A Novel Morpholino Oligomer Targeting ISS-N1 Improves Rescue of Severe SMA Transgenic Mice

In the search of the most efficacious antisense oligonucleotides (AO) aimed at inducing SMN2 exon 7 inclusion, we systematically assessed three AOs, PMO25 (-10, -34), PMO18 (-10, -27) and PMO20 (-10, -29), complementary to SMN2 intron 7 splicing silencer (ISS-N1). PMO25 was the most efficacious in augmenting exon7 inclusion in vitro in spinal muscular atrophy (SMA) patients' fibroblasts and in vitro splicing assays. PMO25 and PMO18 were compared further using a severe SMA mouse model. After a single intra cerebral ventricular (ICV) injection in neonatal mice, PMO25 increased the lifespan of severe SMA mice up to thirty-fold, with an average survival greater by three-fold than PMO18 at a dose of 20 µg/g and 2-fold at 40 µg/g. Exon7 inclusion was increased in the central nervous system (CNS) but not in peripheral tissues. Systemic delivery of PMO25 at birth achieved a similar outcome and produced increased exon7 inclusion both in the CNS and peripherally. Systemic administration of 10 µg/g of PMO25 conjugated to an octa-guanidine dendrimer (VMO25) increased the lifespan only two-fold in neonatal type I SMA mice, although prevented tail necrosis in mild SMA mice. Higher doses and ICV injection of VMO25 were associated with toxicity. We conclude that (1) the 25-mer AO is more efficient than the 18-mer and 20-mer in modifying SMN2 splicing in vitro; (2) it is more efficient in prolonging survival in SMA mice and (3) naked Morpholino oligomers are more efficient and safer than vivo-Morpholinos and have potential for future SMA clinical applications.