Review
1.
Introduction
2.
Duchenne muscular dystrophy
3.
Spinal muscular atrophy
Aleksander Touznik, Joshua JA Lee & Toshifumi Yokota†
4.
Dysferlin deficiency
University of Alberta, Faculty of Medicine and Dentistry, Department of Medical Genetics, Alberta, Canada
(dysferlinopathies)
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New developments in exon skipping and splice modulation therapies for neuromuscular diseases
5.
Fukuyama congenital muscular dystrophy
6.
Conclusion
7.
Expert opinion
Introduction: Antisense oligonucleotide (AON) therapy is a form of treatment for genetic or infectious diseases using small, synthetic DNA-like molecules called AONs. Recent advances in the development of AONs that show improved stability and increased sequence specificity have led to clinical trials for several neuromuscular diseases. Impressive preclinical and clinical data are published regarding the usage of AONs in exon-skipping and splice modulation strategies to increase dystrophin production in Duchenne muscular dystrophy (DMD) and survival of motor neuron (SMN) production in spinal muscular atrophy (SMA). Areas covered: In this review, we focus on the current progress and challenges of exon-skipping and splice modulation therapies. In addition, we discuss the recent failure of the Phase III clinical trials of exon 51 skipping (drisapersen) for DMD. Expert opinion: The main approach of AON therapy in DMD and SMA is to rescue (‘knock up’ or increase) target proteins through exon skipping or exon inclusion; conversely, most conventional antisense drugs are designed to knock down (inhibit) the target. Encouraging preclinical data using this ‘knock up’ approach are also reported to rescue dysferlinopathies, including limb-girdle muscular dystrophy type 2B, Miyoshi myopathy, distal myopathy with anterior tibial onset and Fukuyama congenital muscular dystrophy. Keywords: antisense therapy, Duchenne muscular dystrophy, dysferlinopathy, exon skipping, Fukuyama congenital muscular dystrophy, limb-girdle muscular dystrophy 2B, Miyoshi myopathy, spinal muscular atrophy, splice modulation Expert Opin. Biol. Ther. [Early Online]
1.
Introduction
Antisense oligonucleotides (AONs) are synthetic single-stranded DNA-like molecules of variable length (between 15 and 35 nucleobases) that are useful in modulating gene expression [1]. AON-based therapy is a form of treatment for genetic disorders or infections that utilizes these short synthetic DNA-like molecules which are capable of hybridizing in a sequence-specific manner to pre-mRNA [1,2]. The aim of antisense therapeutics is to modulate splicing through the use of AONs. AONs can target pre-mRNA for RNase H-mediated degradation, translational arrest or the modulation of splicing (exon skipping or inclusion). AONs target consensus sequences on target pre-mRNA, preventing the interaction of the spliceosome complex with regions of AON/nucleobase hybridization, resulting in the modulation of splicing. Since the 1980s, Isis Pharmaceuticals, Inc. (Carlsbad, CA, USA) and other companies have focused on clinical applications of AON drugs. However, despite 10.1517/14712598.2014.896335 © 2014 Informa UK, Ltd. ISSN 1471-2598, e-ISSN 1744-7682 All rights reserved: reproduction in whole or in part not permitted
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Recent advancements in antisense oligonucleotide technology pave the way for clinical trials for neuromuscular diseases. New ‘Knock-up’ techniques using AONs allow for clinical trials of neuromuscular diseases that couldn’t be solved using other approaches. Skipping exons 45 -- 55 in DMD may provide an effective treatment that is applicable to ~ 45% of DMD patients. Modified AONs for treating SMA have been granted orphan drug status by the FDA and are currently being tested in Phase II clinical trials.
This box summarizes key points contained in the article.
early promise, the progress of AON drug development has been quite slow. There have been only two FDA-approved antisense drugs to date: fomivirsen sodium (Vitravene; Isis Pharmaceuticals, Inc.) for the treatment of cytomegalovirus retinitis in immunocompromised AIDS patients with HIV infection and Kynamro (Isis Pharmaceuticals, Carlsbad, CA, USA) for the treatment of familial hypercholesterolemia [1]. Importantly, these antisense drugs were designed to ‘knock down’ or reduce target gene expression. Antisense-mediated modulation of splicing is a newer application of AONs pioneered by Dr. Ryszard Kole and colleagues [3]. In contrast to the knock down approach, exon skipping and splice modulation are primarily designed to rescue (‘knock up’ or increase) the target proteins. In 1993, Dominski and Kole employed synthetic antisense 2¢-Omethylribooligonucleotides targeting the cryptic splice site in mutated human b-globin pre-mRNAs (responsible for b-thalassemias) to restore correct splicing of these RNAs in vitro [3]. Current antisense therapeutic approaches for neuromuscular diseases use various AON chemistries, including more novel molecular tools such as the phosphorodiamidate morpholino oligomers (PMOs), the vivo-morpholino (vPMO) and the 2¢O-methylphosphorothioate-modified (2¢OMePS) antisense oligo [4-8]. All AONs are distinct from typical nucleic acids in regard to the composition of their backbones and/or the chemical structures to which their nucleobases are attached. For example, the PMO or morpholino antisense oligo has a backbone formed from a phosphorodiamidate linkage as opposed to a phosphodiester linkage, whereas the nucleobases are attached to morpholine moieties as opposed to ribose/ deoxyribose rings [6]. The vPMOs are PMOs conjugated to octa-guanidine dendrimers which serve as cell-penetrating moieties, increasing the ability of these molecules to penetrate the cell membrane and reach their intracellular targets [6,7]. In terms of their pharmacokinetics, morpholinos have comparably short serum half-lives, owing to the high efficiency with which they are cleared from the kidney. The popular 2¢OMePS AON chemistry contains phosphorothioate linkages and 2¢ 2
modifications at the ribose ring. These phosphorothioate modifications impede kidney clearance, resulting in a longer serum half-life and more efficient tissue uptake. The safety and tolerability of this particular AON has been well characterized through several studies involving its potential therapeutic application for various diseases, especially in Duchenne muscular dystrophy (DMD) [9,10]. Current AONs have been engineered so as to surmount the various hurdles associated with this form of therapy. For example, antisense drug delivery was originally a significant problem, since AONs at that time could not easily penetrate the bilipid layer of the cell [11-13]. Other problems associated with AON therapy include toxicity, activated immune response, intracellular sequestration of oligos and the limited efficiency in silencing of pre-mRNA targets [14-17]. Thanks to their various chemical modifications, modern AONs differ sufficiently from endogenous nucleic acids that they are not easily recognized by DNA/RNA-binding proteins or nucleases, making them much more stable. Further, these modifications have led to reduced toxicity, increased cellular uptake and more efficient hybridization with target nucleic acids. Continued advancements in AON design will be essential in order to facilitate their widespread clinical application. In this review, we will conduct a detailed assessment of current advances in AON-based therapeutic treatments for several degenerative neuromuscular diseases, including DMD, spinal muscular atrophy (SMA), dysferlinopathies and Fukuyama congenital muscular dystrophy (FCMD). 2.
Duchenne muscular dystrophy
DMD is a lethal, recessive X-linked genetic disorder affecting about 1 in 3500 males worldwide [18,19]. Characteristics of the disease include progressive deterioration of the skeletal muscles which results in delayed motor milestones, proximal weakness, hypertrophied calves, markedly elevated serum creatine kinase levels and development of serious muscle weakness [20]. Patients soon develop wheelchair-dependency and the need for assisted ventilation. In addition to the wasting of the skeletal muscles, DMD is also hallmarked by progressive cardiomyopathy which presents clinically during early adolescence [21-24]. The majority of patients affected by DMD pass away during their late teens to mid-thirties. Mortality typically occurs due to cardiac problems or respiratory failure, although improvements in treating respiratory complications that are consequential to DMD, such as through the use of assisted ventilation, have significantly increased the survival of DMD patients [25]. DMD is mostly caused by deletion, duplication or nonsense mutations in the dystrophin (DMD) gene, resulting in an absence of functional dystrophin protein that is normally present in unaffected muscle fibers [26-28]. Small mutations, such as point mutations, splice site mutations and small intra-exon deletions remain difficult to identify. Dystrophin plays an essential role in maintaining muscle fiber integrity by linking
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Exon skipping and splice modulation therapies in neurology
the cytoskeleton of the muscle fiber and the extracellular matrix through the organization of a multiprotein complex known as the dystrophin-associated complex, located near the cell membrane [29-32]. Becker muscular dystrophy (BMD) is also caused by mutations in the DMD gene, but it is five times less frequent and is a milder form of the disease, characterized by an internally deleted (truncated) dystrophin protein that retains both functional ends [27]. BMD commonly arises from in-frame deletions, resulting in some missing exons but producing semi-functional dystrophin protein. The goal of antisense-mediated exon skipping in DMD is to restore the open reading frame by employing AONs to remove exons flanking deletions or to remove in-frame mutated exons to bypass the mutation (Figure 1) [26,33]. To varying extents, exon skipping has been observed to occur spontaneously in both DMD patients and in DMD animal models, resulting in the production of so-called dystrophin revertant fibers [34-36]. In 1996, Pramono et al. reported the first successful antisense-mediated exon 19 skipping in human DMD lymphoblastoid cells in vitro [37]. Mann et al. used 2¢OMePS AONs administered via intramuscular injection into the muscles of dystrophic mdx mice to induce the skipping of mutation-carrying exon 23 in vivo [38]. Systemic administration of AONs resulting in body-wide exon skipping and rescue of dystrophin expression was demonstrated by Lu et al. by using 2¢OMePS in mdx mice [5]. In addition, antisense PMOs induced therapeutically beneficial levels of dystrophin expression in body-wide skeletal muscles in dystrophic dogs [39,40]. Previously concluded and current DMD clinical trials use AON technology to influence exclusion of exons (exon 51 or 53) in the ‘mutation hot spot’, a region of the DMD gene located between exons 45 and 55 in which the majority (~ 63%) of deletion mutations among DMD patients occur [41], producing a truncated dystrophin protein which may retain partial functionality and could ameliorate the severe DMD dystrophic phenotype, as seen in mildly affected patients with BMD. Determining an effective AON-based treatment for most DMD cases has proven difficult because of the high genetic heterogeneity that has been recorded in DMD cases. However, the ‘hot spot’ region between exons 45 and 55 is a promising antisense target for treating the majority of DMD patients. Exon 51 skipping alone could theoretically treat approximately 13% of all cases [42]. Recently, a dose-ranging Phase II clinical study of a PMO (eteplirsen, also called AVI-4658 or B30) targeting exon 51 administered intravenously has been concluded. The results of these investigations have indicated an increase of functional dystrophin in biopsied muscle fibers and ambulatory stabilization as measured by the 6-min walk test in PMO-treated patients. Notably, in addition to the initially treated group, the placebo-controlled group eventually received PMO treatment, but after 24 weeks. Thus, this study compared early treatment versus later treatment, as opposed to treated versus non-treated. Therefore,
duration and not dosage was likely responsible for favorable outcomes [33,43]. The treatments were well tolerated by the patients, who did not display any serious adverse effects. Additionally, a clinical trial of exon 53 skipping using PMOs has been started by Nippon-Shinyaku Co. Ltd in 2013 [1]. Clinical trials for exon 44 and exon 45 skipping by Prosensa are ongoing as well. Although the PMO-based treatments show great potential for future exon skipping and splice modulation therapies for neuromuscular diseases, the recent failure of a Phase III clinical study by GlaxoSmithKline and Prosensa using their 2¢OMePS oligo drisapersen (also known as PRO051 and GSK2402968) may impede the development of such therapies, at least in the near future. This will be discussed in Section 7 below. 3.
Spinal muscular atrophy
SMA is a lethal, autosomal recessive neurodegenerative disorder affecting approximately 1 in 6000 to 1 in 8000 children. SMA patients show progressive impairment of motor neuron function that leads to loss of voluntary movement of the arms and legs and eventually death in early childhood. This impairment is caused by loss of motor neurons which die because they fail to produce sufficient quantities of the survival of motor neuron (SMN) protein. There are four described types of SMA, which are distinguished primarily by the motor milestones achieved by the patient, age of onset, and visible variations on the degree of severity [44]. Type I SMA (Werdnig-Hoffmann disease; acute infantile) is the most severe form of SMA. It affects infants usually between birth and the age of 6 months. SMA type I patients can be classified as non-sitters as they are unable to sit without support. Type II SMA (chronic infantile) is an intermediate form of the disease that manifests after the age of 6 months. Those affected are able to sit up, but phenotypically fall into the non-walker class as they cannot walk or even stand unaided. Type III SMA (Kugelberg-Welander disease; chronic juvenile) can be separated into 2 groups based on age of onset. Type IIIa manifests before the age of 3 and symptoms of type IIIb occur after 3 years of age. It is common for the symptoms to be present around the age of 18 months (after the child is ambulatory). Type IV SMA (adult onset) only shows mild symptoms of muscle weakness and manifests after the age of 30 [45,46]. SMA can be caused by a variety of intragenic deletions, point mutations, nonsense mutations and entire gene deletions of the SMN1 gene located on chromosome 5 [47,48]. SMN protein functions by forming an SMN complex which facilitates the assembly of spliceosomal small nuclear ribonucleoprotein particles [49]. The SMN gene is made up of a large, inverted duplication of a 500 kb element containing the telomeric SMN1 and the centromeric SMN2 which differ only by five nucleotides [50]. The SMN2 is a copy gene (nearly identical gene) of SMN1 and characterized as a major modifier of SMA severity. SMA type I patients have one or two SMN2 copies, but the majority of SMA type
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Restoration of dystrophin with exon 51 skipping therapy
Exon 52 deletion Pre-mRNA
51 53
51
50
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Antisense oligonucleotides
A deletion mutation in of exon 52 leads to out-of-frame mRNA
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Antisense oligos inhibit the splicing of exon 51, leading to skipping exons 51
STOP
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mRNA
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Exon 52 deletion causes frame-shifting. Dystrophin protein is not produced
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Skipping exons 51 restore the reading frame, leading to production of short form dystrophin
Protein
Figure 1. Schematic representation of exon 51 skipping therapy. A deletion mutation of exon 52 in the DMD gene leads to linkage of exons 51 and 53. This causes disruption of reading frame and results in stop signal in exon 53. An antisense oligo can promote skipping exon 51 and restore the reading frame by linking exons 50 and 53. In this scheme, the shape of each exon indicates phasing of each exon. For example, left end of exons: i) vertical line means that the exons begin by the first nucleotide of a codon; ii) arrows toward left mean that the exons begin with the second nucleotide of a codon; and iii) arrows toward right mean that the exon begin with the third nucleotide of a codon. In case the left end and right end fit together, it will be an in-frame mutation.
II patients have three SMN2 copies, and SMA type III patients have three or four copies of the SMN2 gene. Although the coding sequence is nearly identical, SMN2 contains a C-to-T transition on the 6th nucleotide of exon 7 which converts a splicing enhancer into a silencer motif. This change results in the exclusion of exon 7 in the majority of SMN2 mRNA transcripts, producing a non-functional version of SMN protein. Approximately 10% of SMN2 mRNA is capable of escaping the exon 7 exclusion and generates a full-length SMN2 transcript that produces the same protein product as the SMN1 mRNA transcript [51,52]. It is interesting to note that SMA patients retain at least one functional copy of the SMN2 gene, thereby making upregulation of the gene by promoting exon 7 inclusion a promising therapeutic approach [52]. Antisense PMOs that target splice silencing motifs have been used to successfully rescue SMA phenotypes in severe mouse models after intracerebroventricular injections (Figure 2) [53-55]. Even after a single injection, mice showed an increase in lifespan. In human clinical trials, Isis Pharmaceuticals is currently working on the development of a specific oligonucleotide called ISIS-SMNRx, which is designed to work against the splicing silencer in SMN2 to promote the inclusion of exon 7. ISIS-SMNRx is a uniformly 2¢-O-methoxyethyl-modified AON drug that corrects the splicing of SMN2 pre-mRNA, which results in production 4
of fully functional SMN protein. This AON chemistry was developed by Adrian Krainer’s laboratory and used in preclinical trials where it has been shown to notably improve the condition of a severe SMA mouse strain. It has since been granted orphan drug status by the FDA [56]. Isis Pharmaceuticals is now in collaboration with Biogen Indec, Inc., in developing this compound to treat all types of SMA. Phase I clinical trials have been completed and a Phase II study is now being conducted. This trial involves administration of multiple doses of ISIS-SMNRx, administered into the spinal fluid three times over the study’s duration, in patients with infantile-onset SMA. The purpose of this study is to test the safety, tolerability and pharmacokinetics of multiple dose treatments. So far, the drug appears to be well tolerated and treatment has resulted in a notable improvement of muscle function in some patients [57]. 4.
Dysferlin deficiency (dysferlinopathies)
Dysferlin is a ubiquitously expressed plasma membraneassociated protein encoded by the dysferlin (DYSF) gene [58-60]. DYSF is expressed at greater levels in cardiac and skeletal muscle where it plays an important role in vesicle trafficking and calcium-dependent muscle membrane repair via formation of a membrane patch, composed of recruited cytoplasmic
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Exon skipping and splice modulation therapies in neurology
Hypo-functional SMN2 gene
Restoration of SMN with exon inclusion therapy
Intronic splicing silencer site Antisense oligos Exon 6
Exon 7
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Exon 6
Exon 8
Exon 6
Exon 7
Exon 6
Exon 8
Hypo-functional protein
Exon 7
Exon 8
Exon 8
Functional protein
Figure 2. Exon 7 inclusion therapy for SMA with antisense oligos is shown. SMA is caused by a mutation in the SMN1 gene. The SMN2 gene is a paralogue of the SMN1 gene. However, a single nucleotide silence mutation in exon 7 interferes with an exonic splicing enhancer, leading to exon 7 skipping and production of hypo-functional SMN protein (left panel). AONs against the intronic splice silencer site (ISS) facilitate the inclusion of exon 7, leading to the production of functional fulllength SMN protein. AON: Antisense oligonucleotide; SMA: Spinal muscular atrophy; SMN: Survival of motor neuron.
vesicles [61,62]. The DYSF protein contains a C-terminal transmembrane domain, two DYSF domains and seven C2 domains which modulate protein and lipid binding [60,63-66]. Mutations in the DYSF gene are commonly associated with at least three autosomal recessive muscular dystrophies: Miyoshi myopathy (MM), limb-girdle muscular dystrophy type 2B (LGMD2B) and distal myopathy with anterior tibial onset (DMAT; also distal anterior compartment myopathy) [58,59,61,65,67-69]. Like all muscular dystrophies, dysferlinopathies are characterized by progressive muscle wasting, although they differ in terms of which muscle groups are initially affected. In MM, the distal muscles of the limb girdle are mostly affected, such as the gastrocnemius, whereas in LGMD2B, the proximal muscles of the lower limb girdle are initially affected [70,71]. DMAT presents similarly to MM, but with a different distribution of muscle weakness, commencing in the anterior tibial muscles and progressing to the posterior compartment [68,70]. Although such clinical phenotypic distinctions can exist, at least initially, dysferlinopathies manifest across a wide clinical spectrum and several unassigned variants have been described [72-74]. Notwithstanding such clinical heterogeneity, as the disease progresses, the spectrum of pathology narrows to the point that individual disorders are no longer easily distinguishable, with the disease eventually including both proximal and distal muscles. Age of onset is also highly variable, ranging from early teens to 70 years of age [75-77]. Benefiting from recent advances in AON design, as well as encouraging results from clinical trials in DMD patients, the use of AONs remains a promising therapeutic avenue for dysferlinopathies. Sinnreich et al. previously reported a female patient harboring a DYSF mutation resulting in the in-frame skipping of exon 32, who exhibited only a mild dystrophic
phenotype [78]. Following this, AON-mediated exon skipping was shown to effectively modulate mRNA splicing of DYSF exon 32 in vitro using patient-derived cells (Figure 3) [79]. Other groups have demonstrated that truncated DYSF constructs (called mini-DYSF), much like the shortened dystrophin products of exon skipping or gene therapy in DMD (minidystrophin), retained functionality and were able to rescue membrane repair in vitro and in vivo [80,81]. Further evidence supporting the idea that truncated DYSF is capable of retaining some functionality was found in another dysferlinopathy patient whose DYSF product was discovered to have only the last two C2 domains and the C-terminal transmembrane domain; yet, despite some muscle weakness, the patient was ambulatory without assistance at age 41 [82]. In the case of DMD, a large proportion of patients harboring deletion mutations have mutations in the range of exons 45 -- 55; this is known as the ‘mutational hot-spot’. Dysferlinopathy has no such ‘hot-spot’; therefore, future investigations aimed at identifying exons most amenable to exon skipping, those exons which are not essential to proper protein function, will be of great benefit. Since some dysferlinopathy patients with very mild dystrophic phenotypes have presented as lacking one or more C2 domains, it is believed that some of these domains may be functionally redundant and could therefore be possible targets of exon skipping. For example, the mildly affected patient reported by Sinnreich et al. was missing the fourth C2 domain due to the in-frame skipping of exon 32 -- a consequence of a lariat branch point mutation [78]. Not all of the DYSF C2 domains may be appropriate for targeted exon skipping. The dysferlinopathy mouse model SJL/J displays a severe phenotype despite the in-frame skipping of exon 45, which results in a partial deletion of the terminal C2 domain [83,84].
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Restoration of dysferlin with exon 32 skipping therapy Exon 52 nonsense mutation Pre-mRNA
STOP
STOP 30
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32 33
A nonsense mutation in exon 32 leads to a premature stop codon
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33 Antisense oligonucleotides Antisense oligos inhibit the splicing of exon 32, leading to skipping exons 32
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STOP
mRNA
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31
32
33
Exon 32 nonsense mutation causes a stop signal. Dysferlin protein is not produced
30
31
33
Skipping exons 32 restore the reading frame, leading to production of short form dysferlin
Protein
Figure 3. Exon 32 skipping therapy for dysferlin (DYSF) deficiency is shown. A nonsense mutation in exon 32 in the DYSF gene leads to a premature stop codon. This causes disruption of reading frame and results in stop signal in exon 32. An antisense oligo can skip exon 32 and restore the reading frame by linking exons 31 and 33.
In her report, Aartsma-Rus prioritizes the most appropriate DYSF exons for exon skipping therapy based on exons which either have confirmed redundancy, resulting in in-frame translational shifts, or are not implicated in splice site aberrations or otherwise associated with a pathological outcome [33]. Currently, there are no ongoing or pending clinical trials for AON-mediated exon skipping in dysferlinopathy. 5.
Fukuyama congenital muscular dystrophy
FCMD is a form of muscular dystrophy with a high prevalence in Japan (having an incidence of about 1 in 10,000 births), which follows an autosomal recessive pattern of inheritance and exhibits cerebral cortical dysplasia as well as progressive myopathy [85,86]. Symptoms of FCMD typically manifest before 9 months of age as hypotonia and muscle weakness, with infants exhibiting a significant delay in reaching motor development milestones [87]. The upper proximal and lower distal muscles are most affected, and unlike DMD patients, who can maintain limited mobility through childhood, the majority of FCMD patients never walk [88]. The FCMD gene is located on chromosome 9 and encodes the 461-amino acid protein fukutin, a ubiquitously expressed protein which is thought to act as a glycosyltransferase, adding sugar residues to a-dystroglycan, a member of the dystrophinassociated protein complex [89,90]. Fukutin is also believed to be an extracellular protein, located in the extracellular matrix, 6
with potential involvement in maintaining muscle membrane integrity and proper function of the basement membrane in neural tissues [91]. Approximately 87% of affected individuals share a haplotype attributed to a single founder, containing a 3-kbp SINE-VNTR-Alu retrotransposal tandem repeat insertion in the 3¢-untranslated region of the FCMD gene which interferes with proper mRNA splicing and results in the disease phenotype [86,92-94]. This retrotransposal insertion is believed to have originated between 2000 and 2500 years ago during a time when Japan’s indigenous Jomon people mixed with the immigrant Yayoi people of eastern China [87]. Whether the FCMD mutation was originally introduced in Japan as a direct result of migration or whether the mutation originated in the ancient Japanese population has yet to be determined [86]. The molecular mechanism behind FCMD pathology is the induced mRNA splicing errors (exon trapping) generated by a novel splice donor site in exon 10 and a splice acceptor in the retrotransposal insertion which results in the truncation of exon 10 [94]. A therapeutic approach utilizing multiple next-generation AONs (cell-penetrating octa-guanidine dendrimer-conjugated vivo-morpholinos) targeted against exonic and intronic splice enhancer sites was successfully used to restore normal fukutin mRNA and protein in vivo using a murine model, as well as in vitro using human patient cells (Figure 4) [94]. To date, this is the first and the only demonstration of AON-mediated splice modulation therapy as a possible clinical treatment for FCMD;
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Exon skipping and splice modulation therapies in neurology
Exons Normal fukutin 6
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10
1. Fukuyama dystrophy patients New splice donor 6
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3′UTR SVA retrotransposon
3′UTR
Spliced out (exon-trapping)
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New splice accepter
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1 SVA retrotransposon 0
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Aberrant splicing and short exon 10 2. Antisense cocktail treatment
D5 (Intronic splice enhancer) 6
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3′UTR
A3 E3 (exonic splice enhancer)
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Restoration of normal splicing Not to scale
Figure 4. Strategy of antisense therapy for Fukuyama congenital muscular dystrophy (FCMD) is shown. Retrotransposon insertion in the fukutin gene leads to aberrant splicing. A cocktail antisense morpholino injection targeting three splice enhancer sites can rescue the abnormal splicing in FCMD.
no AON-based clinical trials are currently planned for the treatment of FCMD. 6.
Conclusion
Antisense-mediated exon skipping and splice modulation are promising therapeutic approaches for treating neuromuscular diseases. Here, we reviewed the recent progress and challenges of exon skipping and splice modulation therapies for DMD, SMA, dysferlinopathies and FCMD. The recent data from the Phase II DMD clinical trials by Sarepta Therapeutics, Inc. are very promising. Isis Pharmaceuticals has recently started Phase II clinical trials of an AON therapy for SMA. Antisense drugs against FCMD are still in the preclinical stage but showed promising results in an animal model and in human cells. In vitro studies have revealed that dysferlinopathies are also promising targets for exon skipping therapy. In contrast to conventional knockdown strategies, exon skipping and splice modulation therapies are designed to rescue (knock up or increase) the target. It is reported that the knock-up approach is effective at lower efficiencies than a knockdown approach [14]. In a knockdown approach, the goal is typically
to knockdown 90% of the mRNA target to bring protein expression down to 10%. In contrast, for exon skipping in DMD, the goal is to restore protein expression levels to 10%, although the exact efficacy required for SMA, dysferlinopathies and FCMD are currently not well understood [14]. With the current progress of antisense research, new AON drugs for the widespread application of exon skipping and splice modulation therapies will likely be forthcoming. 7.
Expert opinion
Although promising, exon-skipping therapy for DMD is currently at a critical moment. In February 2014, Sarepta Therapeutics announced that their lead candidate drug, eteplirsen, a PMO-based AON targeting exon 51, was successful in demonstrating stability on pulmonary function tests through a 120-week Phase IIb open-label extension study. These findings follow in the wake of previously reported 120-week data which demonstrated stabilization during the 6-min walk test in treated patients. No clinically significant adverse reactions have been reported through week 120.
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Notwithstanding these promising early results, it remains to be seen whether eteplirsen can produce significant clinical benefits over a longer period of time. In September 2013, Prosensa and GSK announced that drisapersen, an exon 51-skipping antisense drug using 2¢OMePS chemistry, did not show statistically significant improvements in the primary outcome measure of the 6-min walking distance test in a Phase III clinical trial, which was carried out with 186 patients [95]. Despite failing to reach the primary end point of this particular clinical trial, drisapersen has had previous successes in other Phase II clinical trials, demonstrating safety and tolerability, novel dystrophin expression in biopsied muscle fibers and improvements in the 6-min walking distance test after 12 weeks of treatment [9,10]. There are plenty of lessons to be learned from these most recent clinical trials. First, a controversy exists over the optimal target of the exon-skipping approach [96]. In terms of the incidence of BMD, other exons such as exons 45, 53 or 55 could be more promising targets [97]. In addition, testing of a multi-exonskipping method (e.g., exons 45 -- 55 skipping), which is expected to lead to milder phenotypes versus the more traditional single exon-skipping approach, is currently underway [41,97]. Remaining challenges include the need to mix 11 AONs, safety and regulatory concerns and the fact that they currently have been shown to work only in mouse models. Second, the efficiency of exon skipping with 2¢OMePS is reported to be lower than that of PMOs in vivo [4,39]. Importantly, the development of new generation antisense oligos (e.g., peptide-conjugated antisense oligos) is ongoing and may augment future AON-based therapies by providing novel, highly effective AON chemistries [98,99]. Third, the efficiency of exon skipping via subcutaneous administration is reported to be 40 -- 80% lower than that achieved via intravenous
administration in the mouse model [4]. Finally, although the 6-min walk test has been shown to be a reliable assessment and is the current standard in clinical assessment, some question its consistency in representing significant clinical outcome [95]. The previous open-label, Phase II extension trial of drisapersen showed stabilization of disease progression measured by serial tests of the 6-min walk test; however, comparing the Phase II and III data, the impressive stabilization of the walking distance previously demonstrated may have been influenced by a training effect of the outcome measure or a placebo effect. Importantly, the Phase II trial was an unblinded open-label study with no control group, and only low levels of dystrophin were detected in muscle biopsies. To date, the interpretation of clinical end points has been challenging. Taken together, exon-skipping therapy that targets different exons, different AON chemistries and/or intravenous administration of antisense drugs may be more effective in producing favorable clinical results for the treatment of DMD.
Declaration of interest This work was supported by the University of Alberta Faculty of Medicine and Dentistry, Parent Project Muscular Dystrophy (USA), The Friends of Garrett Cumming Research Funds, HM Toupin Neurological Science Research Funds, Muscular Dystrophy Canada, Canada Foundation for Innovation, Alberta Enterprise and Advanced Education, the Women and Children’s Health Research Institute and Canadian Institute of Health Research. T Yokota has been and is currently the principal investigator of federal grants aiding the development of morpholino chemistry for exon skipping. The remaining authors have no competing interests to declare.
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Expert Opin. Biol. Ther. (2014) 14(6)
Affiliation Aleksander Touznik1, Joshua JA Lee1 & Toshifumi Yokota†1,2 PhD † Author for correspondence 1 Assistant Professor and Muscular Dystrophy Canada Research Chair, University of Alberta, Faculty of Medicine and Dentistry, Department of Medical Genetics, Edmonton, Alberta, Canada 2 University of Alberta, Faculty of Medicine and Dentistry, Department of Medical Genetics, 829 Medical Sciences Building, Edmonton, Alberta, T6G 2H7, Canada Tel: +780 492 1102; Fax: +780 492 1998; E-mail: [email protected]
11
1.
Introduction
2.
Duchenne muscular dystrophy
3.
Spinal muscular atrophy
Aleksander Touznik, Joshua JA Lee & Toshifumi Yokota†
4.
Dysferlin deficiency
University of Alberta, Faculty of Medicine and Dentistry, Department of Medical Genetics, Alberta, Canada
(dysferlinopathies)
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New developments in exon skipping and splice modulation therapies for neuromuscular diseases
5.
Fukuyama congenital muscular dystrophy
6.
Conclusion
7.
Expert opinion
Introduction: Antisense oligonucleotide (AON) therapy is a form of treatment for genetic or infectious diseases using small, synthetic DNA-like molecules called AONs. Recent advances in the development of AONs that show improved stability and increased sequence specificity have led to clinical trials for several neuromuscular diseases. Impressive preclinical and clinical data are published regarding the usage of AONs in exon-skipping and splice modulation strategies to increase dystrophin production in Duchenne muscular dystrophy (DMD) and survival of motor neuron (SMN) production in spinal muscular atrophy (SMA). Areas covered: In this review, we focus on the current progress and challenges of exon-skipping and splice modulation therapies. In addition, we discuss the recent failure of the Phase III clinical trials of exon 51 skipping (drisapersen) for DMD. Expert opinion: The main approach of AON therapy in DMD and SMA is to rescue (‘knock up’ or increase) target proteins through exon skipping or exon inclusion; conversely, most conventional antisense drugs are designed to knock down (inhibit) the target. Encouraging preclinical data using this ‘knock up’ approach are also reported to rescue dysferlinopathies, including limb-girdle muscular dystrophy type 2B, Miyoshi myopathy, distal myopathy with anterior tibial onset and Fukuyama congenital muscular dystrophy. Keywords: antisense therapy, Duchenne muscular dystrophy, dysferlinopathy, exon skipping, Fukuyama congenital muscular dystrophy, limb-girdle muscular dystrophy 2B, Miyoshi myopathy, spinal muscular atrophy, splice modulation Expert Opin. Biol. Ther. [Early Online]
1.
Introduction
Antisense oligonucleotides (AONs) are synthetic single-stranded DNA-like molecules of variable length (between 15 and 35 nucleobases) that are useful in modulating gene expression [1]. AON-based therapy is a form of treatment for genetic disorders or infections that utilizes these short synthetic DNA-like molecules which are capable of hybridizing in a sequence-specific manner to pre-mRNA [1,2]. The aim of antisense therapeutics is to modulate splicing through the use of AONs. AONs can target pre-mRNA for RNase H-mediated degradation, translational arrest or the modulation of splicing (exon skipping or inclusion). AONs target consensus sequences on target pre-mRNA, preventing the interaction of the spliceosome complex with regions of AON/nucleobase hybridization, resulting in the modulation of splicing. Since the 1980s, Isis Pharmaceuticals, Inc. (Carlsbad, CA, USA) and other companies have focused on clinical applications of AON drugs. However, despite 10.1517/14712598.2014.896335 © 2014 Informa UK, Ltd. ISSN 1471-2598, e-ISSN 1744-7682 All rights reserved: reproduction in whole or in part not permitted
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Recent advancements in antisense oligonucleotide technology pave the way for clinical trials for neuromuscular diseases. New ‘Knock-up’ techniques using AONs allow for clinical trials of neuromuscular diseases that couldn’t be solved using other approaches. Skipping exons 45 -- 55 in DMD may provide an effective treatment that is applicable to ~ 45% of DMD patients. Modified AONs for treating SMA have been granted orphan drug status by the FDA and are currently being tested in Phase II clinical trials.
This box summarizes key points contained in the article.
early promise, the progress of AON drug development has been quite slow. There have been only two FDA-approved antisense drugs to date: fomivirsen sodium (Vitravene; Isis Pharmaceuticals, Inc.) for the treatment of cytomegalovirus retinitis in immunocompromised AIDS patients with HIV infection and Kynamro (Isis Pharmaceuticals, Carlsbad, CA, USA) for the treatment of familial hypercholesterolemia [1]. Importantly, these antisense drugs were designed to ‘knock down’ or reduce target gene expression. Antisense-mediated modulation of splicing is a newer application of AONs pioneered by Dr. Ryszard Kole and colleagues [3]. In contrast to the knock down approach, exon skipping and splice modulation are primarily designed to rescue (‘knock up’ or increase) the target proteins. In 1993, Dominski and Kole employed synthetic antisense 2¢-Omethylribooligonucleotides targeting the cryptic splice site in mutated human b-globin pre-mRNAs (responsible for b-thalassemias) to restore correct splicing of these RNAs in vitro [3]. Current antisense therapeutic approaches for neuromuscular diseases use various AON chemistries, including more novel molecular tools such as the phosphorodiamidate morpholino oligomers (PMOs), the vivo-morpholino (vPMO) and the 2¢O-methylphosphorothioate-modified (2¢OMePS) antisense oligo [4-8]. All AONs are distinct from typical nucleic acids in regard to the composition of their backbones and/or the chemical structures to which their nucleobases are attached. For example, the PMO or morpholino antisense oligo has a backbone formed from a phosphorodiamidate linkage as opposed to a phosphodiester linkage, whereas the nucleobases are attached to morpholine moieties as opposed to ribose/ deoxyribose rings [6]. The vPMOs are PMOs conjugated to octa-guanidine dendrimers which serve as cell-penetrating moieties, increasing the ability of these molecules to penetrate the cell membrane and reach their intracellular targets [6,7]. In terms of their pharmacokinetics, morpholinos have comparably short serum half-lives, owing to the high efficiency with which they are cleared from the kidney. The popular 2¢OMePS AON chemistry contains phosphorothioate linkages and 2¢ 2
modifications at the ribose ring. These phosphorothioate modifications impede kidney clearance, resulting in a longer serum half-life and more efficient tissue uptake. The safety and tolerability of this particular AON has been well characterized through several studies involving its potential therapeutic application for various diseases, especially in Duchenne muscular dystrophy (DMD) [9,10]. Current AONs have been engineered so as to surmount the various hurdles associated with this form of therapy. For example, antisense drug delivery was originally a significant problem, since AONs at that time could not easily penetrate the bilipid layer of the cell [11-13]. Other problems associated with AON therapy include toxicity, activated immune response, intracellular sequestration of oligos and the limited efficiency in silencing of pre-mRNA targets [14-17]. Thanks to their various chemical modifications, modern AONs differ sufficiently from endogenous nucleic acids that they are not easily recognized by DNA/RNA-binding proteins or nucleases, making them much more stable. Further, these modifications have led to reduced toxicity, increased cellular uptake and more efficient hybridization with target nucleic acids. Continued advancements in AON design will be essential in order to facilitate their widespread clinical application. In this review, we will conduct a detailed assessment of current advances in AON-based therapeutic treatments for several degenerative neuromuscular diseases, including DMD, spinal muscular atrophy (SMA), dysferlinopathies and Fukuyama congenital muscular dystrophy (FCMD). 2.
Duchenne muscular dystrophy
DMD is a lethal, recessive X-linked genetic disorder affecting about 1 in 3500 males worldwide [18,19]. Characteristics of the disease include progressive deterioration of the skeletal muscles which results in delayed motor milestones, proximal weakness, hypertrophied calves, markedly elevated serum creatine kinase levels and development of serious muscle weakness [20]. Patients soon develop wheelchair-dependency and the need for assisted ventilation. In addition to the wasting of the skeletal muscles, DMD is also hallmarked by progressive cardiomyopathy which presents clinically during early adolescence [21-24]. The majority of patients affected by DMD pass away during their late teens to mid-thirties. Mortality typically occurs due to cardiac problems or respiratory failure, although improvements in treating respiratory complications that are consequential to DMD, such as through the use of assisted ventilation, have significantly increased the survival of DMD patients [25]. DMD is mostly caused by deletion, duplication or nonsense mutations in the dystrophin (DMD) gene, resulting in an absence of functional dystrophin protein that is normally present in unaffected muscle fibers [26-28]. Small mutations, such as point mutations, splice site mutations and small intra-exon deletions remain difficult to identify. Dystrophin plays an essential role in maintaining muscle fiber integrity by linking
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Exon skipping and splice modulation therapies in neurology
the cytoskeleton of the muscle fiber and the extracellular matrix through the organization of a multiprotein complex known as the dystrophin-associated complex, located near the cell membrane [29-32]. Becker muscular dystrophy (BMD) is also caused by mutations in the DMD gene, but it is five times less frequent and is a milder form of the disease, characterized by an internally deleted (truncated) dystrophin protein that retains both functional ends [27]. BMD commonly arises from in-frame deletions, resulting in some missing exons but producing semi-functional dystrophin protein. The goal of antisense-mediated exon skipping in DMD is to restore the open reading frame by employing AONs to remove exons flanking deletions or to remove in-frame mutated exons to bypass the mutation (Figure 1) [26,33]. To varying extents, exon skipping has been observed to occur spontaneously in both DMD patients and in DMD animal models, resulting in the production of so-called dystrophin revertant fibers [34-36]. In 1996, Pramono et al. reported the first successful antisense-mediated exon 19 skipping in human DMD lymphoblastoid cells in vitro [37]. Mann et al. used 2¢OMePS AONs administered via intramuscular injection into the muscles of dystrophic mdx mice to induce the skipping of mutation-carrying exon 23 in vivo [38]. Systemic administration of AONs resulting in body-wide exon skipping and rescue of dystrophin expression was demonstrated by Lu et al. by using 2¢OMePS in mdx mice [5]. In addition, antisense PMOs induced therapeutically beneficial levels of dystrophin expression in body-wide skeletal muscles in dystrophic dogs [39,40]. Previously concluded and current DMD clinical trials use AON technology to influence exclusion of exons (exon 51 or 53) in the ‘mutation hot spot’, a region of the DMD gene located between exons 45 and 55 in which the majority (~ 63%) of deletion mutations among DMD patients occur [41], producing a truncated dystrophin protein which may retain partial functionality and could ameliorate the severe DMD dystrophic phenotype, as seen in mildly affected patients with BMD. Determining an effective AON-based treatment for most DMD cases has proven difficult because of the high genetic heterogeneity that has been recorded in DMD cases. However, the ‘hot spot’ region between exons 45 and 55 is a promising antisense target for treating the majority of DMD patients. Exon 51 skipping alone could theoretically treat approximately 13% of all cases [42]. Recently, a dose-ranging Phase II clinical study of a PMO (eteplirsen, also called AVI-4658 or B30) targeting exon 51 administered intravenously has been concluded. The results of these investigations have indicated an increase of functional dystrophin in biopsied muscle fibers and ambulatory stabilization as measured by the 6-min walk test in PMO-treated patients. Notably, in addition to the initially treated group, the placebo-controlled group eventually received PMO treatment, but after 24 weeks. Thus, this study compared early treatment versus later treatment, as opposed to treated versus non-treated. Therefore,
duration and not dosage was likely responsible for favorable outcomes [33,43]. The treatments were well tolerated by the patients, who did not display any serious adverse effects. Additionally, a clinical trial of exon 53 skipping using PMOs has been started by Nippon-Shinyaku Co. Ltd in 2013 [1]. Clinical trials for exon 44 and exon 45 skipping by Prosensa are ongoing as well. Although the PMO-based treatments show great potential for future exon skipping and splice modulation therapies for neuromuscular diseases, the recent failure of a Phase III clinical study by GlaxoSmithKline and Prosensa using their 2¢OMePS oligo drisapersen (also known as PRO051 and GSK2402968) may impede the development of such therapies, at least in the near future. This will be discussed in Section 7 below. 3.
Spinal muscular atrophy
SMA is a lethal, autosomal recessive neurodegenerative disorder affecting approximately 1 in 6000 to 1 in 8000 children. SMA patients show progressive impairment of motor neuron function that leads to loss of voluntary movement of the arms and legs and eventually death in early childhood. This impairment is caused by loss of motor neurons which die because they fail to produce sufficient quantities of the survival of motor neuron (SMN) protein. There are four described types of SMA, which are distinguished primarily by the motor milestones achieved by the patient, age of onset, and visible variations on the degree of severity [44]. Type I SMA (Werdnig-Hoffmann disease; acute infantile) is the most severe form of SMA. It affects infants usually between birth and the age of 6 months. SMA type I patients can be classified as non-sitters as they are unable to sit without support. Type II SMA (chronic infantile) is an intermediate form of the disease that manifests after the age of 6 months. Those affected are able to sit up, but phenotypically fall into the non-walker class as they cannot walk or even stand unaided. Type III SMA (Kugelberg-Welander disease; chronic juvenile) can be separated into 2 groups based on age of onset. Type IIIa manifests before the age of 3 and symptoms of type IIIb occur after 3 years of age. It is common for the symptoms to be present around the age of 18 months (after the child is ambulatory). Type IV SMA (adult onset) only shows mild symptoms of muscle weakness and manifests after the age of 30 [45,46]. SMA can be caused by a variety of intragenic deletions, point mutations, nonsense mutations and entire gene deletions of the SMN1 gene located on chromosome 5 [47,48]. SMN protein functions by forming an SMN complex which facilitates the assembly of spliceosomal small nuclear ribonucleoprotein particles [49]. The SMN gene is made up of a large, inverted duplication of a 500 kb element containing the telomeric SMN1 and the centromeric SMN2 which differ only by five nucleotides [50]. The SMN2 is a copy gene (nearly identical gene) of SMN1 and characterized as a major modifier of SMA severity. SMA type I patients have one or two SMN2 copies, but the majority of SMA type
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Exon 52 deletion Pre-mRNA
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Figure 1. Schematic representation of exon 51 skipping therapy. A deletion mutation of exon 52 in the DMD gene leads to linkage of exons 51 and 53. This causes disruption of reading frame and results in stop signal in exon 53. An antisense oligo can promote skipping exon 51 and restore the reading frame by linking exons 50 and 53. In this scheme, the shape of each exon indicates phasing of each exon. For example, left end of exons: i) vertical line means that the exons begin by the first nucleotide of a codon; ii) arrows toward left mean that the exons begin with the second nucleotide of a codon; and iii) arrows toward right mean that the exon begin with the third nucleotide of a codon. In case the left end and right end fit together, it will be an in-frame mutation.
II patients have three SMN2 copies, and SMA type III patients have three or four copies of the SMN2 gene. Although the coding sequence is nearly identical, SMN2 contains a C-to-T transition on the 6th nucleotide of exon 7 which converts a splicing enhancer into a silencer motif. This change results in the exclusion of exon 7 in the majority of SMN2 mRNA transcripts, producing a non-functional version of SMN protein. Approximately 10% of SMN2 mRNA is capable of escaping the exon 7 exclusion and generates a full-length SMN2 transcript that produces the same protein product as the SMN1 mRNA transcript [51,52]. It is interesting to note that SMA patients retain at least one functional copy of the SMN2 gene, thereby making upregulation of the gene by promoting exon 7 inclusion a promising therapeutic approach [52]. Antisense PMOs that target splice silencing motifs have been used to successfully rescue SMA phenotypes in severe mouse models after intracerebroventricular injections (Figure 2) [53-55]. Even after a single injection, mice showed an increase in lifespan. In human clinical trials, Isis Pharmaceuticals is currently working on the development of a specific oligonucleotide called ISIS-SMNRx, which is designed to work against the splicing silencer in SMN2 to promote the inclusion of exon 7. ISIS-SMNRx is a uniformly 2¢-O-methoxyethyl-modified AON drug that corrects the splicing of SMN2 pre-mRNA, which results in production 4
of fully functional SMN protein. This AON chemistry was developed by Adrian Krainer’s laboratory and used in preclinical trials where it has been shown to notably improve the condition of a severe SMA mouse strain. It has since been granted orphan drug status by the FDA [56]. Isis Pharmaceuticals is now in collaboration with Biogen Indec, Inc., in developing this compound to treat all types of SMA. Phase I clinical trials have been completed and a Phase II study is now being conducted. This trial involves administration of multiple doses of ISIS-SMNRx, administered into the spinal fluid three times over the study’s duration, in patients with infantile-onset SMA. The purpose of this study is to test the safety, tolerability and pharmacokinetics of multiple dose treatments. So far, the drug appears to be well tolerated and treatment has resulted in a notable improvement of muscle function in some patients [57]. 4.
Dysferlin deficiency (dysferlinopathies)
Dysferlin is a ubiquitously expressed plasma membraneassociated protein encoded by the dysferlin (DYSF) gene [58-60]. DYSF is expressed at greater levels in cardiac and skeletal muscle where it plays an important role in vesicle trafficking and calcium-dependent muscle membrane repair via formation of a membrane patch, composed of recruited cytoplasmic
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Hypo-functional SMN2 gene
Restoration of SMN with exon inclusion therapy
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Exon 6
Exon 8
Exon 6
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Exon 6
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Figure 2. Exon 7 inclusion therapy for SMA with antisense oligos is shown. SMA is caused by a mutation in the SMN1 gene. The SMN2 gene is a paralogue of the SMN1 gene. However, a single nucleotide silence mutation in exon 7 interferes with an exonic splicing enhancer, leading to exon 7 skipping and production of hypo-functional SMN protein (left panel). AONs against the intronic splice silencer site (ISS) facilitate the inclusion of exon 7, leading to the production of functional fulllength SMN protein. AON: Antisense oligonucleotide; SMA: Spinal muscular atrophy; SMN: Survival of motor neuron.
vesicles [61,62]. The DYSF protein contains a C-terminal transmembrane domain, two DYSF domains and seven C2 domains which modulate protein and lipid binding [60,63-66]. Mutations in the DYSF gene are commonly associated with at least three autosomal recessive muscular dystrophies: Miyoshi myopathy (MM), limb-girdle muscular dystrophy type 2B (LGMD2B) and distal myopathy with anterior tibial onset (DMAT; also distal anterior compartment myopathy) [58,59,61,65,67-69]. Like all muscular dystrophies, dysferlinopathies are characterized by progressive muscle wasting, although they differ in terms of which muscle groups are initially affected. In MM, the distal muscles of the limb girdle are mostly affected, such as the gastrocnemius, whereas in LGMD2B, the proximal muscles of the lower limb girdle are initially affected [70,71]. DMAT presents similarly to MM, but with a different distribution of muscle weakness, commencing in the anterior tibial muscles and progressing to the posterior compartment [68,70]. Although such clinical phenotypic distinctions can exist, at least initially, dysferlinopathies manifest across a wide clinical spectrum and several unassigned variants have been described [72-74]. Notwithstanding such clinical heterogeneity, as the disease progresses, the spectrum of pathology narrows to the point that individual disorders are no longer easily distinguishable, with the disease eventually including both proximal and distal muscles. Age of onset is also highly variable, ranging from early teens to 70 years of age [75-77]. Benefiting from recent advances in AON design, as well as encouraging results from clinical trials in DMD patients, the use of AONs remains a promising therapeutic avenue for dysferlinopathies. Sinnreich et al. previously reported a female patient harboring a DYSF mutation resulting in the in-frame skipping of exon 32, who exhibited only a mild dystrophic
phenotype [78]. Following this, AON-mediated exon skipping was shown to effectively modulate mRNA splicing of DYSF exon 32 in vitro using patient-derived cells (Figure 3) [79]. Other groups have demonstrated that truncated DYSF constructs (called mini-DYSF), much like the shortened dystrophin products of exon skipping or gene therapy in DMD (minidystrophin), retained functionality and were able to rescue membrane repair in vitro and in vivo [80,81]. Further evidence supporting the idea that truncated DYSF is capable of retaining some functionality was found in another dysferlinopathy patient whose DYSF product was discovered to have only the last two C2 domains and the C-terminal transmembrane domain; yet, despite some muscle weakness, the patient was ambulatory without assistance at age 41 [82]. In the case of DMD, a large proportion of patients harboring deletion mutations have mutations in the range of exons 45 -- 55; this is known as the ‘mutational hot-spot’. Dysferlinopathy has no such ‘hot-spot’; therefore, future investigations aimed at identifying exons most amenable to exon skipping, those exons which are not essential to proper protein function, will be of great benefit. Since some dysferlinopathy patients with very mild dystrophic phenotypes have presented as lacking one or more C2 domains, it is believed that some of these domains may be functionally redundant and could therefore be possible targets of exon skipping. For example, the mildly affected patient reported by Sinnreich et al. was missing the fourth C2 domain due to the in-frame skipping of exon 32 -- a consequence of a lariat branch point mutation [78]. Not all of the DYSF C2 domains may be appropriate for targeted exon skipping. The dysferlinopathy mouse model SJL/J displays a severe phenotype despite the in-frame skipping of exon 45, which results in a partial deletion of the terminal C2 domain [83,84].
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Restoration of dysferlin with exon 32 skipping therapy Exon 52 nonsense mutation Pre-mRNA
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Exon 32 nonsense mutation causes a stop signal. Dysferlin protein is not produced
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Skipping exons 32 restore the reading frame, leading to production of short form dysferlin
Protein
Figure 3. Exon 32 skipping therapy for dysferlin (DYSF) deficiency is shown. A nonsense mutation in exon 32 in the DYSF gene leads to a premature stop codon. This causes disruption of reading frame and results in stop signal in exon 32. An antisense oligo can skip exon 32 and restore the reading frame by linking exons 31 and 33.
In her report, Aartsma-Rus prioritizes the most appropriate DYSF exons for exon skipping therapy based on exons which either have confirmed redundancy, resulting in in-frame translational shifts, or are not implicated in splice site aberrations or otherwise associated with a pathological outcome [33]. Currently, there are no ongoing or pending clinical trials for AON-mediated exon skipping in dysferlinopathy. 5.
Fukuyama congenital muscular dystrophy
FCMD is a form of muscular dystrophy with a high prevalence in Japan (having an incidence of about 1 in 10,000 births), which follows an autosomal recessive pattern of inheritance and exhibits cerebral cortical dysplasia as well as progressive myopathy [85,86]. Symptoms of FCMD typically manifest before 9 months of age as hypotonia and muscle weakness, with infants exhibiting a significant delay in reaching motor development milestones [87]. The upper proximal and lower distal muscles are most affected, and unlike DMD patients, who can maintain limited mobility through childhood, the majority of FCMD patients never walk [88]. The FCMD gene is located on chromosome 9 and encodes the 461-amino acid protein fukutin, a ubiquitously expressed protein which is thought to act as a glycosyltransferase, adding sugar residues to a-dystroglycan, a member of the dystrophinassociated protein complex [89,90]. Fukutin is also believed to be an extracellular protein, located in the extracellular matrix, 6
with potential involvement in maintaining muscle membrane integrity and proper function of the basement membrane in neural tissues [91]. Approximately 87% of affected individuals share a haplotype attributed to a single founder, containing a 3-kbp SINE-VNTR-Alu retrotransposal tandem repeat insertion in the 3¢-untranslated region of the FCMD gene which interferes with proper mRNA splicing and results in the disease phenotype [86,92-94]. This retrotransposal insertion is believed to have originated between 2000 and 2500 years ago during a time when Japan’s indigenous Jomon people mixed with the immigrant Yayoi people of eastern China [87]. Whether the FCMD mutation was originally introduced in Japan as a direct result of migration or whether the mutation originated in the ancient Japanese population has yet to be determined [86]. The molecular mechanism behind FCMD pathology is the induced mRNA splicing errors (exon trapping) generated by a novel splice donor site in exon 10 and a splice acceptor in the retrotransposal insertion which results in the truncation of exon 10 [94]. A therapeutic approach utilizing multiple next-generation AONs (cell-penetrating octa-guanidine dendrimer-conjugated vivo-morpholinos) targeted against exonic and intronic splice enhancer sites was successfully used to restore normal fukutin mRNA and protein in vivo using a murine model, as well as in vitro using human patient cells (Figure 4) [94]. To date, this is the first and the only demonstration of AON-mediated splice modulation therapy as a possible clinical treatment for FCMD;
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Exons Normal fukutin 6
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Restoration of normal splicing Not to scale
Figure 4. Strategy of antisense therapy for Fukuyama congenital muscular dystrophy (FCMD) is shown. Retrotransposon insertion in the fukutin gene leads to aberrant splicing. A cocktail antisense morpholino injection targeting three splice enhancer sites can rescue the abnormal splicing in FCMD.
no AON-based clinical trials are currently planned for the treatment of FCMD. 6.
Conclusion
Antisense-mediated exon skipping and splice modulation are promising therapeutic approaches for treating neuromuscular diseases. Here, we reviewed the recent progress and challenges of exon skipping and splice modulation therapies for DMD, SMA, dysferlinopathies and FCMD. The recent data from the Phase II DMD clinical trials by Sarepta Therapeutics, Inc. are very promising. Isis Pharmaceuticals has recently started Phase II clinical trials of an AON therapy for SMA. Antisense drugs against FCMD are still in the preclinical stage but showed promising results in an animal model and in human cells. In vitro studies have revealed that dysferlinopathies are also promising targets for exon skipping therapy. In contrast to conventional knockdown strategies, exon skipping and splice modulation therapies are designed to rescue (knock up or increase) the target. It is reported that the knock-up approach is effective at lower efficiencies than a knockdown approach [14]. In a knockdown approach, the goal is typically
to knockdown 90% of the mRNA target to bring protein expression down to 10%. In contrast, for exon skipping in DMD, the goal is to restore protein expression levels to 10%, although the exact efficacy required for SMA, dysferlinopathies and FCMD are currently not well understood [14]. With the current progress of antisense research, new AON drugs for the widespread application of exon skipping and splice modulation therapies will likely be forthcoming. 7.
Expert opinion
Although promising, exon-skipping therapy for DMD is currently at a critical moment. In February 2014, Sarepta Therapeutics announced that their lead candidate drug, eteplirsen, a PMO-based AON targeting exon 51, was successful in demonstrating stability on pulmonary function tests through a 120-week Phase IIb open-label extension study. These findings follow in the wake of previously reported 120-week data which demonstrated stabilization during the 6-min walk test in treated patients. No clinically significant adverse reactions have been reported through week 120.
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Notwithstanding these promising early results, it remains to be seen whether eteplirsen can produce significant clinical benefits over a longer period of time. In September 2013, Prosensa and GSK announced that drisapersen, an exon 51-skipping antisense drug using 2¢OMePS chemistry, did not show statistically significant improvements in the primary outcome measure of the 6-min walking distance test in a Phase III clinical trial, which was carried out with 186 patients [95]. Despite failing to reach the primary end point of this particular clinical trial, drisapersen has had previous successes in other Phase II clinical trials, demonstrating safety and tolerability, novel dystrophin expression in biopsied muscle fibers and improvements in the 6-min walking distance test after 12 weeks of treatment [9,10]. There are plenty of lessons to be learned from these most recent clinical trials. First, a controversy exists over the optimal target of the exon-skipping approach [96]. In terms of the incidence of BMD, other exons such as exons 45, 53 or 55 could be more promising targets [97]. In addition, testing of a multi-exonskipping method (e.g., exons 45 -- 55 skipping), which is expected to lead to milder phenotypes versus the more traditional single exon-skipping approach, is currently underway [41,97]. Remaining challenges include the need to mix 11 AONs, safety and regulatory concerns and the fact that they currently have been shown to work only in mouse models. Second, the efficiency of exon skipping with 2¢OMePS is reported to be lower than that of PMOs in vivo [4,39]. Importantly, the development of new generation antisense oligos (e.g., peptide-conjugated antisense oligos) is ongoing and may augment future AON-based therapies by providing novel, highly effective AON chemistries [98,99]. Third, the efficiency of exon skipping via subcutaneous administration is reported to be 40 -- 80% lower than that achieved via intravenous
administration in the mouse model [4]. Finally, although the 6-min walk test has been shown to be a reliable assessment and is the current standard in clinical assessment, some question its consistency in representing significant clinical outcome [95]. The previous open-label, Phase II extension trial of drisapersen showed stabilization of disease progression measured by serial tests of the 6-min walk test; however, comparing the Phase II and III data, the impressive stabilization of the walking distance previously demonstrated may have been influenced by a training effect of the outcome measure or a placebo effect. Importantly, the Phase II trial was an unblinded open-label study with no control group, and only low levels of dystrophin were detected in muscle biopsies. To date, the interpretation of clinical end points has been challenging. Taken together, exon-skipping therapy that targets different exons, different AON chemistries and/or intravenous administration of antisense drugs may be more effective in producing favorable clinical results for the treatment of DMD.
Declaration of interest This work was supported by the University of Alberta Faculty of Medicine and Dentistry, Parent Project Muscular Dystrophy (USA), The Friends of Garrett Cumming Research Funds, HM Toupin Neurological Science Research Funds, Muscular Dystrophy Canada, Canada Foundation for Innovation, Alberta Enterprise and Advanced Education, the Women and Children’s Health Research Institute and Canadian Institute of Health Research. T Yokota has been and is currently the principal investigator of federal grants aiding the development of morpholino chemistry for exon skipping. The remaining authors have no competing interests to declare.
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Affiliation Aleksander Touznik1, Joshua JA Lee1 & Toshifumi Yokota†1,2 PhD † Author for correspondence 1 Assistant Professor and Muscular Dystrophy Canada Research Chair, University of Alberta, Faculty of Medicine and Dentistry, Department of Medical Genetics, Edmonton, Alberta, Canada 2 University of Alberta, Faculty of Medicine and Dentistry, Department of Medical Genetics, 829 Medical Sciences Building, Edmonton, Alberta, T6G 2H7, Canada Tel: +780 492 1102; Fax: +780 492 1998; E-mail: [email protected]
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