Making Sense of Antisense: The Power of Gene Silencing

The human genome contains around 20,000 protein coding genes, each a template for a protein that plays a role in the body’s structure, function and regulation. [1, 2] A small percentage of gene variants, whether hereditary or non-inherited, trigger the production of disease-causing faulty proteins, leading to disease. [3]

Diseases caused by changes in proteins range from mild to severe, and include rare diseases, cardiovascular disease, cancer and other disorders. Treatment approaches for genetic diseases have focused on managing symptoms through small molecule and biologic therapeutics. Newer approaches target the genetic mutations themselves, by replacing (gene therapy), removing (gene editing) or muting (gene silencing) the genetic code. [4] These target the molecular causes of disease, rather than just treating the symptoms.

Making sense of antisense

The gene silencing approach works by targeting mutations and preventing or reducing expression of disease-causing proteins, and the naturally occurring mechanisms include antisense oligonucleotides (ASOs) and RNA interference (RNAi) strategies. [5, 6] 

ASOs are stabilised single- or double-stranded synthetic oligonucleotides that bind to the target DNA, triggering the cleaving of the target mRNA (messenger RNA) by RNase. In the RNAi process, after administration, small interfering ribonucleic acids (siRNAs) and microRNAs (miRNAs) bind to the mRNA, either inhibiting the translation of the mRNA into protein, or inducing its degradation. [5, 6] With both approaches, each oligonucleotide is specific to a single mutation, but mixtures could be used to treat a disorder associated with a number of genetic variants.

Fomivirsen (Vitravene) was the first ASO to be approved by the US Food and Drugs Administration (FDA), in 1998. It was developed by Isis Pharmaceuticals and licensed to Novartis for the treatment of cytomegalovirus (CMV) retinitis in people who were HIV-positive. The drug was withdrawn in 2001 as the success of antiretroviral therapy reduced demand. It however provided proof-of-concept for antisense oligonucleotides, with the next drug, Ionis/Genzyme’s mipomersen (Kynamro), being approved in 2013 for the treatment of homozygous familial hypercholesterolemia. [7, 8] 

As of March 2024, the FDA and European Medicines Agency (EMA) had approved 12 antisense oligonucleotides and six siRNAs. [9] The global market for antisense oligonucleotides and RNAi was worth around $4.4 billion in 2023 and is expected to grow at 18.2% CAGR to reach $19.7 billion in 2023. This is driven by the increasing prevalence of neurodegenerative and genetic disorders, growing investments in research related to gene expression and delivery technologies, and the growth in regulatory approvals for antisense therapeutics as the key drivers behind this growth. [10] 

Research into ASOs is thriving, particularly in genetic disorders, neurological diseases, and cancer. As an example, SynaptixBio is developing an ASO to treat H-ABC, the most severe form of the rare disease TUBB4A leukodystrophy. This genetic neurodegenerative disorder affects around 1600 babies and children worldwide every year and makes up around 9% of all leukodystrophies. The gain of function mutation in the TUBB4A gene leads to a failure to lay down myelin around the nerves, resulting in seizures, muscle contractions, uncontrollable limb movements and issues with speech. Life expectancy depends on severity of the disease and many die by their late teens. [11]

SynaptixBio’s H-ABC ASO, SB-19642, is a DNA molecule with RNA ‘wings’ that binds to the mRNA. It is highly specific, targeting only the aberrant tubulin, allowing the other tubulins to continue to play their physiological roles. The company is currently selecting a candidate ASO to begin Phase I clinical trials. [12]

Biogen’s ASO BIIB080, the first gene silencing approach to Alzheimer’s disease, has been assessed in a UK Phase II trial. The ASO targets the Tau protein gene, one of two proteins known to be prevalent in patients with Alzheimer's disease. The trials have recently been extended after initial success. [13]

Trials are underway for gene silencing ASOs with potential in Parkinson’s disease, motor neurone disease and Duchenne muscular dystrophy. ASOs can also be used in combination with vaccines and drugs to target a number of disease mechanisms concurrently. As an example, mipomersen is used in combination with cholesterol-lowering drugs and lifestyle changes in familial hypercholesterolemia. [10, 14]

The benefits and challenges

‘Gene silencing’ is different from gene editing in that the gene itself in untouched; only its expression as a protein is affected. ASOs are highly targeted, with a low potential for off-target effects. Gene silencing therapeutics require repeat treatments in contrast with the gene therapy or gene editing ‘one and done’ approach. While this might appear to be a disadvantage, it can make the approach more flexible, as the dose can be reduced or increased to tailor the effect, or stopped if there are unwanted side effects. [12, 15]

Gene-based therapeutics can be highly effective, but their costs can be very high. As an example, nusinersen (Spinraza), an ASO developed to treat spinal muscular atrophy, costs around $2 million for a course of three doses.[16, 17] 

While the costs for manufacturing ASOs are relatively low, the small population size means that is can be hard to recruit patients for clinical trials, more difficult to show an effect in the trials, and the market is limited. Paediatric clinical trials are also more challenging and higher cost. [12] 

The future of ASOs and gene silencing

ASOs are highly targeted drugs and so can only be effective if they are stable, can reach the required location, and are taken up into cells in sufficient quantities. They are large molecules so do not easily pass through the cell membrane or the blood-brain barrier and are easily broken down in the body. [18]

Local delivery to the eye or the spinal cord, as well as delivery to the liver, has proved successful. Delivery to other parts of the body will require delivery systems, and there are a variety of approaches in development: [7, 18, 19]

· Chemical modification – adjusting the drug-like properties of oligonucleotides to improve pharmacokinetics, pharmacodynamics and biodistribution

· Bioconjugation – adding lipids, peptides, aptamers, antibodies and sugars to improve targeting, cell penetration and internalisation

· Nanocarriers – optimising biophysical and biological properties to cross biological barriers and enable transmembrane intracellular delivery

· Liposomes – using lipid bilayers with added components to protect the oligonucleotides, ensure stability, improve biocompatibility and toxicity, and trigger uptake into cells

· Exosomes – creating encapsulated vesicles to target specific organs, tissues and cell types, and cross biological membranes.

· Nanoparticles – engineering particles to improve tissue specificity and uptake.

Work on the human genome has allowed researchers to discover vast numbers of genes. The next step is a need for a greater understanding of the role of these genes in health and disease, and how gene-based interventions, including gene silencing, can make a difference.

With thanks to Dan Williams, CEO, executive director and co-founder of SynaptixBio

References

1. Green, E. Gene. National Human Genome Research Institute. Last accessed: 23 January 2025. Available from: https://www.genome.gov/genetics-glossary/Gene.

2. Amaral, P., et al., The status of the human gene catalogue. Nature, 2023. 622(7981): p. 41-47.

3. Variants and health. MedlinePlus. Last accessed: 23 January 2025. Available from: https://medlineplus.gov/genetics/understanding/mutationsanddisorders/.

4. Braga, L.A.M., C.G. Conte Filho, and F.B. Mota, Future of genetic therapies for rare genetic diseases: what to expect for the next 15 years? Ther Adv Rare Dis, 2022. 3: p. 26330040221100840.

5. Blom, D.J., et al., RNA-based therapy in the management of lipid disorders: a review. Lipids Health Dis, 2022. 21(1): p. 41.

6. Yosipovitch, G., Understanding the Mechanism and Applications of Gene Silencing. Epigenetics Research: Open Access, 2023(1): p. 1000131.

7. Types of antisense oligonucleotides: Advancing therapeutic frontiers. CAS, 10 May 2024. Available from: https://www.cas.org/resources/cas-insights/types-antisense-oligonucleotides-advancing-therapeutic.

8. Bradley, C.A., First antisense drug is approved with fleeting success. Nature Milestones, 14 November 2019. Available from: https://www.nature.com/articles/d42859-019-00080-6.

9. Vinjamuri, B.P., J. Pan, and P. Peng, A Review on Commercial Oligonucleotide Drug Products. J Pharm Sci, 2024. 113(7): p. 1749-1768.

10. Antisense and RNAi Therapeutics Market – Technology (RNA Interference, Antisense RNA), Route of Administration (Intrathecal, Intravenous, Subcutaneous), Application (Neurodegenerative, Cancer, Skin, Genetic Disorders) – Global Forecast (2024 – 2032). Global Market Insights. May 2024. Available from: https://www.gminsights.com/industry-analysis/antisense-and-rnai-therapeutics-market.

11. Zehr, L., SynaptixBio targeting rare disease, TUBB4A leukodystrophy, virtually. BioTuesdays, 2 May 2023. Available from: https://biotuesdays.com/2023/05/02/synaptixbio-targeting-rare-disease-tubb4a-leukodystrophy-virtually/.

12. Dan Williams, CEO, executive director and co-founder of SynaptixBio.

13. UCLH trial of world-first treatment for Alzheimer’s disease progresses into larger trial. University College London Hospitals. Last accessed: 17 April 2024. Available from: https://www.uclh.nhs.uk/news/uclh-trial-world-first-treatment-alzheimers-disease-progresses-larger-trial.

14. Dhuri, K., et al., Antisense Oligonucleotides: An Emerging Area in Drug Discovery and Development. J Clin Med, 2020. 9(6).

15. ‘Gene silencing’ therapies market set to boom, says leading biotech. PharmiWeb.com, 20 November 2024. Available from: https://www.pharmiweb.com/press-release/2024-11-20/gene-silencing-therapies-market-set-to-boom-says-leading-biotech.

16. Elvidge, S., Making Cell and Gene Therapy Affordable. Pharma Sources: An eye on the biopharma industry, 4 November 2024. Available from: https://www.pharmasources.com/industryinsights/making-cell-and-gene-therapy-affordable-76789.html.

17. Qiu, J., et al., History of development of the life-saving drug "Nusinersen" in spinal muscular atrophy. Front Cell Neurosci, 2022. 16: p. 942976.

18. Roberts, T.C., R. Langer, and M.J.A. Wood, Advances in oligonucleotide drug delivery. Nat Rev Drug Discov, 2020. 19(10): p. 673-694.

19. Gagliardi, M. and A.T. Ashizawa, The Challenges and Strategies of Antisense Oligonucleotide Drug Delivery. Biomedicines, 2021. 9(4).