Repeat length as a key determinant for disease severity and antisense oligonucleotide activity in myotonic dystrophy...

Myotonic dystrophy type 1 (DM1) is caused by a (CTG) _n expansion in the DMPK gene, leading to a multisystemic manifestation and broad disease presentation. Although the DM1 phenotype and onset correlate with expansion length, understanding DM1 etiology and developing effective therapies remains challenging. Here, we investigated the contribution of repeat length on aberrant splicing and response to antisense oligonucleotides (ASOs). In primary DM1 myoblasts bearing repeat lengths of 800, 1,200, or >3,000, DMPK downregulation was achieved by blocking and gapmer ASOs, though splicing correction was inefficient. Further analyses revealed profound differences in DMPK mRNA levels. To exclude such confounding effects, we generated an isogenic myoblast panel with repeats from 0 to 2,900 triplets using a repeat-targeted CRISPR/Cas9 nickase approach. This panel revealed repeat-length dependency of aberrant splicing and nuclear MBNL1 abundance. While the blocker ASO marginally induced DMPK downregulation with longer repeats, its effect on splicing correction was evident, though decreased as repeat length increased. The gapmer ASO led to substantial downregulation and essentially normalized splicing levels throughout. Our study demonstrates that repeat length is central to therapeutic effectiveness, but this correlation may be obscured by genetic background, underscoring the need to consider genotypic heterogeneity in DM1 clinical trials.