Unlocking the Potential of Bioconjugation Enhancing Oligonucleotide Delivery

The delivery potential of oligonucleotides can be enhanced through direct covalent conjugation of various moieties that promote intracellular uptake, target the drug to specific cells/tissues or reduce clearance from the circulation. These include lipids (for example, cholesterol that facilitates interactions with lipoprotein particles in the circulation), peptides (for cell targeting and/or cell penetration), α-tocopherol, aptamers, antibodies, fatty acids and sugars (for example, N-acetylgalactosamine (GalNAc)). Bioconjugation chemistry is at the centerpiece of this therapeutic oligonucleotide revolution, and significant opportunities exist for development of new modification chemistries, for mechanistic studies at the chemical-biology interface, and for translating such agents to the clinic.

Challenges for Oligonucleotide Delivery

Achieving effective delivery of oligonucleotide therapeutics to many tissues remains a major translational challenge. Oligonucleotides are typically large, hydrophilic polyanions (single-stranded ASOs are ~4-10 kDa, double-stranded siRNAs are ~14 kDa), properties that mean they do not readily pass through the plasma membrane. For activity, systemically injected nucleic acid drugs must resist nuclease degradation in the extracellular space, bypass renal clearance, evade non-productive sequestration by certain plasma proteins, avoid removal by the reticuloendothelial system (that is, mononuclear phagocytes, liver sinusoidal endothelial cells and Kupffer cells), cross the capillary endothelium at the desired target cell(s) within an organ/tissue by paracellular or transcellular routes, traverse the plasma membrane, escape the endolysosomal system before lysosomal degradation or re-export via exocytosis and arrive at the correct intracellular site of action. Systemic delivery to the central nervous system (CNS) presents an additional obstacle, as oligonucleotide-based therapeutics are generally not able to traverse the blood-brain barrier (BBB).

To date, the majority of oligonucleotide therapeutics (and almost all of the approved nucleic acid drugs) have focused on either local delivery (for example, to the eye or spinal cord) or delivery to the liver. Although other highly vascularized tissues with discontinuous or fenestrated endothelia, such as the kidneys and spleen, are also sites for oligonucleotide accumulation, the development of effective technologies for extrahepatic systemic delivery remains a major goal for the oligonucleotide therapeutics field.

Fig. 1 Tissue barriers to oligonucleotide delivery. (Juliano, R. L., 2016)

Role of Oligonucleotide Bioconjugation

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 (relative to nanoparticle approaches), meaning that they generally exhibit favorable biodistribution profiles (on account of being able to reach tissues beyond those with discontinuous or fenestrated endothelia). A common theme in oligonucleotide bioconjugation approaches is the promotion of interactions between the conjugate and its corresponding cell surface receptor protein, leading to subsequent internalization by receptor-mediated endocytosis. Therefore, oligonucleotide conjugates provide a means to enhance tissue targeting, cell internalization, endosomal escape, target binding specificity, resistance to nucleases, and more.

Bioconjugation Strategies to Enhance Delivery

GalNAc conjugates

GalNAc is a carbohydrate moiety that binds to the highly liver-expressed asialoglycoprotein receptor 1 (ASGR1, ASPGR) with high affinity (Kd = 2.5 nM) and facilitates the uptake of PO ASOs and siRNAs into hepatocytes by endocytosis. ASGR1 is very highly expressed in the liver, and is rapidly recycled to the cell membrane, making it an ideal receptor for effective liver-targeted delivery. The interaction between GalNAc and ASGR1 is pH-sensitive, such that dissociation of the receptor and oligonucleotide conjugate occurs during acidification of the endosome. The GalNAc moiety is subsequently subject to enzymatic degradation that liberates the oligonucleotide.

GalNAc conjugation enhanced ASO potency by ~7-fold in mouse, specific to the liver, and by ~30-fold in human patients. As such, GalNAc conjugation is now one of the leading strategies for delivering experimental oligonucleotide drugs currently in development, given its high liver silencing potential, small size relative to nanoparticle complexes, defined chemical composition and low cost of synthesis.

Fig. 2 Schematics of various delivery strategies for small interfering RNAs (siRNAs) and antisense oligonucleotides (ASOs). (Roberts, T. C., et al, 2022)

Antibody conjugates

Specific interactions between an antibody and a cell surface receptor have the potential to enable delivery to tissues and/or cell subpopulations that are not accessible using other technologies. Various receptors have been successfully targeted for siRNA delivery, including the HIV gp protein, HER2, CD7, CD71 and TMEFF2. Similarly, ASOs have also been conjugated with antibodies against CD44, EPHA2 and EGFR. In these cases, the ASO was delivered as a duplex with a DNA carrier strand to which the antibody was attached via click chemistry. Such a design allows the DNA passenger to be degraded after cellular entry, thereby releasing the ASO from the complex.

Aptamer conjugates

Aptamers can be considered 'chemical antibodies' that bind to their respective target proteins with high affinity, but present numerous advantages over antibodies as they are simple and inexpensive to manufacture (that is, by chemical synthesis), are smaller in size and exhibit lower immunogenicity.

Peptide conjugates

Peptides are an attractive source of ligands that may confer tissue/cell-targeting, cell-penetrating (that is, CPPs) or endosomolytic properties onto therapeutic oligonucleotide conjugates. Peptide conjugation has been explored for siRNA delivery. For example, the cyclic RGD peptide (recognized by αvβ3 integrin receptors) has successfully been used to deliver anti-VEGFR2 siRNA conjugates to mouse tumors. Similarly, the CPPs TAT (48-60) and penetratin have been utilized as siRNA conjugates for delivery to the lung via the intratracheal route.

References:

Juliano, R. L. The delivery of therapeutic oligonucleotides. Nucleic acids research. 2016, 44(14): 6518-6548.
Roberts, T. C.; et al. Advances in oligonucleotide drug delivery. Nature Reviews Drug Discovery. 2020, 19(10): 673-694.

* For research use only. Not intended for any clinical use.

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Unlocking the Potential of Bioconjugation Enhancing Oligonucleotide Delivery

Challenges for Oligonucleotide Delivery

Role of Oligonucleotide Bioconjugation

Bioconjugation Strategies to Enhance Delivery

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