Determination of DLC1 isoform 1 (DLC1-i1) as a gene therapy for the treatment of spinal muscular atrophy

Abstract

Spinal muscular atrophy (SMA) is a motor neuron (MN) disease caused by loss of the ubiquitously expressed survival motor neuron (SMN) spliceosome protein, resulting in selective degeneration of spinal motor neurons (MNs) but the mechanism underlying the specific loss of MNs remains unknown. A previous report showed that Deleted in Liver Cancer 1 (DLC1) is the most down-regulated gene in MNs derived from a SMA patient but its roles in MN development and SMA pathogenesis remain to be elucidated. Here, we found a specific increase in the expression level of DLC1 isoform 1 (DLC1-i1) in human induced pluripotent stem cells (hiPSCs)-derived MNs compared to other isoforms. Knockdown of DLC1-i1 in MN progenitors resulted in reduced MN formation, axonal outgrowth, synapse number and cell survival, whereas overexpression of DLC1-i1 promoted MN differentiation with extensive neurite projections. Consistent with previous findings, we detected downregulated expression of DLC1-i1 in MNs derived from SMA patients’ urine-derived iPSCs compared with healthy controls, likely due to intron retention of DLC1-i1 pre-mRNA. In addition, SMA patients-derived neuromuscular organoids (NMOs) showed reduced MNs and skeletal muscles generation, resulting in defective formation of neuromuscular junctions. Overexpression of DLC1-i1 partly restored both neural and muscle fibre formation in SMA NMOs. Similarly, DLC1-i1 expression was markedly reduced in the lumber spinal cord of SMA mice compared with that of wild-type, implying conservation of dysregulated DLC1-i1 expression in both the human SMA MNs and the spinal neurons of SMA mice. Injection of AAV9-SMN into the brain of SMA mice not only restored their weight and locomotor function but also DLC1-i1 expression level in the lumber spinal cord. Importantly, SMA mice treated with AAV9-DLC1-i1 together with AAV9-SMN resulted in synergistic improvement of their motor activity and lifespan compared with single treatment. Altogether, these findings demonstrate that MN-specific DLC1-i1 functions downstream of SMN to regulate MN maturation, survival, and neuromuscular junctions formation, revealing a new therapeutic target for the treatment of SMA.