From Fragile Oligonucleotides to an Established Therapeutic Modality
Nucleic acid therapeutics (NATs) have evolved from an intriguing scientific concept into a major pharmaceutical modality. More than two decades after the first FDA approval of an oligonucleotide drug, therapeutic oligonucleotides now treat rare diseases, such as spinal muscular atrophy and hereditary transthyretin amyloidosis, to widespread diseases such as hypercholesterolemia and geographic atrophy. The success of these medicines was enabled primarily by advances in oligonucleotide chemistry that improved metabolic stability, tissue distribution, potency, safety, and manufacturability.
The fundamental idea behind nucleic acid therapeutics is straightforward. Unlike conventional small molecules or antibodies, oligonucleotides recognize their targets through sequence complementarity or through binding structures formed by folded nucleic acids. By designing sequences that interact selectively with RNA or protein targets, researchers can reduce gene expression, alter RNA splicing, inhibit translation, activate immune pathways, or modulate protein function.
From a medicinal chemistry perspective, the most important observation is that the earliest successful NATs did not succeed because of novel mechanisms. RNase H-mediated antisense activity had been demonstrated long before the first antisense drug was approved. What changed was the chemistry: phosphorothioate backbones, MOE sugars, phosphorodiamidate morpholino backbones, GalNAc conjugation, and optimized siRNA modification patterns progressively solved stability, PK, uptake, and safety limitations. The evolution of NATs has therefore been driven at least as much by advances in chemistry and delivery as by advances in mechanisms of action.
The first challenge was nuclease degradation. Natural DNA and RNA molecules are rapidly cleaved in biological fluids and intracellular environments, and early unmodified oligonucleotides often displayed plasma half-lives measured in minutes. The first major breakthrough was phosphorothioate backbone chemistry, in which one non-bridging oxygen atom of the phosphate group is replaced by sulfur. Phosphorothioate linkages substantially increased nuclease resistance while also increasing binding to plasma proteins, improving biodistribution and enabling therapeutically useful dosing. However, the PS backbone destabilized the duplex, which correlates with lower potency. First generation 2’ modification 2’-O-methyl (2’-OMe), modestly increased duplex stability against an RNA target and nuclease resistance. The 2’-fluoro (2’ F) modification significantly improved both duplex stability and nuclease resistance, leading to a large increase in potency in vitro for gapmers with 2’-F in the wings and a gap of DNA in the middle of the sequence to recruit RNase H. However, the fluorine substituents substantially increased the ASO binding to proteins, leading to high toxicity in vivo. The second-generation sugar modification 2′-O-methoxyethyl (2’-MOE) increased duplex stability, potency, and nuclease resistance with no increase in toxicity, leading to a larger therapeutic window. Higher potency and great duplex stability enabled shorter sequences, lower doses, and improved pharmacological activity. This modification became a foundational component of numerous antisense oligonucleotide drugs and remain central to modern therapeutic design.
Table 1 provides a chronological summary of FDA-approved NAT drugs. Remarkably, despite the rapid growth of the field, relatively few new chemical modifications have been introduced into approved products. Mipomersen, approved in 2013, introduced 5-methylcytosine (5-Me-C), a modification commonly combined with 2′-MOE sugars to increase duplex stability and attenuate innate immune responses. Eteplirsen, approved in 2016, marked the first approval of a phosphorodiamidate morpholino (PMO) backbone. Since that time, only a single additional backbone chemistry, the thiophosphoramidate backbone approved in 2024, has reached the market, and no new nucleotide analogs have been incorporated into FDA-approved NAT therapeutics as of this writing. However, the PMO and 5-Me-C have been widely adopted in NATs. For the PMO backbone, the unparalleled nuclease resistance and extremely low toxicity allowed the development of a new therapeutic modality other than RNase H-mediated gene knockdowns – splicing modulation for the treatment of Duchenne Muscular Dystrophy and Spinal Muscular Atrophy. 5-Me-C is now a standard base modification in the wings of gapmers for RNase H-mediated gene knockdown. However, the greatest impact in the last ten years came from advances in the delivery of ASOs and especially the design and delivery of siRNAs to target tissues and cells by the introduction of the triantennary GalNAc.
FDA-Approved Oligonucleotide Therapeutics
| Drug | Target | Modality | Backbone / Sugar Modifications | Conjugate / Delivery | Indication | Year FDA Approval |
| Fomivirsen | CMV IE2 | RNase H ASO | Full PS; 2′-deoxy | Intravitreal | Cytomegalovirus retinitis (AIDS-related) | 1998 |
| Pegaptanib | VEGF165 | Aptamer | 2′-F RNA; 2′-OMe RNA | 40-kDa PEG; intravitreal | Wet Age-Related Macular Degeneration | 2004 |
| Mipomersen | APOB | RNase H gapmer ASO | PS; 2′-MOE wings; 5-Me-C | (SC) | Homozygous Familial Hypercholesterolemia | 2013 |
| Eteplirsen | DMD exon 51 | Exon-skipping ASO | PMO | (IV) | Duchenne Muscular Dystrophy | 2016 |
| Nusinersen | SMN2 | Exon-inclusion ASO | Fully PS; fully 2′-MOE; 5-Me-C | Intrathecal | Spinal Muscular Atrophy | 2016 |
| Inotersen | TTR | RNase H gapmer ASO | PS; 2′-MOE wings; 5-Me-C | (SC) | Hereditary transthyretin-mediated amyloid polyneuropathy | 2018 |
| Patisiran | TTR | siRNA | 2′-OMe-modified siRNA; dTdT overhangs | LNP (IV) | Hereditary transthyretin-mediated amyloid polyneuropathy | 2018 |
| Golodirsen | DMD exon 53 | Exon-skipping ASO | PMO | (IV) | Duchenne Muscular Dystrophy | 2019 |
| Givosiran | ALAS1 | siRNA | 2′-F; 2′-OMe; PS linkages | GalNAc (SC) | Acute hepatic porphyria | 2019 |
| Lumasiran | HAO1 | siRNA | 2′-F; 2′-OMe; PS linkages | GalNAc (SC) | Primary Hyperoxaluria Type 1 | 2020 |
| Viltolarsen | DMD exon 53 | Exon-skipping ASO | PMO | (IV) | Duchenne Muscular Dystrophy | 2020 |
| Casimersen | DMD exon 45 | Exon-skipping ASO | PMO | (IV) | Duchenne Muscular Dystrophy | 2021 |
| Inclisiran** | PCSK9 | siRNA | 2′-F; 2′-OMe; PS linkages, | GalNAc (SC) | Hypercholesterolemia | 2021 |
| Vutrisiran | TTR | siRNA | 2′-F; 2′-OMe; PS linkages, | GalNAc (SC) | hereditary transthyretin-mediated amyloidosis; ATTR cardiomyopathy | 2022 |
| Tofersen | SOD1 | RNase H gapmer ASO | PS; 2′-MOE wings; 5-Me-C | Intrathecal | SOD1-associated amyotrophic lateral sclerosis | 2023 |
| Avacincaptad pegol** | C5 | Aptamer | 2′-F RNA; 2′-OMe RNA | PEG; intravitreal | Geographic atrophy | 2023 |
| Nedosiran | LDHA | siRNA | 2′-F; 2′-OMe; PS linkages | GalNAc (SC) | Primary hyperoxaluria Type 1 | 2023 |
| Eplontersen | TTR | RNase H gapmer ASO | PS; 2′-MOE wings; 5-Me-C | GalNAc (SC) | Hereditary transthyretin-mediated amyloid polyneuropathy | 2023 |
| Imetelstat* | hTR | Steric-blocking ASO | N3’→P5′ thio-phosphoramidate | 5′-palmitoyl lipid (IV) | Low to intermediate-risk Myelodysplastic Syndrome | 2024 |
| Olezarsen | APOC3 | RNase H gapmer ASO | PS; 2′-MOE | GalNAc (SC) | Familial chylomicronemia syndrome | 2024 |
| Fitusiran | Antithrombin | siRNA | 2′-F; 2′-OMe; PS linkages | GalNAc (SC) | Hemophilia A/B | 2025 |
| Donidalorsen | Prekallikrein | RNase H gapmer ASO | PS; 2′-MOE | GalNAc (SC) | Hereditary angioedema | 2025 |
*Indicates treatment for non-rare disease; **Indicates treatment for a widespread disease.
RNA Interference, Delivery, and the GalNAc Era
The emergence of RNA interference as a functional NAT represented a major milestone. Small interfering RNAs (siRNAs) exploit a natural intracellular pathway in which the RNA-induced silencing complex (RISC) uses complementary RNA guides to direct targeted cleavage of RNA. Because RISC acts catalytically, siRNAs can produce durable target suppression. Therapeutic implementation, however, required extensive chemical optimization: early siRNAs exhibited poor stability, innate immune activation, and unfavorable pharmacokinetics. Patterned 2′-fluoro, 2′-O-methyl modifications and PS backbone at both termini improved stability and tolerability while maintaining compatibility with the RNAi machinery.
Although improved chemistry increased stability and potency, tissue delivery remained a central obstacle. Unlike PS-modified ASOs, siRNAs have negligible protein binding which prevents them ‘hitching a ride’ on cell-surface proteins into the cell via the endocytic pathway. The nucleobases of the siRNA are largely sequestered within the A-form siRNA duplex, while the exterior of the rod-like helix is dominated by the negatively charged phosphate backbone and its associated hydration shell. As a result, the duplex presents relatively little exposed hydrophobic surface for nonspecific protein interactions. As a result, the uptake of siRNAs into cells is minimal without receptor-mediated endocytosis.
One solution emerged through lipid nanoparticle delivery systems (LNPs). Encapsulation within lipid nanoparticles enabled efficient hepatic delivery and supported the approval of patisiran, the first siRNA therapeutic. LNPs showed that extracellular barriers could be overcome, while also introducing manufacturing complexity and infusion-related considerations.
The second, transformative solution, however, was ligand-conjugated delivery. Triantennary N-acetylgalactosamine conjugates exploit the abundantly expressed asialoglycoprotein receptor on hepatocytes, enabling efficient receptor-mediated uptake into the liver after subcutaneous (SC) administration. Modern GalNAc-conjugated siRNAs can achieve durable target suppression with infrequent dosing, explaining why many successful contemporary oligonucleotide drugs target liver-expressed genes. With the exception of patisiran, all FDA-approved siRNA therapies use a triantennary GalNAc ligand on the 3’ of the passenger (sense) strand of the siRNA. There is an issue with using the GalNAc ligand for siRNA delivery – the therapy is limited to hepatocytes.
One might ask why use siRNA when ASOs are also effective. The reason is that once loaded into the RISC complex, the RNA is protected from nucleases and stays localized within the cytoplasm, allowing for a much longer duration of effect. There are two disadvantages of siRNAs – first, they are much more prone to off-target knockdowns due to the rather promiscuous seed region and second, while siRNAs can target nuclear RNAs, they are generally less effective than gapmer ASOs against nuclear-retained transcripts such as MALAT1, making ASOs the preferred therapeutic class for many nuclear RNA targets.
Next-Generation Chemistry
The newest generation of oligonucleotide chemistry seeks to further optimize the therapeutic index. Described in Table 2 are stereopure – phosphorothioate, phosphoryl guanidine, mesyl phosphoramidites backones; sugar modifications such as constrained ethyl (cEt), tricyclo-DNA (tcDNA), and other emerging chemistries aim to increase potency while minimizing toxicity. Antibody-oligonucleotide conjugates (AOCs), cell-penetrating peptides (CPPs), and lipophilic conjugates are being developed to extend delivery beyond the liver to tissues such as muscle, heart, lung, and the central nervous system.
| Modification | Advantage over current standards | Status* |
| Stereopure (chirally-controlled) backbones | Fixing the Rp/Sp configuration at each phosphorothioate linkage instead of the random mixture of stereoisomers in today’s drugs, helps improve potency, nuclease stability and the therapeutic window. | Clinical |
| Phosphoryl guanidine (PN) linkages | Neutral, highly nuclease-resistant linkages that improve RNase-H activity, cellular uptake and duration of effect. | Clinical |
| Constrained / bridged nucleic acids (cEt, tcDNA, AmNA) | Higher target affinity than 2′-MOE or LNA; tricyclo-DNA in particular shows markedly better muscle and CNS uptake. | Clinical / preclinical |
| Mesyl phosphoramidate (MsPA) backbone | Strong nuclease resistance while still supporting RNase-H cleavage, a potential successor to phosphorothioate. | Preclinical / early |
| Antibody–oligonucleotide conjugates (AOCs) | An antibody carries the oligo to muscle and other extrahepatic tissue the GalNAc, liver route cannot reach. | Late clinical |
| Peptide-conjugated oligos (PPMO, CPP) | Cell-penetrating peptides sharply raise uptake into muscle, heart and the CNS. | Clinical |
| Lipophilic / fatty-acid conjugates (e.g., C16, Palmitoyl) | Enable CNS and other extrahepatic delivery of siRNA without a lipid nanoparticle. | Recently FDA approved (Imetelstat) |
New Clinical-Stage NAT Chemistries and Architectures
| Candidate | Company | Novel Chemistry/ Architecture | Target | Indication | Phase | NCT |
| WVE-003 | Wave Life Sciences | Stereopure PN backbone ASO | Mutant HTT | Huntington disease | Phase 1b/2a | NCT05032196 |
| WVE-N531 | Wave Life Sciences | Stereopure PN platform ASO | DMD exon 53 splicing | Duchenne muscular dystrophy | Phase 1b/2 | NCT04906460 |
| SRP-5051 (vesleteplirsen) | Sarepta | PPMO | Dystrophin exon 51 | Duchenne muscular dystrophy | Phase 2 | NCT04004065 |
| Del-desiran (AOC 1001) | Avidity Biosciences | Antibody-oligonucleotide conjugate (AOC); TfR1 mAb-siRNA | DMPK mRNA | Myotonic dystrophy type 1 | Phase 1/2 completed; advanced to Phase 3 development | NCT05027269 |
Conclusion: Chemistry Made the Modality
Today, nucleic acid therapeutics represent one of the most rapidly advancing areas of drug development. The field has progressed from simple phosphorothioate oligonucleotides with modest activity to highly engineered molecular systems incorporating optimized backbone chemistry, sugar modifications, targeting ligands, and advanced delivery technologies. This evolution demonstrates that clinical success depends not only on biological insight but also on chemical innovation.
Every major advance in the field, from improved stability and potency to targeted delivery and reduced toxicity, has ultimately been driven by chemistry. As new modifications and conjugation strategies continue to emerge, the next generation of nucleic acid medicines is likely to reach tissues and diseases that have been inaccessible, further establishing oligonucleotides as a central therapeutic modality in modern medicine.
From Sequence Design to Clinical Candidate
How Synoligo can help with newer chemistry
New modifications are only useful if they can be made reliably, conjugated cleanly, and tested quickly. Synoligo offers custom synthesis that includes non-standard backbones, sugars, and bases; conjugation of peptides, lipids, carbohydrates, and targeting groups (on-resin or after synthesis) for tissue-specific delivery; and high-throughput screening to compare designs. As these newer modifications develop, we can help partners design, make, and test them, from the first version through to a preclinical candidate. See synoligo.com/therapeutic-oligos.
References
- Dhuri K, et al. (2020). Antisense Oligonucleotides: An Emerging Area in Drug Discovery and Development. J Clin Med 9(6):2004. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7355792/
- AlShaer D, et al. (2026). 2025 FDA TIDES (Peptides and Oligonucleotides) Harvest. Pharmaceuticals 19(2):244. https://doi.org/10.3390/ph19020244
- FDA News Release (Dec 23, 2016). FDA Approves First Drug for Spinal Muscular Atrophy. https://www.fda.gov/news-events/press-announcements/fda-approves-first-drug-spinal-muscular-atrophy
- Synoligo — Therapeutic Oligos. https://synoligo.com/therapeutic-oligos/
