21 weeks of voluntary exercise largely preserves diaphragm gene-expression improvements after AAV therapy in mdx mice
Background: Examining exercise after gene therapy
In mdx mice, a model of Duchenne muscular dystrophy (DMD), improvements in diaphragm gene-expression patterns following AAV microdystrophin gene therapy were largely maintained with 21 weeks of voluntary exercise. These findings do not establish exercise safety in patients or complete recovery of muscle function; they describe transcriptomic changes observed under specific animal-experiment conditions.
In DMD, dystrophin deficiency makes muscle-cell membranes vulnerable to mechanical stress, and repeated cycles of injury and regeneration sustain inflammation. Delivering microdystrophin, a shortened form of dystrophin, using an adeno-associated virus (AAV) vector aims to lessen the decline in muscle function. This study asked whether molecular improvements persist when physical activity accompanies treatment.
The researchers analyzed diaphragm tissue collected in an earlier animal experiment. They selected the diaphragm because it is severely affected in mdx mice and shows features resembling muscle pathology in people with DMD.
Key findings: No significant exercise-associated difference in 723 of 774 genes with improved expression
Seven-week-old mice were assigned to five groups: sedentary wild-type, exercising wild-type, untreated sedentary mdx, treatment-only mdx, and treatment-plus-exercise mdx. Treated mice received an AAV9 microdystrophin vector by tail-vein injection at a dose of 2×10¹⁴ vector genomes per kg of body weight. Exercise groups ran voluntarily on wheels for 21 weeks; sedentary groups were given locked wheels. RNA sequencing (RNA-seq) included eight mice per group, except for the untreated mdx and exercising wild-type groups, which each included seven.
Comparison of untreated sedentary mdx mice with sedentary wild-type mice identified differential expression in 2,881 genes. These genes had expression levels greater than twice or less than half the wild-type level, with an adjusted p-value below 0.05. They were associated with metabolic and muscle-function processes as well as inflammation. In the treatment-only group, expression of 774 of these genes improved toward wild-type levels.
When the treatment-plus-exercise group was compared with the treatment-only group, 723 of the 774 genes with improved expression, or 93.41%, did not meet the study’s differential-expression criteria. This does not mean that all of them had returned to normal levels. Of these 723 genes, the researchers classified 104 as fully rescued and 619 as partially rescued. The remaining 51 of the 774 genes were differentially expressed between the treatment-plus-exercise and treatment-only groups.
Full rescue required no differential expression in either of two comparisons—treatment-only versus sedentary wild-type, and treatment-plus-exercise versus exercising wild-type—together with differential expression between the treatment-plus-exercise and untreated mdx groups. This, too, is a statistical classification defined by the study. Expression of inflammation-related genes Tlr2, Il6, and Il1b also improved toward wild-type levels after treatment, with no differential expression detected when exercise was added. Pathway analysis pointed to attenuation of inflammation-related signaling, but this was a prediction based on expression data.
Meaning and outlook: Distinguishing transcriptomic maintenance from exercise prescriptions
These findings provide preclinical evidence that treatment-associated transcriptomic improvements were largely maintained under specific voluntary-exercise conditions. The absence of detected differential expression alone cannot establish equivalence between the two conditions or confirm prevention of tissue damage. No diaphragm tissue was available for additional histopathological analyses to support the RNA-seq results.
Functional effects were not uniform. In an earlier experiment summarized in this paper, adding exercise improved whole-body endurance, but diaphragm force and power were lower than with treatment alone. The researchers interpreted this decrease as related to changes in muscle-fiber properties associated with endurance exercise. This is why maintained gene-expression improvements should be distinguished from improved function in every muscle.
This study alone cannot determine appropriate exercise for people with DMD who have received treatment. Further research should assess differences by exercise type, intensity, duration, and frequency, as well as disease status and age. The study was published in Molecular Therapy Advances; detailed results and methods are available in the full paper.
Source: Skeletal Muscle & Gene Therapy Review, May 2026.
Summary: Utilizing transcriptomic analysis of the diaphragm in mdx mice, this study elucidates the molecular impact of long-term voluntary exercise following AAV-microdystrophin gene therapy. RNA sequencing revealed that AAV treatment rescued 774 dysregulated genes associated with inflammatory and fibrotic pathways. Crucially, 93% of these rescued genes maintained their normalized expression profiles despite 21 weeks of continuous physical activity, proving that mechanically induced stress does not abrogate the therapeutic efficacy of AAV-mediated gene transfer in dystrophic muscle.
This study provides a comparative framework for follow-up preclinical experiments assessing physical activity after gene therapy. For example, researchers could compare inflammation-related gene expression in treatment-only and treatment-plus-exercise groups while measuring whole-body endurance, diaphragm force and power, and tissue damage. Endurance may improve even as force in a specific muscle declines, so these outcomes should be interpreted separately. The 93.41% figure describes a comparison of expression among genes that improved under the conditions used here. It cannot be applied directly as a safety standard for exercise in patients or as evidence of sustained efficacy of other AAV therapies. Evaluating clinical application will require further functional and tissue-level validation across exercise conditions.