Specificity and exon target space of splicing modifying compounds
- Felina Lenkeit
- Judith Knehr
- Marc Altorfer
- Andrea Byrnes
- Wenjing Li
- Jack Hsiao
- Connie Wu
- Priti Gaitonde
- Philip R. Skaanderup
- Steve Mullin
- Elizaveta Solovyeva
- Michal Pikusa
- Andrew T. Krueger
- Johannes Ottl
- Caroline Gubser Keller
- Christian Kolter
- Ulrike Naumann
- Philipp Ottis
- Alejandro Reyes
2026-07-04
Modulation of splicing is an established therapeutic strategy with clinical applications and potential to target specific exons to influence gene expression. Small-molecule splicing modifiers such as Risdiplam and Branaplam induce inclusion of exons typically skipped due to weak $${5}^{{\prime} }$$ 5 ′ splice sites. Risdiplam preferentially induces exons with an N −3 G −2 A −1 sequence at the $${3}^{{\prime} }$$ 3 ′ exon end, whereas Branaplam favors A −3 G −2 A −1 -ending exons. However, determinants of specificity remain unclear, as many motif-matching exons are not induced. Here, we investigate the molecular basis of splicing-modulator specificity. Using biochemical assays, transcriptome analyses, and genetic perturbations, we identify sequence-dependent features that determine exon responsiveness to splicing-modulator induction. We further demonstrate that specificity can be reprogrammed through manipulation of U1 snRNA. These findings refine the determinants of splicing-modulator target space and may support identification of additional target exons and compounds.