OPTIMIZATION OF AAV9 GENE THERAPY FOR SPINAL MUSCULAR ATROPHY WITH RESPIRATORY DISTRESS TYPE 1 USING IN VIVO DISEASE MODELS

Spinal Muscular Atrophy with Respiratory Distress type 1 (SMARD1) is a rare autosomal recessive motoneuron disease with infantile onset, with an estimated incidence of 1/100’000. It is caused by mutations in the immunoglobulin mu-binding protein 2 (IGHMBP2) gene, which lead to a deficient amount of the encoded protein. The main clinical symptoms are distal muscular atrophy associated with diaphragmatic palsy. Nowadays no therapies are approved. In this work, we compared the efficiency of two adeno‐associated virus serotype 9 (AAV9)-IGHMBP2 vectors, carrying different promoters, by administering them intracerebroventricularly in SMARD1 mice model (nmd), during the presymptomatic phase of the disease at postnatal day 1. This comparison allowed us to determine which is the best construct to be translated to the clinic. Then, with the aim to precisely define the therapeutic window, we evaluated AAV9-based treatment in already symptomatic mice at P7 by systemic subcutaneous injection. Expression analysis at P20 on mice treated at the presymptomatic stage demonstrated a significant increase in the IGHMBP2 protein expression level compared to the control. Treatments also resulted in an extended survival time, higher body weight, and improvement in motor behaviors. Histopathological analysis showed in muscles increased innervation of the neuromuscular junctions and enlargement of fibers diameter and, in spinal cords, increased motoneurons number with reduced gliosis and astrogliosis. We also found a restoration of the IGF1 axis, altered in nmd mice. To support the translatability of the therapy, we confirmed the lack of a significant alteration of the major toxicity biomarkers after the treatment and the rescue of the one pathologically altered. Long-term effects were also evaluated at P200 demonstrating differences between the two constructs in maintaining the motoneurons number and mediating cardiac fibrosis amelioration. Results of similar analysis performed on delayed treated mice cohort showed equivalent results, demonstrating that also a treatment in the symptomatic phase could partially rescue the SMARD1 phenotype. Our presymptomatic treatment results lay the foundation for the launch of the ongoing gene therapy-mediated phase I/IIa clinical trial for IGHMBP2-related disease, a crucial step forward in the authorization of the first therapy for SMARD1 pathology, which is up to now treated only with palliative and supportive care. Good results were obtained also delaying the treatment in the early symptomatic phase, but further studies are needed to assess the therapeutic window of this extremely promising approach.