CRISPR-Generated Animal Models of Duchenne Muscular Dystrophy

Duchenne muscular dystrophy (DMD) is a fatal X-linked recessive neuromuscular disorder most commonly caused by mutations disrupting the reading frame of the dystrophin (DMD) gene. DMD codes for dystrophin, which is critical for maintaining the integrity of muscle cell membranes. Without dystrophin, muscle cells receive heightened mechanical stress, becoming more susceptible to damage. An active body of research continues to explore therapeutic treatments for DMD as well as to further our understanding of the disease. These efforts rely on having reliable animal models that accurately recapitulate disease presentation in humans. While current animal models of DMD have served this purpose well to some extent, each has its own limitations. To help overcome this, clustered regularly interspaced short palindromic repeat (CRISPR)-based technology has been extremely useful in creating novel animal models for DMD. This review focuses on animal models developed for DMD that have been created using CRISPR, their advantages and disadvantages as well as their applications in the DMD field.

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CRISPR-Generated Animal Models of Duchenne Muscular Dystrophy

10.20944/preprints202003.0048.v1 ◽

2020 ◽

Author(s):

Kenji Rowel Q. Lim ◽

Quynh Nguyen ◽

Kasia Dzierlega ◽

Yiqing Huang ◽

Toshifumi Yokota

Keyword(s):

Duchenne Muscular Dystrophy ◽

Muscular Dystrophy ◽

Animal Models ◽

Mechanical Stress ◽

Muscle Cell ◽

Muscle Cells ◽

Neuromuscular Disorder ◽

Reading Frame ◽

Advantages And Disadvantages ◽

Dmd Gene

Duchenne muscular dystrophy (DMD) is a fatal X-linked recessive neuromuscular disorder most commonly caused by mutations disrupting the reading frame of the dystrophin (DMD) gene. DMD codes for dystrophin, which is critical for maintaining the integrity of muscle cell membranes. Without dystrophin, muscle cells receive heightened mechanical stress, becoming more susceptible to damage. An active body of research continues to explore therapeutic treatments for DMD as well as to further our understanding of the disease. These efforts rely on having reliable animal models that accurately recapitulate disease presentation in humans. While current animal models of DMD have served this purpose quite well, each comes with their own limitations. To help overcome this, clustered regularly interspaced short palindromic repeats (CRISPR)-based technology has been extremely useful in creating novel animal models for DMD. This review focuses on animal models developed for DMD that have been created using CRISPR, their advantages and disadvantages as well as their applications in the DMD field.

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Genotype–Phenotype Correlations in Duchenne and Becker Muscular Dystrophy Patients from the Canadian Neuromuscular Disease Registry

Journal of Personalized Medicine ◽

10.3390/jpm10040241 ◽

2020 ◽

Vol 10 (4) ◽

pp. 241

Author(s):

Kenji Rowel Q. Lim ◽

Quynh Nguyen ◽

Toshifumi Yokota

Keyword(s):

Muscular Dystrophy ◽

Neuromuscular Disease ◽

Becker Muscular Dystrophy ◽

Neuromuscular Disorder ◽

Test Results ◽

Disease Registry ◽

Reading Frame ◽

Negative Results ◽

Genetic Test Results ◽

Dmd Gene

Duchenne muscular dystrophy (DMD) is a fatal neuromuscular disorder generally caused by out-of-frame mutations in the DMD gene. In contrast, in-frame mutations usually give rise to the milder Becker muscular dystrophy (BMD). However, this reading frame rule does not always hold true. Therefore, an understanding of the relationships between genotype and phenotype is important for informing diagnosis and disease management, as well as the development of genetic therapies. Here, we evaluated genotype–phenotype correlations in DMD and BMD patients enrolled in the Canadian Neuromuscular Disease Registry from 2012 to 2019. Data from 342 DMD and 60 BMD patients with genetic test results were analyzed. The majority of patients had deletions (71%), followed by small mutations (17%) and duplications (10%); 2% had negative results. Two deletion hotspots were identified, exons 3–20 and exons 45–55, harboring 86% of deletions. Exceptions to the reading frame rule were found in 13% of patients with deletions. Surprisingly, C-terminal domain mutations were associated with decreased wheelchair use and increased forced vital capacity. Dp116 and Dp71 mutations were also linked with decreased wheelchair use, while Dp140 mutations significantly predicted cardiomyopathy. Finally, we found that 12.3% and 7% of DMD patients in the registry could be treated with FDA-approved exon 51- and 53-skipping therapies, respectively.

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miRNome profiling in Duchenne Muscular Dystrophy; Identification of Asymptomatic and Manifesting Female Carriers

Bioscience Reports ◽

10.1042/bsr20211325 ◽

2021 ◽

Author(s):

Nahla O Mousa ◽

Ahmed A Sayed ◽

Nagia Fahmy ◽

Mariam G Elzayat ◽

Usama Bakry ◽

...

Keyword(s):

Duchenne Muscular Dystrophy ◽

Muscular Dystrophy ◽

Family Members ◽

Neurological Complications ◽

Neuromuscular Disorder ◽

Muscular Weakness ◽

Mirna Species ◽

Dmd Gene ◽

Circulating Levels ◽

Female Carriers

Duchenne muscular dystrophy (DMD) is a fatal neuromuscular disorder that occurs due to inactivating mutations in DMD gene, leading to muscular dystrophy. Prediction of pathological complications of DMD and the identification of female carriers are important research points that aim to reduce disease burden. Herein, we describe a case of a late DMD patient and his immediate female family members, who all carry same DMD mutation and exhibited varied degrees of symptoms. In our study, we sequenced the whole miRNome in leukocytes and plasma of the family members and results were validated using Real-Time PCR. Our results highlighted the role of miR-409-3p, miR-424-5p, miR-144-3p as microRNAs that show correlation with the extent of severity of muscular weakness and can be used for detection of asymptomatic carriers. Cellular and circulating levels of miR-494-3p had showed significant increase in symptomatic carriers, which may indicate significant roles played by this miRNA in the onset of muscular weakness. Interestingly, circulating levels of miR-206 and miR-410-3p were significantly increased only in the severely symptomatic carrier. In conclusion, our study highlighted several miRNA species, which could be used in predicting the onset of muscle and/or neurological complications in DMD carriers.

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Cardiac Myoediting Attenuates Cardiac Abnormalities in Human and Mouse Models of Duchenne Muscular Dystrophy

Circulation Research ◽

10.1161/circresaha.121.319579 ◽

2021 ◽

Author(s):

Ayhan Atmanli ◽

Andreas C Chai ◽

Miao Cui ◽

Zhaoning Wang ◽

Takahiko Nishiyama ◽

...

Keyword(s):

Duchenne Muscular Dystrophy ◽

Muscular Dystrophy ◽

Dilated Cardiomyopathy ◽

Genome Editing ◽

Rna Sequencing ◽

Reading Frame ◽

Transcriptional Dysregulation ◽

Cardiac Abnormalities ◽

Dmd Gene ◽

Novel Therapeutic

Rationale: Absence of dystrophin in Duchenne muscular dystrophy (DMD) results in the degeneration of skeletal and cardiac muscles. Owing to advances in respiratory management of DMD patients, cardiomyopathy has become a significant aspect of the disease. While CRISPR/Cas9 genome editing technology holds great potential as a novel therapeutic avenue for DMD, little is known about the potential of DMD correction using CRISPR/Cas9 technology to mitigate cardiac abnormalities in DMD. Objective: To define the effects of CRISPR/Cas9 genome editing on structural, functional and transcriptional abnormalities in DMD-associated cardiac disease. Methods and Results: We generated induced pluripotent stem cells (iPSCs) from a patient with a deletion of exon 44 of the DMD gene (ΔEx44) and his healthy brother. We targeted exon 45 of the DMD gene by CRISPR/Cas9 genome editing to generate corrected DMD (cDMD) iPSC lines, wherein the DMD open reading frame was restored via reframing (RF) or exon skipping (ES). While DMD cardiomyocytes (CMs) demonstrated morphologic, structural and functional deficits compared to control CMs, CMs from both cDMD lines were similar to control CMs. Bulk RNA-sequencing of DMD CMs showed transcriptional dysregulation consistent with dilated cardiomyopathy, which was mitigated in cDMD CMs. We then corrected dysfunctional DMD CMs by adenoviral delivery of Cas9/gRNA and showed that correction of DMD CMs post-differentiation reduces their arrhythmogenic potential. Single-nucleus RNA-sequencing of hearts of DMD mice showed transcriptional dysregulation in CMs and fibroblasts, which in corrected mice was reduced to similar levels as wildtype mice. Conclusions: We show that CRISPR/Cas9-mediated correction of DMD ΔEx44 mitigates structural, functional and transcriptional abnormalities consistent with dilated cardiomyopathy irrespective of how the protein reading frame is restored. We show that these effects extend to postnatal editing in iPSC-CMs and mice. These findings provide key insights into the utility of genome editing as a novel therapeutic for DMD-associated cardiomyopathy.

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New Approach for Antisense Oligonucleotide-Mediated Exon Skipping in Duchenne Muscular Dystrophy

Journal of Advanced Computational Intelligence and Intelligent Informatics ◽

10.20965/jaciii.2012.p0521 ◽

2012 ◽

Vol 16 (4) ◽

pp. 521-526

Author(s):

Yoshitsugu Aoki ◽

◽

Tetsuya Nagata ◽

Shin’ichi Takeda

Keyword(s):

Duchenne Muscular Dystrophy ◽

Muscular Dystrophy ◽

Antisense Oligonucleotide ◽

Antisense Oligonucleotides ◽

Exon Skipping ◽

New Approach ◽

Reading Frame ◽

Promising Therapeutic Approach ◽

Dmd Gene ◽

Dystrophin Protein

Duchenne Muscular Dystrophy (DMD) is a lethalmuscle disorder characterized by mutations in the DMD gene. These mutations primarily disrupt the reading frame, resulting in the absence of functional dystrophin protein. Exon skipping, which involves the use of antisense oligonucleotides is a promising therapeutic approach for DMD, and clinical trials on exon skipping are currently underway in DMD patients. Recently, stable and less-toxic antisense oligonucleotides with higher efficacy have been developed in mouse and dog models of DMD. This review highlights a new approach for antisense oligonucleotide-based therapeutics for DMD, particularly for exon skipping-based methods.

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Adeno-Associated Virus (AAV)-Mediated Gene Therapy for Duchenne Muscular Dystrophy: The Issue of Transgene Persistence

Frontiers in Neurology ◽

10.3389/fneur.2021.814174 ◽

2022 ◽

Vol 12 ◽

Author(s):

Arianna Manini ◽

Elena Abati ◽

Andi Nuredini ◽

Stefania Corti ◽

Giacomo Pietro Comi

Keyword(s):

Gene Therapy ◽

Duchenne Muscular Dystrophy ◽

Muscular Dystrophy ◽

Genetic Material ◽

Premature Death ◽

Gene Replacement ◽

Neuromuscular Disorder ◽

Adeno Associated Virus ◽

Dmd Gene

Duchenne muscular dystrophy (DMD) is an X-linked recessive, infancy-onset neuromuscular disorder characterized by progressive muscle weakness and atrophy, leading to delay of motor milestones, loss of autonomous ambulation, respiratory failure, cardiomyopathy, and premature death. DMD originates from mutations in the DMD gene that result in a complete absence of dystrophin. Dystrophin is a cytoskeletal protein which belongs to the dystrophin-associated protein complex, involved in cellular signaling and myofiber membrane stabilization. To date, the few available therapeutic options are aimed at lessening disease progression, but persistent loss of muscle tissue and function and premature death are unavoidable. In this scenario, one of the most promising therapeutic strategies for DMD is represented by adeno-associated virus (AAV)-mediated gene therapy. DMD gene therapy relies on the administration of exogenous micro-dystrophin, a miniature version of the dystrophin gene lacking unnecessary domains and encoding a truncated, but functional, dystrophin protein. Limited transgene persistence represents one of the most significant issues that jeopardize the translatability of DMD gene replacement strategies from the bench to the bedside. Here, we critically review preclinical and clinical studies of AAV-mediated gene therapy in DMD, focusing on long-term transgene persistence in transduced tissues, which can deeply affect effectiveness and sustainability of gene replacement in DMD. We also discuss the role played by the overactivation of the immune host system in limiting long-term expression of genetic material. In this perspective, further studies aimed at better elucidating the need for immune suppression in AAV-treated subjects are warranted in order to allow for life-long therapy in DMD patients.

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Duchenne Muscular Dystrophy Animal Models

10.5772/intechopen.96738 ◽

2021 ◽

Author(s):

Tatiana V. Egorova ◽

Ivan I. Galkin ◽

Yulia V. Ivanova ◽

Anna V. Polikarpova

Keyword(s):

Duchenne Muscular Dystrophy ◽

Muscular Dystrophy ◽

Animal Models ◽

Structural Protein ◽

Gene Deletions ◽

Advantages And Disadvantages ◽

Potential Applications ◽

Fundamental Research ◽

Therapy Development ◽

The Moment

Duchenne muscular dystrophy is a complex and severe orphan disease. It develops when the organism lacks the expression of dystrophin - a large structural protein. Dystrophin is transcribed from the largest gene in the human genome. At the moment, there is no cure available. Dozens of groups all over the world search for cure. Animal models are an important component of both the fundamental research and therapy development. Many animal models reproducing the features of disease were created and actively used since the late 80’s until present. The species diversity spans from invertebrates to primates and the genetic diversity of these models spans from single mutations to full gene deletions. The models are often non-interchangeable; while one model may be used for particular drug design it may be useless for another. Here we describe existing models, discuss their advantages and disadvantages and potential applications for research and therapy development.

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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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Have Duchenne Muscular Dystrophy Patients an Increased Cancer Risk?

Journal of Neuromuscular Diseases ◽

10.3233/jnd-210676 ◽

2021 ◽

pp. 1-5

Author(s):

Gian Luca Vita ◽

Luisa Politano ◽

Angela Berardinelli ◽

Giuseppe Vita

Keyword(s):

Duchenne Muscular Dystrophy ◽

Muscular Dystrophy ◽

Systematic Investigation ◽

Mdx Mice ◽

True Incidence ◽

Patients With Cancer ◽

Dmd Gene ◽

Age Range ◽

Prevalence Of Cancer

Background: Increasing evidence suggests that Duchenne muscular dystrophy (DMD) gene is involved in the occurrence of different types of cancer. Moreover, development of sarcomas was reported in mdx mice, the murine model of DMD, in older age. So far, nine isolated DMD patients were reported with concomitant cancer, four of whom with rhabdomyosarcoma (RMS), but no systematic investigation was performed about the true incidence of cancer in DMD. Methods: All members of the Italian Association of Myology were asked about the occurrence of cancer in their DMD patients in the last 30 years. Results: Four DMD patients with cancer were reported after checking 2455 medical records. One developed brain tumour at the age of 35 years. Two patients had alveolar RMS at 14 and 17 years of age. The fourth patient had a benign enchondroma when 11-year-old. Conclusion: Prevalence of cancer in general in the Italian DMD patients does not seem to be different from that in the general population with the same age range. Although the small numbers herein presented do not allow definitive conclusion, the frequent occurrence of RMS in DMD patients raises an alert for basic researchers and clinicians. The role of DMD gene in cancer merits further investigations.

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