SMN2 splicing modifiers improve motor function and longevity in mice with spinal muscular atrophy

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Science  08 Aug 2014:
Vol. 345, Issue 6197, pp. 688-693
DOI: 10.1126/science.1250127

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  1. Fig. 1 Small molecules modulate SMN2 alternative splicing and increase SMN protein levels in cells from SMA patients.

    (A) Chemical structures of SMN-C1, SMN-C2, and SMN-C3 small molecules. (B) RT-PCR analysis of SMN2 mRNAs. (Top) SMN-C1 treatment for 24 hours increases the level of FL mRNA and reduces the level of mRNA lacking exon 7 (Δ7) in SMA type I patient fibroblasts. (Bottom) The effects of SMN-C1, SMN-C2, and SMN-C3 on FL and Δ7 mRNA levels in SMA type I patient fibroblasts are concentration-dependent. (C) (Left) Western blot of SMN protein in SMA type I patient fibroblasts after 48 hours of continuous treatment with SMN-C3. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. (Right) The effects of SMN-C1, SMN-C2, and SMN-C3 on SMN protein abundance are concentration-dependent. GAPDH and actin were used as loading controls. (D) HTRF analysis (48) of the SMN protein after 48 hours of continuous treatment with SMN-C1 (left) and SMN-C2 (right) in fibroblasts from SMA type I, II, and III patients or from an unaffected control (carrier). (E) (Left) Motoneuron cultures generated from iPSCs derived from a SMA type I patient. Cells were treated with DMSO (control) or SMN-C3 (300 nM) and immunostained for SMN and Islet-1. Scale bar, 100 μm. (Right) The effect of SMN-3 on SMN protein levels in Islet-1–positive motoneurons and Islet-1–negative cells is concentration-dependent. (B to E) Data represent mean ± SEM (error bars) of three or four independent samples per data point. Concentration-dependence data were fitted to a Hill equation.

  2. Fig. 2 SMN splicing modifiers demonstrate high specificity as assessed by RNA sequencing.

    (A) Difference in total transcript expression of SMN-C3 (500 nM) versus DMSO-treated SMA type I patient fibroblasts (set to 1 for each transcript) for 11,714 human genes with an RPKM (reads per kilobase per million reads) ≥ 1 in either DMSO or SMN-C3 treatment conditions. Abundance of mRNA is shown as log2 fold change (Log2FC) values (0 = no change, +1 = doubling, –1 = reduction by half). SMN2 is highlighted by the arrow, showing no significant change in total mRNA abundance. (B) Differential effects of treatment on individual splice junctions in human transcripts. For each splice junction, spanning reads were counted in both treated and control conditions. Affected splice junctions are characterized by either absolute difference in counts (Δ) or relative changes (Log2FC). The product p = Δ x Log2FC was used to rank splice junctions (up-regulated in blue, down-regulated in red). The top 114 splice junctions with p > 100 are shown (~300,000 splice junctions analyzed in total). Twenty splice junctions showed p > 300. Note that two SMN2 transcript variants [NM_022875 (var a) and NM_022877 (var c)] share the critical target splice junction 5′ of intron 7. For STRN3, a switch between variants NM_014574 and NM_001083893 is observed. Fifteen splice junctions belong to PDXDC1 with all splice junctions similarly affected. PDXDC2P is a pseudogene, probably identified due to the strong similarity between the PDXDC1 and PDXDC2P transcripts around that junction (not resolved by the mapping algorithm). Data represent analysis of RNA sequencing data from three different experimental samples per group.

  3. Fig. 3 SMN splicing modifiers increase SMN protein expression and provide therapeutic benefit to Δ7 mice.

    (A) Relative expression of SMN2 FL and Δ7 mRNA in whole blood after once daily oral doses of SMN-C3 (10 mg/kg per day) on day 1 (blue) and day 10 (red) in adult C/C-allele mice. (B) Levels of SMN-C3 in plasma of C/C-allele mice after SMN-C3 treatment (10 mg/kg per day) on days 1 and 10. (C) SMN protein levels in quadriceps muscle (open circles) and brain (solid circles) of C/C-allele mice after once daily oral doses of SMN-C3 (10 mg/kg) on days 1 and 10. (D) SMN protein in the brain, spinal cord, quadriceps, and longissimus muscle of C/C-allele mice and heterozygous littermates after 10 daily oral doses of vehicle or SMN-C3 at 10 mg/kg. (E and F) SMN protein levels in the brain (E) and quadriceps muscle (F) of Δ7 mice after seven daily intraperitoneal doses (P3 through P9) of vehicle or SMN-C3 (0.1, 0.3, or 1 mg/kg). (G to J) Mice were treated from P3 to P23 once daily with vehicle or SMN-C3 by intraperitoneal injection at 0.3, 1, or 3 mg/kg, and thereafter once daily by oral gavage with 1, 3, or 10 mg/kg. (G) Appearance of a vehicle-treated Δ7 mouse (Δ7 Veh), a SMN-C3–treated Δ7 mouse (Δ7 SMN-C3), and a vehicle-treated heterozygous mouse (HET Veh). (H) Body weight from P3 through P60. Numbers at right indicate survivors at P60 among 10 (HET) or 16 (Δ7) mice per group. (I) Righting reflex of Δ7 mice. Shown is the mean time (from three trials) for a mouse to right itself after being put onto its back, assessed at P9 and P16 (also see movie S1). (J) Kaplan-Meier survival curves from P3 to P65. (A to F and H to J) RT-PCR and protein data represent means ± SEM (error bars) of four to six animals per data point; data in (G) to (J) from 16 Δ7 mice and 10 HET mice per group. **P < 0.01 and ***P < 0.001, as assessed by one-way analysis of variance (ANOVA) followed by Bonferroni test (D) or Dunnett’s test using vehicle-treated animals (E and F) or HET mice (I) as control.

  4. Fig. 4 SMN splicing modifiers prevent neuromuscular pathology in Δ7 mice.

    Animals were treated from P3 through P14 once daily with vehicle or SMN-C3 by intraperitoneal injection at 0.3 or 3 mg/kg. The number of ventral horn motoneurons in lumbar segments three to five (L3 to L5), the innervation of splenius capitis NMJs, and EDL muscle cross-sectional area were assessed at P14 by immunohistochemical methods. (A) Immunomicrographs showing L3-to-L5 motoneurons labeled with choline acetyltransferase antibodies. Scale bar, 100 μm. (B) Quantification of the number of motoneurons in L3-to-L5 spinal cord segments. (C) Immunomicrographs showing the innervation pattern of the splenius capitis muscle. NMJs are labeled with α-bungarotoxin for acetylcholine receptor clusters (red) and anti-synaptophysin and neurofilament antibodies for presynaptic nerve terminals (green). Scale bars, 40 μm (upper panel); 20 μm (lower panel). Dashed boxes indicate NMJs shown at a higher magnification in the lower panel. (D) Quantification of NMJ innervation. (E) Cross sections of EDL muscles. Scale bar, 100 μm. (F) Quantification of the total cross-sectional area of EDL muscles.​ (B, D, and F) Data represent means ± SEM (error bars) of five to seven mice per group. **P < 0.01 and ***P < 0.001, as assessed by one-way ANOVA followed by Dunnett’s test using vehicle-treated Δ7 mice as a control.

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