Oligonucleotide therapeutics represent a rapidly expanding area within biotechnology. To date, the FDA and EMA have approved over 20 drugs in this class. Despite their successes, oligonucleotides have inherent limitations, including:
To address these limitations, conjugation to various molecules, such as oligosaccharides, antibodies, proteins, peptides, lipids, and polymers, is actively being explored to enhance the therapeutic potential of oligonucleotides.
Built on the discovery of the asialoglycoprotein receptor (ASGPR) (1), the conjugation of GalNAc moieties to oligonucleotides exemplifies a successful targeted oligonucleotide delivery to the liver. ASGPR, which is located in the liver plays a key role in glycoprotein clearance and has been effectively leveraged as a targeting mechanism for GalNAc-conjugated oligonucleotides.
Alnylam Pharmaceuticals pioneered this strategy for siRNA-GalNAc conjugates (2), and Isis Pharmaceuticals (now Ionis Pharmaceuticals) for ASO-GalNAc conjugates (3). The GalNAc conjugation strategy has led to the approval of seven therapeutics to date. Current preclinical research is expanding the application of GalNAc conjugates, focusing on identifying new liver gene targets and delivering novel oligonucleotide payloads, such as small activating RNAs (saRNAs) (4).
Inspired by the success of GalNAc conjugation, extensive research is underway to develop extra-hepatic oligonucleotide drug delivery methods using diverse oligonucleotide conjugates (Figure 1).
Inspired by the success of antibody-drug conjugates (ADCs), AOCs are being actively explored. Avidity Biosciences' delpacibart etedesiran, the first AOC to enter clinical trials, is currently in Phase III trials.
Lipid conjugation enhances penetration of biological membranes and endosomal release (5), as well as improves therapeutic plasma half-life (6). Imetelstat, a first-in-class anti-cancer telomerase inhibitor, features a palmitoyl lipid group conjugated to the oligonucleotide (7).
Conjugation to polymers like PEG enhances stability and increases the half-life of oligonucleotides, as demonstrated by the approved pegylated aptamers avacincaptad pegol and pegaptanib.
Conjugation of oligonucleotides to CPPs is being explored to enhance endosomal escape and plasma membrane penetration (8).
Targeting peptides, such as glucagon-like peptide-1 (GLP-1), can improve targeted delivery of oligonucleotides (9).
Small molecular weight compounds are also being explored as conjugation moieties at the pre-clinical stage. Anisamide, anandamide, folate, carbachol, and estrone are being explored as receptor-targeting ligands, while cholesterol, α-tocopherol, squalene, and various fatty acids are being investigated as membrane penetration enhancers (5).
Design effective structural characterization studies for oligonucleotide therapeutics.
Example data from the analysis of unmodified Inotersen and Inotersen.
Structural characterization of oligonucleotide conjugates presents a complex task. It often requires analyzing the products of individual conjugation steps, confirming the final conjugate structure, monitoring structural or chemical changes during bioconjugation, and profiling product- and process-related impurities (Figure 2).
The sequence of the oligonucleotide must be determined using appropriate mass spectrometry techniques (Blog 2).
For antibody, protein, or peptide conjugates, amino acid sequence determination is essential, with a focus on oligonucleotide attachment sites. The choice of conjugation chemistry (e.g., site-specific vs. random) influences the determination of number of oligonucleotides per protein (e.g. oligonucleotide-to-antibody ratio/O-A-R) and their attachment positions, which can affect the bioconjugate's physiochemical and biological properties.
Conjugation chemistry may induce structural changes to amino acid side chains, such as deamidation or oxidation, and potentially affect glycosylation patterns (as seen in monoclonal antibodies). For instance, the loss of sialic acid residues, known to be relatively labile, is a common concern. The use of reducing agents in cysteine-based conjugation may lead to disulfide bond scrambling, necessitating disulfide bridge analysis.
Conjugation processes can impact HOS elements like protein α-helices and β-sheets, which can be analyzed using techniques such as Circular Dichroism (CD), Fourier Transform Infra-Red spectroscopy (FT-IR), Microfluidic Modulation Spectroscopy (MMS), fluorescence analysis (intrinsic and extrinsic), and 1D/2D Nuclear Magnetic Resonance (NMR) (Blog 4).
Hydrophobic lipid or CPP conjugation can lead to aggregate formation, which can be assessed by sedimentation velocity-analytical ultracentrifugation (SV-AUC) and size exclusion chromatography with multiangle laser light scattering (SEC-MALS).
Following the success of GalNAc-oligonucleotide conjugates, significant effort is directed toward discovering new conjugation strategies for targeted non-liver delivery, as well as efficient uptake and endosomal escape of oligonucleotide therapeutics. While a universal "magic" moiety remains elusive, numerous conjugates are being evaluated in clinical and preclinical settings, each with a unique mechanism of action.
From a structural characterization perspective, each conjugate poses specific challenges, requiring careful consideration of the effects of conjugation chemistry and confirmation of successful conjugation. This comprehensive analysis can be achieved using a variety of analytical techniques available across BioPharmaSpec’s global laboratories.
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