The potential of oligonucleotide therapeutics is vast, as illustrated by the growing number of drugs coming to market and the increase in noise and interest in this field. Oligonucleotide therapeutics hold promise to silence aberrant expression, modulate gene splicing or activate the expression of previously undruggable targets across a range of diseases. As the field has progressed, increased understanding has allowed researchers to overcome significant challenges in therapeutic function, from half-life control and extension to target specificity. Despite these advances, the primary challenge remains of targeted delivery to a specific cell type or tissue.
Encapsulation to direct conjugation:
A new dawn in targeted delivery
Many have focused delivery around viral vectors and lipid nanoparticles (LNP) to encapsulate the oligonucleotide and protect it from degradation during transportation to the tissue of interest. Modulating the LNP surface can tune the delivery to various tissues and organs to improve targeting.1
The advent of N-acetylgalactosamine (GalNAc) conjugates offered a breakthrough in targeted delivery of oligonucleotide therapeutics. Oligonucleotide therapeutics could be conveniently conjugated to a triantennary GalNAc molecule to achieve targeted delivery to hepatocytes.2 The GalNAc structure binds asialoglycoprotein receptor (ASGPR1), which is highly expressed on hepatocytes with approximately 500,000 copies per hepatocyte and minimal expression in non-hepatic tissues.3
GalNAc molecules have been designed to bind to ASGPR1 with high specificity and affinity, triggering hepatocyte-specific uptake of conjugates. For antisense oligonucleotides (ASO) GalNAc conjugation improved targeted delivery to hepatocytes and increased the levels of the ASO within the hepatocytes. With small inhibiting RNA (siRNA), GalNAc use has now largely replaced LNPs for hepatocyte-targeted drugs. Most siRNAs in the clinic for the treatment of liver disease now use GalNAc-targeting strategies.4
Following the discovery of GalNAc, the race is on to find equivalent molecules that allow specific targeting for alternative tissues and/or disease states. For example, targeting solid tumors for precision chemotherapy approaches, targeting cardiomyocytes for cardiac conditions, targeting muscle for muscular dystrophies or targeting across the blood-brain barrier (BBB) for CNS diseases. Yet without prior knowledge of tissue-specific biomarkers for each disease state or organ, identifying a candidate target and the correct moiety to approach this target is the drug developer’s needle in a haystack.
Biomarker and delivery vehicle co-discovery
For targeted delivery across the BBB, many researchers are targeting the Transferrin Receptor 1 (TfR1).5-7 TfR is highly expressed by brain capillary endothelial cells forming the blood-brain barrier, and monoclonal antibodies and aptamers binding this target have shown the ability to penetrate the CNS as delivery vehicles for liposomes and as bispecific aptamers.7,8 However, this receptor is also expressed in other tissues, such as muscle, where companies such as Avidity Bioscience and Dyne Therapeutics have active clinical programs for the targeted delivery of oligonucleotide therapeutics for muscular dystrophies using TfR1.9 Modulating dosing levels and administration may allow increased tissue selectivity for this target, but the challenge of identifying tissue-specific biomarkers for targeted delivery remains.
Hypothesis-free ligand discovery approaches offer new methods for overcoming this knowledge gap. Rather than pursue specific biomarkers for a target tissue or cell type, the Optimer platform’s hypothesis-free discovery method involves screening large libraries of ligands against the desired cell type with counter-screening against multiple alternative cell types (Figure 1).10
Once an Optimer ligand is selected with the required cell specificity and internalization profile, the biomarker can be identified using the selected Optimer to isolate the target ligand and analyzing via mass spectrometry. This strategy opens the door to identifying new biomarkers for improved tissue targeting of oligonucleotide therapeutics while offering a single discovery strategy for both novel biomarkers and their appropriate targeting ligand. The Optimer binders can be subsequently conjugated to oligonucleotide therapeutics or generated as a single oligonucleotide through solid phase synthesis of both the Optimer ligand and oligonucleotide drug as a single strand.
Targeted administration for targeted delivery
Alternative strategies under development include delivery routes to specific tissues. Earlier this year, Alnylam published work showing stable siRNA silencing in the CNS via intrathecal delivery, in the eye via intravitreal delivery, and in the lung via intranasal delivery.11 Eli Lilly, among others, is working on nose-to-brain delivery of siRNA using drug-filled gels placed at the back of the nasal cavity.12 The drug component can diffuse into the CNS via the olfactory pathway.
These methods appear highly effective and could offer patient treatment strategies where currently few exist. Drawbacks of such direct administration to the target tissue include the highly invasive nature of delivery (particularly with methods such as intrathecal delivery). Invasive administration methods are associated with low patient tolerability and the need for patient hospitalization, which increases costs on already overburdened healthcare systems.
The evidence supporting the low-toxicity and long-term effects of oligonucleotide therapeutics continues to grow, demonstrating the potential of these drugs to treat conditions from cancer to monogenic diseases. Nonetheless, delivery and internalization remain a major challenge for this field. A range of possibilities are now on the horizon to overcome this challenge, holding promise for new strategies, new delivery vehicles and, ultimately, new oligonucleotide therapies to improve patients’ treatments. The goal for the field will be to offer the most effective and tolerable delivery format for patients, which I predict will involve multiple iterations of oligonucleotide therapies, moving through targeted administration towards targeted delivery using new tissue-selective biomarkers.
- Wang, X. et al. Preparation of selective organ-targeting (SORT) lipid nanoparticles (LNPs) using multiple technical methods for tissue-specific mRNA delivery. Nat Protoc (2022) https://doi.org/10.1038/s41596-022-00755-x
- Biessen, E.A.L. et al. Design of a targeted peptide nucleic acid prodrug to inhibit hepatic human microsomal triglyceride transfer protein expression in hepatocytes. Bioconjug Chem 13 (2), 295-302 (2002).
- Li, Y. et al. Targeted delivery of macromolecular drugs: asialoglycoprotein receptor (ASGPR) expression by selected hepatoma cell lines used in antiviral drug development. Curr Drug Deliv 5(4), 299-302 (2008).
- Debacker, A.J. et al. Delivery of Oligonucleotides to the Liver with GalNAc: From Research to Registered Therapeutic Drug. Mol Ther 28(8), 1759-1771 (2020).
- Bicycle Therapeutics. Bicycle Therapeutics Enters Exclusive License and Collaboration Agreement with Ionis to Develop Targeted Oligonucleotide Therapeutics. Accessible at: https://investors.bicycletherapeutics.com/news-releases/news-release-details/bicycle-therapeutics-enters-exclusive-license-and-collaboration (2021).
- Bourassa, P. et al. Transferrin Receptor-Mediated Uptake at the Blood-Brain Barrier Is Not Impaired by Alzheimer’s Disease Neuropathology. Mol Pharm 16(2), 583-594 (2019).
- MacDonald, J. et al. Development of a Bifunctional Aptamer Targeting the Transferrin Receptor and Epithelial Cell Adhesion Molecule (EpCAM) for the Treatment of Brain Cancer Metastases. ACM Chem Neurosci 8(4), 777-784 (2017).
- Thomsen, M.S. et al. Blood–Brain Barrier Transport of Transferrin Receptor-Targeted Nanoparticles. Pharmaceutics. 14(10), 2237 (2022).
- Mullard, A. Antibody–oligonucleotide conjugates enter the clinic. Nat Rev Drug Discov 21, 6-8 (2021).
- Aptamer Group. Customer case studies – Optimer®-enabled delivery of therapeutic cargo to a specific hepatic cell population. Accessible at: https://aptamergroup.com/optimer-binders/case-studies/ (2022).
- Brown, K.M. et al. Expanding RNAi therapeutics to extrahepatic tissues with lipophilic conjugates. Nat Biotech 40, 1500–1508 (2022).
- Hall, M. State of the art, frontiers and current research. Eli Lilly presentation at RNA Leaders USA, 18th October 2022.
David Bunka is the chief technical officer of Aptamer Group. He holds a Ph.D. in molecular biology and has spent almost 20 years developing nucleic acid aptamers against an array of targets including small molecules, disease-associated proteins, several cancer-associated cell-lines, viruses and tissue biopsies. Bunka has authored several peer-reviewed research articles, invited review articles and a book chapter on aptamer-based therapeutics. He has also given many guest seminars covering aptamer-based applications at prominent universities and international conferences.