This is the dawning of the age of antisense
* Adapted from Aquarius/Let the Sunshine In
Despite four FDA approved oligonucleotide therapeutics, (fomivirsen – Vitravene, approved in 1998; pegaptanib – Macugen, 2004; mipomersen – Kynamro, 2013; eteplirsen – Exondys 51, 2016), the field has yet to achieve recognition as a valuable drug category alongside gene and cell therapy, small molecules and biologics.
This is set to change with the approval of nusinersen (Spinraza) for the treatment of spinal muscular atrophy (SMA) on the 23rd of December 2016. SMA is the most common genetic cause for infant mortality and up until now, there were no approved treatments available. As will be discussed in this article, nusinersen has shown unprecedented activity in ameliorating SMA in clinical trials. Perhaps most promising is preliminary data from the ongoing NURTURE study in pre-symptomatic infants suggesting that treated patients achieved motor milestones at developmentally normal time points.
SMA is inherited in an autosomal recessive manner and is the result of deletion or loss of function of the survival of motor neuron 1 (SMN1) gene with ensuing degeneration of spinal motor neurons and skeletal muscle atrophy. In severe cases (classified as Type 0 and Type I), this is followed by symmetric limb paralysis, respiratory failure, and death within 6 months and 2 years, respectively. Intermediate (Type II – children can sit but not walk independently or Type III – children may walk for a few years) and mild adult-onset (Type IV – muscle weakness) phenotypes of the disease also exist.
In general, severity of the disease can be significantly ameliorated depending on the expression level of the full-length SMN2 gene product. SMN2 is a nearly identical copy of SMN1 with a C to T nucleotide change in exon 7 which results in exclusion of this exon from 80-90% of the SMN2 transcripts and thus produces a shortened, non-functional protein that is quickly degraded. Both SMN genes are located in the 5q13.2 chromosomal region which underwent a segmental duplication during Homo sapiens evolution and is therefore enriched in pseudogenes and repetitive sequences. This pre-disposes the region to re-arrangements, duplications and gene conversions. Large scale deletions, particular those that include deletions of SMN2 in addition to SMN1 are associated with more severe phenotypes, while SMN1 to SMN2 gene conversion is associated with milder disease. This is because every SMN2 copy will generate around 10-20% of exon 7 included transcripts, so extra SMN2 copies result in additional functional SMN protein. (1) Thus, patients with bi-allelic deletion/mutation of SMN1 with 1 or 2 copies of SMN2 are likely to have the most severe disease phenotypes (Type 0 or Type I, respectively). Patients with SMN1 to SMN2 gene conversions or SMN2 duplications that result in 3-4 copies of SMN2 generally have the milder Type II and III phenotypes while patients with >4 copies of SMN2 are likely Type IV. (1)
The newly approved SMA treatment Spinraza is an antisense oligonucleotide (previously known as nusinersen, ISIS-SMNrx, ISIS 396443 and ASO-10-27) that increases levels of functional SMN protein produced from the SMN2 copies by boosting exon 7 inclusion in SMN2 mRNAs. Nusinersen has the nucleotide sequence TCACTTTCATAATGCTGG and is fully 2′-O-methoxyethyl (MOE) modified on a phosphorothioate backbone. It targets the intronic splicing silencer N1 (ISS-N1) which was originally described by Singh et al. in 2006. (2) Blocking the ISS-N1 with nusinersen results in the destabilization of an inhibitory stem-loop RNA structure in intron 7 close to the end of exon 7 and thus leads to increased exon inclusion.
In vivo studies in a humanized SMN2 transgenic mouse model of severe SMA demonstrated that injection of nusinersen into cerebral lateral ventricles increased SMN2 exon 7 inclusion 4-6 fold with a concomitant increase in SMN protein (3). Nusinersen also moderately increased survival and improved motor function. In addition, pharmacologically relevant tissue concentrations could be achieved in non-human primates after intrathecal infusion. (3) Unfortunately, non-human primates and un-modified mice lack the 5q13 duplication and hence do not have the SMN2 gene. Consequently, pharmacodynamics could only be studied in humanized transgenic SMN2 mouse models.
Surprisingly, systemic delivery of nusinersen was necessary for extended survival in this mouse model, suggesting that, at least in these mice, SMN expression in tissues other than brain was essential for survival. (4) In 2015 a study from the same group demonstrated that restoring SMN expression in the CNS was not required for rescue of the SMA phenotype and that increasing SMN levels in peripheral tissues alone was sufficient for complete rescue. (5) The paper suggested that the moderately increased survival seen with direct CNS delivery was likely due to partial clearance of the ASO into the blood and subsequent uptake by peripheral tissues. Latest results indicate that extended survival with systemic ASO delivery may be due to correction of defects in liver and spleen development in the SMA mouse models. (6, 7)
However, based on the available data in 2011 which suggested some amelioration of the disease with intrathecal delivery of nusinersen, Ionis Pharmaceuticals (at the time known as Isis Pharmaceuticals) initiated a phase I study in human patients (NCT01494701). Results of this study and its open-label extension (NCT01780246) were published in March of this year by Chiriboga et al. (8) and have already been discussed in this column (here). In summary, a single intrathecal injection in children with type II and III SMA aged 2-14 years lead to a significant increase (32.8%; p=0.008; n=8) in Hammersmith Functional Motor Scale Expanded (HFMSE) scores 9-14 months after treatment at the highest dose (9 mg). This study also confirmed findings from non-human primates that nusinersen half-life in CSF was 4-6 months after initial clearance, supporting infrequent administration. A second open-label extension study (NCT02052791) showed continuing improvement in the Type II patients as well as improvement in some measurements and stabilisation in others in the Type III patients. (9)
Results published in November 2016 from a phase II study (NCT01839656) in infants with Type I SMA younger than 7 months were even more encouraging. (10) Four participants received loading doses of 6 mg on days 1, 15, and 85, and then 12 mg on day 253 and every 4 months thereafter while 16 received 12 mg doses on the same schedule. As of the cut-off point on Jan 26, 2016, the median age to death or permanent ventilation had not been reached and comparison with a natural history study revealed a statistically significant difference (p=0.0014). Measurements of developmental motor milestones using the Hammersmith Infant Neurological Exam—Part 2 (HINE-2) and the Children’s Hospital of Philadelphia Infant Test of Neuromuscular Disorders (CHOP-INTEND) also showed statistically significant improvements (p=0.0002 and p=0.008, respectively) compared to natural history. These improvements translated into clinically meaningful benefit: for example, eight participants developed the ability to sit, five were able to stand and two even started walking. However, the authors clearly stated “that treatment with nusinersen has not restored normal age-appropriate function.”
As of the cut-off point, 4 of the 20 study participants had died and 3 were on permanent ventilation. In support of the research necessary to develop this new treatment, the families of three of the deceased infants permitted tissue samples to be taken. This made it possible to demonstrate that nusinersen concentrations in all areas of the spinal cord were greater than 10 μg/g, the predicted concentration required for pharmacological effects. The research also showed that in nusinersen-treated infants, 50-69% of SMN2 mRNA contained exon 7, a 2.6-fold increase over untreated controls.
Based on the encouraging results from these initial clinical studies, Ionis Pharmaceuticals together with Biogen Inc. initiated a randomised, double-blind sham-procedure controlled phase III study (ENDEAR; NCT02193074) in 111 infants with Type I SMA in July 2014. The study incorporated more frequent dosing with 12 mg at the start of treatment (days 1, 15, 29 and 64 and then at 6 and 10 months) in an attempt to inhibit the early stages of atrophy as much as possible. (11) The study included a pre-planned interim efficacy analysis after 80 participants had been assessed at the 6-month treatment appointment.
The primary end point specified for the interim analysis was the proportion of motor milestone responders defined as improvement in more HINE categories than worsening. Improvement and worsening were measured as ≥2-point difference in ability to kick (or maximal score) or ≥1-point difference in any other milestone excluding voluntary grasp. As was first reported in a press release in August 2016 (12), the trial was stopped early and all patients were transferred over into the treatment arm because a pre-specified interim analysis showed that 40% of treated patients achieved the primary endpoint versus 0% of patients in the control arm (p<0.0001). (13) The interim analysis also demonstrated that 23% of treated patients died, compared to 43% of patients in the control arm. (13) The companies also announced that they would initiate regulatory filings and were working on setting up an expanded access program.
A second phase III study (CHERISH; NCT02292537) in 126 non-ambulatory patients with later-onset SMA (consistent with Type 2; age 2-12) was initiated in November 2014. Again, a pre-specified interim analysis was included in the trial design to potentially accelerate the development timeline. According to the press release from the 7th of November 2016 (14), patients in the treatment arm showed a difference of 5.9 points on the HFMSE scale over the sham-controlled arm after 15 months of treatment (p= 0.0000002).
The accumulating data from the ongoing trials suggested that the earlier SMA patients were treated with nusinersen, the more benefit they would gain. To investigate this hypothesis, a phase II study (NURTURE; NCT02386553) in pre-symptomatic, genetically diagnosed SMA patients with 2 or 3 SMN2 copies and less than 6 weeks old at the start of treatment was initiated in May 2015. Participants are dosed with 12 mg nusinersen on days 1, 15, 29 and 64 and then maintenance doses every 4 months. Preliminary data presented at the International Congress of the World Muscle Society on the 8th of October 2016 showed that all infants were alive and none required chronic respiratory support. (15) As of the data cut-off point in June 2016, ten participants were older than 6 months. At the same time point in the ENDEAR phase III study, 23% of nusinersen treated and 43% of untreated infants had died. In addition, most NURTURE participants achieved motor milestones consistent with normal development. Although still speculative, these data suggest that treatment as early as possible before SMA symptoms are evident will normalise development and that a genetic screening program in infants should be introduced as soon as possible.
Based on this accumulated data, Spinraza was approved with amazing speed by the FDA for all types of SMA, only 3 months after Biogen and Ionis completed their New Drug Application (13) with the EMA and other agencies sure to follow suit soon.
Naturally, Spinraza has been hailed as a breakthrough for such a devastating disease with no other treatment options. At the same time, the question now arises if the non-neuronal pathologies seen in SMA mouse models will become an issue in patients, now that Type I SMA carriers are likely to live significantly longer. In this respect it is noteworthy that nusinersen was present in peripheral tissues such as liver and kidney in the autopsy samples from participants in the phase II trial, consistent with limited clearance from the CSF into the systemic circulation. (10) It remains to be seen if the drug concentration in peripheral tissues is high enough and localized to the correct cells within the tissues to alleviate non-neuronal pathologies if they do appear. Another as yet unanswered question is if continued life-long treatment with Spinraza will be necessary, or if treatment can be reduced in frequency and/or dose after a certain developmental stage. This may also be possible by increasing overall SMN2 protein expression with a second ASO targeted to an inhibitory lncRNA transcribed from the SMN antisense strand. (16)
Nusinersen has without doubt demonstrated the feasibility of administering antisense oligonucleotides directly to the CNS via semi-regular intrathecal injections (3 times a year after loading period). Research into oligonucleotide therapeutics for neurological diseases that have so far proven refractory to small molecule and biologic drugs such as amyotrophic lateral sclerosis, Huntingtons disease, frontotemporal dementia, Angelman syndrome, Spinocerebellar Ataxia Type 2 and even Alzheimers disease is already ongoing. Truly, this is the dawning of the age of antisense.
- Copy Number Variations in the Survival Motor Neuron Genes: Implications for Spinal Muscular Atrophy and Other Neurodegenerative Diseases.
Front Mol Biosci. 2016 Mar 10;3:7.
- Splicing of a critical exon of human Survival Motor Neuron is regulated by a unique silencer element located in the last intron.
Singh NK, Singh NN, Androphy EJ, Singh RN.
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- Antisense oligonucleotides delivered to the mouse CNS ameliorate symptoms of severe spinal muscular atrophy.
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- Peripheral SMN restoration is essential for long-term rescue of a severe spinal muscular atrophy mouse model.
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- Motor neuron cell-nonautonomous rescue of spinal muscular atrophy phenotypes in mild and severe transgenic mouse models.
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- Survival Motor Neuron (SMN) protein is required for normal mouse liver development.
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- Survival of motor neurone protein is required for normal postnatal development of the spleen.
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- Results from a phase 1 study of nusinersen (ISIS-SMN(Rx)) in children with spinal muscular atrophy.
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- Treatment of infantile-onset spinal muscular atrophy with nusinersen: a phase 2, open-label, dose-escalation study.
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- The Antisense Transcript SMN-AS1 Regulates SMN Expression and Is a Novel Therapeutic Target for Spinal Muscular Atrophy.
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