Neuronal targeted AAV Micro-Dystrophin restores neurobehavioural co-morbidities, grip strength and motor coordination in mdx52 mouse model of Duchenne Muscular Dystrophy

Duchenne muscular dystrophy (DMD) is a X-linked disorder caused by mutations in the DMD gene, which disrupts production of multiple isoforms of dystrophin in multiple organs namely muscle, heart and brain. While progressive muscle disease and cardiomyopathy are the hallmarks of DMD, over 40% of individuals also experience significant neurobehavioral comorbidities, including autism spectrum disorder (ASD), attention deficit hyperactivity disorder (ADHD), obsessive compulsive disorder (OCD) and intellectual disability. These deficits are linked to the loss of brain isoforms and approximately 90% of DMD individuals have loss of either Dp427 or both Dp427 and Dp140 in the brain. We studied the mdx52 mouse model, which lacks these isoforms and exhibits severe fear and anxiety-like behaviours, suitable to evaluate the therapeutic efficacy on neuro-comorbidities after delivering a neuronal-targeted adeno-associated virus (AAV) micro-dystrophin ({micro}Dys) therapy. We compared two delivery routes intravenous (IV) and intracerebroventricular (ICV) in neonatal mdx52 male mice to assess impact on an extensive range of neurobehavioural aspects including emotional reactivity, neurocognitive, OCD and motor coordination deficits. While both routes successfully reduced emotional reactivity and anxiety-related behaviours, IV delivery emerged as the superior therapeutic strategy addressing a more comprehensive spectrum of DMD related brain co-morbidities. Critically, significant improvements in cognitive deficits and OCD-like behaviours were achieved only through IV delivery. This was associated with a widespread lower transduction pattern across the brain, including hindbrain and cerebellum, which were less effectively targeted by ICV injection, although forebrain transduction with ICV delivery was higher. Brain {micro}Dys expression successfully restored dystrophin interactors dystroglycan, syntrophin and pre- and post-synaptic functional interactors VGLUT1, gephyrin, GABAAR with both delivery methods. These results demonstrate that while ICV gene therapy results in improved emotional reactivity and anxiety-related behaviour in the mdx52, only the systemic, neuronal-targeted gene therapy efficiently transduced the central nervous system restoring neuronal synaptic functional complexes of both Dp427 and 140 isoforms and simultaneously restored peripheral NMJ dystrophin deficiency. Beyond cognitive restoration, while both routes improved aspects of gait on CatWalk XT, only IV delivery significantly enhanced motor coordination on the Beam walk and, unexpectedly, normalised grip strength. This was specifically linked to the selective expression of {micro}Dys at neuromuscular junctions (NMJs), which corrected post-synaptic electrophysiological dysfunction of mdx52 mice. Our findings establish a significant foundation for incorporating brain-directed strategies into the future therapeutic approaches for DMD, offering a holistic approach to treating DMD as a multisystemic disease.