mCherry on Top: A Positive Read-Out Cellular Platform for Screening DMD Exon Skipping Xenopeptide-PMO Conjugates.

Phosphorodiamidate morpholino oligomers (PMOs) are a special type of antisense oligonucleotides (ASOs) that can be used as therapeutic modulators of pre-mRNA splicing. Application of nucleic-acid-based therapeutics generally requires suitable delivery systems to enable efficient transport to intended tissues and intracellular targets. To identify potent formulations of PMOs, we established a new screening platform based on mdx exon 23 skipping. Here, a new positive read-out system (mCherry-DMDEx23) is presented that is sensitive toward the PMO(Ex23) sequence mediating DMD exon 23 skipping and, in this model, functional mCherry expression. After establishment of the reporter system in HeLa cells, a set of amphiphilic, ionizable xenopeptides (XPs) was screened in order to identify potent carriers for PMO delivery. The identified best-performing PMO formulation with high splice-switching activity at nanomolar concentrations was then translated to trials, where exon 23 skipping in different organs of healthy BALB/c mice was confirmed. The predesigned - workflow enables evaluation of PMO(Ex23) carriers without change of the PMO sequence and formulation composition. Furthermore, the identified PMO-XP conjugate formulation was found to induce highly potent exon skipping and redistributed PMO activity in different organs .