Antisense oligonucleotide selection scheme for rare Duchenne muscular dystrophy mutations: Application to DMD exon 16 skipping.

Over the last decades, antisense oligonucleotides (AONs) have emerged as powerful tools to modulate alternative splicing, allowing exclusion of a specific exon from a target mRNA to bypass mutations or restore the reading frame, enabling production of a functional protein. In Duchenne muscular dystrophy (DMD), a lethal X-linked disorder affecting 1:5,000 males, four AON therapies have received accelerated approval. However, these treatments are applicable to only 27% of patients, leaving most individuals with rare mutations without therapeutic options. Here, we outline a methodology to identify relevant AONs for such populations. This approach aims to prequalify candidates and eliminate unsuitable leads before animal testing, reducing development time and costs. Following high-throughput screening, selected AONs are assessed for intravenous delivery compatibility and serum protein binding to support model selection and forecast human pharmacokinetics. As proof of concept, we applied this strategy to tricyclo-DNA AONs targeting an ultra-rare DMD exon 16 mutation. From a 59-member library, we defined a lead compound with efficient exon 16 skipping, favorable pharmacokinetic and safety profiles, able to rescue dystrophic transcriptomic and proteomic signatures. This study demonstrates the feasibility of developing a safe and effective lead compound within a reasonable time frame and cost, supporting compassionate use and future n = 1 trials.