Abstract
GalNAc oligonucleotide conjugates demonstrate improved potency in vivo due to selective and efficient delivery to hepatocytes in the liver via receptor-mediated endocytosis. GalNAc-siRNA and GalNAc-antisense oligonucleotides are at various stages of clinical trials, while the first two drugs were already approved by FDA. Also, GalNAc conjugates are excellent tools for functional genomics and target validation in vivo. The number of GalNAc residues in a conjugate is crucial for delivery as cooperative interaction of several GalNAc residues with asialoglycoprotein receptor enhances delivery in vitro and in vivo. Here we provide a robust protocol for the synthesis of triple GalNAc CPG solid support and GalNAc phosphoramidite, synthesis and purification of RNA conjugates with multiple GalNAc residues either to 5′-end or 3′-end and siRNA duplex formation.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Bajan S, Hutvagner G (2020) RNA-based therapeutics: from antisense oligonucleotides to miRNAs. Cells 9:E137. https://doi.org/10.3390/cells9010137
Zamecnik PC, Stephenson ML (1978) Inhibition of Rous sarcoma virus replication and cell transformation by a specific oligodeoxynucleotide. Proc Natl Acad Sci U S A 75:280–284
Chernikov IV, Vlassov VV, Chernolovskaya EL (2019) Current development of siRNA bioconjugates: from research to the clinic. Front Pharmacol 10:444. https://doi.org/10.3389/fphar.2019.00444
Prakash TP, Mullick AE, Lee RG et al (2019) Fatty acid conjugation enhances potency of antisense oligonucleotides in muscle. Nucleic Acids Res 47:6029–6044. https://doi.org/10.1093/nar/gkz354
Biscans A, Coles A, Haraszti R et al (2019) Diverse lipid conjugates for functional extra-hepatic siRNA delivery in vivo. Nucleic Acids Res 47:1082–1096. https://doi.org/10.1093/nar/gky1239
Huang X, Leroux JC, Castagner B (2017) Well-defined multivalent ligands for hepatocytes targeting via asialoglycoprotein receptor. Bioconjug Chem 28(2):283–295. https://doi.org/10.1021/acs.bioconjchem.6b00651
Sharma VK, Osborn MF, Hassler MR et al (2018) Novel cluster and monomer-based GalNAc structures induce effective uptake of siRNAs in vitro and in vivo. Bioconjug Chem 29(7):2478–2488. https://doi.org/10.1021/acs.bioconjchem.8b00365
Scott LJ (2020) Givosiran: first approval. Drugs 80(3):335–339. https://doi.org/10.1007/s40265-020-01269-0
Garrelfs S, Frishberg Y, Hulton S et al (2020) Illuminate-A, a phase 3 study of lumasiran, an investigational RNAi therapeutic, in children and adults with primary hyperoxaluria type 1 (PH1). Nephrol Dial Transplant 35(S3):LB002. https://doi.org/10.1093/ndt/gfaa146.LB002
Ray KK, Wright RS, Kallend D et al (2020) Two phase 3 trials of inclisiran in patients with elevated LDL cholesterol. N Engl J Med 382(16):1507–1519. https://doi.org/10.1056/NEJMoa1912387
Cardona CM, Gawley RE (2002) An improved synthesis of a trifurcated newkome-type monomer and orthogonally protected two-generation dendrons. J Org Chem 67(4):1411–1413. https://doi.org/10.1021/jo0161678
Yamaguchi K, Tsuda Y, Shimakage TA et al (1998) Syntheses of phospholipids containing 2-nitrobenzyl ester. Moieties at the terminals of alkyl chains and properties of photodegradable liposomes from the lipids. Bull Chem Soc Jpn 71:1923–1929. https://doi.org/10.1246/bcsj.71.1923
Pujol AM, Cuillel M, Jullien AS et al (2012) A sulfur tripod glycoconjugate that releases a high-affinity copper chelator in hepatocytes. Angew Chem Int Ed 51(30):7445–7448. https://doi.org/10.1002/anie.201203255
Azhayev AV, Antopolsky ML (2001) Amide group assisted 3′-dephosphorylation of oligonucleotides synthesized on universal A-supports. Tetrahedron 57(23):4977–4986. https://doi.org/10.1016/S0040-4020(01)00409-4
Nair JK, Willoughby JL, Chan A et al (2014) Multivalent N-acetylgalactosamine-conjugated siRNA localizes in hepatocytes and elicits robust RNAi-mediated gene silencing. J Am Chem Soc 136(49):16958–16961. https://doi.org/10.1021/ja505986a
Acknowledgements
Development of this protocol was supported by Russian Science Foundation (RSF 19-44-04111).
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2021 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Ulashchik, E.A., Martynenko-Makaev, Y.V., Akhlamionok, T.P., Melnik, D.M., Shmanai, V.V., Zatsepin, T.S. (2021). Synthesis of GalNAc-Oligonucleotide Conjugates Using GalNAc Phosphoramidite and Triple-GalNAc CPG Solid Support. In: Ditzel, H.J., Tuttolomondo, M., Kauppinen, S. (eds) Design and Delivery of SiRNA Therapeutics. Methods in Molecular Biology, vol 2282. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1298-9_7
Download citation
DOI: https://doi.org/10.1007/978-1-0716-1298-9_7
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-1297-2
Online ISBN: 978-1-0716-1298-9
eBook Packages: Springer Protocols