The Need for Variant-Specific Splicing Assessment in Developing Antisense Oligonucleotide Therapies for Duchenne Muscular Dystrophy: Lessons from the DMD c.4675−2A>G Variant

Duchenne muscular dystrophy is caused by pathogenic variants in the DMD gene. Splice-site variants can generate aberrant transcripts whose consequences cannot always be predicted by the classical reading-frame rule. Here, we characterized the c.4675−2A>G variant at the exon 34 splice acceptor site for the development of an exon-skipping therapy. A minigene analysis demonstrated that c.4675−2A>G activated a cryptic splice acceptor within exon 34 rather than inducing skipping of the entire exon, resulting in deletion of the first 7 nucleotides, a frameshift, and a premature termination codon. To restore the reading frame by promoting skipping of the entire exon 34, we used antisense oligonucleotides (ASOs) targeting distinct splice-regulatory regions. Among the ASO treatments tested, including individual ASOs and ASO combinations, A1&E4&D1 produced the highest exon exclusion and consistently promoted exon 34 skipping across the examined −2A mutant genotypes. A1&E4&D1 consists of ASOs A1, E4, and D1. A1 targeted the cryptic 3′ splice acceptor region, E4 targeted a predicted exonic splicing enhancer region, and D1 targeted the 5′ splice donor region. These findings illustrate a stepwise, variant-directed strategy in which characterization of aberrant splicing guides the design of a tailor-made ASO intervention.