Morpholino-Mediated Exon Inclusion for Spinal Muscular Atrophy (SMA)

The application of antisense oligonucleotides (AONs) to modify pre-messenger RNA splicing has great potential for treating genetic diseases. The strategies used to redirect splicing for therapeutic purposes involve the use of AONs complementary to splice motifs, enhancer or silencer sequences. AONs to block intronic splicing silencer motifs can efficiently augment exon 7 inclusion in the survival motor neuron 2 (SMN2) gene and have demonstrated robust therapeutic effects in both pre-clinical studies and clinical trials in spinal muscular atrophy (SMA), which has led to the approval of nusinersen. AONs with phosphoroamidate morpholino (PMO) backbone have shown target engagement with restoration of the defective protein in Duchenne muscular dystrophy (DMD) and their safety profile lead to the approval of four DMD AON drugs. PMO AONs are also effective in correcting SMN2 exon 7 splicing and rescuing SMA transgenic mice. Here we provide the details of methods that our lab has used to evaluate PMO-mediated SMN2 exon 7 inclusion in the in vivo studies conducted in SMA transgenic mice. The methods comprise mouse experiment procedures, assessment of PMOs on exon 7 inclusion at RNA levels by reverse transcription PCR and quantitative real-time PCR. In addition, we present methods for protein quantification using western blot in mouse tissues, for neuropathology assessment of skeletal muscle (e.g. muscle pathology and neuromuscular junction staining) as well as behaviour test in the SMA mice (e.g. righting reflex).