GalNAc Conjugates for Hepatic Targeted Delivery of siRNA
Liver-Targeted Delivery of siRNA
The liver is the largest internal organ in the human body and plays an important role in metabolism, detoxification, iron homeostasis, and synthesis and secretion of major plasma proteins. Thus, the liver is an attractive organ for the development of small interfering RNA (siRNA) therapeutics, which is suitable for both passive and targeted delivery of siRNA drugs. Passive delivery takes advantage of the inherent tendency of liposomes and lipid nanoparticles to accumulate in the filtering organs of the reticulated endothelial system, mainly the liver, but also lymph nodes, kidneys, and spleen. Targeted delivery of siRNAs exploits asialoglycoprotein receptor (ASGPR), which is highly expressed on the surface of hepatocytes. One of representative delivery systems for the development of siRNA therapies for the treatment of liver disease is N-acetylgalactosamine (GalNAc)-siRNA conjugates.
GalNAc-siRNA Conjugates
Conjugation of GalNAc has become a major strategy for delivery of oligonucleotides to hepatocytes. Such conjugates are efficiently internalized by binding to the ASGPR, which is conserved across species and highly expressed by hepatocytes and can mediate clathrin-involved endocytosis. ASGPR is formed by a major 48 kDa (ASGPR-1) and a minor 40 kDa (ASGPR-2) subunits. The primary role of ASGPR is to bind, internalize, and subsequently remove glycoproteins containing terminal galactose or GalNAc residues from the circulation. Ligand binding to ASGPR depends on Ca2+, the position of terminal galactose residues, and pH above 6.5.
GalNAc-siRNA conjugates shows good efficiency and safety for targeted delivery to hepatocytes in vivo. GalNAc can rapidly bind to ASGPR to be internalized into cells and re-display on the cell surface. Single GalNAc residue binds to the receptor with relatively low affinity. However, cooperative interactions with several GalNAc of multimerized receptor subunit clusters show higher affinity for ASGPR compared to single GalNAc. Among them, the triantennary GalNAc conjugate with a mutual distance of ~20 Å exhibits the highest affinity with ASGPR. By conjugating three GalNAc moieties to the siRNA with proper linker chemistry, siRNA can be efficiently delivered to hepatocytes by subcutaneous injection. Benefiting from this enhanced stabilization modification, GalNAc-siRNA conjugates can remain in the circulation system and cytoplasm for a long time, resulting in long-term gene silencing and therapeutic effects in vivo.
GalNAc-CPG and GalNAc Azide
Amerigo Scientific offers triantennary GalNAc (tri-GalNAc) CPG for the synthesis of siRNA-GalNAc conjugates. 500Å and 1000Å are available. We also offer tri-GalNAc azide that can be bound to oligonucleotides via click chemistry.
RNA Silencing
RNA silencing is a highly conserved gene regulation mechanism mediated by siRNA. siRNA is a duplex composed of the guide and passenger strands and it silences gene expression by either suppressing transcription or by triggering a sequence-specific degradation of target mRNA. The siRNA is loaded onto the Argonaute (AGO) protein, a core component of the RNA-induced silencing complex (RISC). Subsequently, the passenger strand of siRNA is ejected and the guide strand remains loading on the AGO. The guide strand then pairs with the target mRNA in perfect sequence complementarity, resulting in AGO cleavage to repress gene expression.
The therapeutic prospects of N-acetylgalactosamine-siRNA conjugates. Front Pharmacol. 2022 Dec 14;13:1090237.
siRNA Therapy Development
siRNA has innate advantages over small molecule therapeutics and monoclonal antibody drugs, because siRNA achieves its function through complete base pairing with mRNA, whereas small molecules and monoclonal antibodies require recognition of the complex spatial conformation of target proteins. Many diseases cannot be treated by small molecule and antibody drugs because target molecules with high activity, affinity, and specificity cannot be identified. However, theoretically, any gene can be targeted by siRNA, simply by selecting the correct nucleotide sequence of the targeted mRNA. This advantage enables the development of siRNA therapeutics with a shorter development span and a broader therapeutic field than the development of small molecule or antibody drugs.
Despite the promise of siRNAs in drug development, naked, unmodified siRNAs have some disadvantages, such as unsatisfactory stability, poor pharmacokinetic behavior, and possible induction of off-target effects, making their clinical application difficult. Off-target effects of siRNA can occur through several different mechanisms, one of which is the binding of the passenger strand of siRNA to undesired mRNA. In addition, the seed regions of the siRNA passenger or guide strand recognize a certain mRNA sequence, particularly at the 30-untranslation region (UTR), and silence gene expression in a "miRNA-like" manner. The passenger or guide strand of siRNA binds to toll-like receptors (TLRS) to stimulate the immune response and affect the expression of downstream genes.
In addition to the off-target effect, the difficulty of naked siRNA molecules to cross the cell membrane due to their high molecular weight, hydrophilic and polyanionic properties is also a major limitation for the development of siRNA therapeutics. Various delivery systems have been developed to deliver siRNA to desired tissues and cells. These systems utilize chemically synthesized materials (such as liposomes, polymers, dendrimers, and inorganic nanoparticles), biological agents (such as peptides, antibodies, aptamers, CpG, 3-Way junctions, and exosomes), and physical methods (such as electroporation, microneedle poking, hydrodynamic injections, and microfluidic extrusion).
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