Antisense Oligonucleotides Shed New Light on the Pathogenesis and Treatment of Spinal Muscular Atrophy

Spinal muscular atrophy (SMA) is one of the most common autosomal recessive disorders that cause infant mortality. SMA is caused by loss of the survival of motor neuron (SMN) protein, resulting in the degeneration of motor neurons in the spinal cord.^1,2 In type 1 SMA, the most severe form, onset of disease occurs before 6 months of age and death occurs within the first 2 years of life. There is currently no effective treatment for SMA. Using systematic administration of an antisense oligonucleotide (ASO) to neonatal mice, Hua et al. now show that this approach has a robust ability to rescue a mouse model of severe SMA, significantly extending the life span of affected mice.³ The new data also reveal a role for liver-produced insulin-like growth factor 1 (IGF1) in the development of SMA pathogenesis. These findings not only raise questions concerning the involvement of peripheral tissues, such as liver, in SMA pathogenesis but also provide new leads for developing potential drug candidates to treat this devastating disease.

The SMN protein has diverse functions in RNA metabolism and is expressed ubiquitously throughout the body. However, it remains unknown why and how a deficiency in SMN protein primarily affects the motor system. In addition to its involvement in forming nuclear “gem” structures and participating in RNA splicing, SMN protein is also present in the cytoplasm, where it forms complexes with various ribonucleoproteins and messenger RNAs (mRNAs) and is crucial for mRNA transport in axons.^1,2 Whether motor neuron degeneration is caused by impaired SMN function during RNA splicing, mRNA transport, or other cellular processes is unknown, and the downstream targets of SMN remain to be identified. The human genome contains two SMN genes—SMN1 and SMN2—and SMA is often caused by homozygous loss of SMN1. Although SMN2 encodes a functional protein, the gene contains a single silent nucleotide transition in exon 7, leading to frequent skipping of this exon during RNA splicing. As a consequence, the SMN2 gene generates only approximately 10% of the correctly spliced transcript, a level insufficient to support long-term motor neuron survival.^1,2

One strategy for developing a potential treatment for SMA is to boost the production of correctly spliced transcripts from the SMN2 gene.² An intronic splicing silencer, termed ISS-N1, in intron 7 of the SMN gene has previously been identified.⁴ ASOs that blocked ISS-N1 were shown to increase the inclusion of exon 7 in SMN2 in patient fibroblasts. In a previous study, Hua et al. identified an optimal 18-mer termed ASO-10–27 that targeted ISS-N1 and promoted efficient inclusion of SMN2 exon 7 (ref. 5). They demonstrated that ASO-10–27, when infused via intracerebroventricular (i.c.v.) injection into mice with mild SMA, increased inclusion of SMN2 exon 7 in spinal cord motor neurons. Hua et al. have now extended their work by administering ASO-10–27 via subcutaneous (s.c.) injection into neonatal mice with severe SMA.³ Two doses of ASO-10–27 injected between postnatal day 0 (P0; newborn) and P3 extended the median life span of the mice from 10 days to 108 days. One additional injection on both P5 and P7 further extended the life span to 137 days. These changes in life span were dose dependent; at the highest dose (two injections of 160 mg of ASO-10–27 per gram of body weight), the median life span was extended to 248 days, a 25-fold increase relative to the untreated control. In addition, early intervention was important: delay of ASO-10–27 treatment to between P5 and P7, even when an elevated dose was administered, increased the life span only marginally. This finding is similar to what was observed in two previous SMA gene therapy studies that used mouse models of severe SMA.^6,7 In those studies, expression of wild-type SMN in the central nervous system (CNS) of neonatal mice through adeno-associated virus (AAV)–mediated gene delivery extended the median life span from 15 days to 157 days in one study and by more than 250 days in another. These studies therefore raise hope that real progress can be made to treat this as yet untreatable disease.

In addition to extended life span, Hua et al. showed that many disease-associated symptoms, including reduced myofiber size and altered neuromuscular junction architecture, were improved in the treated mice.³ Behavior and motor function in ASO-treated mice were also improved in a dose-responsive manner. However, although ASO-treated mice continued to gain body weight, they never reached the same weight as the control heterozygous mice. Moreover, the fraction of mice with ear and tail necrosis continued to increase with age. Finally, unlike most of the heterozygous control mice, the majority of ASO-treated mice with SMA failed to survive beyond 500 days. The exhibited growth abnormality and lasting necrosis may ultimately contribute to the reduced long-term survival benefit provided by ASO for these mice. One can argue that the treatment only transiently elevates SMN expression as a result of the relatively short half-life of ASO in vivo (~22 days in liver).³ However, in an AAV gene therapy study for mice with SMA, treated mice exhibited a similar premature death,⁷ suggesting that ASO injection or AAV-mediated gene delivery is not sufficient to completely correct the disease phenotype.

Molecular analysis of SMN2 expression in Hua and colleagues' study showed that s.c. injection led to dose-responsive inclusion of exon 7 in various tissues, with the strongest effect occurring in the liver.³ Exon 7 inclusion increased only moderately in the CNS. In contrast, i.c.v. injection of the same ASO led to exon 7 inclusion exclusively in the CNS, not in other tissues. The fact that systemic ASO administration resulted in a much more prolonged life span than i.c.v. injection suggests that tissues other than the CNS are involved in SMA pathogenesis. Because all mice with severe SMA exhibited reduced body weight, and the liver is the major organ that produces IGF1, which modulates animal growth, Hua et al. examined serum IGF1 levels in SMA mice.³ Indeed, IGF1 levels in SMA mice were significantly reduced relative to those of heterozygous controls. Close examination revealed, however, that the expression of IGF-binding protein acid-labile subunit (IGFALS) rather than the expression of IGF1 itself was affected in SMA mice. IGFALS binds to IGF1 and IGF-binding protein 3 to form a stable ternary complex, extending the half-life of IGF1 from 10 minutes to more than 12 hours.⁸ ASO administration rescued expression of Igfals in the liver and restored serum IGF1 levels. Because Igfals expression correlated with SMN deficiency, Hua et al. hypothesize that an early deficiency in circulating IGF1 may contribute to SMA pathogenesis.³ This hypothesis is supported by observations that local increases in IGF1 in either the spinal cord or muscle increase the survival of mice with severe SMA.^9,10 Igf1-null mice are also phenotypically similar to SMA mice, exhibiting a similar small size, severe muscle dystrophy, and early death.¹¹ The gain in animal body weight in the current study of Hua et al. appeared to be greater than that observed in the two AAV gene therapy studies, suggesting a more efficient rescue of SMN2 splicing in the liver and increased IGF1 production. The current study thus suggests that IGF1 signaling in the liver is also involved in SMA pathogenesis.

ASO injection during the neonatal period clearly provides therapeutic benefits to the SMA mice. Because only two injections were sufficient to generate a long-term survival advantage, the current data suggest that a transient early restoration of SMN expression can have a profound effect on treated animals. This raises the prospect for translation into large animals and clinical application. A study using AAV-mediated gene delivery in nonhuman primates is encouraging.⁶ A newborn cynomolgus macaque transduced with intravenous AAV injection showed robust expression of the reporter gene within the dorsal root ganglia and motor neurons along the entire neuraxis. ASO administration via i.c.v. or s.c. injection may have similar potential for success in large animals. However, the window for therapeutic intervention during development may be limited. In mice, only early intervention between P0 and P3 resulted in significant correction of phenotypic abnormalities.³ Formation of the mouse blood–brain barrier could block efficient CNS delivery of ASO via s.c. administration. Whether it is possible to introduce the ASO into the CNS of older animals via other routes and whether such a maneuver could be carried out in time to rescue already damaged motor neurons in patients are some of the issues that must be resolved.

The new strategy demonstrated by Hua et al. offers some obvious advantages over AAV-mediated gene delivery; it would be much easier and more cost-effective to prepare good manufacturing practice–grade ASO than AAV in sufficient quantities for clinical application. In addition, the application of ASO in the clinic would not be burdened by regulatory issues concerning vector safety. Whereas immune responses against AAV capsid protein could prevent repeated virus administration, ASO is not expected to elicit such responses. Considering the importance of timing for therapeutic intervention, prenatal diagnosis or screening of newborns for SMN mutations remains critical for the success of this strategy. One issue raised by the current study is why the CNS correction of SMN2 splicing by i.c.v. injection extended the life span of SMA mice for only 6 days, whereas s.c. injection extended the animal life span by more than 200 days. Is a transient increase in SMN expression in multiple tissues besides the CNS necessary to achieve long-lasting correction of the disease phenotype? Another issue is whether IGF1 levels are reduced in the livers of SMA patients and how Igfals expression is affected by SMN deficiency. Is it mediated through defective RNA splicing because of the lack of functional SMN protein or through other mechanisms, such as reduced transcription or IGFALS protein stability? Deriving hepatocytes from induced pluripotent stem cells from SMA patients may provide a powerful tool for resolving this issue.^12,13

In summary, Hua et al. have identified a new and potentially easier approach than viral vector–mediated gene delivery to treat SMA. This study also uncovers potential functional roles of peripheral organs such as the liver in the progression of the disease. Further refinement of these discoveries should improve our understanding of SMA and allow development of new approaches to treat this disease in humans. Because aberrant alternative splicing has been shown to play critical roles in the pathogenesis of several other human diseases,¹⁴ the strategy described in this study may also be applicable for the treatment of those splicing-relevant diseases.