GalNAc Technology for Oligonucleotide Drug Delivery

Overview of oligonucleotide therapy

Nucleic acid technology can be widely used in the treatment or prevention of genetic diseases, tumors, viral infections, sensory organs, and other diseases. It's a new generation of therapy after small molecule drugs and antibody drugs. At present, the research on nucleic acid drugs can be roughly divided into two categories: oligonucleotides and mRNA.

The mechanism of mRNA drugs is to produce the target proteins by coding. Oligo therapeutics mainly regulate gene expression through a series of mechanisms, such as gene silencing, non-coding RNA inhibition, and gene activation, based on the base complementary pairing principle proposed by Watson-Crick and pairing with DNA, mRNA, or pre-mRNA. Inter-nucleotide chains and ribose can be modified to improve drug properties, including serum stability, protein binding, potency, and low immunogenicity. However, oligos are negatively charged macromolecules with poor in vitro stability and cannot freely diffuse on the cell membrane like lipophilic small molecule drugs. Moreover, the therapeutic target of oligos is in the cell, so the precise delivery of oligos into the target cells remains a major challenge, limiting the therapeutic development of oligos.

GalNAc for oligo drug delivery

• Oligo drug delivery system

Chemical modification of oligo drugs can improve stability and reduce immunogenicity to a certain extent, but the exposed oligos bring many problems, e.g.,

• It's easy to be degraded by nucleases, resulting in a short half-life in the blood, making it difficult to maintain good efficacy
• It's unable to carry out precise treatment of diseased tissues and cells because of no active targeting
• The large molecular weight and net negative charge make it difficult to cross the cell membrane
• It is easily trapped in the endosome and cannot be released after entering the cell

Oligo drugs need to rely on reliable delivery systems to achieve better stability, safety, and efficacy in vivo. If the targeted delivery system is developed, the dose can be further decreased to reduce off-target effects and toxic side effects.

Oligo drug delivery strategies can be divided into two categories:

• Formulate drugs into nanocarriers for transfection into target cells, such as lipid nanoparticles, inorganic nanoparticles, polymer nanocarriers, and exosomes.
• Conjugation of drugs with target ligands to specific high-capacity receptors on target cells, like small molecule ligand conjugation, antibody, and antibody fragment conjugation.

To date, a relatively mature delivery technology is lipid nanoparticle (LNP) coupled with N-acetylgalactoamine (GalNAc). Promoted by mRNA COVID-19 vaccines, LNP is mainly used to deliver siRNA and mRNA, while GalNAc conjugation technology is applied for liver-targeted delivery of oligos.

• GalNAc conjugated delivery system

GalNAc conjugated delivery system is a breakthrough in the development of oligo drugs. GalNAc, a galactosaminoglycan derivative, is a high-affinity targeting ligand for the asialoglycoprotein receptor (ASGPR). ASGPR can specifically highly express on the surface of hepatocytes (about 106 per hepatocyte) while other receptors express only 104-105 or even lower on the surface of hepatocytes.

The selectivity of ASGPR is closely related to the type of sugar molecules, the number of antennae, the spatial distance, and other factors. The three-antenna GalNAc was finally optimized and used as a high-affinity ASGPR targeting ligand. In addition to the quantitative advantage of high expression, ASGPR is also a highly efficient endocytic cycling receptor with a cycling rate of about 15min, while the cycling time of other cell-surface recyclable receptors is usually 90min. Given the two advantages of the large number and short circulation time of ASGPR, GalNAc-siRNA conjugates can achieve efficient cellular internalization. ASGPR and clathrin-mediated endocytosis can efficiently transport GalNAc from the cell surface to the cytoplasm. Additionally, GalNAc conjugation technology has been applied in both ASO drugs and siRNA drugs as well, becoming the most efficient oligo drug delivery system at present, though it can only target liver cells.

• In vivo mechanism of GalNAC-siRNA conjugate

The structure of GalNAC-siRNA conjugate consists of three parts: trivalent GalNAc target head, linker arm, and siRNA molecule. The GalNAc target head is covalently coupled to the 3' end of the sense strand of siRNA in a trivalent manner to form the GalNAC-siRNA conjugate.

Fig. 1 Structure of GalNAc-siRNA conjugates

When the GalNAC-siRNA conjugate binds to ASGPR, it can enter the endocytosis via clathrin-mediated endocytosis. After the conjugation into EarlyEndosomes (EE), ASGPR is separated from the GalNAC-siRNA conjugate at low pH and returned to the surface of hepatocytes for circulation. As EE acidify and mature, they will gradually turn into Late Endosomes or Multivesicular bodies (MVB), and only < 0.01% of siRNA can escape from the Late Endosomes or MVB to the cytoplasm. However, the efficient cellular uptake allows nearly 1 million siRNA every 15 minutes to enter the early endosomes, and the amount of siRNA reaching the cytoplasm far exceeds the threshold of RNAi reaction, which can better meet the needs of drug administration and make short-term drug efficacy possible. Retention of siRNA molecules in an acidic environment is necessary to maintain long-term efficacy. Studies have shown that the long-term effect of GalNAC-siRNA conjugation is due to the enhanced metabolic stability of siRNA after chemical modification, which can greatly improve the survival rate of siRNA in an acidic environment and form an intracellular siRNA reservoir. siRNA in these repositories can be slowly released into the cytoplasm from an acidic environment and then loaded into the RNA-induced silencing complex (RISC) to prolong the pharmacological persistence of GalNAC-siRNA conjugation.

Fig. 2 Delivery of GalNAc-siRNA conjugates into hepatocytes (Springer AD, 2018)

Although LNP and GalNAc can both accumulate and take up well in the liver, the delivery strategy developed based on GalNAc is more advantageous than LNP. Firstly, intravenous LNP can cause infusion-related reactions, which should be combined with antihistamines, acetaminophen, and dexamethasone. However, GalNAc conjugated nucleic acid drugs can be administered subcutaneously, which avoids the safety problems caused by the immunogenicity of lipid molecules and PEG during LNP use. In addition, compared with LNP, products developed based on GalNAc are easier to scale up and benefit for dose and frequency of administration. Compared with LNPs, the synthesis and refining of GalNAC-siRNA conjugates are much simpler. In addition, the GalNAc conjugate is convenient for clinical use and can be self-administered subcutaneously, with the advantages of rapid absorption, high absorption rate, and long half-life. Unlike LNPs, GalNAc conjugation is safe and does not require anti-inflammatory therapy before infusion. At present, although there are many nucleic acid drugs developed based on LNP, RNA drugs developed based on GalNAc conjugation technology are more widely used in clinical trials.