Oligonucleotide modalities are nucleic acid polymers able to act at the RNA level to treat a wide range of diseases. Despite their tremendous potential in therapeutics, they have poor pharmacokinetics and pharmacodynamics properties. A major challenge that has held back the therapeutic application of oligonucleotides is their delivery to the diseased cells since the therapeutic RNA target is inside the cell.

The delivery potential of antisense oligonucleotides (ASOs) and small interfering RNAs (siRNAs) can be enhanced through direct covalent conjugation of various moieties that promote intracellular uptake, target the drug to specific cells or reduce clearance from the circulation. Bioconjugates constitute distinct, homogeneous, single-component molecular entities with precise stoichiometry, meaning that high-scale synthesis is relatively simple and their pharmacokinetic properties are well defined. Furthermore, bioconjugates are typically of small size, and generally exhibit favorable bio-distribution profiles. The great asset of the bioconjugation is the internalization of oligonucleotides through the interaction of the conjugate and its corresponding cell surface receptor protein, which leads to a receptor-mediated endocytosis. The interaction of bioconjugates with cell type-associated receptors thereby enables targeted delivery to specific tissues, or cell types within a tissue.

Graham et al. showed in 1998 that phosphorothioate-modified ASOs are preferentially internalized by endothelial cells lining the liver sinusoids and by Kupffer cells resulting in little ASO accumulation in hepatocytes. 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. The ASGPR is a high-capacity, rapidly internalizing receptor with approximately 500,000 copies per hepatocyte. Trimeric N-acetylgalactosamine (GalNAc) ligands for the ASGPR have been developed, and these were first utilized to deliver oligonucleotides to the liver more than 20 years ago. Biessen et al. showed that linking phosphorothioate oligonucleotides to a multivalent N-acetylgalactosamine cluster, a synthetic ligand for the ASGPR, results in an ASO that accumulates mostly in hepatocytes.

Role of GalNAc and its application

ASGPR is a transmembrane protein that plays a critical role in serum glycoprotein homeostasis by mediating the endocytosis and lysosomal degradation of glycoproteins with exposed terminal galactose or N‐acetylgalactosamine. This receptor is highly expressed on hepatocytes while extra‐hepatic expression is minimal. Therefore, due to its specificity and high capacity to internalize its specific substrate from circulation, ASGPR is the ideal receptor candidate for targeted delivery of therapeutics to the liver. This hepatocyte-specific receptor is comprised of the highly homologous major ASGPR1 and minor ASGPR2 subunits. Each subunit consists of a cytosolic N-terminal domain, a single transmembrane segment, a stalk domain, and a Ca²⁺-dependent carbohydrate recognition domain (CRD) at the C-terminus. The CRD is known to mediate binding of non-reducing terminal β-D-galactose or GalNAc residues with high affinity.

Scheme 1 – One structure of Trimeric GalNAc ligand

GalNAc (Scheme 1) is a carbohydrate moiety that binds to the highly liver-expressed asialoglycoprotein receptor 1 (ASGR1) with high affinity (Kd = 2.5 nM) (1). The avidity of the receptor is dependent on the number of ligands that is attached. 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. Thus GalNAc ligands have been designed to bind to the ASGPR with high specificity and affinity, thereby triggering hepatocyte-specific uptake of conjugates. GalNAc conjugates bind to the ASGPR and are taken up in endosomes, where the conjugate dissociates from the receptor. The interaction between GalNAc and ASGPR1 is pH-sensitive, such that dissociation of the receptor and oligonucleotide conjugate occurs during acidification during endosomal maturation. 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 GalNAc moiety is subsequently subject to enzymatic degradation that liberates the oligonucleotide (2).

GalNAc conjugates (Scheme 2) have become a breakthrough approach in the therapeutic oligonucleotide field with enormous potential (3). The ligands derived from GalNAc are compatible with solid-phase oligonucleotide synthesis and deprotection conditions, with synthesis yields comparable to those of standard oligonucleotides (1b). A complete GalNAc-siRNA can be synthesized on a solid-state oligonucleotide synthesizer and chemically defined by mass spectrometry. Additionally, conjugation methods on the 5’ end of ASOs have been reported in the literature (4). Similarly to siRNAs, conjugation of ASOs to GalNAc ligands has been shown to improve potency of ASOs in hepatocytes (2).

Scheme 2 – Schematic representation of oligonucleotide-GalNAc conjugates (light blue and orange spheres represent different type of 2’-modified nucleotides, green triangle can be different type of phosphoro -linkage)

siRNA GalNAc conjugates:

For siRNAs, conjugation to the passenger strand is generally preferred so as not to hinder the on-target silencing activity of the guide strand and, conversely, to diminish the off-target gene silencing potential of the passenger strand. GalNAc can be placed either at the 3′ or 5′ ends of the siRNA sense strand (1). Usually, siRNAs are made up of patterns of alternating of 2’-O-methyl and 2’-O-fluoro nucleotides with insertion of phosphorothioate bonds (PS) at the extremities of the strands to enhance pharmacokinetics properties. 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 GalNAc conjugates. This protects the end of the siRNA from degradation and impeding the cell to phosphorylate the double strand prior its insertion into the RISC (RNA induced silencing complex). The latter effect can increase the potency of the siRNA up to 10 folds.

ASO GalNAc conjugates:

GalNAc conjugation on both the 3’ and 5’-end of the oligonucleotide has been evaluated with the 5’ having slightly enhanced potency in cells and in animals (4). The unconjugated ASOs are mainly delivered to the liver when injected systematically. However, the conjugation of a GalNAc ligand to the 5’ end of an ASO increases the potency by 10-fold for hepatocyte targets in rodents (2) and up to 20-fold when locked nucleic acids were used on the wings of a gapmer. These advances have translated to improvements in potency up to 30-fold in the clinic.

Conclusion

The development of GalNAc oligonucleotide conjugates for the targeted delivery of RNA interference therapeutics to liver enabled great advances in the oligonucleotide potency. With the successful translation in human clinical trials, this strategy has been widely adopted for a variety of nucleic acid therapeutics, including ASOs and anti-microRNAs (anti-miRs). The attachment of the GalNAc moiety to ASOs led to targeted delivery to the hepatocytes and increased ASO drug levels in the hepatocytes. As described previously, the GalNAc conjugates have also been used for siRNA delivery, and have now largely replaced the lipid nanoparticle (LNP) delivery for liver targets diseases. Most siRNAs in the clinic for treatment of liver disease now use the GalNAc-targeting strategy. Alnylam Pharmaceuticals, who have pioneered siRNA therapy, had received in 2019 their first GalNAc US Food and Drug Administration (FDA) drug approval, Givosiran, for acute hepatic porphyria. Furthermore, many others are on clinical trials.

The rapid emergence of a robust pipeline of GalNAc-conjugated oligonucleotides for a wide range of liver-based diseases represents a true innovation for the field of oligonucleotide therapeutics. It starts to encourage research for other extrahepatic ligand-based delivery systems. For example, a GLP1R agonist peptide has been shown to effectively target an ASO to the GLP1R present on pancreatic insulin-secreting beta cells, leading to target knockdown. With our strong expertise in API manufacturing, we support pharmaceutical companies in their development of new oligonucleotide modalities. To meet the demand of our partners in the oligonucleotide field, we have expanded our manufacturing platform to include the custom production of oligonucleotides. At Bachem, technological leadership and innovative strength have been the cornerstones of our success since the very beginning of our company.

References

(1) a) Janas, M. M. et al. Nat. Commun. 9, 723 (2018) ; b) Nair, J. K. et al. J. Am. Chem. Soc. 136, 16958–16961 (2014).

(2) Prakash, T. P. et al. Nucleic Acids Res. 42, 8796–8807 (2014).

(3) Sehgal, A. et al. Nat. Med. 21, 492–497 (2015).

(4) Østergaard, M. E. et al. Bioconj. Chem. 26, 1451–1455 (2015).

For reviews on the topic:

Debacker, A. J. et al Mol. Therapy, 28, 1759–1771 (2020).

Khvorova, A. & Watts, J. K. Nat. Biotechnol. 35, 238–248 (2017).