Enhanced antisense oligonucleotide delivery reveals that transcript turnover impacts apparent splicing rescue in...

Steric-blocking antisense oligonucleotides rescue myotonic dystrophy type 1 phenotypes in preclinical models and are under evaluation in clinical trials. However, the rationale for biomarker selection remains a topic of debate. Here, we show that a cyclic cell-penetrating peptide that escapes endosomes enhances muscle delivery of a phosphorodiamidate morpholino oligonucleotide designed to block pathogenic CUG repeat expansions in HSA^LR mice. A single systemic administration rescued mis-splicing and eliminated myotonia 1 week post-injection, with partial splicing rescue evident after 24 h. Interestingly, some exons showed more robust rescue than others, but the relationship between muscleblind-like (MBNL) protein concentration ([MBNL]) and percent spliced in (Ψ) did not fully explain the extent of rescue. We hypothesized that since pre-existing transcripts must be degraded to reveal the full drug effect, rates of transcript replacement might account for these discrepancies. We formulated a mathematical framework and used Bayesian inference to model how apparent Ψ lags behind nascent Ψ as a function of time; faster rates of replacement result in shorter lags. In vivo 5-ethynyl uridine labeling followed by RNA sequencing (RNA-seq) validated these predictions. Overall, we show that transcript turnover influences Ψ during periods of dynamically changing [MBNL] and recommend considering this when selecting splicing biomarkers and interpreting responses to therapeutic interventions.