Introduction of sugar-modified nucleotides into CpG-containing antisense oligonucleotides inhibits TLR9 activation.
Antisense oligonucleotides
Innate immunity
Sugar-modified nucleotides
Toll-like receptors
Journal
Scientific reports
ISSN: 2045-2322
Titre abrégé: Sci Rep
Pays: England
ID NLM: 101563288
Informations de publication
Date de publication:
21 May 2024
21 May 2024
Historique:
received:
18
03
2024
accepted:
08
05
2024
medline:
22
5
2024
pubmed:
22
5
2024
entrez:
21
5
2024
Statut:
epublish
Résumé
Antisense oligonucleotides (ASOs) are synthetic single-stranded oligonucleotides that bind to RNAs through Watson-Crick base pairings. They are actively being developed as therapeutics for various human diseases. ASOs containing unmethylated deoxycytidylyl-deoxyguanosine dinucleotide (CpG) motifs are known to trigger innate immune responses via interaction with toll-like receptor 9 (TLR9). However, the TLR9-stimulatory properties of ASOs, specifically those with lengths equal to or less than 20 nucleotides, phosphorothioate linkages, and the presence and arrangement of sugar-modified nucleotides-crucial elements for ASO therapeutics under development-have not been thoroughly investigated. In this study, we first established SY-ODN18, an 18-nucleotide phosphorothioate oligodeoxynucleotide with sufficient TLR9-stimulatory activity. We demonstrated that an unmethylated CpG motif near its 5'-end was indispensable for TLR9 activation. Moreover, by utilizing various sugar-modified nucleotides, we systematically generated model ASOs, including gapmer, mixmer, and fully modified designs, in accordance with the structures of ASO therapeutics. Our results illustrated that introducing sugar-modified nucleotides in such designs significantly reduces TLR9-stimulatory activity, even without methylation of CpG motifs. These findings would be useful for drug designs on several types of ASOs.
Identifiants
pubmed: 38773176
doi: 10.1038/s41598-024-61666-3
pii: 10.1038/s41598-024-61666-3
doi:
Substances chimiques
TLR9 protein, human
0
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
11540Subventions
Organisme : Japan Agency for Medical Research and Development
ID : JP15ak010103
Organisme : Japan Agency for Medical Research and Development
ID : JP17ak0101073
Organisme : Japan Agency for Medical Research and Development
ID : JP21ae0121022
Organisme : Japan Agency for Medical Research and Development
ID : JP20mk0101187
Informations de copyright
© 2024. The Author(s).
Références
Crooke, S. T., Baker, B. F., Crooke, R. M. & Liang, X. H. Antisense technology: An overview and prospectus. Nat. Rev. Drug Discov. 20, 427–453 (2021).
doi: 10.1038/s41573-021-00162-z
pubmed: 33762737
Khvorova, A. & Watts, J. K. The chemical evolution of oligonucleotide therapies of clinical utility. Nat. Biotechnol. 35, 238–248 (2017).
doi: 10.1038/nbt.3765
pubmed: 28244990
pmcid: 5517098
Wan, W. B. & Seth, P. P. The medicinal chemistry of therapeutic oligonucleotides. J. Med. Chem. 59, 9645–9667 (2016).
doi: 10.1021/acs.jmedchem.6b00551
pubmed: 27434100
Takakusa, H. et al. Drug metabolism and pharmacokinetics of antisense oligonucleotide therapeutics: Typical profiles, evaluation approaches, and points to consider compared with small molecule drugs. Nucleic Acid Ther. 33, 83–94 (2023).
doi: 10.1089/nat.2022.0054
pubmed: 36735616
pmcid: 10066781
Islam, M. A. & Obika, S. Bridged Nucleic Acids for Therapeutic Oligonucleotides. In Handbook of Chemical Biology of Nucleic Acids (ed. Sugimoto, N.) 497–542 (Springer Nature Singapore, 2023). https://doi.org/10.1007/978-981-19-9776-1_18 .
doi: 10.1007/978-981-19-9776-1_18
Obika, S. et al. Synthesis of 2′-O,4′-C-methyleneuridine and-cytidine. Novel bicyclic nucleosides having a fixed C3′-endo sugar puckering. Tetrahedron Lett. 38, 8735–8738 (1997).
doi: 10.1016/S0040-4039(97)10322-7
Koshkin, A. A. et al. 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 (1998).
doi: 10.1016/S0040-4020(98)00094-5
Morita, K. et al. 2′-O,4′-C-ethylene-bridged nucleic acids (ENA): Highly nuclease-resistant and thermodynamically stable oligonucleotides for antisense drug. Bioorg. Med. Chem. Lett. 12, 73–76 (2002).
doi: 10.1016/S0960-894X(01)00683-7
pubmed: 11738576
Crooke, S. T., Liang, X. H., Baker, B. F. & Crooke, R. M. Antisense technology: A review. J. Biol. Chem. 296, 100416 (2021).
doi: 10.1016/j.jbc.2021.100416
pubmed: 33600796
pmcid: 8005817
Egli, M. & Manoharan, M. Chemistry, structure and function of approved oligonucleotide therapeutics. Nucleic Acids Res. 51, 2529–2573 (2023).
doi: 10.1093/nar/gkad067
pubmed: 36881759
pmcid: 10085713
de la Torre, B. G. & Albericio, F. The pharmaceutical industry in 2023: An analysis of FDA drug approvals from the perspective of molecules. Molecules 29, 585 (2024).
doi: 10.3390/molecules29030585
pubmed: 38338330
pmcid: 10856271
Täubel, J. et al. Novel antisense therapy targeting microRNA-132 in patients with heart failure: Results of a first-in-human Phase 1b randomized, double-blind, placebo-controlled study. Eur. Heart J. 42, 178–188 (2021).
doi: 10.1093/eurheartj/ehaa898
pubmed: 33245749
Thum, T., Batkai, S. & Foinquinos, A. U.S. Patent 11208651. (2021).
Pfeiffer, N. et al. First-in-human phase I study of ISTH0036, an antisense oligonucleotide selectively targeting transforming growth factor beta 2 (TGF-β2), in subjects with open-angle glaucoma undergoing glaucoma filtration surgery. PLoS One 12, e0188899 (2017).
doi: 10.1371/journal.pone.0188899
pubmed: 29190672
pmcid: 5708654
Hill, A. C. & Hall, J. The MOE modification of RNA: Origins and widescale impact on the oligonucleotide therapeutics field. Helvetica Chimica Acta https://doi.org/10.1002/hlca.202200169 (2023).
doi: 10.1002/hlca.202200169
Hemmi, H. et al. A Toll-like receptor recognizes bacterial DNA. Nature 408, 740–745 (2000).
doi: 10.1038/35047123
pubmed: 11130078
Bauer, S. et al. Human TLR9 confers responsiveness to bacterial DNA via species-specific CpG motif recognition. Proc. Natl. Acad. Sci. USA 98, 9237–9242 (2001).
doi: 10.1073/pnas.161293498
pubmed: 11470918
pmcid: 55404
Krieg, A. M. et al. CpG motifs in bacterial DNA trigger direct B-cell activation. Nature 374, 546–549 (1995).
doi: 10.1038/374546a0
pubmed: 7700380
Hartmann, G. et al. Delineation of a CpG phosphorothioate oligodeoxynucleotide for activating primate immune responses in vitro and in vivo. J. Immunol. 164, 1617–1624 (2000).
doi: 10.4049/jimmunol.164.3.1617
pubmed: 10640783
Pohar, J., Kužnik Krajnik, A., Jerala, R. & Benčina, M. Minimal sequence requirements for oligodeoxyribonucleotides activating human TLR9. J. Immunol. 194, 3901–3908 (2015).
doi: 10.4049/jimmunol.1402755
pubmed: 25780037
Pollak, A. J. et al. Insights into innate immune activation via PS-ASO-protein-TLR9 interactions. Nucleic Acids Res. 50, 8107–8126 (2022).
doi: 10.1093/nar/gkac618
pubmed: 35848907
pmcid: 9371907
Vollmer, J. et al. Modulation of CpG oligodeoxynucleotide-mediated immune stimulation by locked nucleic acid (LNA). Oligonucleotides 14, 23–31 (2004).
doi: 10.1089/154545704322988021
pubmed: 15104893
Ohto, U. et al. Toll-like receptor 9 contains two DNA binding sites that function cooperatively to promote receptor dimerization and activation. Immunity 48, 649–658 (2018).
doi: 10.1016/j.immuni.2018.03.013
pubmed: 29625894
Rahman, S. M. et al. Design, synthesis, and properties of 2’,4’-BNA(NC): A bridged nucleic acid analogue. J. Am. Chem. Soc. 130, 4886–4896 (2008).
doi: 10.1021/ja710342q
pubmed: 18341342
Obika, S. et al. Stability and structural features of the duplexes containing nucleoside analogues with a fixed N-type conformation, 2′-O,4′-C-methyleneribonucleosides. Tetrahedron Lett. 39, 5401–5404 (1998).
doi: 10.1016/S0040-4039(98)01084-3
Obika, S. et al. 2’-O,4’-C-Methylene bridged nucleic acid (2’,4’-BNA): Synthesis and triplex-forming properties. Bioorg. Med. Chem. 9, 1001–1011 (2001).
doi: 10.1016/S0968-0896(00)00325-4
pubmed: 11354656
Shimo, T. et al. Design and evaluation of locked nucleic acid-based splice-switching oligonucleotides in vitro. Nucleic Acids Res. 42, 8174–8187 (2014).
doi: 10.1093/nar/gku512
pubmed: 24935206
pmcid: 4081108
Ohto, U. et al. Structural basis of CpG and inhibitory DNA recognition by Toll-like receptor 9. Nature 520, 702–705 (2015).
doi: 10.1038/nature14138
pubmed: 25686612
Pohar, J. et al. Short single-stranded DNA degradation products augment the activation of Toll-like receptor 9. Nat. Commun. 8, 15363 (2017).
doi: 10.1038/ncomms15363
pubmed: 28530246
pmcid: 5458134
Putta, M. R. et al. Immune-stimulatory dinucleotide at the 5’-end of oligodeoxynucleotides is critical for TLR9-mediated immune responses. ACS Med. Chem. Lett. 4, 302–305 (2013).
doi: 10.1021/ml300482z
pubmed: 24900663
pmcid: 4027476