Peripheral SMN restoration is essential for long-term rescue of a severe spinal muscular atrophy mouse model

Abstract

Spinal muscular atrophy (SMA) is a motor neuron disease and the leading genetic cause of infant mortality; it results from loss-of-function mutations in the survival motor neuron 1 (SMN1) gene. Humans have a paralogue, SMN2, whose exon 7 is predominantly skipped, but the limited amount of functional, full-length SMN protein expressed from SMN2 cannot fully compensate for a lack of SMN1. SMN is important for the biogenesis of spliceosomal small nuclear ribonucleoprotein particles, but downstream splicing targets involved in pathogenesis remain elusive. There is no effective SMA treatment, but SMN restoration in spinal cord motor neurons is thought to be necessary and sufficient. Non-central nervous system (CNS) pathologies, including cardiovascular defects, were recently reported in severe SMA mouse models and patients, reflecting autonomic dysfunction or direct effects in cardiac tissues. Here we compared systemic versus CNS restoration of SMN in a severe mouse model. We used an antisense oligonucleotide (ASO), ASO-10-27, that effectively corrects SMN2 splicing and restores SMN expression in motor neurons after intracerebroventricular injection. Systemic administration of ASO-10-27 to neonates robustly rescued severe SMA mice, much more effectively than intracerebroventricular administration; subcutaneous injections extended the median lifespan by 25 fold. Furthermore, neonatal SMA mice had decreased hepatic Igfals expression, leading to a pronounced reduction in circulating insulin-like growth factor 1 (IGF1), and ASO-10-27 treatment restored IGF1 to normal levels. These results suggest that the liver is important in SMA pathogenesis, underscoring the importance of SMN in peripheral tissues, and demonstrate the efficacy of a promising drug candidate.

Figure 1

Systemic versus ICV ASO-10-27 injections in SMA mice. a, Survival curves after ICV administration at P1. 20 μg ASO (ICV20, n=14) or saline (ICV0, n=18) gave mean survivals of 17 and 10 d, respectively (P<0.001). ASO-treated heterozygotes (Het-ICV, n=15) served as controls. Spinal-cord RNA and protein samples (n=3) were analyzed at P7 by radioactive RT-PCR (b) or immunoblotting with SMN-KH mAb (c). FL: full-length mRNA; Δ7, exon-7-skipped mRNA; incl, exon 7 inclusion. d, Survival curves after SC administration with saline (SC0, n=26) or ASO (SC50, n=12) twice between P0 and P3. SC50-SC50 (n=14) mice received two additional SC injections at P5-P7. Het-SC-ICV (n=13) and SC50-ICV20 (n=18) were heterozygous and SMA mice, respectively, that received combined P1 ICV and P0-P3 SC injections. Each SC injection dose was 50 μg/g. P<0.0001 for all groups versus SC0. e, Dose-dependent survival after two SC injections at P0-P3 with 40 (n=26), 80 (n=18), or 160 (n=14) μg/g/injection. Saline-treated SMA (SC0, n=23) or heterozygous mice (Het, n=18) served as controls. P<0.0001 for all groups versus SC0.

Figure 2

SMN2 splicing and protein expression in mouse tissues after SC ASO injection. a, Radioactive RT-PCR of RNA from P7 SMA mice after two SC injections between P0 and P3 at 0, 40, 80, or 160 μg/g/injection. b, Statistics of exon 7 inclusion (n=3). c, Protein samples from P7 SMA mice (n=3) treated with 160 μg/g/injection were analyzed by immunoblotting with SMN-KH mAb (Supplementary Fig. 7a). d, RT-PCR of liver RNA from P10, P30, and P180 SMA mice shows decreasing effect of ASO-10-27 over time. e, Statistics of data in panel d. *P<0.05, **P<0.001, ***P<0.0001 compared to saline controls. Means ± s.d. are shown.

Figure 3

Evaluation of affected tissues and motor-function. H&E-stained tissues from P9 ASO-treated SMA mice (SC160, n=6, two SC injections at 160 μg/g/injection at P0-P3) versus saline controls (SC0, n=6) and untreated heterozygotes (Het, n=6) (Supplementary Fig. 14). Saline-treated mice were ambulant at P9, and were expected to live for another 3–5 d. α-motor-neuron counts in each cross-section of L1-L2 spinal cord (a), mean fiber-cross-sectional area (200 fibers) of rectus femoris muscle (b), heart weight (c), and thickness of the heart inter-ventricular septum (IVS) and left ventricular wall (LVW) (d) significantly improved in ASO-treated mice. e, Arborisation complexity of NMJs was restored in ASO-treated mice. P90 SC40 (n=12, 124 trials), SC80 (n=13, 137 trials), SC160 (n=11, 117 trials) and untreated heterozygous mice (Het, n=12, 135 trials) were tested 3–5 times/d for 3 d on a Rotarod, using an acceleration profile. The mean times for staying on the spinning rod (f), and the number of no-fall-trials and of mice with ≥1 no-fall trial (g) are shown. h, Grip strength (grams-force) of SC160 mice (n=6) evaluated at 5 and 9 months (m) reached ~80% of that of heterozygous mice (n=6). *P<0.05; **P<0.01. Means ± s.d. are shown.

Figure 4

IGF1 system is disrupted in SMA mice. Treated mice received two SC ASO injections at 80 μg/g/injection between P0 and P3 (SC80). a, IGF1 serum levels in P6 and P9 SMA mice (SC0), measured by ELISA (mean of three measurements per sample), were strikingly lower than in heterozygous littermates (Het) or treated SMA mice (P<0.001 for all samples). b, Total liver RNA from P1 and P5 SMA mice and heterozygous littermates was analyzed by radioactive RT-PCR to measure Igf1, Igfals, and Igfbp3 expression, with Gapdh as control. c, ASO treatment restored hepatic Igfals expression, assayed at P5. d, Quantitation of hepatic Igfals expression; *P<0.01 versus heterozygous or ASO-treated SMA samples. Means ± s.d. are shown.