Duchenne muscular dystrophy (DMD) is a fatal genetic disorder associated with mutations in the dystrophin gene that lead to expression of a non-functional protein, resulting in progressive loss of muscle function. Current treatments, such as gene therapies that express a synthetic micro-dystrophin protein missing many essential protein domains, have shown limited improvement to disease prognosis and have not met critical functional endpoints. The long-term efficacy of micro-dystrophin gene therapies remains uncertain, particularly in pediatric patients where muscle turnover and growth can dilute transgene expression. Alternatively, a gene editing approach could allow for lifelong benefits of muscle retention and function by permanently correcting the root cause of the disease. PBGENE-DMD is a gene editing therapy that delivers two ARCUS nucleases using a single AAV to excise exons 45-55 in the dystrophin gene and restore expression of a functional protein. This functional dystrophin is found in a subset of Becker Muscular Dystrophy patients who express mild symptoms or are asymptomatic. Up to 60% of DMD patients have mutations in the 45-55 mutation hotspot targeted by PBGENE-DMD, allowing for treatment of a broad patient population. Here, we demonstrate long-term efficacy improvement and safety after PBGENE-DMD treatment in two studies utilizing a humanized DMD mouse model. In the first study evaluating PBGENE-DMD efficacy, we observed continual increases in dystrophin protein and dystrophin positive cells across skeletal and cardiac muscle tissues over 9 months. Functional analysis of muscle force output (MFO) showed significant improvement over 9 months, reaching 92% of the MFO of non-diseased mice. Additionally, we conducted a Good Laboratory Practice (GLP) toxicology and pharmacokinetic mouse study and observed improvements in clinical chemistry and muscle injury pathology after treatment with PBGENE-DMD. These nonclinical studies support the potential long-term efficacy of PBGENE-DMD and demonstrate an acceptable safety and tolerability profile for clinical trials.