Exploring Science
Targeting RNA: A New Frontier in Medicine
Life's central dogma involves DNA transcription to RNA and subsequent translation into proteins. Traditionally, RNA was thought to serve solely as a messenger of genetic information from DNA to the ribosomes. However, discovering non-regulatory RNAs has revealed the vast repertoire of RNA-mediated processes. As a result, it is now considered the only macromolecule that can execute all biological procedures, making it a critical target for therapeutic intervention. The prevailing approach for preventing or treating diseases is established by targeting molecular, biological, or cellular processes. Currently, small molecules and antibodies are mainly used in drug discovery targeting proteins.
Nonetheless only 1,5% of the human gene pool encodes for proteins (approximately 20.000) 16, disorder-associated account for 10-15% of these proteins (2.000- 3.000 proteins) and less than 700 proteins are successfully targeted with approved drugs . Therefore, aiming at proteins can only provide narrow possibilities for developing novel therapeutic modalities.
In this scenario, ASOs and siRNAs play a crucial role in expanding our capability to target a greater spectrum of diseases by acting upstream the protein level, at the gene expression. This is achieved by ASO/siRNA binding to mRNA or pre mRNA via Watson–Crick base pairing and exerting their effect through various mechanisms of actions, further explained in Mechanisms of Action of Oligonucleotides.
Antisense technology has started to fulfil its potential, as demonstrated by the approval of several RNA-targeted drugs for clinical application, including single-strand antisense drugs (ASOs) and double-strand ASOs (siRNAs).
Confusion in classifying these modalities (ASOs and siRNAs) stems from ambiguous definitions. Here, antisense oligonucleotides are operationally defined as 8–50 mer, single or double stranded oligonucleotides that bind cellular or nuclear mRNA (or pre mRNA) through Watson–Crick pairing.
Chemistry and Mechanisms of Action (MOA) of Oligonucleotides
Small interfering RNAs (siRNAs) are double stranded molecules ( ~20 nucleotides) that silence gene expression through a process known as RNA interference (RNAi). Chemically speaking, they are composed of a sense (or passenger ) strand and an antisense ( or guide) strand. Once inside the cell, the siRNA gets loaded into the RISC complex (RNA Induced Silencing Complex), where the passenger strand unwinds and only the guide strand remains incorporated in the RISC. This guide strand serves to guide the RISC to the targeted mRNA sequence, allowing it to be cleaved and degrading the targeted mRNA, thus preventing protein production.
siRNAs are commonly used in functional genomics research, target validation and therapeutic gene silencing.
Antisense Oligonucleotides (ASOs) are single stranded molecules ( ~18 nucleotides) (usually DNA-like or RNA-like) that modulate gene expression in various ways by bind specifically to a target RNA molecule through complementary base pairing. Gene expression can be altered so to be silenced/inhibited, activated or modulated via acting at the mRNA splicing level.
The diverse modulations of gene expression can be achieved by several MOA. Silencing can be achieved by blocking translation of a messenger RNA (mRNA) or promoting degradation of the RNA. Activation occus by sequestring miRNA that reduces/inhibits gene expression. Modulation of the mRNA splicing can arise by ASOs hindering splicing sites required for naturally occuring splicing. For ease of interpretation, see Figure 1.
ASOs are widely studied and used in treatments for genetic and neurological diseases.
For ASOs three different mechanism of action exist which exert two different effects: gene silencing or degradation. Gene silencing can be achieved by (A) steric blocking; ASO binds to the targeted mRNA, preventing ribosomes to bind and translating it to proteins or by (B) degradation; achieved by Gapmer ASOs where binding to the targeted sequence ( mRNA or premRNA ) leads to the cleavage of the formed double strand by the RNase H1 and thus to its degradation.
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Figure 1. Summary of the mechanisms of action for both ASOs ( blue left side ) and siRNAs (red right side) . siRNAs are double stranded molecules ( ~20 nucleotides). They are composed of a sense (or passenger ) strand and an antisense ( or guide) strand. Once inside the cell, the siRNA gets loaded into the RISC complex (RNA Induced Silencing Complex), where the passenger strand unwinds and is removed. The remaining guide strand guides the complex to the targeted mRNA sequence, cleaves it and promotes its degradation. For ASOs three different mechanism of action exist which exert two different effects: gene silencing or degradation. Gene silencing can be achieved by (A) steric blocking; ASO binds to the targeted mRNA, preventing ribosomes to bind and translating it to proteins or by (B) degradation; achieved by Gapmer ASOs where binding to the targeted sequence ( mRNA or premRNA ) leads to the cleavage of the formed double strand by the RNase H1 and thus to its degradation.
Despite their potential and mechanistic specificity, we face challenges when translating them into therapies . Unlike conventional small‑molecule drugs, they are large, polyanionic macromolecules with poor membrane permeability, leading to inefficient cellular uptake. They are also susceptible to nuclease‑mediated degradation, which shortens systemic and intracellular half‑life and reduces pharmacodynamic effects. Overall the tool is holds great potential but reaching its target tissue remains difficult- the so called delivery issue.
The technology has been around for decades—nearly thirty years have passed since the arrival of "Fomivirsen", the first ever approved oligonucleotide in the market. Consequently, we now have greater confidence in the underlying science and in the chemistries we can apply to enhance their drug‑like properties. Rational chemical modification of the oligonucleotide backbone, sugar, and bases can improve nuclease resistance and tune pharmacokinetic and biodistribution profiles (see Table 1).
Table 1 Figure 2 illustrates a range of chemical modifications applied to improve the drug-like properties of these compounds. In practical terms, these modifications are introduced to address key liabilities such as susceptibility to nuclease-mediated degradation and rapid systemic clearance. As noted previously, unmodified compounds can be readily degraded by nucleases and eliminated from the body before reaching their intended target. One important chemical modification is the replacement of a non-bridging oxygen atom in the phosphate backbone with sulfur, generating a phosphorothioate linkage. This modification reduces recognition by exonucleases, thereby increasing molecular stability and prolonging the time the compound remains intact in biological systems. In addition, phosphorothioate modifications enhance binding to plasma proteins, which can reduce renal clearance and extend the compound’s circulating half-life. Beyond backbone modifications, sugar modifications can further improve the properties of antisense oligonucleotides (ASOs), particularly by increasing their binding affinity for the target RNA. Together, these examples illustrate how chemical modification strategies contribute fundamentally to the development of oligonucleotide therapeutics. To put it simply, even a seemingly small structural change, such as the substitution of a single atom, can alter the conformation final compound, thereby influencing its compatibility with biological components such as proteins, nucleases, and the target nucleic acid itself.
Additionally to chemically stabilize the oligonucleotide strand , conjugation to various chemical moieties has been proven to be a successfull/useful strategy . This is clearly seen in the GalNac case, being GalNac a chemical conjugation which allows truly efficient delivery of the oligonucleotide to the liver. Real life example is Givosiran (siRNA GalNac conjugate) approved in 2018 and since then we already have 5 drugs on the market with this conjugation. It is like we found the key for a tissue and now we opened the liver door (see Table 2 ).
Table 2:Commercially available antisense oligonucleotides. The table also shows the diseases for which they are used, their chemical modifications, and their year of approval.
It would be exciting if we had the keys to other organs as well, and this is what the community aspires to achieve. This is also what this network is working on. Each of us is working to generate more information on how to access a specific organ for the development of the corresponding keys to each of these doors.
The future prospects for these modalities remain highly promising. ClinicalTrials.gov currently lists hundreds of ongoing clinical trials involving antisense agents, the majority of which are in Phase 1 or Phase 2, with many others progressing into Phase 3. This suggests that additional antisense therapeutics are likely to reach the market in the coming years.
The literature indicates that antisense technologies represent a particularly valuable approach for rare diseases. However, the development of inclisiran, an approved therapy for hypercholesterolemia, also demonstrates their potential in the treatment of more common disorders. Overall, antisense therapeutics appear to hold substantial promise for neurodegenerative diseases such as Huntington’s disease, muscular disorders such as Duchenne muscular dystrophy, and liver diseases with an underlying genetic basis.
This was a brief exploration of antisense technology: a simple yet holistic view of the strengths that make this field so promising, and the challenges that continue to shape its path forward. We hope it has shown why antisense inspires such enthusiasm. If there is one message to take away; what we see today is only the tip of the iceberg.
Stay tuned: in the next part, we will take a closer look at the European landscape surrounding antisense technology and the many players helping shape its progress!!!!
