Publications by authors named "Rika Maruyama"

41 Publications

eSkip-Finder: a machine learning-based web application and database to identify the optimal sequences of antisense oligonucleotides for exon skipping.

Nucleic Acids Res 2021 07;49(W1):W193-W198

Department of Medical Genetics, University of Alberta Faculty of Medicine and Dentistry, 8613-114 St, Edmonton, AB, Canada.

Exon skipping using antisense oligonucleotides (ASOs) has recently proven to be a powerful tool for mRNA splicing modulation. Several exon-skipping ASOs have been approved to treat genetic diseases worldwide. However, a significant challenge is the difficulty in selecting an optimal sequence for exon skipping. The efficacy of ASOs is often unpredictable, because of the numerous factors involved in exon skipping. To address this gap, we have developed a computational method using machine-learning algorithms that factors in many parameters as well as experimental data to design highly effective ASOs for exon skipping. eSkip-Finder (https://eskip-finder.org) is the first web-based resource for helping researchers identify effective exon skipping ASOs. eSkip-Finder features two sections: (i) a predictor of the exon skipping efficacy of novel ASOs and (ii) a database of exon skipping ASOs. The predictor facilitates rapid analysis of a given set of exon/intron sequences and ASO lengths to identify effective ASOs for exon skipping based on a machine learning model trained by experimental data. We confirmed that predictions correlated well with in vitro skipping efficacy of sequences that were not included in the training data. The database enables users to search for ASOs using queries such as gene name, species, and exon number.
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http://dx.doi.org/10.1093/nar/gkab442DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8265194PMC
July 2021

Antisense Oligonucleotide Treatment in a Humanized Mouse Model of Duchenne Muscular Dystrophy and Highly Sensitive Detection of Dystrophin Using Western Blotting.

Methods Mol Biol 2021 ;2224:203-214

Faculty of Medicine and Dentistry, Department of Medical Genetics, University of Alberta, Edmonton, AB, Canada.

Duchenne muscular dystrophy (DMD) is a devastating X-linked muscle disorder affecting many children. The disease is caused by the lack of dystrophin production and characterized by muscle wasting. The most common causes of death are respiratory failure and heart failure. Antisense oligonucleotide-mediated exon skipping using a phosphorodiamidate morpholino oligomer (PMO) is a promising therapeutic approach for the treatment of DMD. In preclinical studies, dystrophic mouse models are commonly used for the development of therapeutic oligos. We employ a humanized model carrying the full-length human DMD transgene along with the complete knockout of the mouse Dmd gene. In this model, the effects of human-targeting AOs can be tested without cross-reaction between mouse sequences and human sequences (note that mdx, a conventional dystrophic mouse model, carries a nonsense point mutation in exon 23 and express the full-length mouse Dmd mRNA, which is a significant complicating factor). To determine if dystrophin expression is restored, the Western blotting analysis is commonly performed; however, due to the extremely large protein size of dystrophin (427 kDa), detection and accurate quantification of full-length dystrophin can be a challenge. Here, we present methodologies to systemically inject PMOs into humanized DMD model mice and determine levels of dystrophin restoration via Western blotting. Using a tris-acetate gradient SDS gel and semi-dry transfer with three buffers, including the Concentrated Anode Buffer, Anode Buffer, and Cathode Buffer, less than 1% normal levels of dystrophin expression are easily detectable. This method is fast, easy, and sensitive enough for the detection of dystrophin from both cultured muscle cells and muscle biopsy samples.
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http://dx.doi.org/10.1007/978-1-0716-1008-4_15DOI Listing
April 2021

Immortalized Canine Dystrophic Myoblast Cell Lines for Development of Peptide-Conjugated Splice-Switching Oligonucleotides.

Nucleic Acid Ther 2021 04 9;31(2):172-181. Epub 2021 Feb 9.

Department of Molecular Therapy, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Kodaira, Japan.

Duchenne muscular dystrophy (DMD) is a severe muscle-wasting disease caused by frameshift or nonsense mutations in the gene, resulting in the loss of dystrophin from muscle membranes. Exon skipping using splice-switching oligonucleotides (SSOs) restores the reading frame of pre-mRNA by generating internally truncated but functional dystrophin protein. To potentiate effective tissue-specific targeting by functional SSOs, it is essential to perform accelerated and reliable screening-based assessment of novel oligonucleotides and drug delivery technologies, such as cell-penetrating peptides, before their pharmacokinetic and toxicity evaluation. We have established novel canine immortalized myoblast lines by transducing murine cyclin-dependent kinase-4 and human telomerase reverse transcriptase genes into myoblasts isolated from beagle-based wild-type or canine X-linked muscular dystrophy in Japan (CXMD) dogs. These myoblast lines exhibited improved myogenic differentiation and increased proliferation rates compared with passage-15 primary parental myoblasts, and their potential to differentiate into myotubes was maintained in later passages. Using these dystrophin-deficient immortalized myoblast lines, we demonstrate that a novel cell-penetrating peptide (Pip8b2)-conjugated SSO markedly improved multiexon skipping activity compared with the respective naked phosphorodiamidate morpholino oligomers. screening using immortalized canine cell lines will provide a basis for further pharmacological studies on drug delivery tools.
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http://dx.doi.org/10.1089/nat.2020.0907DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7997716PMC
April 2021

A Genotype-Phenotype Correlation Study of Exon Skip-Equivalent In-Frame Deletions and Exon Skip-Amenable Out-of-Frame Deletions across the Gene to Simulate the Effects of Exon-Skipping Therapies: A Meta-Analysis.

J Pers Med 2021 Jan 14;11(1). Epub 2021 Jan 14.

Department of Medical Genetics, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, AB T6G 2H7, Canada.

Dystrophinopathies are caused by mutations in the gene. Out-of-frame deletions represent most mutational events in severe Duchenne muscular dystrophy (DMD), while in-frame deletions typically lead to milder Becker muscular dystrophy (BMD). Antisense oligonucleotide-mediated exon skipping converts an out-of-frame transcript to an in-frame one, inducing a truncated but partially functional dystrophin protein. The reading frame rule, however, has many exceptions. We thus sought to simulate clinical outcomes of exon-skipping therapies for exons from clinical data of exon skip-equivalent in-frame deletions, in which the expressed quasi-dystrophins are comparable to those resulting from exon-skipping therapies. We identified a total of 1298 unique patients with exon skip-equivalent mutations in patient registries and the existing literature. We classified them into skip-equivalent deletions of each exon and statistically compared the ratio of DMD/BMD and asymptomatic individuals across the gene. Our analysis identified that five exons are associated with significantly milder phenotypes than all other exons when corresponding exon skip-equivalent in-frame deletion mutations occur. Most exon skip-equivalent in-frame deletions were associated with a significantly milder phenotype compared to corresponding exon skip-amenable out-of-frame mutations. This study indicates the importance of genotype-phenotype correlation studies in the rational design of exon-skipping therapies.
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http://dx.doi.org/10.3390/jpm11010046DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7830903PMC
January 2021

DUX4 Transcript Knockdown with Antisense 2'-O-Methoxyethyl Gapmers for the Treatment of Facioscapulohumeral Muscular Dystrophy.

Mol Ther 2021 02 15;29(2):848-858. Epub 2020 Oct 15.

Department of Medical Genetics, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, AB T6G2H7, Canada; Muscular Dystrophy Canada Research Chair, Edmonton, AB T6G2H7, Canada. Electronic address:

Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant disorder characterized by a progressive, asymmetric weakening of muscles, starting with those in the upper body. It is caused by aberrant expression of the double homeobox protein 4 gene (DUX4) in skeletal muscle. FSHD is currently incurable. We propose to develop a therapy for FSHD using antisense 2'-O-methoxyethyl (2'-MOE) gapmers, to knock down DUX4 mRNA expression. Using immortalized patient-derived muscle cells and local intramuscular injections in the FLExDUX4 FSHD mouse model, we showed that our designed 2'-MOE gapmers significantly reduced DUX4 transcript levels in vitro and in vivo, respectively. Furthermore, in vitro, we observed significantly reduced expression of DUX4-activated downstream targets, restoration of FSHD signature genes by RNA sequencing, significant improvements in myotube morphology, and minimal off-target activity. This work facilitates the development of a promising candidate therapy for FSHD and lays down the foundation for in vivo systemic treatment studies.
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http://dx.doi.org/10.1016/j.ymthe.2020.10.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7854280PMC
February 2021

Molecular Diagnosis and Novel Therapies for Neuromuscular Diseases.

J Pers Med 2020 Sep 16;10(3). Epub 2020 Sep 16.

Department of Medical Genetics, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, AB T6G 2H7, Canada.

With the development of novel targeted therapies, including exon skipping/inclusion and gene replacement therapy, the field of neuromuscular diseases has drastically changed in the last several years. Until 2016, there had been no FDA-approved drugs to treat Duchenne muscular dystrophy (DMD), the most common muscular dystrophy. However, several new personalized therapies, including antisense oligonucleotides eteplirsen for DMD exon 51 skipping and golodirsen and viltolarsen for DMD exon 53 skipping, have been approved in the last 4 years. We are witnessing the start of a therapeutic revolution in neuromuscular diseases. However, the studies also made clear that these therapies are still far from a cure. Personalized genetic medicine for neuromuscular diseases faces several key challenges, including the difficulty of obtaining appropriate cell and animal models and limited its applicability. This Special Issue "Molecular Diagnosis and Novel Therapies for Neuromuscular/Musculoskeletal Diseases" highlights key areas of research progress that improve our understanding and the therapeutic outcomes of neuromuscular diseases in the personalized medicine era.
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http://dx.doi.org/10.3390/jpm10030129DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7564006PMC
September 2020

Inotersen for the Treatment of Hereditary Transthyretin Amyloidosis.

Methods Mol Biol 2020 ;2176:87-98

Department of Medical Genetics, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, AB, Canada.

Hereditary transthyretin amyloidosis (hATTR) is a rare autosomal dominant condition in which mutations in the transthyretin gene cause amyloid fibrils to develop and deposit into tissues, affecting primarily the nerves and heart causing polyneuropathy and cardiomyopathy respectively. Standard treatment has been liver transplants to try and eliminate the mutated transthyretin products as the liver is the main source of transthyretin production. A new drug named inotersen (brand name Tagsedi), also known as IONIS-TTR, has been approved by the United States Food and Drug Agency, Health Canada, and European Commission in 2018, and introduced to the market for patients in stage 1 and stage 2 hATTR polyneuropathy. Inotersen is a second-generation antisense oligonucleotide with 2'-O-methoxyethyl modification designed to bind to the 3' untranslated region of the transthyretin mRNA in the nucleus of the liver cells. By doing so, it prevents the production of the mutant and wild-type forms of transthyretin, impeding the progression of the disease. In this article, the mechanism of action and safety profile of inotersen will be discussed along with some future directions following its approval.
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http://dx.doi.org/10.1007/978-1-0716-0771-8_6DOI Listing
March 2021

Knocking Down Long Noncoding RNAs Using Antisense Oligonucleotide Gapmers.

Methods Mol Biol 2020 ;2176:49-56

Department of Medical Genetics, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, AB, Canada.

Long noncoding RNAs (lncRNAs) are a class of RNA with 200 nucleotides or longer that are not translated into protein. lncRNAs are highly abundant; a study estimates that at least four times more lncRNAs are typically present than coding RNAs in humans. However, function of more than 95% of human lncRNAs are still unknown. Synthetic antisense oligonucleotides called gapmers are powerful tools for lncRNA loss-of-function studies. Gapmers contain a central DNA part, which activates RNase H-mediated RNA degradation, flanked by modified oligonucleotides, such as 2'-O-methyl RNA (2'OMe), 2'-O-methoxyethyl RNA (2'MOE), constrained ethyl nucleosides (cEt), and locked nucleic acids (LNAs). In contrast to siRNA or RNAi-based methods, antisense oligonucleotide gapmer-based knockdown is often more effective against nuclear-localized lncRNA targets, since RNase H is mainly localized in nuclei. As such, gapmers are also potentially a powerful tool for therapeutics targeting lncRNAs in various diseases, including cancer, cardiovascular diseases, lung fibrosis, and neurological/neuromuscular diseases. This chapter will discuss the development and applications of gapmers for lncRNA loss-of-function studies and tips to design effective antisense oligonucleotides.
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http://dx.doi.org/10.1007/978-1-0716-0771-8_3DOI Listing
March 2021

Membrane Repair Deficit in Facioscapulohumeral Muscular Dystrophy.

Int J Mol Sci 2020 Aug 4;21(15). Epub 2020 Aug 4.

Research Center for Genetic Medicine, Children's National Hospital, 111 Michigan Ave NW, Washington, DC 20010, USA.

Deficits in plasma membrane repair have been identified in dysferlinopathy and Duchenne Muscular Dystrophy, and contribute to progressive myopathy. Although Facioscapulohumeral Muscular Dystrophy (FSHD) shares clinicopathological features with these muscular dystrophies, it is unknown if FSHD is characterized by plasma membrane repair deficits. Therefore, we exposed immortalized human FSHD myoblasts, immortalized myoblasts from unaffected siblings, and myofibers from a murine model of FSHD () to focal, pulsed laser ablation of the sarcolemma. Repair kinetics and success were determined from the accumulation of intracellular FM1-43 dye post-injury. We subsequently treated FSHD myoblasts with a -targeting antisense oligonucleotide (AON) to reduce expression, and with the antioxidant Trolox to determine the role of expression and oxidative stress in membrane repair. Compared to unaffected myoblasts, FSHD myoblasts demonstrate poor repair and a greater percentage of cells that failed to repair, which was mitigated by AON and Trolox treatments. Similar repair deficits were identified in myofibers. This is the first study to identify plasma membrane repair deficits in myoblasts from individuals with FSHD, and in myofibers from a murine model of FSHD. Our results suggest that expression and oxidative stress may be important targets for future membrane-repair therapies.
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http://dx.doi.org/10.3390/ijms21155575DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7432481PMC
August 2020

Inhibition of expression with antisense LNA gapmers as a therapy for facioscapulohumeral muscular dystrophy.

Proc Natl Acad Sci U S A 2020 07 29;117(28):16509-16515. Epub 2020 Jun 29.

Department of Medical Genetics, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, AB T6G2H7, Canada;

Facioscapulohumeral muscular dystrophy (FSHD), characterized by progressive muscle weakness and deterioration, is genetically linked to aberrant expression of in muscle. DUX4, in its full-length form, is cytotoxic in nongermline tissues. Here, we designed locked nucleic acid (LNA) gapmer antisense oligonucleotides (AOs) to knock down in immortalized FSHD myoblasts and the FSHD mouse model. Using a screening method capable of reliably evaluating the knockdown efficiency of LNA gapmers against endogenous messenger RNA in vitro, we demonstrate that several designed LNA gapmers selectively and effectively reduced expression with nearly complete knockdown. We also found potential functional benefits of AOs on muscle fusion and structure in vitro. Finally, we show that one of the LNA gapmers was taken up and induced effective silencing of upon local treatment in vivo. The LNA gapmers developed here will help facilitate the development of FSHD therapies.
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http://dx.doi.org/10.1073/pnas.1909649117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7368245PMC
July 2020

Exons 45-55 Skipping Using Mutation-Tailored Cocktails of Antisense Morpholinos in the DMD Gene.

Mol Ther 2019 11 26;27(11):2005-2017. Epub 2019 Jul 26.

Department of Medical Genetics, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, AB T6G 2H7, Canada; Muscular Dystrophy Canada Research Chair, Edmonton, AB T6G 2H7, Canada. Electronic address:

Mutations in the dystrophin (DMD) gene and consequent loss of dystrophin cause Duchenne muscular dystrophy (DMD). A promising therapy for DMD, single-exon skipping using antisense phosphorodiamidate morpholino oligomers (PMOs), currently confronts major issues in that an antisense drug induces the production of functionally undefined dystrophin and may not be similarly efficacious among patients with different mutations. Accordingly, the applicability of this approach is limited to out-of-frame mutations. Here, using an exon-skipping efficiency predictive tool, we designed three different PMO cocktail sets for exons 45-55 skipping aiming to produce a dystrophin variant with preserved functionality as seen in milder or asymptomatic individuals with an in-frame exons 45-55 deletion. Of them, the most effective set was composed of select PMOs that each efficiently skips an assigned exon in cell-based screening. These combinational PMOs fitted to different deletions of immortalized DMD patient muscle cells significantly induced exons 45-55 skipping with removing 3, 8, or 10 exons and dystrophin restoration as represented by western blotting. In vivo skipping of the maximum 11 human DMD exons was confirmed in humanized mice. The finding indicates that our PMO set can be used to create mutation-tailored cocktails for exons 45-55 skipping and treat over 65% of DMD patients carrying out-of-frame or in-frame deletions.
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http://dx.doi.org/10.1016/j.ymthe.2019.07.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6838919PMC
November 2019

Efficacy of Multi-exon Skipping Treatment in Duchenne Muscular Dystrophy Dog Model Neonates.

Mol Ther 2019 01 19;27(1):76-86. Epub 2018 Oct 19.

Department of Medical Genetics, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, AB T6G2H7, Canada; Muscular Dystrophy Canada Research Chair, Edmonton, AB T6G2H7, Canada. Electronic address:

Duchenne muscular dystrophy (DMD) is caused by mutations in DMD, which codes for dystrophin. Because the progressive and irreversible degeneration of muscle occurs from childhood, earlier therapy is required to prevent dystrophic progression. Exon skipping by antisense oligonucleotides called phosphorodiamidate morpholino oligomers (PMOs), which restores the DMD reading frame and dystrophin expression, is a promising candidate for use in neonatal patients, yet the potential remains unclear. Here, we investigate the systemic efficacy and safety of early exon skipping in dystrophic dog neonates. Intravenous treatment of canine X-linked muscular dystrophy in Japan dogs with a 4-PMO cocktail resulted in ∼3%-27% in-frame exon 6-9 skipping and dystrophin restoration across skeletal muscles up to 14% of healthy levels. Histopathology was ameliorated with the reduction of fibrosis and/or necrosis area and centrally nucleated fibers, significantly in the diaphragm. Treatment induced cardiac multi-exon skipping, though dystrophin rescue was not detected. Functionally, treatment led to significant improvement in the standing test. Toxicity was not observed from blood tests. This is the first study to demonstrate successful multi-exon skipping treatment and significant functional improvement in dystrophic dogs. Early treatment was most beneficial for respiratory muscles, with implications for addressing pulmonary malfunction in patients.
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http://dx.doi.org/10.1016/j.ymthe.2018.10.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6318815PMC
January 2019

Identification of Novel Antisense-Mediated Exon Skipping Targets in DYSF for Therapeutic Treatment of Dysferlinopathy.

Mol Ther Nucleic Acids 2018 Dec 11;13:596-604. Epub 2018 Oct 11.

Department of Medical Genetics, University of Alberta, Edmonton, AB, Canada; The Friends of Garrett Cumming Research & Muscular Dystrophy Canada HM Toupin Neurological Science Research Chair, Edmonton, AB T6G 2H7, Canada. Electronic address:

Dysferlinopathy is a progressive myopathy caused by mutations in the dysferlin (DYSF) gene. Dysferlin protein plays a major role in plasma-membrane resealing. Some patients with DYSF deletion mutations exhibit mild symptoms, suggesting some regions of DYSF can be removed without significantly impacting protein function. Antisense-mediated exon-skipping therapy uses synthetic molecules called antisense oligonucleotides to modulate splicing, allowing exons harboring or near genetic mutations to be removed and the open reading frame corrected. Previous studies have focused on DYSF exon 32 skipping as a potential therapeutic approach, based on the association of a mild phenotype with the in-frame deletion of exon 32. To date, no other DYSF exon-skipping targets have been identified, and the relationship between DYSF exon deletion pattern and protein function remains largely uncharacterized. In this study, we utilized a membrane-wounding assay to evaluate the ability of plasmid constructs carrying mutant DYSF, as well as antisense oligonucleotides, to rescue membrane resealing in patient cells. We report that multi-exon skipping of DYSF exons 26-27 and 28-29 rescues plasma-membrane resealing. Successful translation of these findings into the development of clinical antisense drugs would establish new therapeutic approaches that would be applicable to ∼5%-7% (exons 26-27 skipping) and ∼8% (exons 28-29 skipping) of dysferlinopathy patients worldwide.
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http://dx.doi.org/10.1016/j.omtn.2018.10.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6234522PMC
December 2018

Morpholino-Mediated Exon Skipping Targeting Human ACVR1/ALK2 for Fibrodysplasia Ossificans Progressiva.

Methods Mol Biol 2018 ;1828:497-502

Department of Medical Genetics, University of Alberta Faculty of Medicine and Dentistry, Edmonton, AB, Canada.

Fibrodysplasia ossificans progressiva (FOP) is a rare autosomal-dominant disorder characterized by progressive heterotopic ossification. More than 95% of cases are caused by a recurrent mutation (617G>A; R206H) of ACVR1/ALK2, a bone morphogenetic protein (BMP) type I receptor. Recent studies revealed that ACVR1 induces heterotopic ossification by aberrant activation in response to activin A. Because ACVR1 is a hyperactive receptor, a promising therapeutic strategy is to decrease the activity of ACVR1 in patients. Here, we describe a method to reduce ACVR1 expression in FOP patient cells by exon skipping in ACVR1 mRNAs using phosphorodiamidate morpholino oligomers (PMOs). This strategy can be applied to the screen to select antisense oligomers to knockdown not only ACVR1 but also genes which cause other autosomal-dominant genetic diseases.
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http://dx.doi.org/10.1007/978-1-4939-8651-4_32DOI Listing
April 2019

Systemic and ICV Injections of Antisense Oligos into SMA Mice and Evaluation.

Methods Mol Biol 2018 ;1828:455-465

Department of Medical Genetics, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, AB, Canada.

Spinal muscular atrophy (SMA) is the most common genetic cause of infantile death caused by mutations in the SMN1 gene. Nusinersen (Spinraza), an antisense therapy-based drug with the 2'-methoxyethoxy (2'MOE) chemistry approved by the FDA in 2016, brought antisense drugs into the spotlight. Antisense-mediated exon inclusion targeting SMN2 leads to SMN protein expression. Although effective, 2'MOE has weaknesses such as the inability to cross the blood-brain barrier and the high cost of treatment. To investigate new chemistries of antisense oligonucleotides (ASOs), SMA mouse models can serve as an important source. Here we describe methods to test the efficacy of ASOs, such as phosphorodiamidate morpholino oligomers (PMOs), in a severe SMA mouse model.
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http://dx.doi.org/10.1007/978-1-4939-8651-4_28DOI Listing
April 2019

In Vitro Evaluation of Antisense-Mediated Exon Inclusion for Spinal Muscular Atrophy.

Methods Mol Biol 2018 ;1828:439-454

Department of Medical Genetics, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, AB, Canada.

Spinal muscular atrophy (SMA), the most common gentic cause of infantile death caused by mutations in the SMN1 gene, presents a unique case in the field of splice modulation therapy, where a gene (or lack of) is responsible for causing the disease phenotype but treatment is not focused around it. Antisense therapy targeting SMN2 which leads to SMN protein expression has been at the forefront of research when it comes to developing a feasible therapy for treating SMA. Recent FDA approval of an antisense-based drug with the 2'-methoxyethoxy (2'MOE) chemistry, called nusinersen (Spinraza), brought antisense drugs into the spotlight. The 2'MOE, although effective, has weaknesses such as the inability to cross the blood-brain barrier and the high cost of treatment. This propelled the research community to investigate new chemistries of antisense oligonucleotides (ASOs) that may be better in both treatment and cost efficiency. Here we describe two types of ASOs, phosphorodiamidate morpholino oligomers (PMOs) and locked nucleic acids (LNA)-DNA mixmers, being investigated as potential treatments for SMA, and methods used to test their efficacy, including quantitative RT-PCR, Western blotting, and immunofluorescence staining to detect SMN in nuclear gems/Cajal bodies, in type I SMA patient fibroblast cell lines.
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http://dx.doi.org/10.1007/978-1-4939-8651-4_27DOI Listing
April 2019

In Vivo Evaluation of Multiple Exon Skipping with Peptide-PMOs in Cardiac and Skeletal Muscles in Dystrophic Dogs.

Methods Mol Biol 2018 ;1828:365-379

Department of Medical Genetics, University of Alberta Faculty of Medicine and Dentistry, Edmonton, AB, Canada.

Exon skipping is an emerging approach to treating Duchenne muscular dystrophy (DMD), one of the most common lethal genetic disorders. Exon skipping uses synthetic antisense oligonucleotides (AONs) to splice out frame-disrupting exon(s) of DMD mRNA to restore the reading frame of the gene products and produce truncated yet functional proteins. The FDA conditionally approved the first exon-skipping AON, called eteplirsen (brand name ExonDys51), targeting exon 51 of the DMD gene, in late 2016. Using a cocktail of AONs, multiple exons can be skipped, which can theoretically treat 80-90% of patients with DMD. Although the success of multiple exon skipping in a DMD dog model has made a significant impact on the development of therapeutics for DMD, unmodified AONs such as phosphorodiamidate morpholino oligomers (PMOs) have little efficacy in cardiac muscles. Here, we describe our technique of intravenous injection of a cocktail of peptide-conjugated PMOs (PPMOs) to skip multiple exons, exons 6 and 8, in both skeletal and cardiac muscles in dystrophic dogs and the evaluation of the efficacy and toxicity.
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http://dx.doi.org/10.1007/978-1-4939-8651-4_23DOI Listing
April 2019

Systemic Injection of Peptide-PMOs into Humanized DMD Mice and Evaluation by RT-PCR and ELISA.

Methods Mol Biol 2018 ;1828:263-273

Faculty of Medicine and Dentistry, Department of Medical Genetics, University of Alberta, Edmonton, AB, Canada.

Duchenne muscular dystrophy (DMD) is an X-linked recessive disorder due to the lack of dystrophin production. The disease is characterized by muscle wasting, with the most common causes of death being respiratory failure or heart failure. Recently, exon skipping using a phosphorodiamidate morpholino oligomer (PMO) is used as an FDA approved treatment for DMD. Peptide-conjugated PMOs (PPMOs) are used to increase exon skipping efficacy in the heart and are a promising therapy for DMD. Researchers have previously relied on high-performance liquid chromatography (HPLC) or liquid chromatography-mass spectrometry (LC/MS) methods for detecting PPMO uptake, but an enzyme-linked immunosorbent assay (ELISA) has been shown to have greater sensitivity. Here, we present methodologies to determine the uptake efficiency of a PPMO into the heart and efficacy of exon 51 skipping by a PPMO injected retro-orbitally into a humanized DMD mouse model via ELISA and RT-PCR, respectively.
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http://dx.doi.org/10.1007/978-1-4939-8651-4_16DOI Listing
April 2019

Creation of DMD Muscle Cell Model Using CRISPR-Cas9 Genome Editing to Test the Efficacy of Antisense-Mediated Exon Skipping.

Methods Mol Biol 2018 ;1828:165-171

Department of Medical Genetics, University of Alberta Faculty of Medicine and Dentistry, Edmonton, AB, Canada.

Duchenne muscular dystrophy (DMD) is a devastating muscle disorder caused by mutations in the DMD gene. Antisense-mediated exon skipping is a promising strategy to treat DMD. The approval of Exondys 51 (eteplirsen) targeting exon 51 was the most noteworthy accomplishment in 2016. To evaluate and optimize the sequence of antisense oligonucleotides (AOs), muscle cell lines with DMD mutations are useful tools. However, there are only several immortalized muscle cell lines with DMD mutations available that can be used to test the efficacy of exon skipping in vitro. In addition, an invasive muscle biopsy is required to obtain muscle cells from patients. Furthermore, many DMD mutations are very rare and it is hard to find a patient with a specific mutation for muscle biopsy in many cases. Here, we describe a novel approach to create an immortalized muscle cell line with a DMD deletion mutation using the human rhabdomyosarcoma (RD) cell line and the CRISPR/Cas9 system that can be used to test the efficacy of exon skipping.
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http://dx.doi.org/10.1007/978-1-4939-8651-4_10DOI Listing
April 2019

Tips to Design Effective Splice-Switching Antisense Oligonucleotides for Exon Skipping and Exon Inclusion.

Methods Mol Biol 2018 ;1828:79-90

Department of Medical Genetics, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, AB, Canada.

Antisense-mediated exon skipping and exon inclusion have proven to be powerful tools for treating neuromuscular diseases. The approval of Exondys 51 (eteplirsen) and Spinraza (nusinersen) for the treatment of patients with Duchenne muscular dystrophy (DMD) and spinal muscular atrophy (SMA) was the most noteworthy accomplishment in 2016. Exon skipping uses short DNA-like molecules called antisense oligonucleotides (AONs) to correct the disrupted reading frame, allowing the production of functional quasi-dystrophin proteins, and ameliorate the progression of the disease. Exon inclusion for SMA employs an AON targeting an intronic splice silencer site to include an exon which is otherwise spliced out. Recently, these strategies have also been explored in many other genetic disorders, including dysferlin-deficient muscular dystrophy (e.g., Miyoshi myopathy; MM, limb-girdle muscular dystrophy type 2B; LGMD2B, and distal myopathy with anterior tibial onset; DMAT), laminin α2 chain (merosin)-deficient congenital muscular dystrophy (MDC1A), sarcoglycanopathy (e.g., limb-girdle muscular dystrophy type 2C; LGMD2C), and Fukuyama congenital muscular dystrophy (FCMD). A major challenge in exon skipping and exon inclusion is the difficulty in designing effective AONs. The mechanism of mRNA splicing is highly complex, and the efficacy of AONs is often unpredictable. We will discuss the design of effective AONs for exon skipping and exon inclusion in this chapter.
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http://dx.doi.org/10.1007/978-1-4939-8651-4_5DOI Listing
April 2019

Nusinersen in the Treatment of Spinal Muscular Atrophy.

Methods Mol Biol 2018 ;1828:69-76

Department of Medical Genetics, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, AB, Canada.

Spinal muscular atrophy (SMA) is one of the most common genetic causes of infantile death arising due to mutations in the SMN1 gene and the subsequent loss of motor neurons. With the discovery of the intronic splicing silencer N1 (ISS-N1) as a potential target for antisense therapy, several antisense oligonucleotides (ASOs) are being developed to include exon 7 in the final mRNA transcript of the SMN2 gene and thereby increasing the production of spinal motor neuron (SMN) proteins. Nusinersen (spinraza), a modified 2'-O-methoxyethyl (MOE) antisense oligonucleotide is the first drug to be approved by Food and Drug Agency (FDA) in December of 2016. Here we briefly review the pharmacological relevance of the drug, clinical trials, toxicity, and future directions following the approval of nusinersen.
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http://dx.doi.org/10.1007/978-1-4939-8651-4_4DOI Listing
April 2019

Evaluation of Exon Inclusion Induced by Splice Switching Antisense Oligonucleotides in SMA Patient Fibroblasts.

J Vis Exp 2018 05 11(135). Epub 2018 May 11.

Department of Medical Genetics, University of Alberta Faculty of Medicine and Dentistry; Muscular Dystrophy Canada Research Chair, Department of Medical Genetics, University of Alberta Faculty of Medicine and Dentistry;

Spinal muscular atrophy (SMA), a lethal neurological disease caused by the loss of SMN1, presents a unique case in the field of antisense oligonucleotide (AON)-mediated therapy. While SMN1 mutations are responsible for the disease, AONs targeting intronic splice silencer (ISS) sites in SMN2, including FDA-approved nusinersen, have been shown to restore SMN expression and ameliorate the symptoms. Currently, many studies involving AON therapy for SMA focus on investigating novel AON chemistries targeting SMN2 that may be more effective and less toxic than nusinersen. Here, we describe a protocol for in vitro evaluation of exon inclusion using lipotransfection of AONs followed by reverse transcription polymerase chain reaction (RT-PCR), quantitative polymerase chain reaction (qPCR), and Western blotting. This method can be employed for various types of AON chemistries. Using this method, we demonstrate that AONs composed of alternating locked nucleic acids (LNAs) and DNA nucleotides (LNA/DNA mixmers) lead to efficient SMN2 exon inclusion and restoration of SMN protein at a very low concentration, and therefore, LNA/DNA mixmer-based antisense oligonucleotides may be an attractive therapeutic strategy to treat splicing defects caused by genetic diseases. The in vitro evaluation method described here is fast, easy, and sensitive enough for the testing of various novel AONs.
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http://dx.doi.org/10.3791/57530DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6101168PMC
May 2018

Cell Membrane Repair Assay Using a Two-photon Laser Microscope.

J Vis Exp 2018 01 2(131). Epub 2018 Jan 2.

Department of Medical Genetics, University of Alberta Faculty of Medicine and Dentistry;

Numerous pathophysiological insults can cause damage to cell membranes and, when coupled with innate defects in cell membrane repair or integrity, can result in disease. Understanding the underlying molecular mechanisms surrounding cell membrane repair is, therefore, an important objective to the development of novel therapeutic strategies for diseases associated with dysfunctional cell membrane dynamics. Many in vitro and in vivo studies aimed at understanding cell membrane resealing in various disease contexts utilize two-photon laser ablation as a standard for determining functional outcomes following experimental treatments. In this assay, cell membranes are subjected to wounding with a two-photon laser, which causes the cell membrane to rupture and fluorescent dye to infiltrate the cell. The intensity of fluorescence within the cell can then be monitored to quantify the cell's ability to reseal itself. There are several alternative methods for assessing cell membrane response to injury, as well as great variation in the two-photon laser wounding approach itself, therefore, a single, unified model of cell wounding would beneficially serve to decrease the variation between these methodologies. In this article, we outline a simple two-photon laser wounding protocol for assessing cell membrane repair in vitro in both healthy and dysferlinopathy patient fibroblast cells transfected with or without a full-length dysferlin plasmid.
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http://dx.doi.org/10.3791/56999DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5908433PMC
January 2018

Skipping Multiple Exons to Treat DMD-Promises and Challenges.

Biomedicines 2018 Jan 2;6(1). Epub 2018 Jan 2.

Department of Medical Genetics, Faculty of Medicine and Dentistry, University of Alberta, 8812-112 St. Edmonton, AB T6G 2H7, Canada.

Duchenne muscular dystrophy (DMD) is a lethal disorder caused by mutations in the gene. Antisense-mediated exon-skipping is a promising therapeutic strategy that makes use of synthetic nucleic acids to skip frame-disrupting exon(s) and allows for short but functional protein expression by restoring the reading frame. In 2016, the U.S. Food and Drug Administration (FDA) approved eteplirsen, which skips DMD exon 51 and is applicable to approximately 13% of DMD patients. Multiple exon skipping, which is theoretically applicable to 80-90% of DMD patients in total, have been demonstrated in animal models, including dystrophic mice and dogs, using cocktail antisense oligonucleotides (AOs). Although promising, current drug approval systems pose challenges for the use of a cocktail AO. For example, both exons 6 and 8 need to be skipped to restore the reading frame in dystrophic dogs. Therefore, the cocktail of AOs targeting these exons has a combined therapeutic effect and each AO does not have a therapeutic effect by itself. The current drug approval system is not designed to evaluate such circumstances, which are completely different from cocktail drug approaches in other fields. Significant changes are needed in the drug approval process to promote the cocktail AO approach.
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http://dx.doi.org/10.3390/biomedicines6010001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5874658PMC
January 2018

Designing Effective Antisense Oligonucleotides for Exon Skipping.

Methods Mol Biol 2018 ;1687:143-155

Department of Medical Genetics, Faculty of Medicine and Dentistry, University of Alberta, 8613-114 St, Edmonton, AB, Canada.

During the past 10 years, antisense oligonucleotide-mediated exon skipping and splice modulation have proven to be powerful tools for correction of mRNA splicing in genetic diseases. In 2016, the US Food and Drug Administration (FDA)-approved Exondys 51 (eteplirsen) and Spinraza (nusinersen), the first exon skipping and exon inclusion drugs, to treat patients with Duchenne muscular dystrophy (DMD) and spinal muscular atrophy (SMA), respectively. The exon skipping of DMD mRNA aims to restore the disrupted reading frame using antisense oligonucleotides (AONs), allowing the production of truncated but partly functional dystrophin proteins, and slow down the progression of the disease. This approach has also been explored in several other genetic disorders, including laminin α2 chain-deficient congenital muscular dystrophy, dysferlin-deficient muscular dystrophy (e.g., Miyoshi myopathy and limb-girdle muscular dystrophy type 2B), sarcoglycanopathy (limb-girdle muscular dystrophy type 2C), and Fukuyama congenital muscular dystrophy. Antisense-mediated exon skipping is also a powerful tool to examine the function of genes and exons. A significant challenge in exon skipping is how to design effective AONs. The mechanism of mRNA splicing is highly complex with many factors involved. The selection of target sites, the length of AONs, the AON chemistry, and the melting temperature versus the RNA strand play important roles. A cocktail of AONs can be employed to skip multiples exons. In this chapter, we discuss the design of effective AONs for exon skipping.
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http://dx.doi.org/10.1007/978-1-4939-7374-3_10DOI Listing
June 2018

Quantitative Antisense Screening and Optimization for Exon 51 Skipping in Duchenne Muscular Dystrophy.

Mol Ther 2017 11 28;25(11):2561-2572. Epub 2017 Jul 28.

Department of Medical Genetics, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, AB T6G 2H7, Canada; Muscular Dystrophy Canada Research Chair, Edmonton, AB T6G 2H7, Canada. Electronic address:

Duchenne muscular dystrophy (DMD), the most common lethal genetic disorder, is caused by mutations in the dystrophin (DMD) gene. Exon skipping is a therapeutic approach that uses antisense oligonucleotides (AOs) to modulate splicing and restore the reading frame, leading to truncated, yet functional protein expression. In 2016, the US Food and Drug Administration (FDA) conditionally approved the first phosphorodiamidate morpholino oligomer (morpholino)-based AO drug, eteplirsen, developed for DMD exon 51 skipping. Eteplirsen remains controversial with insufficient evidence of its therapeutic effect in patients. We recently developed an in silico tool to design antisense morpholino sequences for exon skipping. Here, we designed morpholino AOs targeting DMD exon 51 using the in silico tool and quantitatively evaluated the effects in immortalized DMD muscle cells in vitro. To our surprise, most of the newly designed morpholinos induced exon 51 skipping more efficiently compared with the eteplirsen sequence. The efficacy of exon 51 skipping and rescue of dystrophin protein expression were increased by up to more than 12-fold and 7-fold, respectively, compared with the eteplirsen sequence. Significant in vivo efficacy of the most effective morpholino, determined in vitro, was confirmed in mice carrying the human DMD gene. These findings underscore the importance of AO sequence optimization for exon skipping.
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http://dx.doi.org/10.1016/j.ymthe.2017.07.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5675502PMC
November 2017

LNA/DNA mixmer-based antisense oligonucleotides correct alternative splicing of the SMN2 gene and restore SMN protein expression in type 1 SMA fibroblasts.

Sci Rep 2017 06 16;7(1):3672. Epub 2017 Jun 16.

Department of Medical Genetics, University of Alberta Faculty of Medicine and Dentistry, Edmonton, Alberta, Canada.

Spinal muscular atrophy (SMA) is an autosomal recessive disorder affecting motor neurons, and is currently the most frequent genetic cause of infant mortality. SMA is caused by a loss-of-function mutation in the survival motor neuron 1 (SMN1) gene. SMN2 is an SMN1 paralogue, but cannot compensate for the loss of SMN1 since exon 7 in SMN2 mRNA is excluded (spliced out) due to a single C-to-T nucleotide transition in the exon 7. One of the most promising strategies to treat SMA is antisense oligonucleotide (AON)-mediated therapy. AONs are utilized to block intronic splicing silencer number 1 (ISS-N1) on intron 7 of SMN2, which causes exon 7 inclusion of the mRNA and the recovery of the expression of functional SMN protein from the endogenous SMN2 gene. We developed novel locked nucleic acid (LNA)-based antisense oligonucleotides (LNA/DNA mixmers), which efficiently induce exon 7 inclusion in SMN2 and restore the SMN protein production in SMA patient fibroblasts. The mixmers are highly specific to the targeted sequence, and showed significantly higher efficacy than an all-LNA oligonucleotide with the equivalent sequence. These data suggest that use of LNA/DNA mixmer-based AONs may be an attractive therapeutic strategy to treat SMA.
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http://dx.doi.org/10.1038/s41598-017-03850-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5473822PMC
June 2017

Effects of systemic multiexon skipping with peptide-conjugated morpholinos in the heart of a dog model of Duchenne muscular dystrophy.

Proc Natl Acad Sci U S A 2017 04 3;114(16):4213-4218. Epub 2017 Apr 3.

Department of Medical Genetics, School of Human Development, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, AB, Canada T6G 2H7;

Duchenne muscular dystrophy (DMD) is a lethal genetic disorder caused by an absence of the dystrophin protein in bodywide muscles, including the heart. Cardiomyopathy is a leading cause of death in DMD. Exon skipping via synthetic phosphorodiamidate morpholino oligomers (PMOs) represents one of the most promising therapeutic options, yet PMOs have shown very little efficacy in cardiac muscle. To increase therapeutic potency in cardiac muscle, we tested a next-generation morpholino: arginine-rich, cell-penetrating peptide-conjugated PMOs (PPMOs) in the canine X-linked muscular dystrophy in Japan (CXMD) dog model of DMD. A PPMO cocktail designed to skip exons 6 and 8 was injected intramuscularly, intracoronarily, or intravenously into CXMD dogs. Intravenous injections with PPMOs restored dystrophin expression in the myocardium and cardiac Purkinje fibers, as well as skeletal muscles. Vacuole degeneration of cardiac Purkinje fibers, as seen in DMD patients, was ameliorated in PPMO-treated dogs. Although symptoms and functions in skeletal muscle were not ameliorated by i.v. treatment, electrocardiogram abnormalities (increased Q-amplitude and Q/R ratio) were improved in CXMD dogs after intracoronary or i.v. administration. No obvious evidence of toxicity was found in blood tests throughout the monitoring period of one or four systemic treatments with the PPMO cocktail (12 mg/kg/injection). The present study reports the rescue of dystrophin expression and recovery of the conduction system in the heart of dystrophic dogs by PPMO-mediated multiexon skipping. We demonstrate that rescued dystrophin expression in the Purkinje fibers leads to the improvement/prevention of cardiac conduction abnormalities in the dystrophic heart.
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http://dx.doi.org/10.1073/pnas.1613203114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5402437PMC
April 2017

Systemic Delivery of Morpholinos to Skip Multiple Exons in a Dog Model of Duchenne Muscular Dystrophy.

Methods Mol Biol 2017 ;1565:201-213

Department of Medical Genetics, University of Alberta Faculty of Medicine and Dentistry, Edmonton, AB, Canada.

Exon-skipping therapy is an emerging approach that uses synthetic DNA-like molecules called antisense oligonucleotides (AONs) to splice out frame-disrupting parts of mRNA, restore the reading frame, and produce truncated yet functional proteins. Multiple exon skipping utilizing a cocktail of AONs can theoretically treat 80-90% of patients with Duchenne muscular dystrophy (DMD). The success of multiple exon skipping by the systemic delivery of a cocktail of AONs called phosphorodiamidate morpholino oligomers (PMOs) in a DMD dog model has made a significant impact on the development of therapeutics for DMD, leading to clinical trials of PMO-based drugs. Here, we describe the systemic delivery of a cocktail of PMOs to skip multiple exons in dystrophic dogs and the evaluation of the efficacies and toxicity in vivo.
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http://dx.doi.org/10.1007/978-1-4939-6817-6_17DOI Listing
February 2018

Eteplirsen in the treatment of Duchenne muscular dystrophy.

Drug Des Devel Ther 2017 28;11:533-545. Epub 2017 Feb 28.

Department of Medical Genetics, Faculty of Medicine and Dentistry, University of Alberta; The Friends of Garrett Cumming Research & Muscular Dystrophy Canada, HM Toupin Neurological Science Research Chair, Edmonton, AB, Canada.

Duchenne muscular dystrophy is a fatal neuromuscular disorder affecting around one in 3,500-5,000 male births that is characterized by progressive muscular deterioration. It is inherited in an X-linked recessive fashion and is caused by loss-of-function mutations in the gene coding for dystrophin, a cytoskeletal protein that stabilizes the plasma membrane of muscle fibers. In September 2016, the US Food and Drug Administration granted accelerated approval for eteplirsen (or Exondys 51), a drug that acts to promote dystrophin production by restoring the translational reading frame of through specific skipping of exon 51 in defective gene variants. Eteplirsen is applicable for approximately 14% of patients with mutations. This article extensively reviews and discusses the available information on eteplirsen to date, focusing on pharmacological, efficacy, safety, and tolerability data from preclinical and clinical trials. Issues faced by eteplirsen, particularly those relating to its efficacy, will be identified. Finally, the place of eteplirsen and exon skipping as a general therapeutic strategy in Duchenne muscular dystrophy treatment will be discussed.
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http://dx.doi.org/10.2147/DDDT.S97635DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5338848PMC
May 2017
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