The advantages of oligonucleotides have led to great interest in these molecules due to their therapeutic potential. Currently, as many as seventeen oligonucleotide drugs received regulatory approval from the FDA and the EMA. In addition, multiple clinical trials with oligonucleotides are ongoing. Industry and regulatory agencies have gained extensive expertise with this product type recently. However, there are regulatory and developmental challenges, both at the product quality/control, nonclinical and clinical levels, that must be considered to develop oligonucleotide therapies successfully.


Oligonucleotide therapies are typically synthetically modified, single-stranded or double-stranded RNAs or RNA/DNA hybrids. Based on their mechanism of action, we can distinguish antisense oligonucleotides (ASOs), small interfering RNAs (siRNAs), anti-microRNAs, and antagomirs. Oligonucleotide therapies are designed to hybridise to complementary targets to modulate specific gene expression across the genome [1].

A main advantage of oligonucleotides is that they can modulate various disease targets, including more than 10,000 proteins in the human genome that have been thought to be undruggable by small molecules. Also, the inherent ease of synthesising new libraries of complementary oligonucleotides targeting the intended protein greatly facilitates these products’ design.

Despite their great therapeutic potential, there are several limitations encountered with oligonucleotides. Drug delivery and toxicity are the major problems faced. After parenteral administration, oligonucleotide drugs must travel through the bloodstream, pass through the biological membranes, and be taken up by the target cells. To overcome these problems, various strategies are employed to enhance the stability of the oligonucleotides and promote their delivery to target tissues. These strategies consist of a) introducing specific structural modifications in the nucleotide backbone or sugar moieties; b) conjugation to different moieties (polymers, peptides, lipids, antibodies, etc.) to direct the oligonucleotide to the target tissue; c) formulation into delivery vectors (i.e. lipid-based nanoparticles) [2]. Safety aspects that should be considered include off-target related toxicities, immune-stimulatory responses, thrombocytopenia, inhibition of coagulation and renal accumulation that leads to renal damage [1, 3].

Up to seventeen oligonucleotide drugs have been approved by the FDA and the EMA (Table 1). These oligonucleotides are antisense oligonucleotides (ASO) or small interference RNAs (siRNAs) to be administered intravenously (IV, to target liver or muscle), subcutaneously (SC, to target liver), intrathecal (to target central nervous system) or intravitreal. Oligonucleotide drugs are generally indicated for orphan conditions, like Duchenne muscular dystrophy, spinal muscular atrophy or hereditary transthyretin amyloidosis. The most recently approved oligonucleotide product was Tofersen (Qalsody), approved by the FDA in April 2023 for treating amyotrophic lateral sclerosis. Tofersen is currently under review by the EMA[1].


Table 1. Oligonucleotide drugs approved by the EMA and FDA as of Jun 2023.

Drug Brand name Approval Indication RoA Target Type
Fomivirsen† Vitravene US, 1998; EU, 1999 Cytomegalovirus retinitis Intravitreal Eye ASO
Pegaptanib Macugen US, 2004; EU, 2006 Neovascular, age-related macular degeneration Intravitreal Eye Aptamer
Mipomersen‡ Kynamro US,2013 Homozygous familial hypercholesterolemia SC Liver ASO
Defibrotide Defitelio US,2016 Hepatic veno-occlusive disease IV Liver ON mixture
Eteplirsen Exondys 51 US, 2016 Duchenne muscular dystrophy IV Muscle ASO
Nusinersen Spinraza US, 2016;  EU, 2017 Spinal muscular atrophy Intrathecal CNS ASO
Inotersen Tegsedi EU, 2018; US, 2018 Human hereditary transthyretin amyloidosis SC Liver ASO
Patisiran Onpattro US, 2018; EU, 2018 Human hereditary transthyretin amyloidosis IV Liver siRNA
Volanesorsen Waylivra EU, 2019 Familial chylomicronemia syndrome SC Liver ASO
Givosiran Givlaari US, 2019; EU, 2020 Acute hepatic porphyria SC Liver siRNA
Golodirsen Vyondys 53 US, 2019 Duchenne muscular dystrophy IV Muscle ASO
Viltolarsen Viltepso US, 2020 Duchenne muscular dystrophy IV Muscle ASO
Lumasiran Oxlumo US, 2020; EU, 2020 Primary hyperoxaluria type 1 SC Liver siRNA
Inclisiran Leqvio EU, 2020; US, 2021 Atherosclerotic cardiovascular disease and heterozygous familial hypercholesterolemia SC Liver siRNA
Casimersen Amondys 45 US, 2021 Duchenne muscular dystrophy IV Muscle ASO
Vutrisiran Amvuttra US, 2022; EU, 2022 Hereditary transthyretin-mediated amyloidosis SC Liver siRNA
Tofersen Qalsody US, 2023 Amyotrophic lateral sclerosis Intrathecal CNS ASO

Abbreviations: ASO, antisense oligonucleotide; CNS, central nervous system; IV: intravenous; SC: subcutaneous; siRNA: small interference RNA. Based on Igarashi et al., 2021 and Thakur, 2022. Updated as of June 9th 2023. †The sale is currently discontinued. ‡It is still available on a very restricted basis due to side effects.


Beyond these approvals, multiple clinical trials with oligonucleotides are ongoing, including Phase III trials in neurological, cardiovascular, metabolic or ophthalmic indications [1]. Industry and regulators have gained thorough expertise on this product type recently. There are, however, challenges that should be considered for the successful development of oligonucleotide therapies.


Regulatory challenges

Only a few specific regulatory guidelines support the development of oligonucleotides.

  • From the CMC point of view, synthetic oligonucleotides are at the interface of small molecules and biologicals. Therefore specific considerations apply to this class of therapeutics. Indeed, oligonucleotides are fully or partially excluded from the scope of ICH Q3A/B, ICH Q6A/B and ICH M7. Of note, a recent concept paper published by the EMA anticipates the publication of a new guideline addressing specific aspects regarding the manufacturing process, characterisation, specifications and analytical control for synthetic oligonucleotides [4].
  • Even if not biological products, the nonclinical development of oligonucleotides should follow recommendations in the ICH S6 guideline Preclinical safety evaluation of biotechnology-derived pharmaceuticals [5]; beyond this regulatory guideline, the Oligonucleotide Safety Working Group (OSWG) has published several articles discussing toxicology assessments for oligonucleotides (genotoxicity, repeat dose toxicity, reproduction toxicity, etc.) that we find very useful [6-8].
  • As for other products, the clinical development plan of oligonucleotide therapies should follow the regulatory guidelines for the intended indication when available. Also, the FDA has recently published a draft guidance for the industry on the Clinical Pharmacology Considerations for the Development of Oligonucleotide Therapeutics. This guideline recommends immunogenicity risk assessments or characterising the impact of hepatic or renal impairment [9].

Also, the FDA recently published three guidelines to support the CMC, nonclinical and clinical development of individualised antisense oligonucleotides developed to treat a severely debilitating or life-threatening disease caused by a unique genetic variant where only a small number of individuals are prospectively identified (usually one or two) [10-12]. While these guidelines can be considered a reference, additional assessments to evaluate efficacy and safety may be expected for products used in wider populations. Moreover, the FDA has also published a draft product-specific guidance for developing generic nusinersen, which provides recommendations for demonstrating the sameness of the active pharmaceutical ingredient (API) for requesting a waiver of in vivo bioequivalence study requirements [13].


CMC Development

For the CMC development of synthetic oligonucleotides, major review issues relate to controlling the starting materials, the drug substance, and the drug product [14, 15]. Some major challenges are as follows:

  • Starting materials are an important part of the overall control strategy. Phosphoramidites, the building blocks used in synthesising oligonucleotides, already have complex chemistry and syntheses. Criticality assessment of their impurity profile is essential, and impurity profiles for phosphoramidites from different suppliers should be compared.
  • A good understanding of the impurity profile in the drug substance should be demonstrated. This is a major challenge as most impurities exist as mixtures of closely related molecules (i.e., diastereomers, n-1, n-2, n+1, n+2), many impurities coelute with the active ingredient, and there is a lack of analytical methods to resolve impurities adequately. Genotoxic impurities need to be comprehensively discussed in the dossier, and the presence of nitrosamines should be controlled and kept as low as possible.
  • Regarding the control of the drug product, usually, ±3 times the standard deviation is adequate for the setting of specifications. Early development batches should not be the basis of specification setting. Indeed post-approval adjustment of specifications may be an option. Usually, no bioassay is expected for antisense and siRNA molecules; however, a justification for the omission of bioassay should be provided.


Nonclinical development

Toxicology packages should be designed to assess, as far as possible, the expected toxicities associated with this therapeutic class (i.e., off-target toxicities, immune stimulation or inhibition of coagulation and renal damage). Importantly, some of these assessments can be done (at least preliminarily) using in silico and in vitro approaches [3].

As for other biological products, a main challenge for the nonclinical development of oligonucleotides is identifying relevant species for toxicology assessments, as oligonucleotide hybridisation to the target sequence is required to assess on-target toxicities. Moreover, the route of administration used in the nonclinical studies should mimic, as far as possible, the intended clinical route, which is an additional challenge for complex routes of administration, like intravitreal or intrathecal.


Clinical development

The therapeutic effect of the oligonucleotides requires a specific level of target engagement in the intended cells. Identifying this therapeutic dose is especially challenging when estimating clinical doses relies on nonclinical data, and differences among human and animal models exist regarding the pharmacokinetic profile and biodistribution of the oligonucleotide.

An unwanted immune response to an oligonucleotide can be generated to the carrier, backbone, oligonucleotide sequence, or any novel epitopes created from the whole drug. The clinical immunogenicity assessment for an oligonucleotide therapeutic should follow a risk-based approach. For clinical immunogenicity assessments, immunogenicity sample collection should coincide with pharmacokinetic and pharmacodynamic sampling time points to evaluate whether anti-drug antibodies (ADAs) impact the pharmacokinetics, pharmacodynamics, and any immune-mediated adverse events of the oligonucleotide therapeutic [9].

An additional challenge often faced during the development of oligonucleotides is demonstrating efficacy based on a relevant clinical outcome. Oligonucleotides are generally developed for treating rare genetic conditions affecting multiple systems, and patients may have very diverse symptoms. Generally, these conditions have a slow and complex evolution, and their natural history is poorly understood. In this scenario, the demonstration of efficacy often relies on surrogated biomarkers that have not always demonstrated clinical relevance. This could be a major issue, as clinical data may fail to demonstrate the positive benefit/risk expected by regulators.



Overall, there are limited regulatory guidelines on oligonucleotide therapies; however, key experience has been gained during the development of the therapies already approved and currently under development. Nevertheless, there are still major challenges to successfully developing these therapies at CMC, nonclinical and clinical levels. In this scenario, it is strongly recommended to discuss the development plan with the regulatory authorities and agree on major aspects, including, among others, specifications (mainly impurities), selection of relevant species, design of the toxicology plan, patient selection, or clinical outcomes of efficacy.


Keen to know how you can design an optimal development plan for your oligonucleotide? Willing to discuss your oligonucleotide program with the regulatory authorities? We can support you!

Building on decades of experience in the EU regulatory requirements, Zwiers Regulatory Consultancy, a ProductLife Group Company, provides up-to-date support for expert advice on drug development and regulatory strategy.


  1. Thakur, S., et al., A perspective on oligonucleotide therapy: Approaches to patient customisation. Front Pharmacol, 2022. 13: p. 1006304.
  2. Anwar, S., F. Mir, and T. Yokota, Enhancing the Effectiveness of Oligonucleotide Therapeutics Using Cell-Penetrating Peptide Conjugation, Chemical Modification, and Carrier-Based Delivery Strategies. Pharmaceutics, 2023. 15(4).
  3. Aurélie Goyenvalle, C.J.-M., Willeke van Roon, Sabine Sewing, Arthur M. Krieg, Virginia Arechavala-Gomeza, and Patrik Andersson, Considerations in the Preclinical Assessment of the Safety of Antisense Oligonucleotides. Nucleic Acid Therapeutics, 2023. 33(1): p. 1-16.
  4. EMA/CHMP/QWP/735423/2022, Concept Paper on the Establishment of a Guideline on the Development and Manufacture of Synthetic Oligonucleotides. 2022.
  5. S6(R1), I., ICH guideline S6 (R1) – preclinical safety evaluation of biotechnology-derived pharmaceuticals. 2011.
  6. Berman, C.L., et al., OSWG Recommendations for Genotoxicity Testing of Novel Oligonucleotide-Based Therapeutics. Nucleic Acid Ther, 2016. 26(2): p. 73-85.
  7. Berman, C.L., et al., Recommendations for safety pharmacology evaluations of oligonucleotide-based therapeutics. Nucleic Acid Ther, 2014. 24(4): p. 291-301.
  8. Marlowe, J.L., et al., Recommendations of the Oligonucleotide Safety Working Group’s Formulated Oligonucleotide Subcommittee for the Safety Assessment of Formulated Oligonucleotide-Based Therapeutics. Nucleic Acid Ther, 2017. 27(4): p. 183-196.
  9. CDER (FDA), Clinical Pharmacology Considerations for the Development of Oligonucleotide Therapeutics – Guidance for Industry. 2022.
  10. CDER (FDA), IND Submissions for Individualised Antisense Oligonucleotide Drug Products for Severely Debilitating or Life-Threatening Diseases: Clinical Recommendations – Draft Guidance for Sponsor-Investigators. 2021.
  11. CDER (FDA), CDER (FDA), IND Submissions for Individualized Antisense Oligonucleotide Drug Products for Severely Debilitating or Life-Threatening Diseases: Chemistry, Manufacturing, and Controls Recommendation. 2021.
  12. CDER (FDA), Nonclinical Testing of Individualized Antisense Oligonucleotide Drug Products for Severely Debilitating or Life-Threatening Diseases – Draft Guidance for Sponsor-Investigators. 2021.
  13. CDER (FDA), Draft Guidance on Nusinersen Sodium. 2022.
  14. Rene Thurmer, EU Regulators’ experience with synthetic oligonucleotides and mRNA technology 2023.
  15. Deyi Zhang, Oligonucleotides: Current Thinking and Analytical Challenges Identified in the Nusinersen PSG Development. 2022.

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Regulatory & development challenges of oligonucleotide therapy