Fixing Faulty Splicing

A study by researchers at the University of Southampton, UK, has explored how antisense oligonucleotides (ASOs) can be custom designed to correct genetic mutations that disrupt RNA splicing. The research focused on tailoring ASOs to specific splicing errors, offering a personalized to treat rare diseases caused by these mutations.

Many rare genetic disorders arise from mutations that interfere with RNA splicing, the process by which genes are edited to create functional proteins. When splicing is disrupted, it can result in incomplete or faulty proteins, leading to severe disease. Many rare disease patients are children and 30 percent of affected individuals will not live to see their fifth birthday.

ASOs can directly target specific RNAs and are already used in approved drugs to modulate splicing. However, splicing errors caused by genetic mutations vary significantly in their nature and location. Each mutation requires a precisely designed ASO, but ASO screening can be difficult and time consuming.

Using minigene models – small, lab-engineered gene segments – as an assay, the team designed custom ASOs based on the mutation location and surrounding sequences that could block incorrect splicing and restore normal RNA processing. They investigated five different types of splicing errors found in patients with rare diseases, including mutations that create faulty splice sites, introduce unwanted RNA segments, or interfere with normal splicing signals.

The team tested three different types of oligonucleotides: morpholinos, Vivo-morpholinos (a modified version for better delivery), and 2′-O-methoxyethyl phosphorothioate (2′-MOE-PS). ASO effectiveness varied depending on the mutation type and chemical composition. In some cases, Vivo-morpholinos were the most effective, while in others, 2′-MOE-PS provided the best correction. Certain ASO designs restored normal RNA processing completely, while others had little effect, highlighting the importance of careful optimization.

The study results show that not all splicing mutations can be corrected with ASOs, particularly those directly affecting essential splicing elements. However, for mutations located away from key splice sites, ASOs could be a fast, efficient, and personalized therapeutic option. The authors write, “In our previous study, where we analyzed aberrant splicing in patient blood RNA, we found that approximately 27% of all splice site-affecting mutations were alternative donor site splicing mutations, making them potential targets for antisense oligonucleotides.”

With newborn genome screening becoming more widespread, the ability to quickly match "actionable" mutations to pre-tested ASOs could help alter the treatment landscape for rare diseases. The study also suggests that a rapid ASO screening pipeline, using minigene models, could accelerate the development of personalized RNA therapies. The authors say, “Our results highlight the potential of single, tailored ASO designs to effectively rescue aberrant splicing across various target groups. This finding holds significant promise for the development of rapid, efficient, and scalable preclinical therapeutic strategies, addressing the pressing need for rare disease therapeutics.”