Applications of CRISPR/Cas9 for the Treatment of Duchenne Muscular Dystrophy

Duchenne muscular dystrophy (DMD) is a fatal X-linked recessive neuromuscular disease prevalent in 1 in 3500 to 5000 males worldwide. As a result of mutations that interrupt the reading frame of the dystrophin gene (DMD), DMD is characterized by a loss of dystrophin protein that leads to decreased muscle membrane integrity, which increases susceptibility to degeneration. CRISPR/Cas9 technology has garnered interest as an avenue for DMD therapy due to its potential for permanent exon skipping, which can restore the disrupted DMD reading frame in DMD and lead to dystrophin restoration. An RNA-guided DNA endonuclease system, CRISPR/Cas9 allows for the targeted editing of specific sequences in the genome. The efficacy and safety of CRISPR/Cas9 as a therapy for DMD has been evaluated by numerous studies in vitro and in vivo, with varying rates of success. Despite the potential of CRISPR/Cas9-mediated gene editing for the long-term treatment of DMD, its translation into the clinic is currently challenged by issues such as off-targeting, immune response activation, and sub-optimal in vivo delivery. Its nature as being mostly a personalized form of therapy also limits applicability to DMD patients, who exhibit a wide spectrum of mutations. This review summarizes the various CRISPR/Cas9 strategies that have been tested in vitro and in vivo for the treatment of DMD. Perspectives on the approach will be provided, and the challenges faced by CRISPR/Cas9 in its road to the clinic will be briefly discussed.

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Methods of CRISPR/Cas9 Exon Skipping for Duchenne Muscular Dystrophy

10.20944/preprints201811.0018.v1 ◽

2018 ◽

Cited By ~ 1

Author(s):

Kenji Rowel Q. Lim ◽

Chantal Yoon ◽

Toshifumi Yokota

Keyword(s):

Duchenne Muscular Dystrophy ◽

Muscular Dystrophy ◽

Wide Spectrum ◽

Membrane Integrity ◽

Exon Skipping ◽

Muscle Membrane ◽

Reading Frame ◽

Long Term Treatment

Duchenne muscular dystrophy (DMD) is a fatal X-linked recessive neuromuscular disease prevalent in 1 in 3500 to 5000 males worldwide. As a result of mutations that interrupt the reading frame of the dystrophin gene (DMD), DMD is characterized by a loss of dystrophin protein which leads to decreased muscle membrane integrity, which increases susceptibility to degeneration. CRISPR/Cas9 technology has garnered interest as an avenue for DMD therapy due to its potential for permanent exon skipping, which can restore the disrupted DMD reading frame in DMD and lead to dystrophin restoration. An RNA-guided DNA endonuclease system, CRISPR/Cas9 allows for the targeted editing of specific sequences in the genome. The efficacy and safety of CRISPR/Cas9 as a therapy for DMD has been evaluated by numerous studies in vitro and in vivo, with varying rates of success. Despite the potential of CRISPR/Cas9-mediated gene editing for the long-term treatment of DMD, its translation into the clinic is currently challenged by issues such as off-targeting, immune response activation, and sub-optimal in vivo delivery. Its nature as being mostly a personalized form of therapy also limits applicability to DMD patients, who exhibit a wide spectrum of mutations. This review summarizes the various CRISPR/Cas9 strategies that have been tested in vitro and in vivo for the treatment of DMD. Perspectives on the approach will be provided, and the challenges faced by CRISPR/Cas9 in its road to the clinic will be briefly discussed.

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Precise correction of Duchenne muscular dystrophy exon deletion mutations by base and prime editing

Science Advances ◽

10.1126/sciadv.abg4910 ◽

2021 ◽

Vol 7 (18) ◽

pp. eabg4910

Author(s):

F. Chemello ◽

A. C. Chai ◽

H. Li ◽

C. Rodriguez-Caycedo ◽

E. Sanchez-Ortiz ◽

...

Keyword(s):

Duchenne Muscular Dystrophy ◽

Muscular Dystrophy ◽

Membrane Integrity ◽

Dystrophin Gene ◽

Muscle Disease ◽

Muscle Membrane ◽

Reading Frame ◽

Split Intein ◽

Virus Serotype

Duchenne muscular dystrophy (DMD) is a fatal muscle disease caused by the lack of dystrophin, which maintains muscle membrane integrity. We used an adenine base editor (ABE) to modify splice donor sites of the dystrophin gene, causing skipping of a common DMD deletion mutation of exon 51 (∆Ex51) in cardiomyocytes derived from human induced pluripotent stem cells, restoring dystrophin expression. Prime editing was also capable of reframing the dystrophin open reading frame in these cardiomyocytes. Intramuscular injection of ∆Ex51 mice with adeno-associated virus serotype-9 encoding ABE components as a split-intein trans-splicing system allowed gene editing and disease correction in vivo. Our findings demonstrate the effectiveness of nucleotide editing for the correction of diverse DMD mutations with minimal modification of the genome, although improved delivery methods will be required before these strategies can be used to sufficiently edit the genome in patients with DMD.

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In vivo non-invasive monitoring of dystrophin correction in a new Duchenne muscular dystrophy reporter mouse

Nature Communications ◽

10.1038/s41467-019-12335-x ◽

2019 ◽

Vol 10 (1) ◽

Cited By ~ 6

Author(s):

Leonela Amoasii ◽

Hui Li ◽

Yu Zhang ◽

Yi-Li Min ◽

Efrain Sanchez-Ortiz ◽

...

Keyword(s):

Duchenne Muscular Dystrophy ◽

Muscular Dystrophy ◽

Genetic Disorder ◽

Dystrophin Gene ◽

Luciferase Reporter ◽

Luciferase Expression ◽

Gene Correction ◽

Reading Frame ◽

Non Invasive

Abstract Duchenne muscular dystrophy (DMD) is a fatal genetic disorder caused by mutations in the dystrophin gene. To enable the non-invasive analysis of DMD gene correction strategies in vivo, we introduced a luciferase reporter in-frame with the C-terminus of the dystrophin gene in mice. Expression of this reporter mimics endogenous dystrophin expression and DMD mutations that disrupt the dystrophin open reading frame extinguish luciferase expression. We evaluated the correction of the dystrophin reading frame coupled to luciferase in mice lacking exon 50, a common mutational hotspot, after delivery of CRISPR/Cas9 gene editing machinery with adeno-associated virus. Bioluminescence monitoring revealed efficient and rapid restoration of dystrophin protein expression in affected skeletal muscles and the heart. Our results provide a sensitive non-invasive means of monitoring dystrophin correction in mouse models of DMD and offer a platform for testing different strategies for amelioration of DMD pathogenesis.

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CRISPR-Cas9 corrects Duchenne muscular dystrophy exon 44 deletion mutations in mice and human cells

Science Advances ◽

10.1126/sciadv.aav4324 ◽

2019 ◽

Vol 5 (3) ◽

pp. eaav4324 ◽

Cited By ~ 55

Author(s):

Yi-Li Min ◽

Hui Li ◽

Cristina Rodriguez-Caycedo ◽

Alex A. Mireault ◽

Jian Huang ◽

...

Keyword(s):

Duchenne Muscular Dystrophy ◽

Muscular Dystrophy ◽

Gene Editing ◽

Dystrophin Gene ◽

Gene Correction ◽

Reading Frame ◽

Deletion Mutations ◽

Significant Step ◽

Induced Pluripotent

Mutations in the dystrophin gene cause Duchenne muscular dystrophy (DMD), which is characterized by lethal degeneration of cardiac and skeletal muscles. Mutations that delete exon 44 of the dystrophin gene represent one of the most common causes of DMD and can be corrected in ~12% of patients by editing surrounding exons, which restores the dystrophin open reading frame. Here, we present a simple and efficient strategy for correction of exon 44 deletion mutations by CRISPR-Cas9 gene editing in cardiomyocytes obtained from patient-derived induced pluripotent stem cells and in a new mouse model harboring the same deletion mutation. Using AAV9 encoding Cas9 and single guide RNAs, we also demonstrate the importance of the dosages of these gene editing components for optimal gene correction in vivo. Our findings represent a significant step toward possible clinical application of gene editing for correction of DMD.

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248. Correction of the Dystrophin Gene Mutation in the mdx5cv Mouse Model of Duchenne Muscular Dystrophy Mediated by Chimeric and DNA Oligonucleotides In Vitro and In Vivo

Molecular Therapy ◽

10.1016/s1525-0016(16)40690-8 ◽

2003 ◽

Vol 7 (5) ◽

pp. S98

Keyword(s):

Duchenne Muscular Dystrophy ◽

Muscular Dystrophy ◽

Mouse Model ◽

Gene Mutation ◽

Dystrophin Gene ◽

Dna Oligonucleotides

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Galectin-3 and N-acetylglucosamine promote myogenesis and improve skeletal muscle function in the mdx model of Duchenne muscular dystrophy

10.1101/203653 ◽

2017 ◽

Cited By ~ 6

Author(s):

Ann Rancourt ◽

Sébastien Dufresne ◽

Guillaume St-Pierre ◽

Julie-Christine Lévesque ◽

Haruka Nakamura ◽

...

Keyword(s):

Skeletal Muscle ◽

Duchenne Muscular Dystrophy ◽

Muscular Dystrophy ◽

Basal Lamina ◽

Dystrophin Gene ◽

Muscle Membrane ◽

Skeletal Muscle Function ◽

Myogenic Stem Cells ◽

Galectin 3

AbstractThe muscle membrane, sarcolemma, must be firmly attached to the basal lamina. The failure of proper attachment results in muscle injury, which is the underlying cause of Duchenne muscular dystrophy (DMD), where mutations in the dystrophin gene disrupts the firm adhesion. In DMD patients, even moderate contraction causes damage, leading to progressive muscle degeneration. The damaged muscles are repaired through myogenesis. Consequently, myogenesis is highly active in DMD patients, and the repeated activation of myogenesis leads to the exhaustion of the myogenic stem cells. Therefore, approaches to reducing the risk of the exhaustion are to develop a treatment that strengthens the interaction between the sarcolemma and the basal lamina, and increases the efficiency of myogenesis. Galectin-3 is an oligosaccharide-binding protein and known to be involved in cell-cell interactions and cell-matrix interactions. Galectin-3 is expressed in myoblasts and skeletal muscle while its function in muscle remains elusive. In this study, we found evidence that galectin-3 and the monosaccharide N-acetylglucosamine, which increases the ligands (oligosaccharides) of galectin-3, promotes myogenesis in vitro. Moreover, in the mdx mouse model of DMD, treatment with N-acetylglucosamine increased the muscle force production. Our results demonstrate that treatment with N-acetylglucosamine can mitigate the burden of DMD.

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Empowering Muscle Stem Cells for the Treatment of Duchenne Muscular Dystrophy

Cells Tissues Organs ◽

10.1159/000514305 ◽

2021 ◽

pp. 1-14

Author(s):

Romina L. Filippelli ◽

Natasha C. Chang

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Duchenne Muscular Dystrophy ◽

Muscular Dystrophy ◽

Cell Function ◽

Critical Role ◽

Membrane Integrity ◽

Muscle Membrane ◽

Muscle Stem Cell ◽

Muscle Stem Cells

Duchenne muscular dystrophy (DMD) is a devastating and debilitating muscle degenerative disease affecting 1 in every 3,500 male births worldwide. DMD is progressive and fatal; accumulated weakening of the muscle tissue leads to an inability to walk and eventual loss of life due to respiratory and cardiac failure. Importantly, there remains no effective cure for DMD. DMD is caused by defective expression of the <i>DMD</i> gene, which encodes for dystrophin, a component of the dystrophin glycoprotein complex. In muscle fibers, this protein complex plays a critical role in maintaining muscle membrane integrity. Emerging studies have shown that muscle stem cells, which are adult stem cells responsible for muscle repair, are also affected in DMD. DMD muscle stem cells do not function as healthy muscle stem cells, and their impairment contributes to disease progression. Deficiencies in muscle stem cell function include impaired establishment of cell polarity leading to defective asymmetric stem cell division, reduced myogenic commitment, impaired differentiation, altered metabolism, and enhanced entry into senescence. Altogether, these findings indicate that DMD muscle stem cells are dysfunctional and have impaired regenerative potential. Although recent advances in adeno-associated vector and antisense oligonucleotide-mediated mechanisms for gene therapy have shown clinical promise, the current therapeutic strategies for muscular dystrophy do not effectively target muscle stem cells and do not address the deficiencies in muscle stem cell function. Here, we discuss the merits of restoring endogenous muscle stem cell function in degenerating muscle as a viable regenerative medicine strategy to mitigate DMD.

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Evaluating the potential of novel genetic approaches for the treatment of Duchenne muscular dystrophy

European Journal of Human Genetics ◽

10.1038/s41431-021-00811-2 ◽

2021 ◽

Author(s):

Vratko Himič ◽

Kay E. Davies

Keyword(s):

Duchenne Muscular Dystrophy ◽

Muscular Dystrophy ◽

Structural Integrity ◽

Viral Vector ◽

Exon Skipping ◽

Dystrophin Gene ◽

Pharmacological Treatments ◽

Genetic Sequencing ◽

Reading Frame ◽

Target Effects

AbstractDuchenne muscular dystrophy (DMD) is an X-linked progressive muscle-wasting disorder that is caused by a lack of functional dystrophin, a cytoplasmic protein necessary for the structural integrity of muscle. As variants in the dystrophin gene lead to a disruption of the reading frame, pharmacological treatments have only limited efficacy; there is currently no effective therapy and consequently, a significant unmet clinical need for DMD. Recently, novel genetic approaches have shown real promise in treating DMD, with advancements in the efficacy and tropism of exon skipping and surrogate gene therapy. CRISPR-Cas9 has the potential to be a ‘one-hit’ curative treatment in the coming decade. The current limitations of gene editing, such as off-target effects and immunogenicity, are in fact partly constraints of the delivery method itself, and thus research focus has shifted to improving the viral vector. In order to halt the loss of ambulation, early diagnosis and treatment will be pivotal. In an era where genetic sequencing is increasingly utilised in the clinic, genetic therapies will play a progressively central role in DMD therapy. This review delineates the relative merits of cutting-edge genetic approaches, as well as the challenges that still need to be overcome before they become clinically viable.

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