There are promising treatment approaches emerging for Duchenne muscular dystrophy (DMD): exon skipping with peptide-conjugated phosphorodiamidate morpholino oligomers (PPMOs) that restore the gene open reading frame, enabling translation of shortened, functional dystrophin proteins; and adeno-associated virus (AAV) vector gene therapy (GT), which systemically delivers a transgene encoding a rationally designed dystrophin protein that retains key functional domains of the wild-type (WT) dystrophin to muscles. Because these modalities produce dystrophin by distinct mechanisms, their potential use in sequential administration is an area of high interest. Using the mouse model of DMD, the safety of sequential PPMO and GT administration and its impact on dystrophin expression was investigated. Animals received 3 doses of PPMO RC-1001 (weeks 8, 12, 16), followed by a single dose of AAVrh74.MHCK7.Mouse-micro-dystrophin GT (week 20), and were euthanized at week 26. Treatment arms (n=8 each) included WT, saline control mdx, PPMO (10 mg/kg; 40 mg/kg), 1.33 × 10¹⁴ vg/kg AAV GT, and PPMO + AAV GT (PPMO 10 mg/kg + AAV GT 1.33 × 10¹⁴ vg/kg; PPMO 40 mg/kg + AAV GT 1.33 × 10¹⁴ vg/kg). Outcomes included serum chemistries (alanine aminotransferase, aspartate aminotransferase, creatinine, blood urea nitrogen, and bilirubin) and dystrophin expression (western blot [WB] and immunofluorescence [IF]). No treatment-related adverse events were observed, including abnormal histopathology. WB/IF evaluation showed that dystrophin expression and localization at the sarcolemma were not adversely impacted by sequential treatment. These preclinical results support the safety of sequential administration and dystrophin expression that is consistent with individual treatment, suggesting that patients may be able to receive continuous exon-skipping therapy prior to GT.