High-throughput discovery of arginine-depleted peptides enables effective antisense delivery for Duchenne muscular dystrophy.

Phosphorodiamidate morpholino oligomers (PMOs) are approved exon-skipping antisense therapeutics for Duchenne muscular dystrophy (DMD), but their clinical utility is limited by poor uptake in muscle tissue, necessitating frequent high-dose administration. Cell-penetrating peptides (CPPs) can enhance intracellular delivery of PMOs, yet conventional arginine-rich CPPs often cause dose-limiting toxicity, including renal damage, which hinders their clinical translation. To address this challenge, we developed a high-throughput, charge-based chromatographic enrichment platform capable of screening over 15,000 synthetic peptides, including sequences with noncanonical (abiotic) amino acids. This approach enabled de novo discovery of arginine-depleted CPPs with improved delivery profiles. Four lead candidates demonstrated efficient nuclear PMO delivery with ~10-fold lower in vitro toxicity compared to standard CPPs such as penetratin. The top-performing peptide, CXP1, showed robust splice-switching activity and favorable tolerability in both cellular and animal models. In dystrophic mdx mice, CXP1-PMO conjugates achieved greater exon skipping compared to PMOs conjugated to R6G at equivalent doses. Tissue levels of CXP1-PMO correlated with exon-skipping efficacy, establishing a clear pharmacokinetic-pharmacodynamic relationship. These findings highlight a mechanistically novel and translationally relevant discovery strategy, demonstrating the potential of high-throughput platforms to generate more effective CPP-based delivery vehicles for antisense therapeutics in DMD and related neuromuscular disorders.