Preclinical evaluation of AAV9-coSMN1 gene therapy for spinal muscular atrophy: efficacy and safety in mouse models and non-human primates.

BACKGROUND: Spinal muscular atrophy (SMA) is a severe neuromuscular disorder caused by the loss of motor neurons in the spinal cord. Our team has initiated clinical trials using adeno-associated virus serotype 9 (AAV9) vectors carrying a codon-optimized human SMN1 (coSMN1) gene, delivered via intrathecal (IT) injection. Here, we present the preclinical research that laid the groundwork for these trials, offering comprehensive data on the efficacy and safety of AAV9-coSMN1 in both murine models and non-human primates. MATERIAL AND METHOD: We developed a codon-optimized hSMN1 expression cassette and analyzed SMN protein levels using Western blot and immunofluorescence. Taiwanese SMA-like mouse model was employed to assess tail length preservation, as well as to examine motor neuron and skeletal muscle pathological phenotypes through immunofluorescence and histopathological staining. Serum biomarkers in both mice and cynomolgus monkeys were measured using a blood chemistry analyzer. The in-vivo biodistribution of AAV9-coSMN1 and toxicological profile were investigated through quantitative Polymerase Chain Reaction(qPCR) and histopathological staining. RESULTS: Codon optimization of hSMN1 led to enhanced gene expression and increased SMN protein levels in vitro. AAV9-coSMN1 demonstrated significant therapeutic efficacy in a Type 3 SMA mouse model, effectively rescuing motor neurons, preserving tail integrity, and improving skeletal muscle histopathology. In vivo studies, both mice and cynomolgus monkeys revealed widespread CNS distribution following a single intracerebroventricular or intrathecal injection, with no observed toxic inflammatory responses in the dorsal root ganglia. Peripheral organs also showed detectable levels of the vector gene, indicating effective systemic distribution. CONCLUSION: The preclinical evaluation confirms that AAV9-coSMN1 is a safe and effective therapeutic candidate for SMA, with potential applicability across various phenotypes. The study provides critical data supporting its advancement to clinical trials, underscoring its promise for broader neurological applications.