Biessen & Berkel recently reviewed the 30-year development of oligonucleotide-based therapeutics focusing on the evolution of the GalNAc technology. Basic research investigating substrate recognition by a liver-specific lectin, the asialoglycoprotein receptor (ASGPR), resulted in the design of synthetic N-acetyl galactosamine (GalNAc) terminated ligands. The ligands are now a key feature of many oligonucleotide therapeutics.
The GalNAc technology enhances the unfavorable pharmacokinetics of oligonucleotide therapeutics for hepatic drug targets. Close to a dozen GalNAc-conjugated oligonucleotides are already approved for clinical use. GalNAc-RNAi drugs' therapeutic half-life is quite long, resulting in monthly to half-yearly dosing regiments.
N-acetylgalactosamine (GalNAc) ligands improve oligonucleotides' cellular uptake and tissue-specific delivery. GalNAc-based delivery is possible because liver cells abundantly and specifically express the asialoglycoprotein receptor (ASGPR). ASGPR binds and uptakes circulating glycoproteins via receptor-mediated endocytosis. Recent years have seen significant progress in the field of GalNAc conjugates.
Figure 1: Structure of a 3’-trianennary GalNAc-based ASGPR ligand. This structure, when conjugated to an siRNA, allows specific delivery of the RNA drug to the liver since it targets the asialoglycoprotein receptor (ASGPR).
The attachment of a trimeric GalNAc moiety to biomolecules selectively directs extracellular molecules into the lysosomes of liver cells. The liver cells degrade the protein if the ligand is attached to a protein. Connecting the ligand to an oligonucleotide drug allows a small amount of the oligonucleotide drug to escape into the cell and block RNA or induce RNA degradation. The commercial availability of GalNAc building blocks, such as 5’-GalNAc C3 phosphoramidite and GalNAc C3 CPG, allows the efficient synthesis of oligonucleotides modified with this molecular compound during or after automated solid phase synthesis.
In 2014, Nair et al. reported the conjugation of small interfering RNAs (siRNAs) to an N-acetylgalactosamine (GalNAc) asialoglycoprotein receptor ligand. This oligonucleotide conjugate facilitates targeted siRNA delivery to liver cells (hepatocytes) in vitro and in vivo. The GalNAc-derived ligands are compatible with solid-phase oligonucleotide synthesis and deprotection conditions allowing their use in standard automated solid-phase synthesis. Nair et al. showed that injections of siRNA-GalNAc conjugates in the fatty tissue, just under the skin, resulted in robust RNAi-mediated gene silencing in the liver.
The discovery of RNA silencing (Nobel prize in 2006 to Mello and Fire) made it evident that small interfering RNA (siRNA) drugs may offer many advantages over small molecular drugs and other protein-based biologicals. Targeting lectins and other receptor systems promise selective and specific delivery of oligonucleotide-based drugs to other cells, not just liver cells. Oligonucleotide drug-target interactions are mainly based on Watson-Crick base pairing but not on the configuration or 3-dimensional structure of the drug and its target, allowing selective intervention of intracellular targets formerly considered undruggable. However, oligonucleotide-based drugs also have some setbacks. Mammalian cells, including human cells, are equipped with several defense mechanisms preventing the invasion and spreading of foreign external DNA and RNA in the host. In general, oligonucleotide-based medicines are not well suited for oral administration. Renal clearance, scavenger receptors of the reticuloendothelial system, and extra- and intracellular enzyme degradation readily eliminate these drugs. Also, oligonucleotides are hydrophilic and do not readily penetrate cell membranes.
Erik A.L. Biessen and Theo J.C. Van Berkel; N-Acetyl Galactosamine Targeting: Paving the Way for Clinical Application of Nucleotide Medicines in Cardiovascular Diseases. Arteriosclerosis, Thrombosis, and Vascular Biology. 2021;41:2855–2865. [Link]
Nair JK, Willoughby JLS, Chan A, Charisse K, Alam MR, Wang Q, Hoekstra M, Kandasamy P, Kel’in AV, Milstein S, 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. [ACS]
Nobel prize 2006: Mello and Fire