Hepatocyte-targeted delivery of therapeutic drugs uses the asialoglycoprotein receptor (ASGPR) targeting ligand, N-Acetyl-D-galactosamine (GalNAc). ASGPR is very abundant in hepatocytes (liver cells), with approximately 0.5 to 1 million copies per cell. The receptor is recycled within 15-20 minutes. The concept of using trivalent-GalNAc clusters for drug delivery to hepatocytes was first demonstrated in 1987 (Juliano, 2016), and for oligonucleotide delivery in 1995 (Nair et al., 2014). The preferred ligand for the ASGR is a triantennary sugar terminating in galactose or N-acetyl galactosamine.
The ASGR accumulates in coated pits after ligand binding, where it rapidly internalizes and is then trafficked to early sorting endosomes. The low pH endomembrane environment results in the dissociation of ligand and receptor, allowing the ASGR to rapidly return to the plasma membrane while the ligand is trafficked to lysosomes. GalNAc-siRNA conjugates are known to silence targeted genes in hepatocytes via subcutaneous administration at single-digit mg/kg doses in mice. Researchers studied a wide variety of chemical approaches to optimize the GalNAc-siRNA conjugate.
Early Work
The pioneering work of Lee and colleagues established the use of trivalent clusters for targeted delivery to the liver. In the late 1960s, Ashwell and colleagues identified a receptor in the liver that could rapidly remove glycoproteins lacking terminal sialic acid residues (Morell et al., 1968). This asialoglycoprotein receptor (ASGP-R), first isolated in 1974, was shown to bind galactose and is now recognized as the first mammalian lectin to be discovered (Stockert et al., 1974; Hudgin et al., 1974). The receptor is a C-type lectin predominantly expressed on the plasma membranes of hepatocytes, where it clears desialylated glycoproteins from the blood. The receptor is an ideal candidate for targeted drug delivery to the liver.
The "trivalent effect", where three GalNAc units significantly increase binding affinity compared to mono- or bivalent forms, was characterized explicitly by Lee et al. in 1983.
Lee et al. showed that the ASGPR has a geometric requirement for the spacing of GalNAc residues. A trivalent "cluster glycoside" with roughly 20 Å spacing between sugars achieved the highest binding affinity (Kumar & Turnbull, 2023; Zhou & Teng, 2021). These findings layed the groundwork for delivery of therapeutic oligonucleotides, proving that multivalency (specifically tri-valency) is essential for effective hepatocyte targeting (Springer & Dowdy, 2018).
| In the 1980s, binding specificity established design rules for effective binding to ASGP-R. Baenziger and Maynard (1980) identified ASGP-R's preference for GalNAc over galactose in studies of glycosidase-treated glycoproteins. Lee and co-workers observed that the GalNAc monosaccharide was 19-fold better than galactose as an inhibitor of the asialoorosomucoid glycoprotein binding to isolated ASGP-R, and 27-fold better for inhibition of the glycoprotein binding to cultured hepatocytes and showed that the number of Gal residues/cluster and their branching mode, which determines the distance between the Gal residues, are significant determinants of binding affinity of the ligand to the mammalian hepatic lectin on the surface of hepatocytes. These observations enabled the development of the first clinical application of synthetic multivalent carbohydrates. In 2019, the US Food and Drug Administration (FDA) approved Givosiran. Givosiran is a small interfering RNA (siRNA) drug targeting hepatocytes via a trivalent carbohydrate ligand based on N-acetylgalactosamine (GalNAc) for the treatment of acute hepatic porphyria. | 
ASGP-R binding. |
This short, double-stranded RNA molecule suppresses gene expression in a sequence-specific manner via RNA interference (RNAi). One of the RNA strands, the guide strand, is complementary to the mRNA sequence targeted for degradation. The guide is the antisense sequence for the target mRNA. When the guide strand is incorporated into the RNA-induced silencing complex (RISC), it can bind to and degrade the targeted mRNA, thereby preventing expression of its encoded protein.
The GalNAc technology catapulted therapeutic oligonucleotide drugs into the clinic (Huang, 2017). However, the development of successful nucleic acid therapeutics also required many advances in hydrolytically stable analogs of phosphodiester linkages. The following focuses on the development of GalNAc-targeted RNA therapeutics.
Timeline of Development
The transition from basic carbohydrate chemistry to clinical drug delivery took several decades:
| Period | Milestone |
| Late 1960s | Ashwell and Morell discover the ASGPR and its role in clearing glycoproteins from circulation (Kumar & Turnbull, 2023). |
| Early 1980s | Y.C. Lee synthesizes the first synthetic "cluster glycosides" and identifies the trivalent GalNAc cluster as the optimal ligand (Kumar & Turnbull, 2023). |
| 2014–2017 | Major biotech firms (like Alnylam and Ionis) publish the first clinical results using Tri-GalNAc conjugated to siRNA and ASOs (Schmidt et al., 2017; Tang & Khvorova, 2024, Nair et al. 2014). The conjugation of optimized, chemically modified siRNAs to an engineered ASGPR ligand resulted in conjugates with systemic stability against nucleases and improved pharmacokinetics relative to the unconjugated siRNAs. These conjugates showed robust and durable silencing of the targeted gene in the liver following single or multiple low-volume SC administrations. |
| 2019 | Givosiran becomes the first FDA-approved siRNA drug utilizing the Tri-GalNAc targeting technology (Kumar & Turnbull, 2023). |
Alnylam Pharmaceuticals developed a high-affinity (Kd = 2.3 nM) trivalent glycocluster ligand for ASGP-R, with GalNAc sugars rather than galactose. The lipidated version of this ligand with a PEG spacer was effective for delivering short interfering RNA (siRNA) molecules in lipid nanoparticles to reduce factor VII expression in mice (Akinc et al., 2010; Jayaraman et al., 2012).
| Triantennary GalNAc (GalNAc3 or TriGalNAc) solid phase synthesis support for the 3’-end and amidite. | siRNA Drug Givosiran |
| 
| 
|
| 
| -- Phosphorothioate, N 2’-O-methyl-ribose, N 2’-Fluororibose The TriGalNAc ligand is conjugated to the 3’end of the sense strand. |
In 2017, Sanhueza reported the structure of X-ray crystal structures of the ASGPR carbohydrate-binding domain with lactose and a compact bicyclic ketal ligand for the receptor (See below.).
| ASGPR carbohydrate-binding domain with lactose (5JPV). | ASGPR carbohydrate-binding domain with bicyclic ketal ligand (5JQ1). |
| 
| 
|
A variety of CPG-Triantennary GalNAc ligands are now commercially available for the production of therapeutic GalNAc-conjugates.
| CPG-TriGalNAcs |
| 
|
| 
|
| TriGalNAc-TEG C7 CPG |
| 
|
| L96 Alnylam original GalNAc ligand |
| 
|
| |
| TriGalNAc-NHS |
| 
|
| |
| TriGalNAc-PRG4 3’-end |
| 
|
| |
| BSI TriGalNAC |
| 
|
| |
| TriGalNAc-PEG5-AmC6 |
| 
|
| |
| TriGalNAc-KK-AMC6 |
| 
|
| 
|
| |
| Trivalent GalNAc TEG |
| 
|
| |
| TriGalNAc KK-Am C6 |
| 
Can be conjugated to AmC6, PSs or POs. |
| |
| Mono GalNAc-COOH |
| 
|
References
Akinc A, Querbes W, De S, Qin J, Frank-Kamenetsky M, Jayaprakash KN, Jayaraman M, Rajeev KG, Cantley WL, Dorkin JR, Butler JS, Qin L, Racie T, Sprague A, Fava E, Zeigerer A, Hope MJ, Zerial M, Sah DW, Fitzgerald K, Tracy MA, Manoharan M, Koteliansky V, Fougerolles Ad, Maier MA. Targeted delivery of RNAi therapeutics with endogenous and exogenous ligand-based mechanisms. Mol Ther. 2010 Jul;18(7):1357-64. Targeted Delivery of RNAi Therapeutics With Endogenous and Exogenous Ligand-Based Mechanisms - PMC, PubMed.
Baenziger JU, Maynard Y. Human hepatic lectin. Physiochemical properties and specificity. J Biol Chem. 1980 May 25;255(10):4607-13. PMID: 7372599. Human hepatic lectin. Physiochemical properties and specificity.
Bissell DM, Anderson KE, Bonkovsky HL. Porphyria. N Engl J Med. 2017 Aug 31;377(9):862-872. PubMed.
Dong Y, Siegwart DJ, Anderson DG. Strategies, design, and chemistry in siRNA delivery systems. Adv Drug Deliv Rev. 2019 Apr;144:133-147. Strategies, Design, and Chemistry in siRNA Delivery Systems - PMC, https://pmc.ncbi.nlm.nih.gov/articles/PMC6745264/
D'Souza A.A., Devarajan P.V. Asialoglycoprotein receptor mediated hepatocyte targeting—strategies and applications. J. Control. Release. 2015;203:126–139. [PubMed]
GalNAc https://www.biosyn.com/tew/N-acetylgalactosamine-or-GalNAc.aspx
GalNAc-conjugated oligonucleotides https://www.biosyn.com/tew/N-Acetylgalactosamine-(GalNAc)-Conjugated-Oligonucleotides-for-Cell-Delivery.aspx
GalNAc custom synthesis https://www.biosyn.com/tew/Custom-synthesis-of-N-acetylgalactosamine-(GalNac)-siRNA.aspx
GalNAc Trivalent TEG https://www.biosyn.com/oligonucleotideproduct/triantennary-galnac,galnac,-trigalnac.aspx
Grewal PK, Uchiyama S, Ditto D, et al. The Ashwell receptor mitigates the lethal coagulopathy of sepsis. Nature Medicine. 2008;14(6):648–655. doi: 10.1038/nm1760. [DOI] [PMC free article] [PubMed] [Google Scholar]
Huang Y. Preclinical and Clinical Advances of GalNAc-Decorated Nucleic Acid Therapeutics. Mol Ther Nucleic Acids. 2017 Mar 17;6:116-132. Preclinical and Clinical Advances of GalNAc-Decorated Nucleic Acid Therapeutics - PMC, PubMed.
Hudgin R. L. Pricer W. E. Ashwell G. J. Biol. Chem. 1974;249:5536–5543. doi: 10.1016/S0021-9258(20)79761-9. [DOI] [PubMed] [Google Scholar]
Ivanova A, Langendonk JG, Kauppinen R, Minder E, Horie Y, Penz C, Chen J, Liu S, Ko JJ, Sweetser MT, Garg P, Vaishnaw A, Kim JB, Simon AR, Gouya L; ENVISION Investigators. Phase 3 Trial of RNAi Therapeutic Givosiran for Acute Intermittent Porphyria. N Engl J Med. 2020 Jun 11;382(24):2289-2301. Phase 3 Trial of RNAi Therapeutic Givosiran for Acute Intermittent Porphyria | New England Journal of Medicine, PubMed.
Jayaraman M, Ansell SM, Mui BL, Tam YK, Chen J, Du X, Butler D, Eltepu L, Matsuda S, Narayanannair JK, Rajeev KG, Hafez IM, Akinc A, Maier MA, Tracy MA, Cullis PR, Madden TD, Manoharan M, Hope MJ. Maximizing the potency of siRNA lipid nanoparticles for hepatic gene silencing in vivo. Angew Chem Int Ed Engl. 2012 Aug 20;51(34):8529-33. https://pmc.ncbi.nlm.nih.gov/articles/PMC3470698/
Juliano RL. The delivery of therapeutic oligonucleotides. Nucleic Acids Res. 2016 Aug 19;44(14):6518-48. [PMC free article] [PubMed]
Khvorova A, Watts JK. The chemical evolution of oligonucleotide therapies of clinical utility. Nat Biotechnol. 2017 Mar;35(3):238-248. The chemical evolution of oligonucleotide therapies of clinical utility - PMC, PubMed.
Kumar, V., & Turnbull, W. B. (2023). Targeted delivery of oligonucleotides using multivalent protein–carbohydrate interactions. Chemical Society Reviews, 52(4), 1273–1287. https://doi.org/10.1039/d2cs00788f
Lee RT, Lee YC. Affinity enhancement by multivalent lectin-carbohydrate interaction. Glycoconj J. 2000 Jul-Sep;17(7-9):543-51. Affinity enhancement by multivalent lectin-carbohydrate interaction - PubMed, CAS PubMed.
Lee RT, Lin P, Lee YC. New synthetic cluster ligands for galactose/N-acetylgalactosamine-specific lectin of mammalian liver. Biochemistry. 1984 Aug 28;23(18):4255-61. PubMed.
Lee RT, Lin P, Lee YC. New synthetic cluster ligands for galactose/N-acetylgalactosamine-specific lectin of mammalian liver. Biochemistry. 1984 Aug 28;23(18):4255-61. PubMed.
Lee YC, Townsend RR, Hardy MR, Lönngren J, Arnarp J, Haraldsson M, Lönn H.; Binding of synthetic oligosaccharides to the hepatic Gal/GalNAc lectin. Dependence on fine structural features. J Biol Chem. 1983 Jan 10;258(1):199-202. PMID: 6848494. Binding of synthetic oligosaccharides to the hepatic Gal/GalNAc lectin. Dependence on fine structural features., https://www.jbc.org/article/S0021-9258(18)33240-X/pdf
Lee, Y.C., and Lee, R.T, Carbohydrate-Protein Interactions: Basis of Glycobiology. Acc. Chem. Res., 1995, 28, 321-327. Carbohydrate-Protein Interactions: Basis of Glycobiology | Accounts of Chemical Research
Li C, Che B, Deng L. Electrochemical Biosensors Based on Carbon Nanomaterials for Diagnosis of Human Respiratory Diseases. Biosensors (Basel). 2022 Dec 22;13(1):12.
Morell A. G. Irvine R. A. Sternlieb I. Scheinberg I. H. Ashwell G. J. Biol. Chem. 1968;243:155–159. doi: 10.1016/S0021-9258(18)99337-3. [DOI] [PubMed] [Google Scholar]
Nair JK, Willoughby JL, Chan A, Charisse K, Alam MR, Wang Q, Hoekstra M, Kandasamy P, Kel'in AV, Milstein S, Taneja N, O'Shea J, Shaikh S, Zhang L, van der Sluis RJ, Jung ME, Akinc A, Hutabarat R, Kuchimanchi S, Fitzgerald K, Zimmermann T, van Berkel TJ, Maier MA, Rajeev KG, Manoharan M. Multivalent N-acetylgalactosamine-conjugated siRNA localizes in hepatocytes and elicits robust RNAi-mediated gene silencing. J Am Chem Soc. 2014 Dec 10;136(49):16958-61. [PubMed]
Sanhueza CA, Baksh MM, Thuma B, Roy MD, Dutta S, Préville C, Chrunyk BA, Beaumont K, Dullea R, Ammirati M, Liu S, Gebhard D, Finley JE, Salatto CT, King-Ahmad A, Stock I, Atkinson K, Reidich B, Lin W, Kumar R, Tu M, Menhaji-Klotz E, Price DA, Liras S, Finn MG, Mascitti V. Efficient Liver Targeting by Polyvalent Display of a Compact Ligand for the Asialoglycoprotein Receptor. J Am Chem Soc. 2017 Mar 8;139(9):3528-3536. https://pmc.ncbi.nlm.nih.gov/articles/PMC6991140/, https://www.ncbi.nlm.nih.gov/Structure/pdb/5JPV
Schmidt, 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. (2017). Characterizing the effect of GalNAc and phosphorothioate backbone on binding of antisense oligonucleotides to the asialoglycoprotein receptor. Nucleic Acids Research, 45(5), 2294–2306. https://doi.org/10.1093/nar/gkx060
Schwartz AL, Fridovich SE, Knowles BB, Lodish HF. Characterization of the asialoglycoprotein receptor in a continuous hepatoma line. The Journal of Biological Chemistry. 1981;256(17):8878–8881. [PubMed] [Google Scholar]
Setten RL, Rossi JJ, Han SP. The current state and future directions of RNAi-based therapeutics. Nat Rev Drug Discov. 2019 Jun;18(6):421-446. doi: 10.1038/s41573-019-0017-4. Erratum in: Nat Rev Drug Discov. 2020 Apr;19(4):291. PubMed.
Sood A, Gerlits OO, Ji Y, Bovin NV, Coates L, Woods RJ. Defining the Specificity of Carbohydrate-Protein Interactions by Quantifying Functional Group Contributions. J Chem Inf Model. 2018 Sep 24;58(9):1889-1901. Defining the Specificity of Carbohydrate–Protein Interactions by Quantifying Functional Group Contributions - PMC, https://pmc.ncbi.nlm.nih.gov/articles/PMC6442460/
Springer, A. D., & Dowdy, S. F. (2018). GalNAc-siRNA Conjugates: Leading the Way for Delivery of RNAi Therapeutics. Nucleic Acid Therapeutics, 28(3), 109–118. https://doi.org/10.1089/nat.2018.0736
Stockert R. J. Morell A. G. Scheinberg I. H. Science. 1974;186:365–366. doi: 10.1126/science.186.4161.365. [DOI] [PubMed] [Google Scholar]
Tang, Q., & Khvorova, A. (2024). RNAi-based drug design: considerations and future directions. Nature Reviews Drug Discovery, 23(5), 341–364. https://doi.org/10.1038/s41573-024-00912-9
Wang F, Li P, Chu HC, Lo PK. Nucleic Acids and Their Analogues for Biomedical Applications. Biosensors (Basel). 2022 Feb 4;12(2):93. https://pmc.ncbi.nlm.nih.gov/articles/PMC8869748/
Zhou, Y., & Teng, P. (2021). Development of Triantennary N-Acetylgalactosamine Conjugates as Degraders for Extracellular Proteins. ChemRxiv. https://doi.org/10.26434/chemrxiv.14234567.v1
---...---