Single Antisense Oligonucleotides Correct Diverse Splicing Mutations in Hotspot Exons.

Area of Science:

• Genetics and Genomics
• Molecular Biology
• Bioinformatics

Background:

• Splicing mutations are implicated in numerous diseases but their precise impact remains incompletely understood.
• Characterizing exonic variants affecting splicing is crucial for understanding disease mechanisms and developing targeted therapies.

Purpose of the Study:

• To analyze the impact of exonic variants on RNA splicing in 71 clinically actionable genes within an asymptomatic population.
• To identify splice-disrupting mutations and their distribution across exons.
• To explore the therapeutic potential of antisense oligonucleotides (ASOs) for correcting splicing defects.

Main Methods:

• Analysis of 32,112 exonic mutations from ClinVar and Geisinger MyCode databases.
• Utilized a minigene reporter assay to experimentally validate splice-disrupting variants.
• Investigated the concentration of mutations in specific exon regions ('hotspot exons').
• Assessed the efficacy of antisense oligonucleotides (ASOs) in reverting splice-disrupting mutations.

Main Results:

• Identified 1,733 splice-disrupting mutations, with highly deleterious variants being more extreme.
• Demonstrated that splice-disrupting mutations are not uniformly distributed but are concentrated in approximately 8% of exons (hotspot exons).
• Showed that multiple splice-disrupting mutations within these hotspot exons can be corrected by single ASOs targeting flanking splice sites.

Conclusions:

• Exonic splicing mutations exhibit a non-random distribution, favoring specific 'hotspot exons'.
• This clustering supports the development of single therapeutic antisense oligonucleotides (ASOs) capable of reverting diverse splice-altering variants within a particular exon.
• Findings pave the way for novel, broadly applicable splice-switching therapies for genetic disorders.