A Novel Morpholino Oligomer Targeting ISS-N1 Improves Rescue of Severe Spinal Muscular Atrophy Transgenic Mice

Abstract

In the search for the most efficacious antisense oligonucleotides (AOs) aimed at inducing SMN2 exon 7 inclusion, we systematically assessed three AOs, PMO25 (-10, -34), PMO18 (-10, -27), and PMO20 (-10, -29), complementary to the SMN2 intron 7 splicing silencer (ISS-N1). PMO25 was the most efficacious in augmenting exon 7 inclusion in vitro in spinal muscular atrophy (SMA) patient fibroblasts and in vitro splicing assays. PMO25 and PMO18 were compared further in a mouse model of severe SMA. After a single intracerebroventricular (ICV) injection in neonatal mice, PMO25 increased the life span of severe SMA mice up to 30-fold, with average survival greater by 3-fold compared with PMO18 at a dose of 20 lg/g and 2-fold at 40 lg/g. Exon 7 inclusion was increased in the CNS but not in peripheral tissues. Systemic delivery of PMO25 at birth achieved a similar outcome and produced increased exon 7 inclusion both in the CNS and peripherally. Systemic administration of a 10-lg/g concentration of PMO25 conjugated to an octaguanidine dendrimer (VMO25) increased the life span only 2-fold in neonatal type I SMA mice, although it 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 the Vivo-Morpholino and have potential for future SMA clinical applications.

Motor neuron cell-nonautonomous rescue of spinal muscular atrophy phenotypes in mild and severe transgenic mouse models

“…Porensky et al (2012) reported no statistically significant peripheral effect on SMN2 splicing after ICV injection of the middle dose; however, this may be attributable to the small sample size (n = 2), as an approximately twofold increase in SMN2 full-length mRNA in the liver and heart was actually observed. A more recent study using 25mer PMO (PMO25) confirmed that systemic delivery at high doses robustly rescues severe SMA mice (Zhou et al 2013). Compared with our previous ICV rescue data, the PMO ASOs appeared to result in stronger rescue (at higher doses) through ICV delivery Passini et al 2011).…”

Alternatively spliced mu opioid receptor C termini impact the diverse actions of morphine

“…This oligo markedly reduced exon 3/exon 7 splicing and expression of exon 7-associated variants and attenuated morphine tolerance without affecting morphine dependence, mimicking the effect of truncation of exon 7-associated C-terminal tails in mE7M-B6 homozygous mice. Antisense morpholino oligos have been successfully used to correct aberrant splicing in a growing number of animal models of human diseases, such as spinal muscular atrophy (74)(75)(76), Duchenne muscular dystrophy (77,78), and β-thalassemia (79,80). Our results provide another example where an in vivo antisense morpholino approach might be of clinical value one day.…”

Pharmacology of a Central Nervous System Delivered 2′-O-Methoxyethyl–Modified Survival of Motor Neuron Splicing Oligonucleotide in Mice and Nonhuman Primates

“…At this time point, we administered 400 mg of the complementary oligonucleotide decoy, a443, by i.c.v. bolus injection and assessed the effects on SMN2 splicing Passini et al, 2011), 29-O-methyl (29-OMe) (Williams et al, 2009), and phosphorodiamidate morpholino (PMO) chemistries (Porensky et al, 2012;Mitrpant et al, 2013;Zhou et al, 2013) (Fig. 3A), which drive SMN2 exon 7 inclusion, have been tested in mice with severe SMA by central administration within the first postnatal day.…”

“…These include small molecules to augment SMN2 transcription, correct SMN2 splicing, cause translational readthrough, and stabilize SMN2 transcripts (Lunn and Wang, 2008); SMN gene delivery to replace SMN protein (Foust et al, 2010;Passini et al, 2010); antisense oligonucleotide (ASO)-based approaches to correct SMN2 splicing (Lim and Hertel, 2001;Miyajima et al, 2002;Cartegni and Krainer, 2003;Skordis et al, 2003;Singh et al, 2006;Hua et al, 2007Hua et al, , 2008Hua et al, , 2011Dickson et al, 2008;Williams et al, 2009;Osman et al, 2012;Porensky et al, 2012;Mitrpant et al, 2013;Zhou et al, 2013); and antisense-producing vector-based strategies (Geib and Hertel, 2009;Meyer et al, 2009), including trans-splicing (Coady et al, 2007;Coady and Lorson, 2010). Some of these strategies have not yet been tested in animal models of SMA, but others have already been shown to be beneficial (Bebee et al, 2012).…”