N-Acetylgalactosamine (GalNAc), an amino sugar derivative of galactose, is a well-defined liver-targeted moiety benefiting from its high affinity with asialoglycoprotein receptor (ASGR). GalNAc (TEG)-CPG is used to synthesize oligonucleotide-GalNAc conjugates. Amerigo Scientific offers GalNAc (TEG)-CPG with pore sizes of 500 Å and 1000 Å. Due to space reasons, the pore size of 1000 Å is suitable for the synthesis of oligonucleotide-GalNAc conjugates with long oligonucleotides, while the pore size of 500 Å is suitable for the synthesis of oligonucleotide-GalNAc conjugates with short oligonucleotides.
Products
Oligonucleotides selectively repress target genes through antisense oligonucleotide (ASO) and RNA interference, and their potential therapeutic applications have attracted significant attention. The oligonucleotide-GalNAc conjugates synthesized using GalNAc (TEG)-CPG have hepatocyte targeting and effective intracellular delivery.
Selectable Pore Sizes
Depending on the desired oligonucleotide sequence length of our customer, different pore sizes are available to facilitate the synthesis and development of oligonucleotide drugs.
GalNAc Conjugates
Nowadays, small molecule drugs, protein drugs, and oligonucleotide drugs are the three main parts of drug development. The liver, as the largest internal organ of the body, plays an important role in metabolism, detoxification, iron homeostasis, and the synthesis and secretion of major plasma proteins. Therefore, many disease targets are present in hepatocytes of the liver that may be sensitive to oligonucleotide drugs.
The GalNAc conjugates, which can bind to the Asialoglycoprotein receptor (ASGR), are a breakthrough approach for the targeted delivery of therapeutic oligonucleotide to hepatocytes. ASGR is a high-volume and rapidly internalized receptor that is expressed in large amounts specifically on the sinusoidal surface of hepatocytes. Human ASGR contains two subunits, ASGR1 and ASGR2, which facilitate the clearance of desialylated serum glycoproteins containing a terminal galactose or GalNAc. However, the ASGR1 subunit alone is considered to be sufficient for the effective uptake of GalNAc-bound oligonucleotides in vitro and in vivo.
Classical Trivalent Structure
The valence and location of GalNAc conjugates are important for the delivery potency and efficacy of GalNAc-linked oligonucleotide drugs. Trivalent GalNAc conjugates can increase the potency of oligonucleotide drugs in mouse hepatocytes by 6 to 10 folds. Bivalent and trivalent GalNAc conjugates bound with the highest affinity to ASGR and showed the best in vitro and in vivo activity. In contrast, monovalent GalNAc conjugates showed 10-fold lower receptor binding affinity but only 2-fold lower in vivo activity compared to trivalent GalNAc conjugates. Taken together, trivalent GalNAc conjugates are widely used as a strategy for GalNAc-oligonucleotide drugs.
GalNAc-mediated Oligonucleotides Delivery to Liver Hepatocytes
Cell surface ASGR interacts with GalNAc-bound oligonucleotides in lectin-coated vesicles of the plasma membrane in the presence of Ca2+ at pH >6 and internalizes the ligand via lectin-dependent receptor-mediated endocytosis. After complete internalization, acidification during endosome maturation separates GalNAc-bound oligonucleotides from ASGR, followed by GalNAc degradation in the lysosome. The free ASGR is further recycled to the plasma membrane of hepatocytes. The large blood flow and endothelial cells of the liver allow for adequate uptake and support the multi-month efficacy of a single injection of oligonucleotides. Because of these advantages, GalNAc conjugates may become the primary method of clinical delivery of oligonucleotides to the liver. Currently, GalNAc-based oligonucleotides feature prominently in the drug development pipeline of several pharmaceutical companies, and in the past two years, three GalNAc-based drugs have received global approval and entered clinical use.
Fig.1 GalNAc-mediated targeted delivery of oligonucleotides to hepatocytes. (Cui, H., et al., 2021)
GalNAc for siRNA and Anti-miR Delivery to Hepatocytes
Currently, all registered GalNAc-based oligonucleotides are siRNA/GalNAc conjugates targeting mRNA for ALAS1 in hepatocytes for acute hepatic porphyria (AHP), mRNA for hydroxyacid oxidase 1 gene (HAO1) in hepatocytes for primary hyperoxaluria type 1 (PH1), and mRNA for Proprotein convertase subtilisin-kexin type 9 gene (PCSK9) in hepatocytes for adult hypercholesterolemia or mixed dyslipidemia, respectively.
GalNAc has also been used for anti-miR (antagomir) delivery. For example, GalNAc conjugated oligonucleotides targeting hepatocyte miR-122 and microRNA-103/107 are developed for the treatment of hepatitis C virus (HCV) and non-alcoholic steatohepatitis (NASH) in patients with type 2 diabetes/pre-diabetes, respectively.
GalNAc for ASO Delivery to Hepatocytes
GalNAc conjugates are also used in the development of ASO drugs. With GalNAc conjugates, GalNAc-ASO shows more than 10-fold greater potency than ASO without conjugates, which supports lower doses and/or less frequent dosing (possibly once a month). The GalNAc-ASO conjugates are optimal for use in a broader patient population as it demonstrates greater convenience for patients, better potency and tolerability, and a higher probability of adherence.
Reference
- Cui, H., Zhu, X., Li, S., Wang, P., & Fang, J. (2021). Liver-targeted delivery of oligonucleotides with N-acetylgalactosamine conjugation. ACS omega, 6(25), 16259-16265.
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