Introduction
Splice-switching oligonucleotides (SSOs) are a class of antisense oligonucleotides (ASOs) designed to modulate pre-mRNA splicing by redirecting the splicing machinery to alter exon inclusion or exclusion [1]. By binding to specific pre-mRNA sequences, SSOs can correct aberrant splicing caused by genetic mutations, restore functional protein expression, or induce therapeutic exon skipping/inclusion in various diseases, including genetic disorders, cancer, and neurological conditions [2,3].
Mechanism of Action of Splice-Switching Oligonucleotides
SSOs typically target splicing regulatory elements such as:
- Exon splicing enhancers (ESEs) – Blocking these sites promotes exon skipping.
- Exon splicing silencers (ESSs) – Masking these regions enhances exon inclusion.
- Intronic splicing motifs – Modulating spliceosome recognition to alter splicing patterns.
Figure 1. Splice-switching oligonucleotides modulate alternative splicing. (top) Diagram of a pre-mRNA transcript with exons depicted as gray boxes and introns as lines. An intronic splicing silencer (ISS, red) and exonic splicing enhancer (ESE, green) are shown bound by a trans-acting inhibitory splicing factor protein (red oval) or stimulatory splicing factor (green oval). These SF proteins either block (−) or promote (+) splicing at splice sites bordering the surrounding exons. (left panel) An SSO that base-pairs to a splicing enhancer sequence creates a steric block to the binding of the stimulatory splicing factor to its cognate enhancer binding site. This block thereby disrupts splicing and results in exon skipping. (right panel) In contrast, an SSO that base-pairs to a splicing silencer sequence element blocks splicing silencer activity by preventing binding of a negatively acting splicing factor. Disruption of the binding of splicing inhibitory proteins to its cognate binding sequence activates splicing at the splice site that is negatively regulated by the silencer element, resulting in exon inclusion.1,2
Upon binding, SSOs interfere with the recruitment of splicing factors (e.g., SR proteins or hnRNPs), leading to modified mRNA processing [1,4]. This approach has been successfully applied in:
- Duchenne muscular dystrophy (DMD) – Exon skipping to restore the dystrophin reading frame.
- Spinal muscular atrophy (SMA) – Modifying SMN2 splicing to increase functional SMN protein.
- Cancer therapy – Altering oncogenic splicing variants (e.g., REST in neuroendocrine tumors) [5].
Therapeutic Applications of SSOs
1. Genetic Disorders
SSOs have shown promise in treating urea cycle disorders by correcting splicing defects in metabolic enzymes [6]. Recent advances in pediatric neurological disorders, including CLN3 Batten disease and USH2A-related retinitis pigmentosa, highlight their potential in neurogenetic diseases [3].
2. Oncology
In osteosarcoma, SSOs targeting aberrant INSR splicing (switching from pro-metastatic IR-A to IR-B isoform) suppress tumor progression [2]. Similarly, in neuroendocrine cancers, REST-directed SSOs restore tumor suppressor function [5].
3. Metabolic & Liver Diseases
SSOs with enhanced endosomal escape (e.g., GalNAc-conjugated SSOs) improve hepatic splice correction efficiency, offering new treatments for metabolic disorders [7].
Advantages of SSO Therapeutics
- High specificity – Precise targeting of splicing defects with minimal off-target effects.
- Durable effects – Single-dose treatments can induce long-lasting splicing modulation.
- Non-genotoxic – Unlike gene editing, SSOs do not permanently alter the genome.
Challenges & Innovations
- Delivery optimization – Chemical modifications (e.g., phosphorodiamidate morpholino oligomers, PMOs) and nanoparticle carriers enhance tissue uptake [7].
- AI/ML-guided design – Computational tools improve SSO efficacy by predicting optimal target sequences [4].
Collaborative Opportunities in SSO Development
For biotech companies, research institutions, and pharmaceutical developers aiming to advance RNA therapeutics, our team provides:
- Custom SSO design services tailored to specific splicing defects.
- AI-powered oligonucleotide sequence optimization for higher efficacy.
- Advanced delivery platform development, including GalNAc and nanoparticle-based systems.
Our expertise bridges the gap between concept and clinical translation, enabling faster, more efficient development of splice-switching oligonucleotide therapies.
Conclusion
SSOs represent a transformative therapeutic strategy for diseases with splicing defects. Ongoing advancements in delivery, AI-driven design, and clinical validation are accelerating their transition into mainstream medicine [1,4,6].
References
- Image retrieved from Figure 1 “Splice-switching oligonucleotides (SSOs) modulate alternative splicing.,” Havens, Mallory A., et al., 2016, used under [CC BY 4.0](https://creativecommons.org/licenses/by/4.0/). The original image was modified by extracting and using only part a, and the title was changed to “Splice-switching oligonucleotides modulate alternative splicing.”
- Havens, Mallory A., and Michelle L. Hastings. “Splice-switching antisense oligonucleotides as therapeutic drugs.” Nucleic acids research 44.14 (2016): 6549-6563.
- Employing splice-switching oligonucleotides and AAVrh74.U7 snRNA to target insulin receptor splicing and cancer hallmarks in osteosarcoma Molecular Therapy – Oncology 2024, DOI: 10.1016/j.omton.2024.200908
- Splice-switching antisense oligonucleotides for pediatric neurological disorders Frontiers in Molecular Neuroscience 2024, DOI: 10.3389/fnmol.2024.1412964
- Development and validation of AI/ML derived splice-switching oligonucleotides Nature Biotechnology 2024, DOI: 10.1038/s44320-024-00034-9
- Splice-switching antisense oligonucleotide controlling tumor suppressor REST is a novel therapeutic medicine for neuroendocrine cancer Molecular Therapy – Nucleic Acids 2024, DOI: 10.1016/j.omtn.2024.102250
- Developing splice-switching oligonucleotides for urea cycle disorder using an integrated diagnostic and therapeutic platform Journal of Hepatology 2025, DOI: 10.1016/j.jhep.2025.02.007
- Accelerated Endosomal Escape of Splice-Switching Oligonucleotides Enables Efficient Hepatic Splice Correction ACS Applied Materials & Interfaces 2024, DOI: 10.1021/acsami.4c19340