. https://doi.org/10.1002/anie.199402261
  21. De Santi, C., Fernandez Fernandez, E., Gaul, R., Vencken, S., Glasgow, A., Oglesby, I. K., Hurley, K., Hawkins, F., Mitash, N., Mu, F., Raoof, R., Henshall, D. C., Cutrona, M. B., Simpson, J. C., Harvey, B. J., Linnane, B., McNally, P., Cryan, S. A., MacLoughlin, R., Swiatecka-Urban, A. and Greene, C. M. (2020) Precise targeting of miRNA sites restores CFTR activity in CF bronchial epithelial cells. Mol. Ther. 28, 1190-1199. https://doi.org/10.1016/j.ymthe.2020.02.001
  22. Djebali, S., Davis, C. A., Merkel, A., Dobin, A., Lassmann, T., Mortazavi, A., Tanzer, A., Lagarde, J., Lin, W., Schlesinger, F., Xue, C., Marinov, G. K., Khatun, J., Williams, B. A., Zaleski, C., Rozowsky, J., Roder, M., Kokocinski, F., Abdelhamid, R. F., Alioto, T., Antoshechkin, I., Baer, M. T., Bar, N. S., Batut, P., Bell, K., Bell, I., Chakrabortty, S., Chen, X., Chrast, J., Curado, J., Derrien, T., Drenkow, J., Dumais, E., Dumais, J., Duttagupta, R., Falconnet, E., Fastuca, M., Fejes-Toth, K., Ferreira, P., Foissac, S., Fullwood, M. J., Gao, H., Gonzalez, D., Gordon, A., Gunawardena, H., Howald, C., Jha, S., Johnson, R., Kapranov, P., King, B., Kingswood, C., Luo, O. J., Park, E., Persaud, K., Preall, J. B., Ribeca, P., Risk, B., Robyr, D., Sammeth, M., Schaffer, L., See, L.-H., Shahab, A., Skancke, J., Suzuki, A. M., Takahashi, H., Tilgner, H., Trout, D., Walters, N., Wang, H., Wrobel, J., Yu, Y., Ruan, X., Hayashizaki, Y., Harrow, J., Gerstein, M., Hubbard, T., Reymond, A., Antonarakis, S. E., Hannon, G., Giddings, M. C., Ruan, Y., Wold, B., Carninci, P., Guigo, R. and Gingeras, T. R. (2012) Landscape of transcription in human cells. Nature 489, 101-108.
  23. Doherty, G. J. and McMahon, H. T. (2009) Mechanisms of endocytosis. Annu. Rev. Biochem. 78, 857-902. https://doi.org/10.1146/annurev.biochem.78.081307.110540
  24. Dowdy, S. F. (2017) Overcoming cellular barriers for RNA therapeutics. Nat. Biotechnol. 35, 222-229. https://doi.org/10.1038/nbt.3802
  25. Echevarria, L., Aupy, P. and Goyenvalle, A. (2018) Exon-skipping advances for Duchenne muscular dystrophy. Hum. Mol. Genet. 27, R163-R172. https://doi.org/10.1093/hmg/ddy171
  26. Eckstein, F. (2000) Phosphorothioate oligodeoxynucleotides: what is their origin and what is unique about them? Antisense Nucleic Acid Drug Dev. 10, 117-121. https://doi.org/10.1089/oli.1.2000.10.117
  27. Epple, S., Thorpe, C., Baker, Y. R., El-Sagheer, A. H. and Brown, T. (2020) Consecutive 5'- and 3'-amide linkages stabilise antisense oligonucleotides and elicit an efficient RNase H response. Chem. Commun. 56, 5496-5499. https://doi.org/10.1039/D0CC00444H
  28. Fire, A., Xu, S., Montgomery, M. K., Kostas, S. A., Driver, S. E. and Mello, C. C. (1998) Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391, 806-811. https://doi.org/10.1038/35888
  29. Freier, S. M. and Altmann, K. H. (1997) The ups and downs of nucleic acid duplex stability: structure-stability studies on chemically-modified DNA:RNA duplexes. Nucleic Acids Res. 25, 4429-4443. https://doi.org/10.1093/nar/25.22.4429
  30. Garber, K. (2016) Big win possible for Ionis/Biogen antisense drug in muscular atrophy. Nat. Biotechnol. 34, 1002-1003. https://doi.org/10.1038/nbt1016-1002
  31. Gaus, H., Miller, C. M., Seth, P. P. and Harris, E. N. (2018) Structural determinants for the interactions of chemically modified nucleic acids with the stabilin-2 clearance receptor. Biochemistry 57, 2061-2064. https://doi.org/10.1021/acs.biochem.8b00126
  32. Gaus, H. J., Gupta, R., Chappell, A. E., Ostergaard, M. E., Swayze, E. E. and Seth, P. P. (2019) Characterization of the interactions of chemically-modified therapeutic nucleic acids with plasma proteins using a fluorescence polarization assay. Nucleic Acids Res. 47, 1110-1122. https://doi.org/10.1093/nar/gky1260
  33. Geary, R. S., Baker, B. F. and Crooke, S. T. (2015) Clinical and preclinical pharmacokinetics and pharmacodynamics of mipomersen (kynamro(®)): a second-generation antisense oligonucleotide inhibitor of apolipoprotein B. Clin. Pharmacokinet. 54, 133-146. https://doi.org/10.1007/s40262-014-0224-4
  34. Geary, R. S., Leeds, J. M., Fitchett, J., Burckin, T., Truong, L., Spain-hour, C., Creek, M. and Levin, A. A. (1997) Pharmacokinetics and metabolism in mice of a phosphorothioate oligonucleotide antisense inhibitor of C-raf-1 kinase expression. Drug Metab. Dispos. 25, 1272-1281.
  35. Geary, R. S., Watanabe, T. A., Truong, L., Freier, S., Lesnik, E. A., Sioufi, N. B., Sasmor, H., Manoharan, M. and Levin, A. A. (2001) Pharmacokinetic properties of 2'-O-(2-methoxyethyl)-modified oligonucleotide analogs in rats. J. Pharmacol. Exp. Ther. 296, 890-897.
  36. Gupta, A., Bahal, R., Gupta, M., Glazer, P. M. and Saltzman, W. M. (2016) Nanotechnology for delivery of peptide nucleic acids (PNAs). J. Control. Release 240, 302-311. https://doi.org/10.1016/j.jconrel.2016.01.005
  37. Gupta, A., Mishra, A. and Puri, N. (2017) Peptide nucleic acids: advanced tools for biomedical applications. J. Biotechnol. 259, 148-159. https://doi.org/10.1016/j.jbiotec.2017.07.026
  38. Hagedorn, P. H., Yakimov, V., Ottosen, S., Kammler, S., Nielsen, N. F., Hog, A. M., Hedtjarn, M., Meldgaard, M., Moller, M. R., Orum, H., Koch, T. and Lindow, M. (2013) Hepatotoxic potential of therapeutic oligonucleotides can be predicted from their sequence and modification pattern. Nucleic Acid Ther. 23, 302-310. https://doi.org/10.1089/nat.2013.0436
  39. Hammond, S. M., Sergeeva, O. V., Melnikov, P. A., Goli, L., Stoodley, J., Zatsepin, T. S., Stetsenko, D. A. and Wood, M. J. A. (2021) Mesyl phosphoramidate oligonucleotides as potential splice-switching agents: impact of backbone structure on activity and intracellular localization. Nucleic Acid Ther. 31, 190-200. https://doi.org/10.1089/nat.2020.0860
  40. Henry, S., Stecker, K., Brooks, D., Monteith, D., Conklin, B. and Bennett, C. F. (2000) Chemically modified oligonucleotides exhibit decreased immune stimulation in mice. J. Pharmacol. Exp. Ther. 292, 468-479.
  41. Hua, Y., Vickers, T. A., Baker, B. F., Bennett, C. F. and Krainer, A. R. (2007) Enhancement of SMN2 exon 7 inclusion by antisense oligonucleotides targeting the exon. PLoS Biol. 5, e73.
  42. Huang, L., Low, A., Damle, S. S., Keenan, M. M., Kuntz, S., Murray, S. F., Monia, B. P. and Guo, S. (2018) Antisense suppression of the nonsense mediated decay factor Upf3b as a potential treatment for diseases caused by nonsense mutations. Genome Biol. 19, 4.
  43. Iversen, P. L., Arora, V., Acker, A. J., Mason, D. H. and Devi, G. R. (2003) Efficacy of antisense morpholino oligomer targeted to c-myc in prostate cancer xenograft murine model and a Phase I safety study in humans. Clin. Cancer Res. 9, 2510-2519.
  44. Juliano, R. L. (2018) Intracellular trafficking and endosomal release of oligonucleotides: what we know and what we don't. Nucleic Acid Ther. 28, 166-177. https://doi.org/10.1089/nat.2018.0727
  45. Juliano, R. L., Wang, L., Tavares, F., Brown, E. G., James, L., Ariyarathna, Y., Ming, X., Mao, C. and Suto, M. (2018) Structure-activity relationships and cellular mechanism of action of small molecules that enhance the delivery of oligonucleotides. Nucleic Acids Res. 46, 1601-1613. https://doi.org/10.1093/nar/gkx1320
  46. Kamola, P. J., Kitson, J. D., Turner, G., Maratou, K., Eriksson, S., Panjwani, A., Warnock, L. C., Douillard Guilloux, G. A., Moores, K., Koppe, E. L., Wixted, W. E., Wilson, P. A., Gooderham, N. J., Gant, T. W., Clark, K. L., Hughes, S. A., Edbrooke, M. R. and Parry, J. D. (2015) In silico and in vitro evaluation of exonic and intronic off-target effects form a critical element of therapeutic ASO gapmer optimization. Nucleic Acids Res. 43, 8638-8650. https://doi.org/10.1093/nar/gkv857
  47. Kim, J., Hu, C., Moufawad El Achkar, C., Black, L. E., Douville, J., Larson, A., Pendergast, M. K., Goldkind, S. F., Lee, E. A., Kuniholm, A., Soucy, A., Vaze, J., Belur, N. R., Fredriksen, K., Stojkovska, I., Tsytsykova, A., Armant, M., DiDonato, R. L., Choi, J., Cornelissen, L., Pereira, L. M., Augustine, E. F., Genetti, C. A., Dies, K., Barton, B., Williams, L., Goodlett, B. D., Riley, B. L., Pasternak, A., Berry, E. R., Pflock, K. A., Chu, S., Reed, C., Tyndall, K., Agrawal, P. B., Beggs, A. H., Grant, P. E., Urion, D. K., Snyder, R. O., Waisbren, S. E., Poduri, A., Park, P. J., Patterson, A., Biffi, A., Mazzulli, J. R., Bodamer, O., Berde, C. B. and Yu, T. W. (2019) Patient-customized oligonucleotide therapy for a rare genetic disease. N. Engl. J. Med. 381, 1644-1652. https://doi.org/10.1056/NEJMoa1813279
  48. Kim, Y. J., Nomakuchi, T., Papaleonidopoulou, F., Yang, L., Zhang, Q. and Krainer, A. R. (2022) Gene-specific nonsense-mediated mRNA decay targeting for cystic fibrosis therapy. Nat. Commun. 13, 2978.
  49. Knerr, L., Prakash, T. P., Lee, R., Drury Iii, W. J., Nikan, M., Fu, W., Pirie, E., Maria, L. D., Valeur, E., Hayen, A., Olwegard-Halvarsson, M., Broddefalk, J., Ammala, C., Ostergaard, M. E., Meuller, J., Sundstrom, L., Andersson, P., Janzen, D., Jansson-Lofmark, R., Seth, P. P. and Andersson, S. (2021) Glucagon Like Peptide 1 Receptor agonists for targeted delivery of antisense oligonucleotides to pancreatic beta cell. J. Am. Chem. Soc. 143, 3416-3429. https://doi.org/10.1021/jacs.0c12043
  50. Koller, E., Vincent, T. M., Chappell, A., De, S., Manoharan, M. and Bennett, C. F. (2011) Mechanisms of single-stranded phosphorothioate modified antisense oligonucleotide accumulation in hepatocytes. Nucleic Acids Res. 39, 4795-4807. https://doi.org/10.1093/nar/gkr089
  51. Kondow-McConaghy, H. M., Muthukrishnan, N., Erazo-Oliveras, A., Najjar, K., Juliano, R. L. and Pellois, J.-P. (2020) Impact of the endosomal escape activity of cell-penetrating peptides on the endocytic pathway. ACS Chem. Biol. 15, 2355-2363. https://doi.org/10.1021/acschembio.0c00319
  52. Koshkin, A. A., Singh, S. K., Nielsen, P., Rajwanshi, V. K., Kumar, R., Meldgaard, M., Olsen, C. E. and Wengel, J. (1998) LNA (Locked Nucleic Acids): synthesis of the adenine, cytosine, guanine, 5-methylcytosine, thymine and uracil bicyclonucleoside monomers, oligomerisation, and unprecedented nucleic acid recognition. Tetrahedron 54, 3607-3630. https://doi.org/10.1016/S0040-4020(98)00094-5
  53. Kotikam, V. and Rozners, E. (2020) Amide-modified RNA: using protein backbone to modulate function of short interfering RNAs. Acc. Chem. Res. 53, 1782-1790. https://doi.org/10.1021/acs.accounts.0c00249
  54. Liang, X.-h., Sun, H., Hsu, C.-W., Nichols, J. G., Vickers, T. A., De Hoyos, C. L. and Crooke, S. T. (2019a) Golgi-endosome transport mediated by M6PR facilitates release of antisense oligonucleotides from endosomes. Nucleic Acids Res. 48, 1372-1391. https://doi.org/10.1093/nar/gkz1171
  55. Liang, X.-h., Sun, H., Shen, W., Wang, S., Yao, J., Migawa, M. T., Bui, H.-H., Damle, S. S., Riney, S., Graham, M. J., Crooke, R. M. and Crooke, S. T. (2017) Antisense oligonucleotides targeting translation inhibitory elements in 5' UTRs can selectively increase protein levels. Nucleic Acids Res. 45, 9528-9546. https://doi.org/10.1093/nar/gkx632
  56. Liang, X. H., Nichols, J. G., Hsu, C. W., Vickers, T. A. and Crooke, S. T. (2019b) mRNA levels can be reduced by antisense oligonucleotides via no-go decay pathway. Nucleic Acids Res. 47, 6900-6916. https://doi.org/10.1093/nar/gkz500
  57. Liang, X. H., Shen, W., Sun, H., Kinberger, G. A., Prakash, T. P., Nichols, J. G. and Crooke, S. T. (2016a) Hsp90 protein interacts with phosphorothioate oligonucleotides containing hydrophobic 2'-modifications and enhances antisense activity. Nucleic Acids Res. 44, 3892-3907. https://doi.org/10.1093/nar/gkw144
  58. Liang, X. H., Shen, W., Sun, H., Migawa, M. T., Vickers, T. A. and Crooke, S. T. (2016b) Translation efficiency of mRNAs is increased by antisense oligonucleotides targeting upstream open reading frames. Nat. Biotechnol. 34, 875-880. https://doi.org/10.1038/nbt.3589
  59. Liang, X. H., Sun, H., Nichols, J. G., Allen, N., Wang, S., Vickers, T. A., Shen, W., Hsu, C. W. and Crooke, S. T. (2018) COPII vesicles can affect the activity of antisense oligonucleotides by facilitating the release of oligonucleotides from endocytic pathways. Nucleic Acids Res. 46, 10225-10245. https://doi.org/10.1093/nar/gky841
  60. Liang, X. H., Sun, H., Shen, W. and Crooke, S. T. (2015) Identification and characterization of intracellular proteins that bind oligonucleotides with phosphorothioate linkages. Nucleic Acids Res. 43, 2927-2945. https://doi.org/10.1093/nar/gkv143
  61. Lim, K. H., Han, Z., Jeon, H. Y., Kach, J., Jing, E., Weyn-Vanhentenryck, S., Downs, M., Corrionero, A., Oh, R., Scharner, J., Venkatesh, A., Ji, S., Liau, G., Ticho, B., Nash, H. and Aznarez, I. (2020) Antisense oligonucleotide modulation of non-productive alternative splicing upregulates gene expression. Nat. Commun. 11, 3501.
  62. Lima, W. F., Prakash, T. P., Murray, H. M., Kinberger, G. A., Li, W., Chappell, A. E., Li, C. S., Murray, S. F., Gaus, H., Seth, P. P., Swayze, E. E. and Crooke, S. T. (2012) Single-stranded siRNAs activate RNAi in animals. Cell 150, 883-894. https://doi.org/10.1016/j.cell.2012.08.014
  63. Lima, W. F., Vickers, T. A., Nichols, J., Li, C. and Crooke, S. T. (2014) Defining the factors that contribute to on-target specificity of antisense oligonucleotides. PLoS One 9, e101752.
  64. Lonn, P., Kacsinta, A. D., Cui, X.-S., Hamil, A. S., Kaulich, M., Gogoi, K. and Dowdy, S. F. (2016) Enhancing endosomal escape for intracellular delivery of macromolecular biologic therapeutics. Sci. Rep. 6, 32301.
  65. Makley, L. N. and Gestwicki, J. E. (2013) Expanding the number of 'druggable' targets: non-enzymes and protein-protein interactions. Chem. Biol. Drug Des. 81, 22-32. https://doi.org/10.1111/cbdd.12066
  66. McDonald, C. M., Shieh, P. B., Abdel-Hamid, H. Z., Connolly, A. M., Ciafaloni, E., Wagner, K. R., Goemans, N., Mercuri, E., Khan, N., Koenig, E., Malhotra, J., Zhang, W., Han, B. and Mendell, J. R. (2021) Open-label evaluation of eteplirsen in patients with Duchenne muscular dystrophy amenable to exon 51 skipping: PROMOVI trial. J. Neuromuscul. Dis. 8, 989-1001. https://doi.org/10.3233/JND-210643
  67. Melton, D. A. (1985) Injected anti-sense RNAs specifically block messenger RNA translation in vivo. Proc. Natl. Acad. Sci. U. S. A. 82, 144-148. https://doi.org/10.1073/pnas.82.1.144
  68. Migawa, M. T., Shen, W., Wan, W. B., Vasquez, G., Oestergaard, M. E., Low, A., De Hoyos, C. L., Gupta, R., Murray, S., Tanowitz, M., Bell, M., Nichols, J. G., Gaus, H., Liang, X. H., Swayze, E. E., Crooke, S. T. and Seth, P. P. (2019) Site-specific replacement of phosphorothioate with alkyl phosphonate linkages enhances the therapeutic profile of gapmer ASOs by modulating interactions with cellular proteins. Nucleic Acids Res. 47, 5465-5479. https://doi.org/10.1093/nar/gkz247
  69. Miller, C. M., Donner, A. J., Blank, E. E., Egger, A. W., Kellar, B. M., Ostergaard, M. E., Seth, P. P. and Harris, E. N. (2016) 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. https://doi.org/10.1093/nar/gkw112
  70. Miller, P. S., Yano, J., Yano, E., Carroll, C., Jayaraman, K. and Ts'o, P. O. (1979) Nonionic nucleic acid analogues. Synthesis and characterization of dideoxyribonucleoside methylphosphonates. Biochemistry 18, 5134-5143. https://doi.org/10.1021/bi00590a017
  71. Miroshnichenko, S. K., Patutina, O. A., Burakova, E. A., Chelobanov, B. P., Fokina, A. A., Vlassov, V. V., Altman, S., Zenkova, M. A. and Stetsenko, D. A. (2019) Mesyl phosphoramidate antisense oligonucleotides as an alternative to phosphorothioates with improved biochemical and biological properties. Proc. Natl. Acad. Sci. U. S. A. 116, 1229-1234. https://doi.org/10.1073/pnas.1813376116
  72. Monia, B. P., Lesnik, E. A., Gonzalez, C., Lima, W. F., McGee, D., Guinosso, C. J., Kawasaki, A. M., Cook, P. D. and Freier, S. M. (1993) Evaluation of 2'-modified oligonucleotides containing 2'-deoxy gaps as antisense inhibitors of gene expression. J. Biol. Chem. 268, 14514-14522. https://doi.org/10.1016/S0021-9258(19)85268-7
  73. Mutisya, D., Hardcastle, T., Cheruiyot, S. K., Pallan, P. S., Kennedy, S. D., Egli, M., Kelley, M. L., Smith, A. V. B. and Rozners, E. (2017) Amide linkages mimic phosphates in RNA interactions with proteins and are well tolerated in the guide strand of short interfering RNAs. Nucleic Acids Res. 45, 8142-8155. https://doi.org/10.1093/nar/gkx558
  74. Nomakuchi, T. T., Rigo, F., Aznarez, I. and Krainer, A. R. (2016) Antisense oligonucleotide-directed inhibition of nonsense-mediated mRNA decay. Nat. Biotechnol. 34, 164-166. https://doi.org/10.1038/nbt.3427
  75. Obika, S., Nanbu, D., Hari, Y., Morio, K.-i., In, Y., Ishida, T. and Imanishi, T. (1997) Synthesis of 2'-O,4'-C-methyleneuridine and -cytidine. Novel bicyclic nucleosides having a fixed C3, -endo sugar puckering. Tetrahedron Lett. 38, 8735-8738. https://doi.org/10.1016/S0040-4039(97)10322-7
  76. Orom, U. A., Kauppinen, S. and Lund, A. H. (2006) LNA-modified oligonucleotides mediate specific inhibition of microRNA function. Gene 372, 137-141. https://doi.org/10.1016/j.gene.2005.12.031
  77. Patutina, O. A., Gaponova Miroshnichenko, S. K., Sen'kova, A. V., Savin, I. A., Gladkikh, D. V., Burakova, E. A., Fokina, A. A., Maslov, M. A., Shmendel, E. V., Wood, M. J. A., Vlassov, V. V., Altman, S., Stetsenko, D. A. and Zenkova, M. A. (2020) Mesyl phosphoramidate backbone modified antisense oligonucleotides targeting miR21 with enhanced in vivo therapeutic potency. Proc. Natl. Acad. Sci. U. S. A. 117, 32370-32379. https://doi.org/10.1073/pnas.2016158117
  78. Plavec, J., Tong, W. and Chattopadhyaya, J. (1993) How do the gauche and anomeric effects drive the pseudorotational equilibrium of the pentofuranose moiety of nucleosides? J. Am. Chem. Soc. 115, 9734-9746. https://doi.org/10.1021/ja00074a046
  79. Prakash, T. P., Graham, M. J., Yu, J., Carty, R., Low, A., Chappell, A., Schmidt, K., Zhao, C., Aghajan, M., Murray, H. F., Riney, S., Booten, S. L., Murray, S. F., Gaus, H., Crosby, J., Lima, W. F., Guo, S., Monia, B. P., Swayze, E. E. and Seth, P. P. (2014) Targeted delivery of antisense oligonucleotides to hepatocytes using triantennary N-acetyl galactosamine improves potency 10-fold in mice. Nucleic Acids Res. 42, 8796-8807. https://doi.org/10.1093/nar/gku531
  80. Sands, H., Gorey-Feret, L. J., Cocuzza, A. J., Hobbs, F. W., Chidester, D. and Trainor, G. L. (1994) Biodistribution and metabolism of internally 3H-labeled oligonucleotides. I. Comparison of a phosphodiester and a phosphorothioate. Mol. Pharmacol. 45, 932-943.
  81. Servais, L., Mercuri, E., Straub, V., Guglieri, M., Seferian, A. M., Scoto, M., Leone, D., Koenig, E., Khan, N., Dugar, A., Wang, X., Han, B., Wang, D. and Muntoni, F. (2022) Long-term safety and efficacy data of golodirsen in ambulatory patients with Duchenne muscular dystrophy amenable to exon 53 skipping: a first-in-human, multicenter, two-part, open-label, phase 1/2 trial. Nucleic Acid Ther. 32, 29-39. https://doi.org/10.1089/nat.2021.0043
  82. Seth, P. P., Siwkowski, A., Allerson, C. R., Vasquez, G., Lee, S., Prakash, T. P., Wancewicz, E. V., Witchell, D. and Swayze, E. E. (2009a) Short antisense oligonucleotides with novel 2'-4' conformationaly restricted nucleoside analogues show improved potency without increased toxicity in animals. J. Med. Chem. 52, 10-13. https://doi.org/10.1021/jm801294h
  83. Seth, P. P., Siwkowski, A., Allerson, C. R., Vasquez, G., Lee, S., Prakash, T. P., Wancewicz, E. V., Witchell, D. and Swayze, E. E. (2009b) Short antisense oligonucleotides with novel 2'-4' conformationaly restricted nucleoside analogues show improved potency without increased toxicity in animals. J. Med. Chem. 52, 10-13. https://doi.org/10.1021/jm801294h
  84. Shen, W., De Hoyos, C. L., Migawa, M. T., Vickers, T. A., Sun, H., Low, A., Bell, T. A., 3rd, Rahdar, M., Mukhopadhyay, S., Hart, C. E., Bell, M., Riney, S., Murray, S. F., Greenlee, S., Crooke, R. M., Liang, X. H., Seth, P. P. and Crooke, S. T. (2019) Chemical modification of PS-ASO therapeutics reduces cellular protein-binding and improves the therapeutic index. Nat. Biotechnol. 37, 640-650. https://doi.org/10.1038/s41587-019-0106-2
  85. Sheng, L., Rigo, F., Bennett, C. F., Krainer, A. R. and Hua, Y. (2020) Comparison of the efficacy of MOE and PMO modifications of systemic antisense oligonucleotides in a severe SMA mouse model. Nucleic Acids Res. 48, 2853-2865. https://doi.org/10.1093/nar/gkaa126
  86. Song, J. J., Smith, S. K., Hannon, G. J. and Joshua-Tor, L. (2004) Crystal structure of Argonaute and its implications for RISC slicer activity. Science 305, 1434-1437. https://doi.org/10.1126/science.1102514
  87. Stec, W. J., Zon, G. and Egan, W. (1984) Automated solid-phase synthesis, separation, and stereochemistry of phosphorothioate analogs of oligodeoxyribonucleotides. J. Am. Chem. Soc. 106, 6077-6079. https://doi.org/10.1021/ja00332a054
  88. Stockert, R. J. (1995) The asialoglycoprotein receptor: relationships between structure, function, and expression. Physiol. Rev. 75, 591-609. https://doi.org/10.1152/physrev.1995.75.3.591
  89. Summerton, J. and Weller, D. (1997) Morpholino antisense oligomers: design, preparation, and properties. Antisense Nucleic Acid Drug Dev. 7, 187-195. https://doi.org/10.1089/oli.1.1997.7.187
  90. Swayze, E. E., Siwkowski, A. M., Wancewicz, E. V., Migawa, M. T., Wyrzykiewicz, T. K., Hung, G., Monia, B. P. and Bennett, C. F. (2007) Antisense oligonucleotides containing locked nucleic acid improve potency but cause significant hepatotoxicity in animals. Nucleic Acids Res. 35, 687-700. https://doi.org/10.1093/nar/gkl1071
  91. Taylor, R. E. and Zahid, M. (2020) Cell penetrating peptides, novel vectors for gene therapy. Pharmaceutics 12, 225.
  92. Teplova, M., Minasov, G., Tereshko, V., Inamati, G. B., Cook, P. D., Manoharan, M. and Egli, M. (1999) Crystal structure and improved antisense properties of 2'-O-(2-methoxyethyl)-RNA. Nat. Struct. Biol. 6, 535-539. https://doi.org/10.1038/9304
  93. Vickers, T. A. and Crooke, S. T. (2016) Development of a quantitative BRET affinity assay for nucleic acid-protein interactions. PLoS One 11, e0161930.
  94. Vickers, T. A., Rahdar, M., Prakash, T. P. and Crooke, S. T. (2019) Kinetic and subcellular analysis of PS-ASO/protein interactions with P54nrb and RNase H1. Nucleic Acids Res. 47, 10865-10880. https://doi.org/10.1093/nar/gkz771
  95. Vickers, T. A., Wyatt, J. R., Burckin, T., Bennett, C. F. and Freier, S. M. (2001) Fully modified 2' MOE oligonucleotides redirect polyadenylation. Nucleic Acids Res. 29, 1293-1299. https://doi.org/10.1093/nar/29.6.1293
  96. Volpi, S., Cancelli, U., Neri, M. and Corradini, R. (2020) Multifunctional delivery systems for peptide nucleic acids. Pharmaceuticals (Basel) 14, 14.
  97. Wagner, K. R., Kuntz, N. L., Koenig, E., East, L., Upadhyay, S., Han, B. and Shieh, P. B. (2021) Safety, tolerability, and pharmacokinetics of casimersen in patients with Duchenne muscular dystrophy amenable to exon 45 skipping: a randomized, double-blind, placebocontrolled, dose-titration trial. Muscle Nerve 64, 285-292. https://doi.org/10.1002/mus.27347
  98. Wang, L., Ariyarathna, Y., Ming, X., Yang, B., James, L. I., Kreda, S. M., Porter, M., Janzen, W. and Juliano, R. L. (2017) A novel family of small molecules that enhance the intracellular delivery and pharmacological effectiveness of antisense and splice switching oligonucleotides. ACS Chem. Biol. 12, 1999-2007. https://doi.org/10.1021/acschembio.7b00242
  99. Ward, A. J., Norrbom, M., Chun, S., Bennett, C. F. and Rigo, F. (2014) Nonsense-mediated decay as a terminating mechanism for antisense oligonucleotides. Nucleic Acids Res. 42, 5871-5879. https://doi.org/10.1093/nar/gku184
  100. Wu, H., Lima, W. F., Zhang, H., Fan, A., Sun, H. and Crooke, S. T. (2004) Determination of the role of the human RNase H1 in the pharmacology of DNA-like antisense drugs. J. Biol. Chem. 279, 17181-17189. https://doi.org/10.1074/jbc.M311683200
  101. Yanai, H., Chiba, S., Ban, T., Nakaima, Y., Onoe, T., Honda, K., Ohdan, H. and Taniguchi, T. (2011) Suppression of immune responses by nonimmunogenic oligodeoxynucleotides with high affinity for high-mobility group box proteins (HMGBs). Proc. Natl. Acad. Sci. U. S. A. 108, 11542-11547. https://doi.org/10.1073/pnas.1108535108
  102. Yang, B., Ming, X., Cao, C., Laing, B., Yuan, A., Porter, M. A., HullRyde, E. A., Maddry, J., Suto, M., Janzen, W. P. and Juliano, R. L. (2015) High-throughput screening identifies small molecules that enhance the pharmacological effects of oligonucleotides. Nucleic Acids Res. 43, 1987-1996. https://doi.org/10.1093/nar/gkv060
  103. Yoshida, T., Morihiro, K., Naito, Y., Mikami, A., Kasahara, Y., Inoue, T. and Obika, S. (2022) Identification of nucleobase chemical modifications that reduce the hepatotoxicity of gapmer antisense oligonucleotides. Nucleic Acids Res. 50, 7224-7234. https://doi.org/10.1093/nar/gkac562
  104. Zamecnik, P. C. and Stephenson, M. L. (1978) Inhibition of Rous sarcoma virus replication and cell transformation by a specific oligodeoxynucleotide. Proc. Natl. Acad. Sci. U. S. A. 75, 280-284. https://doi.org/10.1073/pnas.75.1.280
  105. Zhang, L., Liang, X. H., De Hoyos, C. L., Migawa, M., Nichols, J. G., Freestone, G., Tian, J., Seth, P. P. and Crooke, S. T. (2022) The combination of mesyl-phosphoramidate inter-nucleotide linkages and 2'-O-methyl in selected positions in the antisense oligonucleotide enhances the performance of RNaseH1 active PSASOs. Nucleic Acid Ther. 32, 401-411. https://doi.org/10.1089/nat.2022.0005
  106. Zimmermann, T. S., Karsten, V., Chan, A., Chiesa, J., Boyce, M., Bettencourt, B. R., Hutabarat, R., Nochur, S., Vaishnaw, A. and Gollob, J. (2017) Clinical proof of concept for a novel hepatocyte-targeting GalNAc-siRNA conjugate. Mol. Ther. 25, 71-78. https://doi.org/10.1016/j.ymthe.2016.10.019