Although recent regulatory approval of splice-switching oligonucleotides (SSOs) for the treatment of Duchenne muscular dystrophy (DMD) has been an advance for the field, current SSO chemistries have shown limited clinical benefit due to poor pharmacology. There also remains substantial unmet clinical need for approaches that reach cardiac and respiratory muscles, where restoration of dystrophin may be essential to improve survival. To overcome limitations of existing technologies, we engineered stereopure oligonucleotides with phosphorothioate (PS), phosphodiester (PO), and phosphoryl guanidine-containing (PN) backbones. In survival studies, bi-weekly doses of the PS/PO/PN SSO prevented premature death and improved median survival from 49 days to at least 280 days in the double utrophin/dystrophin knockout mouse, a model with an aggressive dystrophic phenotype. These profound survival benefits were achieved at doses anticipated to be clinically relevant. Importantly, the PS/PO/PN SSO increased dystrophin levels across several muscle types (skeletal, respiratory, and cardiac) and improved both respiratory and skeletal muscle function. Treatment also corresponded with decreased serum biomarkers of muscle damage and improved weight gain. These data demonstrate that chemical optimization can profoundly impact oligonucleotide pharmacology and highlight the therapeutic potential of stereopure PN-containing SSOs in DMD. Further, these data help to support the advancement of WVE-N531, an investigational compound currently in clinical testing (NCT04906460) that incorporates this technology, for the treatment of patients with DMD amenable to exon 53 skipping.