An evolution in new therapeutic strategy that is helping us target deadly rare diseases

In recent years, antisense oligonucleotides (ASOs or AONs), have gained much interest as therapeutic agents. With success stories like Nusinersen (Spinraza) for spinal muscular atrophy (SMA), it seems that ASOs now have an established place on the drug market.

ASO are short single-stranded polymers based on DNA or RNA chemistries that are chemically synthesized. ASOs can regulate gene expression by binding in a sequence-specific manner to an RNA target. The functional activity of ASOs largely depends on the combinations and number of bases in the (target) sequence and the chemical modifications used. The chemical modifications also greatly determine its half-life, pharmacokinetics and toxicity.

The large benefit of ASOs is the gene-modulating ability, and consequently its specific expression modulation (leading to up/down regulation of the affected gene being one example). In this manner ASOs can target the underlying causes of severe inherited diseases for which small molecule or antibody-based drugs cannot be rationally designed.

Over the last 30 years ASOs have evolved to cover a broad range of applications. Early hurdles like low stability (susceptibility to nuclease-mediated breakdown), immunogenicity and cellular uptake have been improved with the discovery and use of novel chemical modifications.

Mechanisms of action

The three most often used applications of ASOs are

  • RNAse H recruitment and subsequent target mRNA degradation
  • Induction of splice switching (e.g. the inclusion or exclusion of an exon from the mRNA)
  • Inhibition of micro RNA (miRNA) by targeting of the miRNA sequence.

Below, the different MOAs are explained in more detail in combination with some examples of clinical applications.

RNAse H recruitment

The most well-known and explored mechanism of action (MOA) of ASOs is RNAse H recruitment and breakdown of the target mRNA, ultimately leading to decreased protein expression. The first US Food and Drug Administration (FDA)- approved ASO using this MOA was Formivirsen (Vitravene) in 1998. Formivirsen was designed for the treatment of human cytomegalovirus (CMV) retinitis. This antisense 21-mer PS ASO has a sequence complementarity to the coding region of the major immediate-early gene of CMV and binding leads to its degradation. Although the drug was successful, it was withdrawn in the EU in 2002 and the US in 2006 because there were cheaper alternatives.

Two other FDA-approved ASOs are Mipomersen (Kynamro) for the use in the treatment of familial hypercholesterolemia and Inotersen (Tegsedi) for the treatment of hereditary transthyretin amyloidosis. Both ASOs are so called “Gapmers”; although they have a fully PS backbone the middle section has no modified ribose groups to allow RNAse H recruitment.

Splice Switching

The second group of ASOs induce alternative splicing and are also called splice-switching ASOs. Normally these motifs attract proteins of the splice machinery that are responsible for the splicing of the pre-mRNA to the mRNA. Binding to the intronic splice silencer  prevents the binding of a negatively acting splicing factor and leads to exon inclusion. On the other hand, binding of ASOs to the splicing enhancer sequence prevents binding of the splice factor and thereby leads to exon skipping.

Splice switching as a therapy was first reported in 2006. Drisapersen (PRO051) was a PS and 2’-OM modified ASO designed to target patients with Duchenne muscular dystrophy (DMD) who are receptive to exon 51 skipping. This skipping results in a slightly shorter DMD transcript that leads to the formation of a protein that can rescue the DMD phenotype to a less severe type of muscular dystrophy. Drisapersen had quite a long road with different clinical studies but was eventually discontinued in 2016 due to a limited positive risk–benefit balance. In contrast, Eteplirsen (EXONDYS 51), a 30-mer PMO with the same target, was approved by the FDA in 2016. PMOs have an altered backbone, resulting in a stronger hybridization to the target RNA and subsequent higher efficiency. PMOs do not allow binding of RNAse H.

In 2016 Nusinersen (Spinraza) was approved for the treatment of SMA. Nusinersen, an 18-mer PS 2’-O-MOE ASO with methylated cytosines induces the inclusion of exon 7 in the mRNA of two genes (SMN1 and SMN2) by targeting and blocking an intron 7 internal splice site. These actions increase SMN protein production and thus improve function.

MiRNA targeting

The last MOA is the miRNA-targeting ASOs. This group of ASOs is used to prevent the binding of a miRNA to its target RNA and thereby inhibiting its function. One of the most commonly used chemical modifications in anti-miRNA ASOs is the LNA. LNAs have a very strong affinity to its target RNA.

Miravirsen (SPC3649), an anti-miRNA drug, is designed for the treatment of chronic hepatitis C infections and is currently in Phase II clinical trials. This LNA ASO is designed to have the complementary sequence to that of mir-122 and forms stable heteroduplexes. Mir-122 is important to the HCV life cycle, and by interfering with mir-122, viral replication can be inhibited.

Opportunities and challenges

In recent years multiple ASOs with different clinical applications have entered the market. However, next to the success stories there are also ASOs that have failed to enter the market. Common reasons for failure include limited added clinical benefit, systemic safety concerns (for example injection-site reactions with Drisapersen), and extremely high costs of manufacturing

In addition, specific peptide conjugates and formulations can increase the delivery and uptake of ASOs in target cells. Therefore, it is of great importance that the type and chemical composition of ASOs used in preclinical studies are carefully selected based on the desired MOA, target gene and target cells/organ to increase the chance for clinical success.


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