Improved Antisense Oligonucleotide Design to Suppress Aberrant SMN2 Gene Transcript Processing: Towards a Treatment for Spinal Muscular Atrophy

Chalermchai Mitrpant,Francesco Muntoni,Arthur H M Burghes,Paul Porensky,Steve D Wilton,Loren Price,Sue Fletcher,Haiyan Zhou,Emanuele Buratti

doi:10.1371/journal.pone.0062114

Abstract

Spinal muscular atrophy (SMA) is caused by loss of the Survival Motor Neuron 1 (SMN1) gene, resulting in reduced SMN protein. Humans possess the additional SMN2 gene (or genes) that does produce low level of full length SMN, but cannot adequately compensate for loss of SMN1 due to aberrant splicing. The majority of SMN2 gene transcripts lack exon 7 and the resultant SMNΔ7 mRNA is translated into an unstable and non-functional protein. Splice intervention therapies to promote exon 7 retention and increase amounts of full-length SMN2 transcript offer great potential as a treatment for SMA patients. Several splice silencing motifs in SMN2 have been identified as potential targets for antisense oligonucleotide mediated splice modification. A strong splice silencer is located downstream of exon 7 in SMN2 intron 7. Antisense oligonucleotides targeting this motif promoted SMN2 exon 7 retention in the mature SMN2 transcripts, with increased SMN expression detected in SMA fibroblasts. We report here systematic optimisation of phosphorodiamidate morpholino oligonucleotides (PMO) that promote exon 7 retention to levels that rescued the phenotype in a severe mouse model of SMA after intracerebroventricular delivery. Furthermore, the PMO gives the longest survival reported to date after a single dosing by ICV.

Highlights

Spinal muscular atrophy (SMA), the second most common autosomal recessive disorder in Caucasians, is the leading genetic cause of death in children under the age of 2 years [1]
Fourteen different phosphorodiamidate morpholino oligonucleotides (PMO) varying in size of 20, 22 and 25 mers were designed to anneal to or near ISS-N1 (-10-25) (Table 1 and Figure S1) and were transfected into fibroblasts derived from an SMA Type I patient using PMO:Leash with lipofection delivery system (Figure 1, Figure 2, Figure 3, Figure 4, Figure 5)
Of the five PMO20mers that annealed between intronic bases 8 to 35 downstream of exon 7, PMO(-10-29) was the most efficient at inducing the full length SMN2 transcript retaining exon 7 (Figure 1)

Summary

Introduction

Spinal muscular atrophy (SMA), the second most common autosomal recessive disorder in Caucasians, is the leading genetic cause of death in children under the age of 2 years [1]. SMA is caused most commonly by loss of the SMN1 gene, resulting in substantial reductions in levels of functional SMN protein. Four groups have produced scAAV9-SMN and reported a remarkable correction of the SMA phenotype with the highest titer virus resulting in mice surviving over 400 days [10,11,12,13]. These studies demonstrated that replacement of SMN, at least early, is an effective therapy; certainly difficulties remain in terms of sufficient vector production if applied to older SMA patients. The scAAV9 does cross the BBB even in older animals in monkeys and can give efficient transduction of motor neurons when introduced intrathecally, which reduces the amount of virus required [14]

Methods

Results

Conclusion