Delivery of Oligonucleotides to the Liver with GalNAc: From Research to Registered Therapeutic Drug

Targeted delivery of oligonucleotides to liver hepatocytes using N-acetylgalactosamine (GalNAc) conjugates that bind to the asialoglycoprotein receptor has become a breakthrough approach in the therapeutic oligonucleotide field. This technology has led to the approval of givosiran for the treatment of acute hepatic porphyria, and there are another seven conjugates in registrational review or phase 3 trials and at least another 21 conjugates at earlier stages of clinical development. This review highlights some of the recent chemical and preclinical advances in this space, leading to a large number of clinical candidates against a diverse range of targets in liver hepatocytes. The review focuses on the use of this delivery system for small interfering RNAs (siRNAs) and antisense molecules that cause downregulation of target mRNA and protein. A number of other approaches such as anti-microRNAs and small activating RNAs are starting to exploit the technology, broadening the potential of this approach for therapeutic oligonucleotide intervention. Targeted delivery of oligonucleotides to liver hepatocytes using N-acetylgalactosamine (GalNAc) conjugates that bind to the asialoglycoprotein receptor has become a breakthrough approach in the therapeutic oligonucleotide field. This technology has led to the approval of givosiran for the treatment of acute hepatic porphyria, and there are another seven conjugates in registrational review or phase 3 trials and at least another 21 conjugates at earlier stages of clinical development. This review highlights some of the recent chemical and preclinical advances in this space, leading to a large number of clinical candidates against a diverse range of targets in liver hepatocytes. The review focuses on the use of this delivery system for small interfering RNAs (siRNAs) and antisense molecules that cause downregulation of target mRNA and protein. A number of other approaches such as anti-microRNAs and small activating RNAs are starting to exploit the technology, broadening the potential of this approach for therapeutic oligonucleotide intervention. Oligonucleotide therapeutics are an emerging class of drugs that have tremendous potential for treating a wide range of diseases.1Levin A.A. Treating disease at the RNA level with oligonucleotides.N. Engl. J. Med. 2019; 380: 57-70Crossref PubMed Scopus (175) Google Scholar There are now nine marketed oligonucleotide products,2Wang F. Zuroske T. Watts J.K. RNA therapeutics on the rise.Nat. Rev. Drug Discov. 2020; (Published online April 27, 2020)https://doi.org/10.1038/d41573-020-00078-0Crossref PubMed Scopus (104) Google Scholar and nusinersen (Biogen/Ionis), which is used to treat spinal muscular atrophy, generated more than $2 billion in sales in 2019. They are designed based on Watson-Crick base pairing and the sequence of the RNA associated with the disease. Modifications to internucleotide linkages and the ribose sugar are introduced to improve drug-like properties, including serum stability, protein binding, potency, and lower immunogenicity.3Craig K. Abrams M. Amiji M. Recent preclinical and clinical advances in oligonucleotide conjugates.Expert Opin. Drug Deliv. 2018; 15: 629-640Crossref PubMed Scopus (32) Google Scholar,4Khvorova A. Watts J.K. The chemical evolution of oligonucleotide therapies of clinical utility.Nat. Biotechnol. 2017; 35: 238-248Crossref PubMed Scopus (621) Google Scholar A major challenge that has held back the therapeutic exploitation of oligonucleotides is their delivery to the diseased cells because the therapeutic RNA target is inside the cell. Oligonucleotides are large, generally negatively charged molecules that do not freely diffuse across cell membranes, unlike lipophilic small molecule drugs.5Juliano R.L. The delivery of therapeutic oligonucleotides.Nucleic Acids Res. 2016; 44: 6518-6548Crossref PubMed Scopus (539) Google Scholar This review focuses on a delivery solution termed N-acetylgalactosamine (GalNAc) and its exploitation by small interfering RNA (siRNA) and antisense oligonucleotides (ASOs), the two leading classes of oligonucleotide therapeutics.1Levin A.A. Treating disease at the RNA level with oligonucleotides.N. Engl. J. Med. 2019; 380: 57-70Crossref PubMed Scopus (175) Google Scholar The approach relies on the fact that liver hepatocytes express the asialoglycoprotein receptor (ASGPR), which binds and clears circulating glycoproteins in which the sialic acid residue has been removed to expose sugar residues.6Steirer L.M. Park E.I. Townsend R.R. Baenziger J.U. The asialoglycoprotein receptor regulates levels of plasma glycoproteins terminating with sialic acid α2,6-galactose.J. Biol. Chem. 2009; 284: 3777-3783Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar,7Bocci V. Pacini A. Pessina G.P. Bargigli V. Russi M. The role of sialic acid in determining the survival of circulating interferon.Experientia. 1977; 33: 164-166Crossref PubMed Scopus (7) Google Scholar The ASGPR is a high-capacity, rapidly internalizing receptor with approximately 500,000 copies per hepatocyte. Trimeric GalNAc ligands for the ASGPR have been developed, and these were first utilized to deliver oligonucleotides to the liver more than 20 years ago.8Hangeland J.J. Flesher J.E. Deamond S.F. Lee Y.C. Ts’O P.O.P. Frost J.J. Tissue distribution and metabolism of the [32P]-labeled oligodeoxynucleoside methylphosphonate-neoglycopeptide conjugate, [YEE(ah-GalNAc)3]-SMCC-AET-pUmpT7, in the mouse.Antisense Nucleic Acid Drug Dev. 1997; 7: 141-149Crossref PubMed Scopus (38) Google Scholar,9Biessen E.A. Vietsch H. Rump E.T. Fluiter K. Kuiper J. Bijsterbosch M.K. van Berkel T.J. Targeted delivery of oligodeoxynucleotides to parenchymal liver cells in vivo.Biochem. J. 1999; 340: 783-792Crossref PubMed Scopus (82) Google Scholar GalNAc conjugates bind to the ASGPR and are taken up in endosomes, where the conjugate dissociates from the receptor. Then, the GalNAc sugars and branches are very quickly lysed from the oligonucleotide before the oligonucleotide escapes to the cytoplasm by a still poorly understood mechanism. The approach for delivering ASOs and siRNAs is highlighted in Figure 1. Several excellent reviews have covered the early history of the field, and the reader is referred to these for more background details.3Craig K. Abrams M. Amiji M. Recent preclinical and clinical advances in oligonucleotide conjugates.Expert Opin. Drug Deliv. 2018; 15: 629-640Crossref PubMed Scopus (32) Google Scholar,10Springer A.D. Dowdy S.F. GalNAc-siRNA conjugates: leading the way for delivery of RNAi therapeutics.Nucleic Acid Ther. 2018; 28: 109-118Crossref PubMed Scopus (264) Google Scholar,11Huang Y. Preclinical and clinical advances of GalNAc-decorated nucleic acid therapeutics.Mol. Ther. Nucleic Acids. 2017; 6: 116-132Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar Following systemic administration, unconjugated ASOs distribute to the liver and other tissues,12Geary R.S. Norris D. Yu R. Bennett C.F. Pharmacokinetics, biodistribution and cell uptake of antisense oligonucleotides.Adv. Drug Deliv. Rev. 2015; 87: 46-51Crossref PubMed Scopus (500) Google Scholar so this raises the question regarding why has GalNAc made such a big impact on ASOs being developed for treating liver disease. A key paper was published in 2014 in which the cellular distribution in the liver in mice after treatment with either a non-conjugated or GalNAc-conjugated ASO to SRB1 was determined.13Prakash T.P. Graham M.J. Yu J. Carty R. Low A. Chappell A. Schmidt K. Zhao C. Aghajan M. Murray H.F. et al.Targeted delivery of antisense oligonucleotides to hepatocytes using triantennary N-acetyl galactosamine improves potency 10-fold in mice.Nucleic Acids Res. 2014; 42: 8796-8807Crossref PubMed Scopus (380) Google Scholar The unconjugated ASO was predominantly (>70%) taken up by the non-parenchymal cellular faction of the liver while, in contrast, the GalNAc-SRB1 ASO was predominantly (>80%) taken up by the hepatocyte fraction of the liver. Thus, the attachment of the GalNAc moiety to the ASO led to targeted delivery to the hepatocytes and increased ASO drug levels in the hepatocytes by about 6- to 7-fold at equivalent doses (Figure 2). This increased delivery to the hepatocytes significantly contributed to the approximately 7-fold increase in potency in vivo (50% knockdown of liver SRB1 mRNA) compared to non-conjugated SRB1 ASO. Other ASO-GalNAc conjugates to hepatocyte targets, that is, FX1, A1AT, APOC3, and transthyretin (TTR), showed similar or slightly greater potency improvements up to 11-fold,13Prakash T.P. Graham M.J. Yu J. Carty R. Low A. Chappell A. Schmidt K. Zhao C. Aghajan M. Murray H.F. et al.Targeted delivery of antisense oligonucleotides to hepatocytes using triantennary N-acetyl galactosamine improves potency 10-fold in mice.Nucleic Acids Res. 2014; 42: 8796-8807Crossref PubMed Scopus (380) Google Scholar demonstrating that this was a general property of GalNAc-ASO conjugates. This improvement in potency in mice has translated very well to the clinic, with typically 10- to 30-fold lower GalNAc-ASO doses being required for functional target knockdown compared to non-targeted ASOs.14Crooke S.T. Baker B.F. Xia S. Yu R.Z. Viney N.J. Wang Y. Tsimikas S. Geary R.S. Integrated assessment of the clinical performance of GalNAc3-conjugated 2′-O-methoxyethyl chimeric antisense oligonucleotides: I. Human volunteer experience.Nucleic Acid Ther. 2019; 29: 16-32Crossref PubMed Scopus (65) Google Scholar For siRNAs, which are double-stranded RNA and typically at least twice the size of ASOs and contain more negative charge, in vivo delivery has been achieved using lipid nanoparticles (LNPs).16Maier M.A. Jayaraman M. Matsuda S. Liu J. Barros S. Querbes W. Tam Y.K. Ansell S.M. Kumar V. Qin J. et al.Biodegradable lipids enabling rapidly eliminated lipid nanoparticles for systemic delivery of RNAi therapeutics.Mol. Ther. 2013; 21: 1570-1578Abstract Full Text Full Text PDF PubMed Scopus (278) Google Scholar Indeed, patisiran, developed for the treatment of hereditary TTR amyloidosis, was the first siRNA drug to receive regulatory approval in 2018 and utilizes an LNP for liver delivery.17Adams D. Gonzalez-Duarte A. O’Riordan W.D. Yang C.C. Ueda M. Kristen A.V. Tournev I. Schmidt H.H. Coelho T. Berk J.L. et al.Patisiran, an RNAi therapeutic, for hereditary transthyretin amyloidosis.N. Engl. J. Med. 2018; 379: 11-21Crossref PubMed Scopus (1424) Google Scholar GalNAc technology has been developed for therapeutic siRNAs and has now largely replaced LNP delivery for liver disease targets.10Springer A.D. Dowdy S.F. GalNAc-siRNA conjugates: leading the way for delivery of RNAi therapeutics.Nucleic Acid Ther. 2018; 28: 109-118Crossref PubMed Scopus (264) Google Scholar,11Huang Y. Preclinical and clinical advances of GalNAc-decorated nucleic acid therapeutics.Mol. Ther. Nucleic Acids. 2017; 6: 116-132Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar Similar to ASO hepatocyte delivery, the importance of having a multivalent GalNAc to deliver siRNAs was reported by Nair et al.18Nair J.K. Willoughby J.L.S. Chan A. Charisse K. Alam M.R. Wang Q. Hoekstra M. Kandasamy P. Kel’in A.V. Milstein S. et al.Multivalent N-acetylgalactosamine-conjugated siRNA localizes in hepatocytes and elicits robust RNAi-mediated gene silencing.J. Am. Chem. Soc. 2014; 136: 16958-16961Crossref PubMed Scopus (672) Google Scholar in 2014 where they demonstrated very effective knockdown in the livers of mice of both APOB100 and TTR mRNA by GalNAc-siRNA conjugates. Most siRNAs in the clinic for treatment of liver disease now use the GalNAc-targeting strategy, and Alnylam Pharmaceuticals, who have pioneered siRNA therapy, recently had their first GalNAc US Food and Drug Administration (FDA) drug approval, givosiran, for acute hepatic porphyria in November 2019. As of February 2020, based on their website, they had another 10 GalNAc conjugates in various stages of clinical development and no additional LNP candidates, demonstrating the impact of GalNAc targeting on the field.19Alnylam Pharmaceuticals. Alnylam development pipeline of investigational RNAi therapeutics. https://www.alnylam.com/alnylam-rnai-pipeline/.Google Scholar This review summarizes some of the advances in GalNAc chemistry, preclinical models used to evaluate GalNAc conjugates, and pharmacokinetic (PK) and safety data, and it provides more details on the clinical experience with GalNAc conjugates, with a focus on information that has emerged during the last 2–3 years. The key breakthrough for the use of GalNAc as a delivery moiety for oligonucleotides was to apply extensive chemical modifications at the 2′ position of the nucleotides and to replace phosphodiester bonds with phosphorothioate (PS) bonds in order to achieve in vivo activity.20Allerson C.R. Sioufi N. Jarres R. Prakash T.P. Naik N. Berdeja A. Wanders L. Griffey R.H. Swayze E.E. Bhat B. Fully 2′-modified oligonucleotide duplexes with improved in vitro potency and stability compared to unmodified small interfering RNA.J. Med. Chem. 2005; 48: 901-904Crossref PubMed Scopus (371) Google Scholar,21Nair J.K. Attarwala H. Sehgal A. Wang Q. Aluri K. Zhang X. Gao M. Liu J. Indrakanti R. Schofield S. et al.Impact of enhanced metabolic stability on pharmacokinetics and pharmacodynamics of GalNAc-siRNA conjugates.Nucleic Acids Res. 2017; 45: 10969-10977Crossref PubMed Scopus (134) Google Scholar These modifications give the conjugates enough nuclease stability to reach the liver after intravenous (i.v.) or subcutaneous injection. Muthiah (Mano) Manoharan was pivotal in the development of GalNAc conjugates.22Oligonucleotide Therapeutics Society 2019 OTS Lifetime Achievement Award winner Muthiah (Mano) Manoharan.https://www.oligotherapeutics.org/muthiah-mano-manoharan-presented-with-2019-ots-lifetime-achievement-award/Date: 2019Google Scholar He received the 2019 Oligonucleotide Therapeutics Society lifetime achievement award for his many contributions to oligonucleotide chemistry, including leading the team at Alnylam that developed the first human therapeutic applications of GalNAc-conjugated siRNA. The structures of the key chemical modifications used in GalNAc conjugates are shown in Figure 3. GalNAc-siRNA conjugates are generally made up of patterns of alternating of 2′-O-methyl and 2′-fluoro nucleotides with insertion of PS bonds at the extremities of the strands (Figure 3).4Khvorova A. Watts J.K. The chemical evolution of oligonucleotide therapies of clinical utility.Nat. Biotechnol. 2017; 35: 238-248Crossref PubMed Scopus (621) Google Scholar In the first generation, siRNA GalNAc conjugates were only partially modified; however, more extensive modifications showed a higher potency and duration of action when tested in vivo.21Nair J.K. Attarwala H. Sehgal A. Wang Q. Aluri K. Zhang X. Gao M. Liu J. Indrakanti R. Schofield S. et al.Impact of enhanced metabolic stability on pharmacokinetics and pharmacodynamics of GalNAc-siRNA conjugates.Nucleic Acids Res. 2017; 45: 10969-10977Crossref PubMed Scopus (134) Google Scholar,23Hassler M.R. Turanov A.A. Alterman J.F. Haraszti R.A. Coles A.H. Osborn M.F. Echeverria D. Nikan M. Salomon W.E. Roux L. et al.Comparison of partially and fully chemically-modified siRNA in conjugate-mediated delivery in vivo.Nucleic Acids Res. 2018; 46: 2185-2196Crossref PubMed Scopus (86) Google Scholar Alnylam estimated the human annual dose required for siRNAs targeting TTR to be 280-fold lower for vutrisiran, a second-generation siRNA that is fully modified and stabilized with PS bonds (called enhanced stabilization chemistry [ESC]), compared to the first-generation chemistry (called standard template chemistry [STC]) for revusiran. The modification of the 5′ end of the antisense strand of siRNA using a stable phosphate analog, vinyl phosphonate, brought even more stability and potency for siRNA conjugates.24Lima 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. et al.Single-stranded siRNAs activate RNAi in animals.Cell. 2012; 150: 883-894Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar The 5′ vinyl phosphonate protects the end of the siRNA from degradation while removing the need for the cell to phosphorylate the double strand prior insertion into the RNA-induced silencing complex (RISC). The latter benefit increased the potency of certain siRNAs conjugates by up to 10-fold.25Parmar R. Willoughby J.L.S. Liu J. Foster D.J. Brigham B. Theile C.S. Charisse K. Akinc A. Guidry E. Pei Y. et al.5′-(E)-vinylphosphonate: a stable phosphate mimic can improve the RNAi activity of siRNA-GalNAc conjugates.ChemBioChem. 2016; 17: 985-989Crossref PubMed Scopus (86) Google Scholar,26Prakash T.P. Kinberger G.A. Murray H.M. Chappell A. Riney S. Graham M.J. Lima W.F. Swayze E.E. Seth P.P. Synergistic effect of phosphorothioate, 5′-vinylphosphonate and GalNAc modifications for enhancing activity of synthetic siRNA.Bioorg. Med. Chem. Lett. 2016; 26: 2817-2820Crossref PubMed Scopus (42) Google Scholar With the siRNA nucleotide content moving away from “natural” nucleotides, and the rise in potency and duration of action, a potential problem of safety came to mind. One of the concerns was that the 2′-fluoro nucleotide contained in the oligonucleotides could cause toxicity due to possible incorporation into genomic DNA and non-specific protein interaction.27Shen W. Liang X.H. Sun H. Crooke S.T. 2′-Fluoro-modified phosphorothioate oligonucleotide can cause rapid degradation of P54nrb and PSF.Nucleic Acids Res. 2015; 43: 4569-4578Crossref PubMed Scopus (77) Google Scholar,28Janas M.M. Jiang Y. Schlegel M.K. Waldron S. Kuchimanchi S. Barros S.A. Impact of oligonucleotide structure, chemistry, and delivery method on in vitro cytotoxicity.Nucleic Acid Ther. 2017; 27: 11-22Crossref PubMed Scopus (28) Google Scholar However, this has not been observed in vivo.29Janas M.M. Zlatev I. Liu J. Jiang Y. Barros S.A. Sutherland J.E. Davis W.P. Liu J. Brown C.R. Liu X. et al.Safety evaluation of 2′-deoxy-2′-fluoro nucleotides in GalNAc-siRNA conjugates.Nucleic Acids Res. 2019; 47: 3306-3320Crossref PubMed Scopus (41) Google Scholar Furthermore, Foster et al.30Foster D.J. Brown C.R. Shaikh S. Trapp C. Schlegel M.K. Qian K. Sehgal A. Rajeev K.G. Jadhav V. Manoharan M. et al.Advanced siRNA designs further improve in vivo performance of GalNAc-siRNA conjugates.Mol. Ther. 2018; 26: 708-717Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar showed that it was possible to reduce the content of 2′-fluoro nucleotides in siRNA to less than 20% for some sequences while maintaining siRNA activity in vivo. Another approach was chosen by Silence Therapeutics involving screening hundreds of modification patterns to identify patterns that used as little modification as possible while maintaining high nuclease stability.31Lindholm M.W. Development of novel therapies using advanced GalNAc-siRNA technology.https://www.silence-therapeutics.com/media/1780/opt-congress-2018-development-of-novel-therapies-using-galnac.pdfDate: 2018Google Scholar The higher potency achieved by the modified siRNA also decreases the chance of off-target toxicity by lowering the required dose. Alnylam has offered an elegant approach to off-target seed interaction by incorporating a glycerol nucleic acid (GNA) and named this new siRNA design ESC+. GNAs have lower affinity to the RNA target than do standard nucleotides, and introducing one at a specific position in the seed region was able to dramatically decrease the off-target toxicity of the sequence while maintaining high potency.32Schlegel M.K. Foster D.J. Kel’in A.V. Zlatev I. Bisbe A. Jayaraman M. Lackey J.G. Rajeev K.G. Charissé K. Harp J. et al.Chirality dependent potency enhancement and structural impact of glycol nucleic acid modification on siRNA.J. Am. Chem. 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These are short 9-mer LNA-PS-modified oligonucleotides conjugated to a GalNAc cluster designed to recognize and bind to the complementary, RISC-bound antisense strand of the siRNA. Injection of the Reversir 7 days after injection of GalNAc-siRNA to TTR was able to fully reverse the silencing of TTR back to normal in 4 days.34Zlatev I. Castoreno A. Brown C.R. Qin J. Waldron S. Schlegel M.K. Degaonkar R. Shulga-Morskaya S. Xu H. Gupta S. et al.Reversal of siRNA-mediated gene silencing in vivo.Nat. Biotechnol. 2018; 36: 509-511Crossref PubMed Scopus (39) Google Scholar Prior to GalNAc conjugation, ASOs were already delivered “naked” in vivo to achieve liver mRNA knockdown. Therefore, the modifications required for good in vivo potency for GalNAc conjugates were similar to unconjugated ASOs. ASO designs depend on their mechanism of action. If the ASO is used to achieve splice switching, the oligonucleotide will be fully modified with 2′-OMe or morpholinos with a high number of PS bonds. In contrast, if the ASO is designed to recruit RNase H to silence mRNA, they are called gapmers and have a central part of just PS-modified DNA and wings on either side containing both PS and modifications at the 2′ position typically using locked nucleic acids (LNAs, cEt) or 2′-OMe/2′-MOE nucleotides (Figures 3 and 4). ASOs are taken up by cells without the need for transfection, and one of the identified mechanisms of uptake of PS ASOs is actually by interacting with the ASGPR.35Schmidt K. Prakash T.P. Donner A.J. Kinberger G.A. Gaus H.J. Low A. Østergaard M.E. Bell M. Swayze E.E. Seth P.P. Characterizing the effect of GalNAc and phosphorothioate backbone on binding of antisense oligonucleotides to the asialoglycoprotein receptor.Nucleic Acids Res. 2017; 45: 2294-2306Crossref PubMed Scopus (65) Google Scholar, 36Donner A.J. Wancewicz E.V. Murray H.M. Greenlee S. Post N. Bell M. Lima W.F. Swayze E.E. Seth P.P. Co-Administration of an excipient oligonucleotide helps delineate pathways of productive and nonproductive uptake of phosphorothioate antisense oligonucleotides in the liver.Nucleic Acid Ther. 2017; 27: 209-220Crossref PubMed Scopus (16) Google Scholar, 37Seth P.P. Tanowitz M. Bennett C.F. Selective tissue targeting of synthetic nucleic acid drugs.J. Clin. Invest. 2019; 129: 915-925Crossref PubMed Scopus (50) Google Scholar Furthermore, while the unconjugated ASOs are mainly delivered to the liver when injected systematically, the conjugation of a GalNAc cluster to the 5′ end of an antisense increases the potency of 2′-OMe and 2′-MOE gapmer ASOs by 10-fold for hepatocyte targets in rodents.13Prakash T.P. Graham M.J. Yu J. Carty R. Low A. Chappell A. Schmidt K. Zhao C. Aghajan M. Murray H.F. et al.Targeted delivery of antisense oligonucleotides to hepatocytes using triantennary N-acetyl galactosamine improves potency 10-fold in mice.Nucleic Acids Res. 2014; 42: 8796-8807Crossref PubMed Scopus (380) Google Scholar These advances have translated to improvements in potency up to 30-fold in the clinic.38Viney N.J. van Capelleveen J.C. Geary R.S. Xia S. Tami J.A. Yu R.Z. Marcovina S.M. Hughes S.G. Graham M.J. Crooke R.M. et al.Antisense oligonucleotides targeting apolipoprotein(a) in people with raised lipoprotein(a): two randomised, double-blind, placebo-controlled, dose-ranging trials.Lancet. 2016; 388: 2239-2253Abstract Full Text Full Text PDF PubMed Scopus (471) Google Scholar A more recent study by Prakash et al.39Prakash T.P. Yu J. Kinberger G.A. Low A. Jackson M. Rigo F. Swayze E.E. Seth P.P. Evaluation of the effect of 2′-O-methyl, fluoro hexitol, bicyclo and morpholino nucleic acid modifications on potency of GalNAc conjugated antisense oligonucleotides in mice.Bioorg. Med. Chem. Lett. 2018; 28: 3774-3779Crossref PubMed Scopus (13) Google Scholar in mice showed that GalNAc conjugation improved potency about 20-fold when LNA and cEt gapmers were used. The ASGPR is formed of two subunits, ASGR1 and ASGR2, assembled in a hetero-oligomer at different ratios.40Meier M. Bider M.D. Malashkevich V.N. Spiess M. Burkhard P. Crystal structure of the carbohydrate recognition domain of the H1 subunit of the asialoglycoprotein receptor.J. Mol. Biol. 2000; 300: 857-865Crossref PubMed Scopus (153) Google Scholar The avidity of the receptor is dependent on the number of ligands attached to the receptor. The affinity of the ASGPR for a trimer of GalNAc is 1,000-fold higher than a dimer and 1,000-fold higher than a monomer, while a tetramer has just a slightly higher affinity for the receptor than a trimer.41Lee Y.C. Lee R.T. Interactions of oligosaccharides and glycopeptides with hepatic carbohydrate receptors.in: Ernst B. Hart G.W. Sinaÿ P. Carbohydrates in Chemistry and Biology: A Comprehensive Handbook. Wiley, 2008: 549-561Google Scholar For this reason, the initial work on GalNAc-conjugated oligonucleotides focused on using a trivalent cluster with the presentation distance between sugar thought to be optimal at 15–20 Å between each GalNAc (Figure 4E).42Khorev O. Stokmaier D. Schwardt O. Cutting B. Ernst B. Trivalent, Gal/GalNAc-containing ligands designed for the asialoglycoprotein receptor.Bioorg. Med. Chem. 2008; 16: 5216-5231Crossref PubMed Scopus (111) Google Scholar In order to achieve this structure, two main strategies have been chosen. One way is to synthesize a trivalent cluster and then link it to the oligonucleotide either by post-synthesis conjugation (e.g., amide coupling, phosphoramidite coupling, or click chemistry) or by coupling the cluster to the solid support prior to the oligonucleotide synthesis (Figures 4A and 4C). Details of synthesis pathways and conjugation conditions can be found in several publications.13Prakash T.P. Graham M.J. Yu J. Carty R. Low A. Chappell A. Schmidt K. Zhao C. Aghajan M. Murray H.F. et al.Targeted delivery of antisense oligonucleotides to hepatocytes using triantennary N-acetyl galactosamine improves potency 10-fold in mice.Nucleic Acids Res. 2014; 42: 8796-8807Crossref PubMed Scopus (380) Google Scholar,18Nair J.K. Willoughby J.L.S. Chan A. Charisse K. Alam M.R. Wang Q. Hoekstra M. Kandasamy P. Kel’in A.V. Milstein S. et al.Multivalent N-acetylgalactosamine-conjugated siRNA localizes in hepatocytes and elicits robust RNAi-mediated gene silencing.J. Am. Chem. 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Novel cluster and monomer-based GalNAc structures induce effective uptake of siRNAs in vitro and in vivo.Bioconjug. Chem. 2018; 29: 2478-2488Crossref PubMed Scopus (20) Google Scholar The second approach to build the cluster is to add monomeric GalNAc sequentially during the oligonucleotide synthesis (Figures 4B and 4D). This latter approach offers more flexibility and is favored for non-trivalent cluster, and it benefits from the easy availability of the monomers. Both approaches have seen comparable activity in vivo, and the main concern when choosing one or the other is manufacturing. GalNAc clusters are preferentially attached to the 3′ end of the sense strand for siRNA and to the 5′ end of the ASOs. 5′ GalNAc-conjugated ASOs show a slight potency advantage compare to 3′ conjugatio