title: Serial incorporation of a monovalent GalNAc phosphoramidite unit into hepatocyte-targeting antisense oligonucleotides.
abstract: The targeting of abundant hepatic asialoglycoprotein receptors (ASGPR) with trivalent N-acetylgalactosamine (GalNAc) is a reliable strategy for efficiently delivering antisense oligonucleotides (ASOs) to the liver. We here experimentally demonstrate the high systemic potential of the synthetically-accessible, phosphodiester-linked monovalent GalNAc unit when tethered to the 5'-terminus of well-characterised 2',4'-bridged nucleic acid (also known as locked nucleic acid)-modified apolipoprotein B-targeting ASO via a bio-labile linker. Quantitative analysis of the hepatic disposition of the ASOs revealed that phosphodiester is preferable to phosphorothioate as an interunit linkage in terms of ASGPR binding of the GalNAc moiety, as well as the subcellular behavior of the ASO. The flexibility of this monomeric unit was demonstrated by attaching up to 5 GalNAc units in a serial manner and showing that knockdown activity improves as the number of GalNAc units increases. Our study suggests the structural requirements for efficient hepatocellular targeting using monovalent GalNAc and could contribute to a new molecular design for suitably modifying ASO.
authors.affiliation: Department of Molecular Innovation in Lipidology, National Cerebral and Cardiovascular Center Research Institute, 5-7-1 Fujishirodai, Suita, Osaka 565-8565, Japan; Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871, Japan. Electronic address: t-yam@phs.osaka-u.ac.jp.
authors.forename: Tsuyoshi
authors.initials: T
authors.lastname: Yamamoto
authors.affiliation: Department of Molecular Innovation in Lipidology, National Cerebral and Cardiovascular Center Research Institute, 5-7-1 Fujishirodai, Suita, Osaka 565-8565, Japan; Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871, Japan.
authors.forename: Motoki
authors.initials: M
authors.lastname: Sawamura
authors.affiliation: Department of Molecular Innovation in Lipidology, National Cerebral and Cardiovascular Center Research Institute, 5-7-1 Fujishirodai, Suita, Osaka 565-8565, Japan; Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871, Japan.
authors.forename: Fumito
authors.initials: F
authors.lastname: Wada
authors.affiliation: Department of Molecular Innovation in Lipidology, National Cerebral and Cardiovascular Center Research Institute, 5-7-1 Fujishirodai, Suita, Osaka 565-8565, Japan.
authors.forename: Mariko
authors.initials: M
authors.lastname: Harada-Shiba
authors.affiliation: Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871, Japan. Electronic address: obika@phs.osaka-u.ac.jp.
authors.forename: Satoshi
authors.initials: S
authors.lastname: Obika
mesh_terms: D000116:Acetylgalactosamine / Q000031:analogs & derivatives* / Q000138:chemical synthesis*; D000410:Alanine Transaminase / Q000097:blood; D000818:Animals; D001055:Apolipoproteins B / Q000235:genetics / Q000378:metabolism; D037263:Asialoglycoprotein Receptor / Q000378:metabolism; D001219:Aspartate Aminotransferases / Q000097:blood; D002784:Cholesterol / Q000097:blood; D055785:Gene Knockdown Techniques; D020868:Gene Silencing; D022781:Hepatocytes / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D008099:Liver / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D020319:Oligodeoxyribonucleotides, Antisense / Q000008:administration & dosage / Q000138:chemical synthesis* / Q000633:toxicity; D009943:Organophosphorus Compounds / Q000138:chemical synthesis*; D012333:RNA, Messenger / Q000378:metabolism
chemical_list: D001055:Apolipoproteins B; D037263:Asialoglycoprotein Receptor; D020319:Oligodeoxyribonucleotides, Antisense; D009943:Organophosphorus Compounds; D012333:RNA, Messenger; D002784:Cholesterol; D001219:Aspartate Aminotransferases; D000410:Alanine Transaminase; D000116:Acetylgalactosamine
keywords: Antisense oligonucleotides; Apolipoproteins; Asialoglycoprotein receptors; Bio-labile linker; Bridged nucleic acid; GalNAc; Hypercholesterolemia; Locked nucleic acid