Scientists have used an antisense oligonucleotide (ASO) to significantly prolong the lifespan and improve motor function in mice with a severe form of the motor neuron disease spinal muscular atrophy (SMA). The approach involved administering an ASO that corrects SMN2 splicing and restores SMN protein expression in motor neurons and other tissues and organs, in particular the liver.
The Cold Spring Harbor Laboratory (CSHL) researchers found that injecting the ASO directly into the CNS of SMA mice efficiently corrected SMN2 splicing, raised SMN protein levels, and led to a modest increase in survival. However, report Adrian R. Krainer, Ph.D., and colleagues at CSHL and Isis Pharmaceuticals, systemic treatment involving two subcutaneous injections of the antisense oligonucleotide boosted survival even further, while combining the two routes of administration increased survival 10-fold compared with CNS administration alone.
The researchers report their findings in Nature. The paper is titled “Peripheral SMN restoration is essential for long-term rescue of a severe spinal muscular atrophy mouse model.”
SMA results from loss-of function mutations in the survival motor neuron 1 (SMN1) gene. Noncentral nervous system pathologies including cardiovascular defects have also recently been reported in severe SMA mouse models and patients, the authors report. Humans do carry a paralogue gene, SMN2, but a splicing glitch means its exon 7 is predominantly missed out, and the limited amount of functional, full length SMN expressed can’t compensate for lack of SMN1.
The team set out to compare the effects of either intracerebroventricular (ICV) injection or systemic administration of an ASO called ASO-10-27 in a severe mouse model of SMA that is deficient in the SMN protein. ASO-10-27 had previously been shown to effectively correct SMN2 splicing and restore SMN expression in motor neurons after ICV injections.
For the latest set of studies SMA mice received either a single ICV injection of ASO-10-27 on postnatal day one (P1) or two separate subcutaneous (SC) injections between P0 and P3. The team in addition evaluated combined treatment regimens in which animals were given both ICV and SC injections or repeated SC injections.
While ICV administration corrected SMN2 splicing in the spinal cord and lead to a marked increase in SMN protein levels, the treatment only extended median survival to 16 days. In contrast, systemic therapy using two SC injections increased median survival to 108 days. Combining an ICV and SC regimen further increased the median survival to 173 days, while giving animals two additional SC injections at P5 and P7—after the initial two SC injections at P0–P3—extended median survival to 137 days.
The SMA mice varied in size, but their average weight was low in comparison with SMN2 heterozygous littermates, and they had much shorter tails, the authors continue. While the majority of rescued SMA mice could run and climb normally, they developed necrotic tails and ears, which were quickly lost: this resembles the phenotype of type III SMA mice, the team adds. Necrosis could be delayed, however, following ICV or SC administration.
A further study was then carried out to evaluate the effects of increasing repeat doses of systemic ASO-10-27 administered twice between P0 and P3. This confirmed that survival time was dose dependent, with the highest dose leading to long-term survival that was comparable to the best results previously reported using adeno-associated expression of the SMN protein in a less severe SMA model. In fact, four out of 32 mice treated systemically (with two injections between P0 and P3) at the two highest ASO-10-27 doses were still alive and active after 500 days.
SC therapy in addition resulted in a dose-dependent rescue of ear and tail necrosis and delays in the development of cataracts and rectal prolapsed. Animals receiving the highest dose of ASO-10-27 had significantly longer tails. Interestingly, adding in two additional SC injections at P5 and P7 only moderately increased survival, “emphasizing the importance of early postnatal therapeutic intervention,” the authors stress.
Analysis of RNA from subcutaneously treated animals confirmed there was a dose-dependent increase in exon 7 inclusion in the spinal cord, brain, liver, heart, kidneys, and skeletal muscle; the strongest effect was seen in the liver. Immunoblotting of spinal cord, liver, and heart tissue samples from mice treated by SC administration showed a corresponding increase in full-length SMN protein.
Exon 7 inclusion in the liver did decrease significantly after P30, but this was consistent with the 20 day half life measurement for ASO in liver. It also emphasizes “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 team adds.
In contrast, while ICV administration of the ASO led to more marked changes in exon 7 inclusion in the brain and spinal cord tissues, it had very limited effects in peripheral tissues. The researchers did find that some ASO accumulated in spinal cord motor neurons after SC administration as well, and there was evidence of moderate SMN2 splicing changes in the CNS. This probably reflected “incomplete closure of the blood-brain barrier in neonates and/or retrograde transport of the ASO,” they note, and also probably contributed to the extended survival.
However, the investigators point out, 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.” Histological examination of SMA-affected tissues and organs in mice given systemic ASO-10-27 at the highest dose confirmed that therapy led to marked benefits spanning a number of tissues. Both spinal cord α-motor neuron counts and heart wall and septum measurements in treated mice were comparable to those of the control heterozygous littermates. Average muscle fibre cross-sectional area also reached 80% of that in heterozygotes, while neuromuscular junction integrity was similar in mice treated with the highest ASO dose to that in the control littermates.
Encouragingly, there was also a dose-dependent benefit of SC ASO-10-27 therapy on motor function: even though three month old mice that had received the two highest doses of ASO didn’t perform as well on a rotorod test as their heterozygous littermates, they still performed better than SMA animals receiving lower doses of the ASO.
Some of the ASO-10-27-treated mice also passed a 30-second acceleration-profile test that many of the heterozygotes failed, the researchers remark. At both five and nine months of age the treated SMA mice demonstrated a forelimb grip that was 80% as strong as that of control mice, while behavioral tests also showed no major difference between treated SMA mice and control animals.
Severe SMA mice tend to be small in relation to wild-type mice, and SC administration of ASO-10-27 has a particularly marked effect on SMN2 splicing in the liver, which contributes about 75% of the circulating IGF1 (a neurotrophic factor that is also involved in cardiac development and function), Dr. Krainer et al. note. These observations prompted the team to look specifically at the effects of ASO on the growth hormone (GH)-IGF1 axis.
While IGF1 was either absent or greatly reduced in serum samples from untreated SMA mice, animals receiving SC ASO-10-27 demonstrated normal IGF1 levels. The lack of IGF1 in SMA was found not to be due to a reduction in hepatic Igf1 messenger RNA but rather was caused by a disease-related reduction in expression of the Igfals gene. Igfals codes for an IGF-binding protein that is stimulated by GH postnatally to bind IGF1 and form a stable complex, which extends IGF1 half life from 10 minutes to 12 hours. Again, SC administration of the ASO rescued Igfals expression in the liver.
“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,” the authors state. Indeed, they note, disruption of the IGF1 system is a common feature of neurodegenerative diseases and Igf1-null mice demonstrate some phenotypic similarity with SMA mice. “Moreover, dysregulation of the IGF1 receptor and its downstream signaling pathway has been observed in patients with type I SMA.” They suggest the latest studies in mice idnicate 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.”