Formulating Oligonucleotide Therapies: siRNA, ASO, and Beyond
A Pipeline That Has Finally Arrived
Oligonucleotide therapies have been in development for decades. The science was compelling from the start: target any gene in the human genome with exquisite specificity by designing a short nucleotide sequence that binds the corresponding mRNA. Switch off disease-causing genes. Restore lost protein expression. Correct splicing errors.
The delivery problem kept most of it on the bench. Oligonucleotides are large, negatively charged, enzymatically unstable molecules that do not cross cell membranes easily. They are rapidly degraded by serum nucleases and cleared by the kidney before they reach their target.
Two decades of formulation innovation have changed that. Lipid nanoparticles, GalNAc conjugation, and chemical modification strategies have collectively unlocked systemic and local delivery of oligonucleotides to a growing range of tissues. The pipeline has followed.
The Three Main Classes and Their Delivery Needs
| Class | Mechanism | Primary Delivery Challenge | Current Delivery Solutions |
| siRNA (small interfering RNA) | Triggers RISC-mediated degradation of complementary mRNA; gene silencing | Negative charge; nuclease degradation; endosomal escape; off-target effects | LNP (ionisable lipid); GalNAc conjugation for hepatic delivery; chemical modifications (2-OMe, PS backbone) |
| Antisense oligonucleotide (ASO) | Binds pre-mRNA or mRNA; blocks translation or triggers RNase H cleavage | Nuclease degradation; tissue distribution; nuclear access for splice-switching ASOs | Naked delivery with chemical modifications (PS backbone; LNA/BNA); GalNAc for liver; local delivery to CNS or lung |
| Aptamer | Folds into 3D structure that binds target protein; blocks or modulates protein function | Rapid renal clearance; short half-life; large-scale synthesis | PEGylation; conjugation to larger carriers; chemical modifications |
| saRNA (self-amplifying RNA) | Encodes an RNA polymerase that amplifies the therapeutic RNA in the cell | Larger molecule than mRNA; requires same endosomal escape strategy; immunogenicity | LNP; same platform as mRNA but larger payload size requires formulation optimisation |
| miRNA mimic or inhibitor | Modulates endogenous miRNA activity; broad gene regulatory effect | Same as siRNA/ASO depending on class; off-target effects from broad miRNA activity | LNP; conjugate delivery; chemical modifications |
GalNAc Conjugation: The Hepatic Delivery Revolution
For oligonucleotides targeting the liver, GalNAc (N-acetylgalactosamine) conjugation has transformed the field. GalNAc is a sugar that binds with high affinity and specificity to the asialoglycoprotein receptor (ASGPR) expressed at high levels on hepatocytes. Conjugating a GalNAc moiety to an ASO or siRNA drives receptor-mediated uptake specifically into liver cells, achieving therapeutic silencing at doses that would be ineffective for the naked oligonucleotide.
GalNAc-conjugated siRNAs (GalNAc-siRNA) are administered as subcutaneous injections, often once monthly or less frequently, with no nanoparticle carrier required. The simplicity of the delivery system relative to LNPs, no lipid formulation, no cold chain, simple subcutaneous administration, has made GalNAc the dominant delivery platform for hepatic oligonucleotide therapies.
The limitation is obvious: it only works for liver targets. For any other tissue, GalNAc does not help.
Chemical Modifications: Stability Before Delivery
Before any delivery system can work, the oligonucleotide must survive long enough to reach the target. Naked, unmodified RNA is degraded by serum nucleases within seconds. Chemical modifications slow that degradation and, in many cases, improve the potency and duration of action of the drug.
The phosphorothioate (PS) backbone modification, where a non-bridging oxygen in the phosphodiester linkage is replaced by sulphur, was the first widely used modification and remains standard in most clinical ASOs. It significantly improves nuclease resistance and increases protein binding in plasma, which extends circulation half-life but also contributes to off-target effects at high doses.
Locked nucleic acid (LNA) modifications constrain the ribose ring in a fixed conformation that increases binding affinity and nuclease resistance. 2-O-methyl and 2-O-methoxyethyl modifications are also widely used. Modern clinical oligonucleotides typically carry a combination of several modification types, optimised for the specific target and delivery context.
Formulation Development for Oligonucleotides
For LNP-delivered siRNAs, the formulation development process is closely analogous to mRNA LNP development: ionisable lipid selection, N:P ratio optimisation, microfluidics manufacture, and physicochemical characterisation of particle size, PDI, encapsulation efficiency, and zeta potential. The key difference is that siRNA is significantly smaller than mRNA, which affects the optimal lipid composition and the encapsulation efficiency assay (Ribogreen versus strand-specific hybridisation assays for siRNA quantification).
For subcutaneous GalNAc-conjugate delivery, the formulation is simpler: the conjugate is dissolved in a compatible aqueous buffer at the target concentration, filtered, and filled into prefilled syringes or vials. The formulation challenges are primarily related to concentration (high concentration formulations can form gels) and stability of the conjugate bond under storage conditions.
Ardena’s Oligonucleotide Platform at Oss
Ardena’s nanomedicine team at Oss has formulation development and GMP manufacturing capability for LNP-delivered oligonucleotides, including siRNA and saRNA payloads. The site’s encapsulation efficiency assays have been adapted for siRNA quantification, and the team has experience optimising LNP formulations for small nucleic acid payloads where the optimal composition differs from mRNA LNP formulations.
