Improved antisense oligonucleotide design to suppress aberrant SMN2 gene transcript processing: towards a treatment for spinal muscular atrophy

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.

Figure 1. a) Splicing pattern of SMN transcripts in type I SMA patient cells transfected with a panel of 20-mer PMOs, unrelated negative control (Sham PMO) using lipofectin or untransfected fibroblasts (Untreated).

b) The lower panel shows the average of percentage of SMN2 exon 7 inclusion, as determined by densitometric analysis of RT-PCR gel images.

Figure 2. a) Splicing pattern of SMN transcript in type I SMA patient cells transfected with PMOs or unrelated negative control (Sham PMO) using lipofectin to compare the efficiency of PMOs of different length.

b) The lower panel shows the average percentage of SMN2 exon inclusion as determined by densitometric analysis of RT-PCR gel images.

Figure 3. a) Splicing pattern of SMN transcript in type I SMA patient cells transfected with a panel of 22-mer PMOs or unrelated negative control (Sham PMO) using lipofectin.

b) The lower panel shows the average percentage of SMN2 exon inclusion as determined by densitometric analysis of RT-PCR gel images.

Figure 4. A titration study of the three lead PMOs: Splicing pattern of SMN transcript in type I SMA patient cells transfected with PMOs or unrelated negative control (Sham PMO) using lipofectin.at concentrations indicated.

The percentage of SMN2 exon inclusion as determined by densitometric analysis of RT-PCR gel images is also shown.

Figure 5. a) Western blot analysis of SMN and ß-tubulin in type I SMA patient cells transfected with PMOs or unrelated negative control (Sham PMO).

b) The lower panel shows the fold change in SMN, normalised against ß-tubulin, as determined by densitometric analysis. P-values indicates statistical significance in SMN expression between PMO(-10-34) and sham PMO.

Figure 6. Western blot analysis of SMN in type I SMA patient fibroblasts nucleofected with PMOs or unrelated negative control (Sham PMO).

The fold change in SMN expression, normalised against ß-tubulin is shown.

Figure 7. Western blot analysis of SMN and ß-actin in a) spinal cord and b) brain of SMA carrier mice (Smn^+/−; SMN2^+/+;Δ7^+/+) intracerebroventricularly injected with 6 mM of PMO(-10-34) or scrambled PMO control.

Tissues were collected at 7, 21 and 65 days after PMO(-10-34) injection and at 21 days after scramble PMO injection.

Figure 8. Survival curve of SMA mice intracerebroventricular injected with different dosage of PMO(-10-34).

Figure 9. Mass of SMA mice intracerebroventricularly injected with PMO(-10-34) at five different doses.