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) gene1. Humans have a paralogue, SMN2, whose exon 7 is predominantly skipped2, 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 particles3, 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 sufficient4. Non-central nervous system (CNS) pathologies, including cardiovascular defects, were recently reported in severe SMA mouse models and patients5,6,7,8, reflecting autonomic dysfunction or direct effects in cardiac tissues. Here we compared systemic versus CNS restoration of SMN in a severe mouse model9,10. We used an antisense oligonucleotide (ASO), ASO-10-27, that effectively corrects SMN2 splicing and restores SMN expression in motor neurons after intracerebroventricular injection11,12. 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.
To compare the effectiveness of ASO-10-27 delivered centrally versus systemically, we administered an intracerebroventricular (ICV) injection of 20 µg ASO-10-27 on postnatal day 1 (P1) to increase SMN expression in CNS tissues, or we administered a subcutaneous (SC) injection of the ASO on two separate days at 50 µg per g of body weight (µg g−1), between P0 and P3 (two doses). These doses were based on our previous studies with this ASO11,13. We also evaluated combined ICV and SC injections, as well as repeated SC injections (Supplementary Table 1). Control heterozygous mice (Smn+/− SMN2+/0) that received ICV and/or SC ASO-10-27 injections had normal survival and behaviour. Severe SMA mice (Smn−/− SMN2+/0) that received ICV and/or SC saline injections survived for 1–2 weeks, with a median survival time of ∼10 days, similar to untreated mice (Fig. 1a, Supplementary Figs 1a and 2a, and Supplementary Movie 1). Delivery of the ASO only into the CNS efficiently corrected SMN2 exon 7 splicing in the spinal cord and led to a striking increase in SMN protein levels, but modestly extended the median survival to 16 days, with a single pup surviving for 1 month (Fig. 1a–c and Supplementary Fig. 2b–d). In marked contrast, systemic treatment with two SC injections resulted in a median survival of 108 days (Fig. 1d). Combining ICV and SC injections of the ASO further increased the median survival to 173 days, and two additional SC injections on P5 and P7, after the initial SC injections at P0–P3, extended the median survival to 137 days (Fig. 1d).
Treated SMA mice varied in size from runts to comparable to their heterozygous littermates; their average weight was low, and their tails were much shorter than normal (Supplementary Figs 3 and 4). The surviving runts slowly gained weight, reaching ∼18 g at ∼3 months. Most rescued SMA mice could run and climb normally; however, their tails and ears developed necrosis and were gradually lost, resembling the phenotype of type III SMA mice (Supplementary Fig. 3e, f). Additional delivery of the ASO either by ICV injection on P1 or repeat SC injections on P5 and P7 delayed necrosis (Supplementary Fig. 3g).
To further characterize the effects of the ASO administered systemically, we carried out a dose–response study with 0 (SC0), 40 (SC40), 80 (SC80) and 160 (SC160) µg g−1 ASO-10-27 as an SC injection, given twice between P0 and P3. Systemic treatment with the ASO resulted in a dose-dependent increase in survival (Fig. 1e), with the median survival increasing from 10 days to 84, 170 and 248 days, respectively. At the highest dose tested, the ASO given systemically resulted in long-term survival comparable to the best results achieved by adeno-associated virus expression of the SMN protein in a slightly less severe mouse model14,15,16. Remarkably, 2 of 14 mice in the SC160 group and 2 of 18 in the SC80 group are still alive and active after >500 days. A similar survival benefit was achieved by intraperitoneal administration (Supplementary Fig. 5). There was no significant difference in weight gain among the three SC-dosing groups; however, mice in the SC160 group had significantly longer tails (Supplementary Fig. 6a–e). We also observed dose-dependent rescue of ear and tail necrosis and dose-dependent delays in the development of cataracts and rectal prolapse (Supplementary Fig. 6f–h). Administration of the ASO in two doses on P5 and P7 resulted in a modest increase in survival, compared with earlier treatment (between P0 and P3), emphasizing the importance of early postnatal therapeutic intervention (Fig. 1d).
To examine SMN2 splicing changes in various tissues after SC injection of the ASO, we performed reverse transcription followed by PCR (RT–PCR) on RNA samples from P7 mice. We detected a dose-dependent increase in exon 7 inclusion in the spinal cord, brain, liver, heart, kidneys and skeletal muscle, with the strongest effect occurring in the liver and the weakest in the kidneys (Fig. 2a, b and Supplementary Fig. 7a, b). By contrast, ICV administration of the ASO resulted in a much more robust change in exon 7 inclusion in the brain and spinal cord tissues but had very limited effects in peripheral tissues (Supplementary Fig. 8a). Immunoblotting of spinal cord, liver and heart tissue samples from mice treated by SC administration showed a corresponding increase in full-length SMN protein (Fig. 2c and Supplementary Figs 7c and 8b). Exon 7 inclusion in the liver significantly decreased after P30 (Fig. 2d, e and Supplementary Fig. 9), consistent with the measured half-(data not shown). These data suggest that transiently increasing SMN expression in peripheral tissues during the first few weeks of life has a profound effect on long-term survival of severe SMA mice.
The SMN2-splicing changes were consistent with the ASO distribution, as assayed by immunohistochemistry, with the apparent exception of the kidneys; however, in the kidneys, most of the ASO had not been internalized by the cells (Supplementary Figs 10 and 11). We also observed some of the ASO accumulating in spinal cord motor neurons (Supplementary Figs 10 and 11). The limited distribution of the ASO and the moderate SMN2-splicing changes in the CNS after systemic administration probably reflect incomplete closure of the blood–brain barrier in neonates17 and/or retrograde transport of the ASO. However, we detected strong cytoplasmic SMN staining and/or a pronounced increase in gem number in spinal cord motor neurons after ICV injection of 20 µg ASO-10-27 but not after two SC injections of ASO-10-27 at 80 µg g−1 between P0 and P3, a dosage that substantially rescued the severe SMA mice (Supplementary Figs 12 and 13). Therefore, the effect on splicing in the CNS after systemic administration probably contributes to the extended survival, which is consistent with the combined ICV and SC treatment resulting in even better survival than SC administration alone (Fig. 1d). However, the striking effects of systemic administration on survival in this severe mouse model cannot be explained solely by a direct effect on SMN2 splicing in the CNS.
The rescue of severe SMA mice by systemic administration of, for example, histone deacetylase inhibitors or adeno-associated virus vectors, has been attributed to the ability of these agents to cross the blood–brain barrier10,15. However, our data indicate that SMN restoration in peripheral tissues, in combination with partial restoration in the CNS, can achieve efficient rescue of severe SMA mice.
In mice that had been treated systemically with 160 µg g−1 ASO-10-27 and sacrificed on P9, histological examination of tissues or organs associated with SMA revealed striking improvements, consistent with the markedly increased survival of mice in the SC160 group. The α-motor neuron counts in the spinal cords of these mice were comparable to those of the control heterozygous littermates, and the mean area of muscle fibre cross-sections was >80% of that of heterozygotes (Fig. 3a, b and Supplementary Fig. 14a). Likewise, the heart weight and the thickness of the interventricular septum and the left ventricular wall were similar in mice in the SC160 group and their heterozygous littermates (Fig. 3c, d and Supplementary Fig. 14b). Finally, staining of the neuromuscular junctions (NMJs) showed that NMJ integrity was similar in mice in the SC160 group and their heterozygous littermates (Fig. 3e).
Most of the mice that were treated systematically with the ASO showed no overt signs of motor dysfunction (Supplementary Movie 2 and Supplementary Table 2). We used three tests to evaluate behaviour and motor function. The first was a rotarod test, which requires limb muscle strength, as well as balance and coordination. Three-month-old mice in the SC80 and SC160 groups could stay on the rotating rod for ∼12 s: that is, for less time than the control heterozygotes but for longer than mice in the SC40 group. Some ASO-10-27-treated mice passed a 30 s acceleration-profile test that many of the heterozygotes failed (Fig. 3f, g and Supplementary Movie 3). Considering that SMA is a neuromuscular disease, this performance represents a remarkable phenotypic improvement. The second test evaluated muscle strength in mice from the SC160 group at 5 and 9 months. At both ages, the forelimb grip strength of treated SMA mice was ∼80% that of the control heterozygous mice (Fig. 3h). The final test used HomeCageScan, a video-based platform for automated high-resolution behaviour analysis18. ASO-10-27-treated SMA mice performed various behaviours similarly to control heterozygous mice, except for rearing, suggesting some hindlimb weakness (Supplementary Fig. 15).
Two observations prompted us to examine the growth hormone (GH)–IGF1 axis. First, all severe SMA mice are small9,10,19, reflecting growth retardation (Supplementary Figs 2a and 3c). Second, the major effect of SC injection of the ASO on SMN2 splicing is in the liver, which contributes ∼75% of the circulating IGF1 (ref. 20). Moreover, restoring IGF1 expression in the liver is sufficient to support normal postnatal growth of Igf1-null mice20. IGF1 is a potent neurotrophic factor21 and is also involved in cardiac development and function22. An enzyme-linked immunosorbent assay (ELISA) of serum samples from SMA mice at P6–P9 showed that IGF1 was undetectable or present at greatly reduced levels compared with the heterozygous controls; SC administration of the ASO restored IGF1 to normal levels in SMA mice (Fig. 4a).
RT–PCR showed that the level of hepatic Igf1 messenger RNA was not reduced in SMA mice compared with the heterozygous controls and that it increased from P1 to P5 in both SMA and control mice (Fig. 4b). IGF-binding protein, acid labile subunit (IGFALS), which is postnatally stimulated by GH, binds to IGF1 and IGF-binding protein 3 (IGFBP3) to form a stable ternary complex, extending the half-life of IGF1 from 10 min to >12 h23. The inactivation of Igfals results in low levels of circulating IGF1 and IGFBP3, as well as impaired postnatal growth23. RT–PCR revealed a marked reduction in the amount of Igfals mRNA in the liver of both P1 and P5 SMA mice compared with the heterozygous controls; moreover, administration of the ASO rescued Igfals expression (Fig. 4c, d). We conclude that the striking reduction in serum IGF1 levels in SMA mice is likely to be caused by decreased Igfals expression, which correlates with SMN deficiency and SMA progression.
Because Igfals expression is decreased on P1, when the pups are still healthy, we propose that the early deficiency in circulating IGF1 may be one of the factors that contribute to the pathogenesis of severe SMA mice (Supplementary Fig. 1b). Although in several mouse mutants, an impaired GH–IGF1 axis results in an increased lifespan24, a severe lack of IGF1 may contribute to SMA progression, together with other defective factors. Consistent with our hypothesis, two recent studies have shown that a local increase in IGF1 in either the spinal cord or muscle increases the survival of severe SMA mice25,26. Indeed, disruption of the IGF1 system is a common feature of neurodegenerative diseases, including Alzheimer’s disease and amyotrophic lateral sclerosis (ALS)27. Igf1-null mice also show some phenotypic similarity to SMA mice, such as small size and severe and generalized muscle dystrophy (including of the diaphragm and heart), with most of them dying at birth28. Moreover, dysregulation of the IGF1 receptor and its downstream signalling pathway has been observed in patients with type I SMA29. However, the results of IGF1 therapy for ALS are not consistent between mice and humans21,30. In light of this inconsistency, it will be crucial to determine the extent to which SMA mouse models accurately mimic human SMA. This will further refine our understanding of the mouse models and influence the development of therapeutics and clinical treatments for SMA.
ASO-10-27 was synthesized as described previously11 and dissolved in 0.9% saline. The severe SMA mouse model was generated from a type III SMA mouse model, as described previously9,10. All mouse protocols were in accordance with the Cold Spring Harbor Laboratory’s Institutional Animal Care and Use Committee guidelines. Treated mice (control and ASO-10-27) were provided with additional gel food. The procedures for neonatal ICV injection, tissue sample collection, RT–PCR, western blotting and the human-specific anti-SMN antibody (SMN-KH) were as described previously11. The primers for the gene expression analysis are shown in Supplementary Table 3. Serum IGF1 levels were analysed with a Mouse/Rat IGF-I Quantikine ELISA kit (R&D Systems).
Mouse spinal cords, quadriceps and hearts were fixed and stained with haematoxylin and eosin as described previously12. α-Motor neurons were counted in serial 10–20-µm cross-sections of the lumbar (L1–L2) spinal cord. The muscle fibre cross-sectional area was calculated by using AxioVision LE velocity software. NMJ staining in toto was performed as described previously12.
For the rotarod (AccuScan Instruments) test, a four-phase profile was used: phase 1, from 1 to 10 r.p.m. in 7.5 s; phase 2, from 10 to 0 r.p.m. in 7.5 s; phase 3, from 0 to 10 r.p.m. in 7.5 s in the opposite direction; and phase 4, from 10 to 0 r.p.m. in 7.5 s. A grip-strength meter (Columbus Instruments) was used for the gripping test. Mice were allowed to grasp a triangular bar with their forelimbs and were pulled back horizontally. The test was repeated five times for each mouse, and the highest value was recorded as the grip force for that animal.
Statistical significance was analysed by two-tailed Student’s t-tests. Kaplan–Meier survival data were analysed with Mantel–Cox tests using the program GraphPad Prism.
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We gratefully acknowledge support from the Muscular Dystrophy Association, the National Institute of General Medical Sciences and St. Giles Foundation. We thank J. Bu and M. Passini for protocols and advice on NMJ staining, and S. Hearn for assistance with microscope imaging.
F.R., G. Hung, and C.F.B. may materially benefit either directly or indirectly through stock options. Y.H., K.S. and A.R.K., along with their employer, could materially benefit if a therapeutic for SMA results from this work.
This file contains Supplementary Tables 1-3, Supplementary Figures 1-15 with legends and an additional reference. (PDF 1843 kb)
This movie shows two P7 pups, both untreated. The smaller one is an SMA mouse and the other one is its heterozygous littermate. (MOV 18216 kb)
This movie shows five 3-week-old newly weaned mice. Three representative SMA mice that had been treated with subcutaneous ASO administration (SC80) between P0-P3 have shorter tails, and two untreated heterozygous mice have normal tails. (MOV 21283 kb)
This movie shows two 3-month-old mice on a Rotarod test with a four-phase acceleration profile. The one that passed the test and has a shorter tail is an SMA mouse from group SC160, and the other mouse is a heterozygote. 36% of SC160 rescued SMA mice passed this test; 33% of heterozygous mice did not pass the test. (MOV 8152 kb)
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Hua, Y., Sahashi, K., Rigo, F. et al. Peripheral SMN restoration is essential for long-term rescue of a severe spinal muscular atrophy mouse model. Nature 478, 123–126 (2011). https://doi.org/10.1038/nature10485
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