The Experts below are selected from a list of 12717 Experts worldwide ranked by ideXlab platform
Toshifumi Yokota - One of the best experts on this subject based on the ideXlab platform.
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Optimization of antisense-mediated Exon Skipping for Duchenne muscular dystrophy
Gene Therapy, 2020Co-Authors: Kasia Dzierlega, Toshifumi YokotaAbstract:Duchenne muscular dystrophy (DMD) is one of the most common lethal muscle-wasting disorders affecting young boys caused by mutations in the DMD gene. Exon Skipping has emerged as a promising therapy for DMD. Antisense oligonucleotides (AONs) are designed to induce the Skipping of Exon(s), in order to restore the reading frame, and therefore, allow for dystrophin expression. Eteplirsen and golodirsen, AONs for DMD Exons 51 and 53 Skipping, have been recently approved by the FDA. Viltolarsen, an AON for DMD Exon 53 Skipping, was approved in Japan earlier this year. Although promising, the efficacy of eteplirsen and AON sequence employed remain controversial. In addition, Exon Skipping faces challenges including the applicability and delivery. This article reviews and discusses Exon Skipping and the current advances being made in the field, on drugs, multi-Exon Skipping, sequence design, and applicability. We also discuss challenges and future directions that will facilitate the development of Exon Skipping therapy.
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Recent advancements in Exon-Skipping therapies using antisense oligonucleotides and genome editing for the treatment of various muscular dystrophies
Expert reviews in molecular medicine, 2019Co-Authors: Jaeho Hwang, Toshifumi YokotaAbstract:Muscular dystrophy is a group of genetic disorders characterised by degeneration of muscles. Different forms of muscular dystrophy can show varying phenotypes with a wide range of age, severity and location of muscle deterioration. Many palliative care options are available for muscular dystrophy patients, but no curative treatment is available. Exon-Skipping therapy aims to induce Skipping of Exons with disease-causing mutations and/or nearby Exons to restore the reading frame, which results in an internally truncated, partially functional protein. In antisense-mediated Exon-Skipping synthetic antisense oligonucleotide binds to pre-mRNA to induce Exon Skipping. Recent advances in Exon Skipping have yielded promising results; the US Food and Drug Administration (FDA) approved eteplirsen (Exondys51) as the first Exon-Skipping drug for the treatment of Duchenne muscular dystrophy, and in vivo Exon Skipping has been demonstrated in animal models of dysferlinopathy, limb-girdle muscular dystrophy type 2C and congenital muscular dystrophy type 1A. Novel methods that induce Exon Skipping utilizing Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) are also being developed where splice site mutations are created within the genome to induce Exon Skipping. Challenges remain as Exon-Skipping agents can have deleterious non-specific effects and different in-frame deletions show phenotypic variance. This article reviews the state of the art of Exon Skipping for treating muscular dystrophy and discusses challenges and future prospects.
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Multiple Exon Skipping in the Duchenne Muscular Dystrophy Hot Spots: Prospects and Challenges
Journal of personalized medicine, 2018Co-Authors: Yusuke Echigoya, Akinori Nakamura, Kenji Rowel Q. Lim, Toshifumi YokotaAbstract:Duchenne muscular dystrophy (DMD), a fatal X-linked recessive disorder, is caused mostly by frame-disrupting, out-of-frame deletions in the dystrophin (DMD) gene. Antisense oligonucleotide-mediated Exon Skipping is a promising therapy for DMD. Exon Skipping aims to convert out-of-frame mRNA to in-frame mRNA and induce the production of internally-deleted dystrophin as seen in the less severe Becker muscular dystrophy. Currently, multiple Exon Skipping has gained special interest as a new therapeutic modality for this approach. Previous retrospective database studies represented a potential therapeutic application of multiple Exon Skipping. Since then, public DMD databases have become more useful with an increase in patient registration and advances in molecular diagnosis. Here, we provide an update on DMD genotype-phenotype associations using a global DMD database and further provide the rationale for multiple Exon Skipping development, particularly for Exons 45–55 Skipping and an emerging therapeutic concept, Exons 3–9 Skipping. Importantly, this review highlights the potential of multiple Exon Skipping for enabling the production of functionally-corrected dystrophin and for treating symptomatic patients not only with out-of-frame deletions but also those with in-frame deletions. We will also discuss prospects and challenges in multiple Exon Skipping therapy, referring to recent progress in antisense chemistry and design, as well as disease models.
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Tips to Design Effective Splice-Switching Antisense Oligonucleotides for Exon Skipping and Exon Inclusion.
Methods of Molecular Biology, 2018Co-Authors: Rika Maruyama, Toshifumi YokotaAbstract: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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Quantitative Evaluation of Exon Skipping in Immortalized Muscle Cells In Vitro.
Methods in molecular biology (Clifton N.J.), 2018Co-Authors: Kenji Rowel Q. Lim, Toshifumi YokotaAbstract:Exon Skipping through the use of antisense oligonucleotides (AOs) is currently one of the most promising approaches for treating Duchenne muscular dystrophy (DMD). While we now have a number of AO drug candidates in clinical trials, we are still faced with issues of poor or controversial efficacy in some of these drugs. This is the case with eteplirsen, an Exon 51-Skipping AO that is the first and only FDA-approved drug for DMD to date. Effective procedures must, therefore, be set up for the in vitro screening of potential AOs for DMD treatment. Here, we describe one such procedure using immortalized DMD patient-derived muscle cells. Aside from allowing for the quantitative evaluation of candidate AOs based on their Exon Skipping efficiency and dystrophin protein rescue levels, these immortalized cells are stable, pure, easy to grow, and not subject to confounding by senescence-related issues. This procedure enables a more reliable screening of AOs prior to their entry in clinical trials and greatly facilitates the search for more efficacious candidate Exon Skipping AOs for DMD treatment.
Steve D. Wilton - One of the best experts on this subject based on the ideXlab platform.
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Precision Medicine through Antisense Oligonucleotide-Mediated Exon Skipping.
Trends in pharmacological sciences, 2018Co-Authors: Frank L. Mastaglia, Sue Fletcher, Steve D. WiltonAbstract:Clinical implementation of two recently approved antisense RNA therapeutics - Exondys51® to treat Duchenne muscular dystrophy (Duchenne MD) and Spinraza® as a treatment for spinal muscular atrophy (SMA) - highlights the therapeutic potential of antisense oligonucleotides (ASOs). As shown in the Duchenne and Becker cases, the identification and specific removal of 'dispensable' Exons by Exon-Skipping ASOs could potentially bypass lethal mutations in other genes and bring clinical benefits to affected individuals carrying amenable mutations. In this review, we discuss the potential of therapeutic alternative splicing, with a particular focus on targeted Exon Skipping using Duchenne MD as an example, and speculate on new applications for other inherited rare diseases where redundant or dispensable Exons may be amenable to Exon-Skipping ASO intervention as precision medicine.
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Multiple Exon Skipping strategies to by-pass dystrophin mutations
Neuromuscular disorders : NMD, 2011Co-Authors: C. Adkin, Sue Fletcher, Abbie M. Adams, Francesco Muntoni, Penelope L. Meloni, Brenda Wong, Steve D. WiltonAbstract:Manipulation of dystrophin pre-mRNA processing offers the potential to overcome mutations in the dystrophin gene that would otherwise lead to Duchenne muscular dystrophy. Dystrophin mutations will require the removal of one or more Exons to restore the reading frame and in some cases, multiple Exon Skipping strategies exist to restore dystrophin expression. However, for some small intra-Exonic mutations, a third strategy, not applicable to whole Exon deletions, may be possible. The removal of only one frame-shifting Exon flanking the mutation-carrying Exon may restore the reading frame and allow synthesis of a functional dystrophin isoform, providing that no premature termination codons are encountered. For these mutations, the removal of only one Exon offers a simpler, cheaper and more feasible alternative approach to the dual Exon Skipping that would otherwise be considered. We present strategies to by-pass intra-Exonic dystrophin mutations that clearly demonstrate the importance of tailoring Exon Skipping strategies to specific patient mutations.
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Evaluation of Exon‐Skipping strategies for Duchenne muscular dystrophy utilizing dystrophin‐deficient zebrafish
Journal of cellular and molecular medicine, 2011Co-Authors: Joachim Berger, Steve D. Wilton, Silke Berger, Arie Jacoby, Peter D. CurrieAbstract:Duchenne muscular dystophy (DMD) is a severe muscle wasting disease caused by mutations in the dystrophin gene. By utilizing antisense oligonucleotides, splicing of the dystrophin transcript can be altered so that Exons harbouring a mutation are excluded from the mature mRNA. Although this approach has been shown to be effective to restore partially functional dystrophin protein, the level of dystrophin protein that is necessary to rescue a severe muscle pathology has not been addressed. As zebrafish dystrophin mutants (dmd) resemble the severe muscle pathology of human patients, we have utilized this model to evaluate Exon Skipping. Novel dmd mutations were identified to enable the design of phenotype rescue studies via morpholino administration. Correlation of induced Exon-Skipping efficiency and the level of phenotype rescue suggest that relatively robust levels of Exon Skipping are required to achieve significant therapeutic ameliorations and that pre-screening analysis of Exon-Skipping drugs in zebrafish may help to more accurately predict clinical trials for therapies of DMD.
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Rational Design of Antisense Oligomers to Induce Dystrophin Exon Skipping
Molecular therapy : the journal of the American Society of Gene Therapy, 2009Co-Authors: Chalermchai Mitrpant, Sue Fletcher, Abbie M. Adams, Penny Meloni, Francesco Muntoni, Steve D. WiltonAbstract:Duchenne muscular dystrophy (DMD), one of the most severe neuromuscular disorders of childhood, is caused by the absence of a functional dystrophin. Antisense oligomer (AO) induced Exon Skipping is being investigated to restore functional dystrophin expression in models of muscular dystrophy and DMD patients. One of the major challenges will be in the development of clinically relevant oligomers and Exon Skipping strategies to address many different mutations. Various models, including cell-free extracts, cells transfected with artificial constructs, or mice with a human transgene, have been proposed as tools to facilitate oligomer design. Despite strong sequence homology between the human and mouse dystrophin genes, directing an oligomer to the same motifs in both species does not always induce comparable Exon Skipping. We report substantially different levels of Exon Skipping induced in normal and dystrophic human myogenic cell lines and propose that animal models or artificial assay systems useful in initial studies may be of limited relevance in designing the most efficient compounds to induce targeted Skipping of human dystrophin Exons for therapeutic outcomes.
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Personalised genetic intervention for Duchenne muscular dystrophy: antisense oligomers and Exon Skipping.
Current molecular pharmacology, 2009Co-Authors: Chalermchai Mitrpant, Sue Fletcher, Steve D. WiltonAbstract:Duchenne muscular dystrophy (DMD) arises from protein-truncating mutations in the large dystrophin gene that preclude synthesis of a functional protein that primarily stabilizes muscle fibre membranes. The absence of dystrophin leads to this most common and serious form of childhood muscle-wasting. Since the identification of the dystrophin gene in 1987, cell and gene repair or replacement therapies have been evaluated for DMD treatment and one genetic intervention, Exon Skipping, is now in clinical trials. Antisense oligomers have been designed to redirect dystrophin splicing patterns so that targeted Exons may be removed from a defective dystrophin pre-mRNA to either restore the reading frame of a deletion, or excise an in-frame Exon corrupted by a nonsense mutation or microinsertion/ deletion. This review discusses the evolution of oligomer induced Exon Skipping, including in vitro applications, evaluation of different oligomer chemistries, the treatment of animal models and alternative Exon Skipping strategies involving viral expression cassettes and ex vivo manipulation of stem cells. The discussion culminates with the current clinical trials and the great challenges that lie ahead. The major obstacle to the implementation of personalised genetic treatments to address the many different mutations that can lead to DMD, are considered to be establishing effective treatments for the different patients and their mutations. Furthermore, the view of regulatory authorities in assessing preclinical data on potentially scores of different but class-specific compounds will be of paramount importance in expediting the clinical application of Exon Skipping therapy for this serious and relentlessly progressive muscle wasting disease.
Annemieke Aartsma-rus - One of the best experts on this subject based on the ideXlab platform.
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What We Have Learned from 10 Years of DMD Exon-Skipping Trials
Muscle Gene Therapy, 2019Co-Authors: Svitlana Pasteuning-vuhman, Annemieke Aartsma-rusAbstract:Duchenne muscular dystrophy (DMD) is an X-linked recessive disorder caused by truncating mutations in the DMD gene. These result in the absence of the muscle fibre stabilizing dystrophin protein and progressive loss of muscle tissue and function. In-frame mutations with partially functional dystrophin generally lead to Becker muscular dystrophy (BMD) with a milder disease phenotype. This was the inspiration for the antisense-mediated Exon-Skipping approach that restores the dystrophin reading frame to allow production of a Becker-type dystrophin. This approach is mutation specific. Since Exon 51 Skipping is applicable to the largest group of DMD patients, two antisense compounds targeting Exon 51 were developed first, i.e. drisapersen and eteplirsen. Ten years have passed since the first Exon-Skipping antisense compound was tested clinically in DMD patients. If objectively evaluated, initial trials were suboptimal with modest clinical success. Major hurdles were that, at the time of trial planning, natural history data and reliable outcome measures to detect clinical benefit were not available. Moreover, the levels of dystrophin that are restored in DMD patients are lower than those observed in BMD patients. This chapter looks back at the lessons that were learned during the development of DMD Exon Skipping so far, to allow for more optimal Exon-Skipping trials in the future.
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Exon Skipping: a first in class strategy for Duchenne muscular dystrophy.
Expert opinion on biological therapy, 2016Co-Authors: Erik H. Niks, Annemieke Aartsma-rusAbstract:Introduction: Exon Skipping is a therapeutic approach for Duchenne muscular dystrophy (DMD) that has been in development for close to two decades. This approach uses antisense oligonucleotides (AON...
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Translational and Regulatory Challenges for Exon Skipping Therapies
Human gene therapy, 2014Co-Authors: Annemieke Aartsma-rus, Anna M. G. Pasmooij, Alessandra Ferlini, Nathalie Goemans, Dominic J. Wells, Katerine Bushby, Elizabeth Vroom, Pavel BalabanovAbstract:Several translational challenges are currently impeding the therapeutic development of antisense-mediated Exon Skipping approaches for rare diseases. Some of these are inherent to developing therapies for rare diseases, such as small patient numbers and limited information on natural history and interpretation of appropriate clinical outcome measures. Others are inherent to the antisense oligonucleotide (AON)-mediated Exon Skipping approach, which employs small modified DNA or RNA molecules to manipulate the splicing process. This is a new approach and only limited information is available on long-term safety and toxicity for most AON chemistries. Furthermore, AONs often act in a mutation-specific manner, in which case multiple AONs have to be developed for a single disease. A workshop focusing on preclinical development, trial design, outcome measures, and different forms of marketing authorization was organized by the regulatory models and biochemical outcome measures working groups of Cooperation of Science and Technology Action: "Networking towards clinical application of antisense-mediated Exon Skipping for rare diseases." The workshop included participants from patient organizations, academia, and members of staff from the European Medicine Agency and Medicine Evaluation Board (the Netherlands). This statement article contains the key outcomes of this meeting.
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The Effect of 6-Thioguanine on Alternative Splicing and Antisense-Mediated Exon Skipping Treatment for Duchenne Muscular Dystrophy
PLoS currents, 2012Co-Authors: Ingrid E.c. Verhaart, Annemieke Aartsma-rusAbstract:The severe muscle wasting disorder Duchenne muscular dystrophy (DMD) is caused by genetic defects in the DMD gene, leading to a complete absence of dystrophin protein. Of the therapeutic approaches addressing the underlying genetic defect, Exon Skipping through antisense oligonucleotides (AONs) is the closest to clinical application. Several strategies to improve the efficiency of this approach are currently being investigated, such as the use of small chemical compounds that improve AONmediated Exon Skipping levels. Recently, enhanced Exon Skipping in combination with a guanine analogue, 6-thioguanine (6TG) was reported for phosphorodiamidate morpholino oligomers (PMO). Here the effect of 6TG on the Exon Skipping efficacy of 2 �-O- methyl phosphorothioate RNA (2OMePS) and PMO AONs in vitro and in vivo was further evaluated, as well as the effect of 6TG by itself. Results confirm an increase of Exon Skipping levels in vitro, however, in contrast to the previous report, no effect was observed in vivo. Importantly, 6TG treatment in vitro resulted in numerous additional DMD Exon Skipping events. This, in combination with the known cytotoxic effects of 6TG after incorporation in DNA, warrants reconsidering of the use of 6TG as enhancer of AON efficiency in DMD, were chronic treatment will be required. Abstract The severe muscle wasting disorder Duchenne muscular dystrophy (DMD) is caused by genetic defects in the DMD gene, leading to a complete absence of dystrophin protein. Of the therapeutic approaches addressing the underlying genetic defect, Exon Skipping through antisense oligonucleotides (AONs) is the closest to clinical application. Several strategies to improve the efficiency of this approach are currently being investigated, such as the use of small chemical compounds that improve AONmediated Exon Skipping levels. Recently, enhanced Exon Skipping in combination with a guanine analogue, 6-thioguanine (6TG) was reported for phosphorodiamidate morpholino oligomers (PMO). Here the effect of 6TG on the Exon Skipping efficacy of 2 �-O- methyl phosphorothioate RNA (2OMePS) and PMO AONs in vitro and in vivo was further evaluated, as well as the effect of 6TG by itself. Results confirm an increase of Exon Skipping levels in vitro, however, in contrast to the previous report, no effect was observed in vivo. Importantly, 6TG treatment in vitro resulted in numerous additional DMD Exon Skipping events. This, in combination with the known cytotoxic effects of 6TG after incorporation in DNA, warrants reconsidering of the use of 6TG as enhancer of AON efficiency in DMD, were chronic treatment will be required. Abstract The severe muscle wasting disorder Duchenne muscular dystrophy (DMD) is caused by genetic defects in the DMD gene, leading to a complete absence of dystrophin protein. Of the therapeutic approaches addressing the underlying genetic defect, Exon Skipping through antisense oligonucleotides (AONs) is the closest to clinical application. Several strategies to improve the efficiency of this approach are currently being investigated, such as the use of small chemical compounds that improve AONmediated Exon Skipping levels. Recently, enhanced Exon Skipping in combination with a guanine analogue, 6-thioguanine (6TG) was reported for phosphorodiamidate morpholino oligomers (PMO). Here the effect of 6TG on the Exon Skipping efficacy of 2 �-O- methyl phosphorothioate RNA (2OMePS) and PMO AONs in vitro and in vivo was further evaluated, as well as the effect of 6TG by itself. Results confirm an increase of Exon Skipping levels in vitro, however, in contrast to the previous report, no effect was observed in vivo. Importantly, 6TG treatment in vitro resulted in numerous additional DMD Exon Skipping events. This, in combination with the known cytotoxic effects of 6TG after incorporation in DNA, warrants reconsidering of the use of 6TG as enhancer of AON efficiency in DMD, were chronic treatment will be required.
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Overview on DMD Exon Skipping.
Methods in molecular biology (Clifton N.J.), 2012Co-Authors: Annemieke Aartsma-rusAbstract:Antisense-mediated Exon Skipping to restore the disrupted dystrophin reading frame is currently in clinical trials for Duchenne muscular dystrophy. This chapter describes the rationale of this approach and gives an overview of in vitro and in vivo experiments with antisense oligonucleotides and antisense genes. Finally, an overview of clinical trials is given and outstanding questions and hurdles are discussed.
Sue Fletcher - One of the best experts on this subject based on the ideXlab platform.
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Precision Medicine through Antisense Oligonucleotide-Mediated Exon Skipping.
Trends in pharmacological sciences, 2018Co-Authors: Frank L. Mastaglia, Sue Fletcher, Steve D. WiltonAbstract:Clinical implementation of two recently approved antisense RNA therapeutics - Exondys51® to treat Duchenne muscular dystrophy (Duchenne MD) and Spinraza® as a treatment for spinal muscular atrophy (SMA) - highlights the therapeutic potential of antisense oligonucleotides (ASOs). As shown in the Duchenne and Becker cases, the identification and specific removal of 'dispensable' Exons by Exon-Skipping ASOs could potentially bypass lethal mutations in other genes and bring clinical benefits to affected individuals carrying amenable mutations. In this review, we discuss the potential of therapeutic alternative splicing, with a particular focus on targeted Exon Skipping using Duchenne MD as an example, and speculate on new applications for other inherited rare diseases where redundant or dispensable Exons may be amenable to Exon-Skipping ASO intervention as precision medicine.
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Multiple Exon Skipping strategies to by-pass dystrophin mutations
Neuromuscular disorders : NMD, 2011Co-Authors: C. Adkin, Sue Fletcher, Abbie M. Adams, Francesco Muntoni, Penelope L. Meloni, Brenda Wong, Steve D. WiltonAbstract:Manipulation of dystrophin pre-mRNA processing offers the potential to overcome mutations in the dystrophin gene that would otherwise lead to Duchenne muscular dystrophy. Dystrophin mutations will require the removal of one or more Exons to restore the reading frame and in some cases, multiple Exon Skipping strategies exist to restore dystrophin expression. However, for some small intra-Exonic mutations, a third strategy, not applicable to whole Exon deletions, may be possible. The removal of only one frame-shifting Exon flanking the mutation-carrying Exon may restore the reading frame and allow synthesis of a functional dystrophin isoform, providing that no premature termination codons are encountered. For these mutations, the removal of only one Exon offers a simpler, cheaper and more feasible alternative approach to the dual Exon Skipping that would otherwise be considered. We present strategies to by-pass intra-Exonic dystrophin mutations that clearly demonstrate the importance of tailoring Exon Skipping strategies to specific patient mutations.
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Rational Design of Antisense Oligomers to Induce Dystrophin Exon Skipping
Molecular therapy : the journal of the American Society of Gene Therapy, 2009Co-Authors: Chalermchai Mitrpant, Sue Fletcher, Abbie M. Adams, Penny Meloni, Francesco Muntoni, Steve D. WiltonAbstract:Duchenne muscular dystrophy (DMD), one of the most severe neuromuscular disorders of childhood, is caused by the absence of a functional dystrophin. Antisense oligomer (AO) induced Exon Skipping is being investigated to restore functional dystrophin expression in models of muscular dystrophy and DMD patients. One of the major challenges will be in the development of clinically relevant oligomers and Exon Skipping strategies to address many different mutations. Various models, including cell-free extracts, cells transfected with artificial constructs, or mice with a human transgene, have been proposed as tools to facilitate oligomer design. Despite strong sequence homology between the human and mouse dystrophin genes, directing an oligomer to the same motifs in both species does not always induce comparable Exon Skipping. We report substantially different levels of Exon Skipping induced in normal and dystrophic human myogenic cell lines and propose that animal models or artificial assay systems useful in initial studies may be of limited relevance in designing the most efficient compounds to induce targeted Skipping of human dystrophin Exons for therapeutic outcomes.
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Personalised genetic intervention for Duchenne muscular dystrophy: antisense oligomers and Exon Skipping.
Current molecular pharmacology, 2009Co-Authors: Chalermchai Mitrpant, Sue Fletcher, Steve D. WiltonAbstract:Duchenne muscular dystrophy (DMD) arises from protein-truncating mutations in the large dystrophin gene that preclude synthesis of a functional protein that primarily stabilizes muscle fibre membranes. The absence of dystrophin leads to this most common and serious form of childhood muscle-wasting. Since the identification of the dystrophin gene in 1987, cell and gene repair or replacement therapies have been evaluated for DMD treatment and one genetic intervention, Exon Skipping, is now in clinical trials. Antisense oligomers have been designed to redirect dystrophin splicing patterns so that targeted Exons may be removed from a defective dystrophin pre-mRNA to either restore the reading frame of a deletion, or excise an in-frame Exon corrupted by a nonsense mutation or microinsertion/ deletion. This review discusses the evolution of oligomer induced Exon Skipping, including in vitro applications, evaluation of different oligomer chemistries, the treatment of animal models and alternative Exon Skipping strategies involving viral expression cassettes and ex vivo manipulation of stem cells. The discussion culminates with the current clinical trials and the great challenges that lie ahead. The major obstacle to the implementation of personalised genetic treatments to address the many different mutations that can lead to DMD, are considered to be establishing effective treatments for the different patients and their mutations. Furthermore, the view of regulatory authorities in assessing preclinical data on potentially scores of different but class-specific compounds will be of paramount importance in expediting the clinical application of Exon Skipping therapy for this serious and relentlessly progressive muscle wasting disease.
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T.P.2.09 Induced Exon Skipping in normal and mdx muscle
Neuromuscular Disorders, 2008Co-Authors: Sue Fletcher, Chalermchai Mitrpant, Penny Meloni, J.p. Steinhaus, Steve D. WiltonAbstract:Induced Exon Skipping to remove or by-pass protein truncating mutations in the dystrophin gene is emerging as a potential therapy for many cases of Duchenne muscular dystrophy. It has been proposed that the compromised sarcolemma of the dystrophic muscle fibres may facilitate uptake of compounds that induce Exon Skipping. If this were the case, then restoration of some functional dystrophin expression may restrict further oligomer uptake, thereby creating a therapeutic ceiling. We addressed this question by systemically treating normal C57BL/10ScSn mice with the same compound that induced substantial dystrophin Exon 23 Skipping and restored dystrophin expression in the mdx mouse model of muscular dystrophy (C57BL/10ScSnmdx). Repeated intraperitoneal injections of a phosphorodiamidate morpholino oligomer coupled to a cell penetrating peptide (PMO-P007), were sufficient to induce readily detectable levels of dystrophin gene transcripts missing Exon 23 in normal skeletal muscle, as detected by RT-PCR. However, Exon 23 Skipping could not be detected in the heart until assay conditions were biased towards generation of shorter PCR products, after which 22% Exon Skipping was apparent in cardiac muscle from treated animals. Detailed protein studies were not possible on the normal dystrophin-positive background, but clearly, the uptake and efficacy of PMO-P007 was not compromised by the normal skeletal muscle sarcolemma. Furthermore, the selective bias that can be achieved to enhance apparent Exon Skipping during RT-PCR assays was such that we recommend molecular testing should be standardised to facilitate valid comparisons between different laboratories and studies.
Le Kang - One of the best experts on this subject based on the ideXlab platform.
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crispr cas9 mediated genome editing induces Exon Skipping by complete or stochastic altering splicing in the migratory locust
BMC Biotechnology, 2018Co-Authors: Dafeng Chen, Jixin Tang, Li Hou, Xianhui Wang, Le KangAbstract:The CRISPR/Cas9 system has been widely used to generate gene knockout/knockin models by inducing frameshift mutants in cell lines and organisms. Several recent studies have reported that such mutants can lead to in-frame Exon Skipping in cell lines. However, there was little research about post-transcriptional effect of CRISPR-mediated gene editing in vivo. We showed that frameshift indels also induced complete or stochastic Exon Skipping by deleting different regions to influence pre-mRNA splicing in vivo. In the migratory locust, the missing 55 bp at the boundary of intron 3 and Exon 4 of an olfactory receptor gene, LmigOr35, resulted in complete Exon 4 Skipping, whereas the lacking 22 bp in Exon 4 of LmigOr35 only resulted in stochastic Exon 4 Skipping. A single sgRNA induced small insertions or deletions at the boundary of intron and Exon to disrupt the 3′ splicing site causing completely Exon Skipping, or alternatively induce small insertions or deletions in the Exon to stochastic alter splicing causing the stochastic Exon Skipping. These results indicated that complete or stochastic Exon Skipping could result from the CRISPR-mediated genome editing by deleting different regions of the gene. Although Exon Skipping caused by CRISPR-mediated editing was an unexpected outcome, this finding could be developed as a technology to investigate pre-mRNA splicing or to cure several human diseases caused by splicing mutations.
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CRISPR/Cas9-mediated genome editing induces Exon Skipping by complete or stochastic altering splicing in the migratory locust
BMC biotechnology, 2018Co-Authors: Dafeng Chen, Jixin Tang, Li Hou, Xianhui Wang, Le KangAbstract:The CRISPR/Cas9 system has been widely used to generate gene knockout/knockin models by inducing frameshift mutants in cell lines and organisms. Several recent studies have reported that such mutants can lead to in-frame Exon Skipping in cell lines. However, there was little research about post-transcriptional effect of CRISPR-mediated gene editing in vivo. We showed that frameshift indels also induced complete or stochastic Exon Skipping by deleting different regions to influence pre-mRNA splicing in vivo. In the migratory locust, the missing 55 bp at the boundary of intron 3 and Exon 4 of an olfactory receptor gene, LmigOr35, resulted in complete Exon 4 Skipping, whereas the lacking 22 bp in Exon 4 of LmigOr35 only resulted in stochastic Exon 4 Skipping. A single sgRNA induced small insertions or deletions at the boundary of intron and Exon to disrupt the 3′ splicing site causing completely Exon Skipping, or alternatively induce small insertions or deletions in the Exon to stochastic alter splicing causing the stochastic Exon Skipping. These results indicated that complete or stochastic Exon Skipping could result from the CRISPR-mediated genome editing by deleting different regions of the gene. Although Exon Skipping caused by CRISPR-mediated editing was an unexpected outcome, this finding could be developed as a technology to investigate pre-mRNA splicing or to cure several human diseases caused by splicing mutations.
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CRISPR/Cas9-mediated genome editing induces Exon Skipping by complete or stochastic altering splicing in the migratory locust
BMC, 2018Co-Authors: Dafeng Chen, Jixin Tang, Li Hou, Xianhui Wang, Le KangAbstract:Abstract Background The CRISPR/Cas9 system has been widely used to generate gene knockout/knockin models by inducing frameshift mutants in cell lines and organisms. Several recent studies have reported that such mutants can lead to in-frame Exon Skipping in cell lines. However, there was little research about post-transcriptional effect of CRISPR-mediated gene editing in vivo. Results We showed that frameshift indels also induced complete or stochastic Exon Skipping by deleting different regions to influence pre-mRNA splicing in vivo. In the migratory locust, the missing 55 bp at the boundary of intron 3 and Exon 4 of an olfactory receptor gene, LmigOr35, resulted in complete Exon 4 Skipping, whereas the lacking 22 bp in Exon 4 of LmigOr35 only resulted in stochastic Exon 4 Skipping. A single sgRNA induced small insertions or deletions at the boundary of intron and Exon to disrupt the 3′ splicing site causing completely Exon Skipping, or alternatively induce small insertions or deletions in the Exon to stochastic alter splicing causing the stochastic Exon Skipping. Conclusions These results indicated that complete or stochastic Exon Skipping could result from the CRISPR-mediated genome editing by deleting different regions of the gene. Although Exon Skipping caused by CRISPR-mediated editing was an unexpected outcome, this finding could be developed as a technology to investigate pre-mRNA splicing or to cure several human diseases caused by splicing mutations