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| THE THERAPEUTIC BOX |
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| Year : 2018 | Volume
: 5
| Issue : 1 | Page : 81-85 |
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Neuromuscular diseases: Recent advances in antisense oligonucleotide therapy
Ashok Verma
Department of Neurology, University of Miami Miller School of Medicine, Miami, FL, Florida
| Date of Web Publication | 28-Jan-2019 |
Correspondence Address:
Ashok Verma
Department of Neurology, Clinical Research Building, 1120 NW 14 Street, Miami, FL - 33136
Florida
 Source of Support: None, Conflict of Interest: None  | Check |
DOI: 10.4103/astrocyte.astrocyte_55_18
Genetic neuromuscular diseases are caused by defective expression of nuclear or mitochondrial genes. Toxic mutant protein may cause cell death, and thus, strategies that reduce mutant gene expression may provide therapeutic benefit. Synthetic antisense oligonucleotide (ASO) has been known to recognize cellular RNA and control gene expression. In recent years, advances in ASO chemistry, creation of designer ASO molecules to enhance their delivery and safety, and design of clinical trials to judge therapeutic efficacy have ushered into an era of plausible application of ASO technology to treat currently incurable neuromuscular diseases. The US Food and Drug Administration has recently approved two ASO therapies in genetic neuromuscular diseases. This brief overview examines the recent advances in ASO technology, evolution, and use of synthetic ASOs as therapeutic platform, and the mechanism of ASO action by exon-inclusion in spinal muscular atrophy and exon-skipping in Duchenne muscular dystrophy, with attention to their advantages and limitations.
Keywords: Dystrophy, Eteplirsen, Nusinersen, oligonucleotide, SMA
How to cite this article:
Verma A. Neuromuscular diseases: Recent advances in antisense oligonucleotide therapy. Astrocyte 2018;5:81-5 |
| Introduction | |  |
Stephenson and Zamecnik in 1978 first used oligonucleotides as laboratory tool to downregulate the expression of specific genes and proposed the potential of using nucleic acid molecules as drugs.[1] These synthetic nucleotides were small in size (15–20 nucleic acid length), and because they were complimentary to sense strand of messenger RNA (mRNA), they were called antisense oligonucleotides (ASOs). In principle, a synthetic nucleotide, if delivered safely to the targeted cellular site, has the potential to bind sequence specifically (Watson–Crick pairing) to the mRNA and, thereby, can control the expression of a gene. In late 1990s, another related technology emerged with the discovery of the small RNA interference (siRNA) pathways that can silence the mutant gene expression and thus the toxic gene product.[2] Duplex siRNAs are potent regulatory agents and have great potential as RNA-based therapeutic platform. However, the application of siRNA to neuromuscular diseases is not as advanced as ASO and will not be discussed in detail in this review.
Although the potential of ASOs as a drug was immediately obvious decades ago, actual development of ASO-based drugs faced many hurdles.[1],[3],[4],[5],[6],[7] First of all, the nucleic acids are highly susceptible to degradation by endogenous nucleases, and ASOs in their native forms have a very short half-life. Furthermore, synthetic ASOs are large (approximately 30 kD) and highly negatively charged molecules and thus do not cross vascular endothelium, dense extracellular matrix, and cell membranes in order to reach to their intracellular targets. Additionally, off-target effect of ASOs may lead to devastating adverse reaction. Finally, synthetic ASOs can be immunogenic. Synthesizing a therapeutic oligonucleotide therefore required historic increment and dozens of chemical steps over the last two decades, each step needing to be almost perfectly efficient, not the least of which was to ensure efficient recognition of targets inside cells, without offside effect.
| Design, Development, and Mechanism of Action of ASO | | |
ASOs are single-stranded deoxyribonucleotide analogs, usually 15–20 bp in length. Their sequence (3′–5′) is antisense and complementary to the sense sequence of the target mRNA. Unmodified oligonucleotides after quick degradation by circulating nucleases are excreted by the kidney; unmodified oligonucleotides are generally too unstable for therapeutic use. Therefore, chemical modification strategies have been developed to overcome this and other obstacles in ASO therapy program.
The rationale for chemically modifying first-generation ASOs in 1980s was to reduce the nuclease degradation. This was achieved by replacing one of the non-bridging oxygen atoms in the phosphate group of nucleotide with either sulfur groups (phosphorothioates), methyl groups (methyl phosphonates), or amines (phosphoramidates).[4] Phosphorothioate substitution (PS) was the earliest and the most commonly used modification that renders the internucleotide linkage resistant to nuclease degradation. Aside from endogenous nuclease resistance, PS modifications have two other distinct advantages.[4] First, it can support endogenous RNAse H activity to degrade the target mRNA or mutant toxic mRNA, thereby diminishing its toxic protein product. Second, PS linkages also improve the pharmacokinetic characteristics by their sequence-independent but length-dependent binding with plasma proteins. Although substantial advantages are conferred by PS backbone modification of nucleotides, such modifications have also been shown to elicit strong platelet activation, aggregation, and thrombi formation in animal models and in-vitro experiments using human platelets.[3],[4],[6]
In order to overcome the various non-sequence-specific side effects of first-generation ASOs and to improve further nuclease resistance and target-binding affinity, second-generation ASOs have been developed.[3],[4] The most commonly used modification in these ASOs is 2′ ribose modifications that include 2′-O-methoxyethyl (2′-OMe) and locked nucleic acid.[3],[4],[8] 2′-OMe modifications are commonly used in a “gapmer” design, a chimeric oligo comprising a DNA sequence core with flanking 2′-MOe nucleotides that enhance the nuclease resistance, in addition to lowering toxicity and increasing hybridization affinities.[4] However, two ASOs that caused severe thrombocytopenia in recent clinical trials have this chimeric ASO design, raising doubts about its safety.[4],[9]
Most recently, third-generation ASOs have been developed to enhance their delivery to the target sites. In this technology, the oligo load is covalently bound to a carrier or ligand, such as lipid particles, liposomes, nanoparticles, and more recently, the sugar N-acetyl galactosamine to enhance safer and enhanced delivery to the target site.[4],[6],[10],[11],[12] Recently approved Eteplirsen (ExonDys 51) for Duchenne muscular dystrophy (DMD) is the third-generation phosphorodiamidate morpholino ASO (see below).
Hybridization of ASOs to the intracellular target mRNA can result in specific inhibition of gene expression by two main mechanisms.[13],[14] The most common mechanism is by induction of endogenous RNAse H activity (ASO–RNase H) that cleaves the mRNA–ASO heteroduplex. This leads to degradation of the target mRNA while leaving the ASO intact. Such antisense effect is thus catalytic and a single ASO can participate in the destruction of many mRNA molecules. The ASOs currently undergoing trials in amyotrophic lateral sclerosis belong to this category (see below). The second group of ASO mechanism of actions includes translational inhibition by steric hindrance, exon skipping, destabilization of pre-mRNA in the nucleus, and targeting destruction of microsomal RNAs that control expression of other genes. Strategies by steric hindrance and modulating splicing do not utilize RNAse H activity and are the main focus of this review.
| Therapeutic ASOs in Clinical Trials | | |
The spectacular advances in ASO technology in recent decades have led to numerous studies investigating the therapeutic potential of ASOs in in-vitro cell models, animal disease models and in human clinical trials. Interestingly however, commercial development of ASOs has been mired with repeated hopeful optimisms and doubtful downturns, chiefly because many clinical studies showed early promise and then faded to disappointment when phase 3 results were revealed. One compound, Fomivirsen, was approved by the US Food and Drug Administration (FDA) in 1998 for treating cytomegalovirus retinitis after intraocular administration.[15] However, this early success was mitigated by the lack of commercial success because anti-retroviral medications reduced cytomegalovirus retinitis as a major health problem.
In 2013, US FDA approved Lomitapide in familial hypercholesterolemia. Lomitapide targets expression of apolipoprotein B and lowers cholesterol in familial hypercholesterolemia.[16] While Lomitapide has not been a commercial success owing to an overall small patient population and competing statin drugs, the demonstration that a systemically administered ASO drug can be successful paved the way to clinical trials of ASOs for treatment of neuromuscular and other diseases.
Fully modified ASO technology that has particularly caught recent scientific and media attention includes the manipulation of alternative spicing where the ASO molecules work as splice switching oligonunucleotides (SSO).[8],[17] In this context, ASOs can be used to modulate the ratio of splicing variants or correct spicing defects, by either inducing exon-skipping or exon-inclusion.[12],[13],[14],[17],[18],[19],[20] Most notably, these advances have been made for neuromuscular diseases, such as SSO technology in spinal muscular atrophy (SMA)[7],[12],[13],[14] and DMD.[18],[19],[20] Although significant confusion still exists with regard to the pathogenicity of many familial amyotrophic lateral sclerosis (fALS)-related genes, the use of ASO–RNAase H technology is the current area of active research in this clinically devastating disease.[21],[22]
| Neuromuscular Disease Targets | | |
Exon-skipping Eteplirsen ASO in DMD
DMD is a fatal neuromuscular disorder affecting approximately 1 in 3500 male births.[23] It is inherited in an X-linked trait and is caused by loss-of-function mutation in DMD gene that codes for dystrophin, a cytoskeletal protein which stabilizes the plasma membrane of muscle fibers.[24] The disease progresses relentlessly, with boys losing ambulation by 12 years of age or before; death often occurs in their 20s, usually from respiratory or cardiac failure.[23] DMD is the largest known human gene, spanning 2.4 Mb in chromosomal Xp21 region with 79 exons and producing a 14-kb transcript.[25],[26] Due to its length, it is highly susceptible to deletion mutations, and certain DMD gene regions are mutation hotspots. Approximately 70% of DMD cases are due to deletion mutations. If DMD gene deletion results in “out-of-frame mutation,” it produces no or negligible dystrophin and results in DMD phenotype [Figure 1]. On the other hand, if DMD gene deletion results in “in-frame mutation,” generating a variant able to produce functional albeit truncated version of dystrophin, it leads to Becker MD (BMD), a milder dystrophinopathy compared to DMD [Figure 1]. Thus, the genetic deletion difference between DMD and BMD presents an important observation: the nature of the deletion determines the severity of the disorder. It is with this underlying principle that exon-skipping ASO has been developed as a therapeutic paradigm for DMD, that is, conversion of DMD to BMD phenotype. | Figure 1: Exon-skipping eteplirsen ASO recognizes DMD exon 51 in pre-mRNA and converts out-of-frame into in-frame smaller transcript of dystrophin. DMD: Duchenne muscular dystrophy; mRNA: messenger RNA; ASO: Antisense oligonucleotide
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Eteplirsen is a 30-nucleotide phosphorodiamidate morpholino oligomer-type third-generation ASO that hybridizes to exon 51 of DMD and causes it to be skipped during splicing;[18],[19],[20] this corrects the translational reading frame in certain DMD gene deletions, resulting in the production of shorted functional dystrophin protein.[18] Eteplirsen ASO therapy is applicable in about 13% cases of DMDs where exon 51 skipping would potentially convert out-of-frame deletion to in-frame deletion in the DMD gene mutations.[18] A related exon-skipping ASO, Drisapersen, in earlier DMD clinical trials used second-generation 2′-OMe PS type ASO for exon 51 skipping strategy.[6],[18]
After obtaining proof of concept in patient-derived cells and animal models, Drisapersen and Eteplirsen ASO chemistries were clinically developed for exon 51 skipping in DMD.[27] US FDA rejected Drisapersen in 2016 due to safety issues associated with the use of the drug and insufficient evidence of clinical utility.[6] The main difference between Drisapersen and Eteplirsen is that in the later, the nucleotide base is attached to a morpholino moiety which is no longer negatively charged and thus makes it safer to use.[6],[18]
On September 19, 2016, Eteplirsen received accelerated approval by the US FDA.[6] Eteplirsen (ExonDys 51) is the first approved ASO for DMD and first approved exon skipping ASO to be used in humans. Although its approval is shrouded in controversy, chiefly from limited data on only 12 cases and from just dystrophin protein as a surrogate marker in muscle biopsies, without proof of clinical improvement, it is hoped that Eteplirsen indeed would work and that longer term treatment will reveal a slower disease progression in DMD patients. Several major urgent challenges in DMD therapeutics however remain, and these include lack of a strategy that can provide a treatment for all patients with DMD, necessity for sustainability and lifelong treatment, and cost of therapy. Eteplirsen (ExonDys 51) is reported to cost in the order of $300,000–400,000 per year per patient.[6]
Exon retention-strategy Nusinersen ASO in SMA
SMA is the most frequent genetic cause of death in children. SMA affects spinal neurons in brainstem and spinal cord and causes progressive muscular atrophy and paralysis.[28] SMA is caused by mutation in the survival motor neuron 1 (SMN1) gene that reduces the level of active SMN protein. Human genome also possesses a second SMN gene, SMN2, but this gene has a C to T mutation in exon 7 that affects splicing and gives rise to an unstable isoform. In fact, SMN2 splices out exon 7 in 90% of transcripts, leading to about 10% of full-length SMN mRNA and low levels of SMN protein. The number of SMN2 copies therefore determines the SMN clinical phenotype; one SMN1 pair gives rise to infantile SMN where the child dies by 2-year age and more than one SMN pair leads to late age-onset SMA (e.g. later childhood and adult SMAs). If this splicing error is corrected, SMN2 gene can produce more active SMN proteins and potentially alleviate symptoms of SMA.
Nusinersen is 2′-OMe phosphoroate ASO that targets intron 7 within SMN2 mRNA, increasing its inclusion and producing functional SMN protein.[7],[12],[28],[29] Following proof of concept in cell and animal models,[30],[31] an open-label study of Nusinersen in type 1 SMA showed significant divergence from natural history for survival and age on permanent ventilation and incremental achievements in motor mile stones.[32] Furthermore, placebo-controlled trial in type II SMA patients revealed that Nusinersen-treated patients had a significantly higher increase in motor function than placebo-treated patients, thus also meeting the prespecified primary end point.[33] Both trials showed a good safety profile and were stopped after interim analysis, enrolling all patents into open-label trials pending regulatory approval. In parallel, an expanded open-access program was initiated for type I SMA to expedite and solidify the trial results.[7] After careful analysis of trial data, US FDA approved Nusinersen ASO just before Christmas on December 23, 2016. So far, Nusinersen is the only FDA-approved medication for SMA.
ASOs generally do not cross blood–brain barrier. Therefore, repeated intrathecal Nusinersen delivery is required which can be invasive and challenging, especially in young and fragile infants. For older type 1 SMA patients, who are already on permanent ventilation, treatment may prolong survival, but it is currently unknown if improvement in muscle function would occur. Furthermore, postmarketing studies will need to be done to investigate both the positive and potentially negative effects after years and decades of Nusinersen treatment. Finally, an ethical issue that parents and clinicians have to grapple with now is when to treat and when not to treat infants with significant SMA paralysis. Similar to Eteplirsen (ExonDys 51), cost of Nusinersen (Spinraza) therapy is high: an estimated $750,000 for the first year, followed by $375,000 annually for life.[7]
| Emerging Target of ASO Therapy in ALS | | |
ALS is currently incurable and invariably fatal disease characterized by the progressive loss of upper and lower motor neurons in cortex, brainstem, and spinal cord. ALS results in progressive paralysis leading to death 3–5 years after disease onset, often from the respiratory failure. The vast majority of ALS cases are sporadic (sALS), whereas 5–10% are familial (fALS), often with autosomal dominant inheritance involving genes that affect RNA metabolism.[34],[35] Although over 25 different genes are now linked to fALS, two genes (SOD1 and C9orf72) account for at least half of these cases.[34],[36]
Extensive research in murine SOD1 models over the last two decades has shown that overexpressed human-mutated SOD1 gene causes progressive motor neuron disease.[34] Although somewhat controversial in SOD1- and C9orf72-linked fALS, toxic gain of function of mutated genes appears to be the proximate cause of neuronal death.[21],[22],[34] Synthetic ASOs designed to destroy the toxic SOD1 and C9orf72 gene transcripts or proteins can thus be a plausible therapeutic strategy in fALS.[22],[37],[38] If successfully and safely delivered to the target site, ASOs can potentially mitigate the toxic effects of SOD1 and C9orf72 transcripts inside the neurons.
The first successful in-vivo oligonucleotide-based study in ALS mouse model used siRNA against human SOD1.[39] Recently, treatment with ASOs in in-vitro C9orf72 showed a positive effect on toxic RNA foci, causing their reduction in fibroblasts [40] and induced-pluripotent neuronal cells.[41] First clinical trial of intrathecal delivery of an ASO (ISIS 333611) in 24 patients with SOD1 fALS was well tolerated in a phase 1 study,[21] opening the field to further clinical trials. The function of C9crf72 gene is still largely unknown, and therefore, the safety of blocking protein expression through ASO-based therapies in C9orf72 fALS cases will have to be carefully monitored.
| Conclusions | | |
The development and recent approval of ASOs that can successfully induce stable alternate splicing of SMN2 and skipping of exon 51 of dystrophin genes are examples of the potential for ASO technology to be applied in ASO-based drug discovery. Experience with Nusinersen demonstrates that ASOs can be safely administered into the central nervous system and they enter target tissues, modulate the intended target, and produce favorable outcome for patients. Because all nucleotide-based ASOs have similar chemical properties, many of the lessons learned during development and refinement of initial ASO therapy platform can be directly transferred to other projects. However, the fact that ASOs do not readily cross an intact blood–brain barrier limits their application via intrathecal injections for central nervous system diseases. Successful ASO development for other neuromuscular diseases that primarily affect peripheral nerves and skeletal muscles will also require efficient delivery methods. Notwithstanding these and other challenges, ASO-based therapeutics do provide the ability to modulate disease causes and pathways and are exciting developments for the currently incurable neuromuscular diseases.
Financial support and sponsorship
Nil.
Conflicts of interest
There are no conflicts of interest.
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