The landscape of nucleic acid therapeutics

With the rapid deployment of the mRNA COVID-19 vaccines and the recent approval of the first CRISPR gene therapy, the range of oligonucleotide-based therapeutics is expanding rapidly. Since the first antisense oligonucleotide was approved in the 1990s, many different approaches have become available to tackle life-changing diseases.  

In this post, we explore some of the key types of nucleic acid therapeutics on the market, covering the critical components of their structures, the modifications that enable them to function and their promise for the future. In a future blog post, we will take a deeper look at the promising technologies in development. 

mRNA

From being a promising yet underexplored approach in the 1990s, mRNA therapeutics have evolved significantly as chemical modifications overcame the challenges of instability and immunotoxicity.¹ 

Tailored mRNA design and synthesis are critical for delivering the therapeutic into the right cells and having the desired efficacy. 

mRNA has five key features in its structure: the 5′ cap, the 3′ poly(A) tail, the open reading frame (ORF), and the 5′ and 3′ untranslated regions (UTRs). Together, these determine the stability and efficiency of the molecule.  

Figure 1. The key elements of a mRNA therapeutic for enhancing stability and effectiveness. 

The COVID-19 mRNA vaccines require ultra-cold storage conditions, which limits distribution and increases costs. This challenge means there is a vast opportunity to advance mRNA therapeutics by developing thermostable products with high clinical efficacy. Research is making progress towards this goal in several ways, including lyophilisation as well as optimisation of mRNA sequence, nanoparticle components and manufacturing processes.² 

Following the mRNA vaccine platform’s success against COVID-19, there has been a surge of interest in applying it to other disease areas. In May 2024, the FDA approved Moderna’s mRESVIA vaccine for RSV. In addition, there is much hope in using mRNA to create personalised cancer vaccines, with a candidate reaching phase III clinical trials.³ 

The technology continues to develop, with the approval of the world’s first self-amplifying mRNA COVID-19 vaccine in Japan in November 2023. ARCT-154 triggers the body to make copies of the vaccine mRNA, leading to more protein production, a stronger immune response and longer duration with lower doses of mRNA.⁴ 

For more information about mRNA development and modifications, see our blog post “How to design mRNA therapeutics for scalable success”. 

Antisense oligonucleotides

Antisense oligonucleotides (ASOs) are small, single-stranded oligodeoxyribonucleotides comprising 8-50 bases that affect the expression of specific target RNAs via two distinct mechanisms: gene expression inhibition and splicing modulation.  

Expression inhibitors have a DNA-like structure and form DNA-RNA heteroduplexes that are selectively degraded by RNase H. Splicing modulators instead bind to the intron-exon junctions and alter splicing events by exon skipping or inclusion.^5,6 

After the approval of the first ASO, fomivirsen, in 1999, the biotechnology community faced a long 14 years before the feat could be repeated. Since then, ASOs have developed as new generations of modifications have offered increased stability and binding affinity. This includes the creation of gapmers – where modified RNA residues flank the DNA to protect it from nucleases and improve binding affinity. 

Therapeutics developers now have a wide range of base and backbone modifications to incorporate and tailor the oligo to its target. This has led to several ASOs being used in the clinic to treat diseases such as spinal muscular atrophy, Duchenne muscular dystrophy and amyotrophic lateral sclerosis (see table 1). 

Table 1. Approved ASOs up to 2023 and the diseases they target. 

For more information about ASO development and emerging technologies, see our blog post “What’s the latest in ASO and siRNA technologies?” 

siRNA  

Small-interfering RNAs (siRNAs) are double-stranded RNAs of 19 to 21 bases that can induce gene silencing by degrading the complementary mRNAs in a sequence-specific manner. In the cell, the siRNA is incorporated into the RNA-induced silencing complex (RISC), where the antisense strand guides RISC to the target mRNA, leading to its degradation.⁶ 

siRNAs are carefully designed to avoid off-target binding, as RISC is tolerant to minor mismatches, and to prevent the activation of toll-like receptors, which could potentially cause systemic toxicity.⁷ 

The size and hydrophilicity of these double-stranded therapeutics make them difficult to deliver into the target cells and are rapidly excreted from the body. Specialised delivery systems, such as lipid or nanoparticle formulations or chemical modifications with cell-surface receptor binding moieties, have allowed these molecules to succeed as approved drugs.⁸ Six siRNA drugs have been approved by the FDA since 2018, with many more in development and testing (see table 2).

After the first successfully approved siRNA, patisiran, all the later therapies took advantage of coupling to N‐acetylgalactosamine (GalNAc) to increase their uptake in liver cells, permitting lower doses. 

Table 2. Approved siRNA therapeutics up to 2023. 

For more information about siRNA development and emerging technologies, see our blog post “What’s the latest in ASO and siRNA technologies?” 

CRISPR

The promise of the CRISPR-Cas9 gene editing system is now proving a reality in the clinic, as the first treatment has become available for sickle cell disease and beta thalassaemia, both genetic conditions that affect red blood cells.

The CRISPR system directs endonucleases known as Cas (CRISPR-associated) proteins to target sequences. Jennifer Doudna and Emmanuelle Charpentier harnessed this system by designing a guide RNA that directs Cas9 to the exact spot in the genome where a cut is desired.9 As the cell attempts to repair the DNA break, a template can be introduced to add or replace a sequence, or an imprecise repair can potentially disable the gene through a frameshift mutation.   Charpentier and Doudna showed that two parts of the guide RNA in the bacterial system could be replaced by a synthetic variant, single guide RNA (sgRNA), that combines their properties. This synthetic format, approximately 100 nucleotides long, gained popularity due to its efficiency and simplicity.

Figure 2. A representation of how the sgRNA guides Cas9 to cleave the target DNA.

By 2023, the first gene therapy based on CRISPR-Cas9 technology received approval for patients. Casgevy precisely edits the genes in bone marrow stem cells to produce fully functional hemoglobin and treat sickle cell disease and beta thalassaemia, both genetic conditions that affect red blood cells.  

The sgRNA’s critical role means that its structure and stability must be carefully considered to ensure therapeutic impact. However, RNA is inherently unstable and highly susceptible to degradation by endo- and exo-nucleases. This instability poses a challenge to effectively use sgRNA in clinical settings, but it can be overcome through its design and production.  

Several modifications can make sgRNA more resistant to nucleases, as well as improve its stability and efficacy. This enhances the applicability of CRISPR-Cas9 as a therapeutic by reducing off-target effects and cytotoxicity.  

For more information about synthesising modified sgRNA for better efficacy, see our blog post “Optimise oligo synthesis and design for effective CRISPR therapeutics”. 

Aptamers

Aptamers are single strands of nucleic acids that bind to a particular target. Despite their many potential benefits as therapeutics – namely high thermal stability, the ability to bind tightly to virtually any target, easy and scalable production, and batch-to-batch uniformity – aptamers have seen little success in the clinic.^10,11 In addition to rapid renal clearance and susceptibility to nucleases, aptamer performance in vitro against purified components has not always translated to similar performance in vivo.¹¹ 

Figure 3. Nucleic acid aptamers are single-stranded oligonucleotides that fold into well-defined three-dimensional structures that can bind to the desired target (e.g. small molecules, proteins, cells and even whole organisms).^12,13 

There are two approved aptamers: pegaptanib (Macugen) and avacincaptad pegol (Izervay), which both treat age-related macular degeneration and are injected directly into the eye. Pegaptanib binds and blocks the vascular endothelial growth factor (VEGF) protein, which is responsible for forming blood vessels. Pegaptanib consists of long polyethylene glycol chains attached to a 28-mer RNA oligonucleotide with 2ʹ-F and -OMe modifications.  

After pegaptanib was approved in 2004, it took 19 years for a second aptamer drug to follow. Avacincaptad pegol is a 39-nucleotide aptamer attached to polyethylene glycol and inhibits complement protein C5. This is used to treat geographic atrophy secondary to age-related macular degeneration. 

Despite a hesitant start to aptamer therapies, recent efforts to harness them as the targeting component of a larger therapeutic may finally see these promising molecules have a greater impact in the clinic. This includes attaching aptamers to target photodynamic therapies, siRNA-containing nanoparticles and thrombin.^14-19 

For more information about aptamer development, see our blog post “Where are all the therapeutic aptamers?” 

The future of oligonucleotide therapeutics

Despite an initial stop-start output of approved drugs, nucleic acid therapeutics are now a firmly established class producing a steady stream of new treatments. The success of the COVID-19 vaccines set a clear example of the unprecedented speed of development that this technology offers and its transformative potential.  

Improved understanding of optimal modifications and delivery mechanisms has led to a rapid pace of innovation. As these existing approaches reach countless patients, the next generation of therapies is waiting in the wings. Stay tuned for our next blog post to find out more about what’s next for nucleic acid therapeutics. 

References

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9. Jinek M et al. (2012) A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337, 816–821. doi:10.1126/science.1225829 
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11. Yan AC, Levy M. Aptamer-Mediated Delivery and Cell-Targeting Aptamers: Room for Improvement. Nucleic Acid Ther. 2018;28(3):194-199. doi:10.1089/nat.2018.0732 
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13. Sun H et al. Oligonucleotide aptamers: new tools for targeted cancer therapy. Molecular Therapy Nucleic Acids 2014; 3, e182, doi:10.1038/mtna.2014.32
    
14. Li L, Zhou B, Xu H et al. Zinc-Loaded Black Phosphorus Multifunctional Nanodelivery System Combined With Photothermal Therapy Have the Potential to Treat Prostate Cancer Patients Infected With COVID-19. Front Endocrinol. 2022;13:872411. doi:10.3389/fendo.2022.872411 
15. Chen L, Hong W, Duan S et al. Graphene quantum dots mediated magnetic chitosan drug delivery nanosystems for targeting synergistic photothermal-chemotherapy of hepatocellular carcinoma. Cancer Biol Ther. 2022;23(1):281-293. doi:10.1080/15384047.2022.2054249 
16. Ibarra LE, Camorani S, Agnello L et al. Selective Photo-Assisted Eradication of Triple-Negative Breast Cancer Cells through Aptamer Decoration of Doped Conjugated Polymer Nanoparticles. Pharmaceutics. 2022;14(3):626. doi:10.3390/pharmaceutics14030626 
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Related content

• What’s the latest in ASO and siRNA technologies?
• How to design mRNA therapeutics for scalable success
• Optimise oligo synthesis and design for effective CRISPR therapeutics
• Where are all the therapeutic aptamers?