Preemptive SOD1 Silencing via Neonatal Intramuscular AAV Therapy Modifies Disease Trajectory in an ALS Mouse Model

Amyotrophic lateral sclerosis (ALS) is a progressive and fatal neurodegenerative disorder with limited therapeutic options. Mutations in the gene encoding superoxide dismutase 1 (SOD1) represent a major genetic cause of familial ALS, driving motor neuron degeneration through toxic gain-of-function mechanisms. Although gene silencing approaches targeting SOD1 show substantial therapeutic potential, their clinical translation remains restricted by suboptimal delivery to the spinal motor neurons and safety concerns linked to conventional viral vectors. This study presents a minimally invasive gene therapy strategy that combines the retrograde transport capability of rAAV2-retro with the safety of an artificial microRNA (miRNA) to achieve pan-spinal SOD1 silencing. A single intramuscular injection of rAAV2-retro-miRNA into neonatal SOD1G93A mice resulted in widespread transduction of spinal motor neurons, significant reduction of mutant SOD1 protein, and multifaceted therapeutic benefits. Treated mice exhibited delayed disease onset, extended lifespan, preserved motor function, reduced neuroinflammation, and protection of neuromuscular junctions and spinal motor neurons. Importantly, the artificial miRNA construct demonstrated a superior safety profile relative to short hairpin RNA (shRNA)-based constructs, which induced marked toxicity and lethality in wild-type mice. These findings establish neonatal intramuscular delivery of rAAV2-retro-miRNA as a safe, efficient, and clinically translatable strategy for preemptive intervention in SOD1-mediated ALS, offering broader applicability to other motor neuron diseases.