Project Summary Duchenne muscular dystrophy (DMD) is a severe disease affecting approximately 1 in 3500 boys, causing profound muscle weakness and degeneration over time. The average life expectancy is 26 years of age and death is typically a result of either cardiomyopathy or respiratory infection. Very limited therapeutic options exist for the treatment of these children. DMD is caused by mutations in the gene encoding the dystrophin protein. One promising approach to treatment involves exon skipping, a process in which an antisense oligonucleotide induces the mutation-containing exon to be spliced out of the final dystrophin mRNA transcript. The FDA recently provisionally approved the first and only DMD-specific therapy, Eteplirsen, which carries out its effect via exon skipping. Eteplirsen belongs to a class of antisense therapeutics known as phosphorodiamidate morpholino oligonucleotide (PMO). Although PMOs are attractive molecules to trigger exon skipping and dystrophin restoration, their clinical efficacy has been limited by poor delivery across the cell membrane and into the nucleus. Peptides have shown promise in facilitating the nuclear delivery of cargoes. However, only a limited number of peptide sequences have been explored for the delivery of PMOs and peptides are prone to proteolytic degradation in serum. Thus, I propose to develop a peptide-based delivery platform to generate new agents for the delivery of PMOs that will enhance nuclear delivery and exon skipping and improve stability in serum. One approach will be to create linear and branched chimeras of peptide sequences, in order to explore how peptide sequence and structural diversity can improve PMO delivery. Simultaneously, a second approach will be to create macrocyclic peptides with fluorine-rich linkers, as macrocycles often confer benefits in terms of stability and delivery. These two approaches will be evaluated both in a cellular green fluorescent protein assay with a reporter PMO and in skeletal muscle cells from a mouse model of DMD with a therapeutic PMO. The compounds generated in this work will be leads for next- generation conjugate DMD therapies with improved clinical efficacy. More generally, given the ease of conjugation of these peptide-based scaffolds to other cargoes of interest, I envision that this work can be readily applied to improve other therapies for congenital diseases in which the major limiting factor is intracellular delivery.