CRISPR Correction of Duchenne Muscular Dystrophy

The ability to efficiently modify the genome using CRISPR technology has rapidly revolutionized biology and genetics and will soon transform medicine. Duchenne muscular dystrophy (DMD) represents one of the first monogenic disorders that has been investigated with respect to CRISPR-mediated correction of causal genetic mutations. DMD results from mutations in the gene encoding dystrophin, a scaffolding protein that maintains the integrity of striated muscles. Thousands of different dystrophin mutations have been identified in DMD patients, who suffer from a loss of ambulation followed by respiratory insufficiency, heart failure, and death by the third decade of life. Using CRISPR to bypass DMD mutations, dystrophin expression has been efficiently restored in human cells and mouse models of DMD. Here, we review recent progress toward the development of possible CRISPR therapies for DMD and highlight opportunities and potential obstacles in attaining this goal.

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Disrupted Calcium Homeostasis in Duchenne Muscular Dystrophy: A Common Mechanism behind Diverse Consequences

International Journal of Molecular Sciences ◽

10.3390/ijms222011040 ◽

2021 ◽

Vol 22 (20) ◽

pp. 11040

Author(s):

Barbara Zabłocka ◽

Dariusz C. Górecki ◽

Krzysztof Zabłocki

Keyword(s):

Duchenne Muscular Dystrophy ◽

Muscular Dystrophy ◽

Calcium Homeostasis ◽

Gene Mutations ◽

Common Mechanism ◽

Developmental Phase ◽

Striated Muscles ◽

Gene Encoding ◽

Dmd Gene ◽

The Impact

Duchenne muscular dystrophy (DMD) leads to disability and death in young men. This disease is caused by mutations in the DMD gene encoding diverse isoforms of dystrophin. Loss of full-length dystrophins is both necessary and sufficient for causing degeneration and wasting of striated muscles, neuropsychological impairment, and bone deformities. Among this spectrum of defects, abnormalities of calcium homeostasis are the common dystrophic feature. Given the fundamental role of Ca2+ in all cells, this biochemical alteration might be underlying all the DMD abnormalities. However, its mechanism is not completely understood. While abnormally elevated resting cytosolic Ca2+ concentration is found in all dystrophic cells, the aberrant mechanisms leading to that outcome have cell-specific components. We probe the diverse aspects of calcium response in various affected tissues. In skeletal muscles, cardiomyocytes, and neurons, dystrophin appears to serve as a scaffold for proteins engaged in calcium homeostasis, while its interactions with actin cytoskeleton influence endoplasmic reticulum organisation and motility. However, in myoblasts, lymphocytes, endotheliocytes, and mesenchymal and myogenic cells, calcium abnormalities cannot be clearly attributed to the loss of interaction between dystrophin and the calcium toolbox proteins. Nevertheless, DMD gene mutations in these cells lead to significant defects and the calcium anomalies are a symptom of the early developmental phase of this pathology. As the impaired calcium homeostasis appears to underpin multiple DMD abnormalities, understanding this alteration may lead to the development of new therapies. In fact, it appears possible to mitigate the impact of the abnormal calcium homeostasis and the dystrophic phenotype in the total absence of dystrophin. This opens new treatment avenues for this incurable disease.

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Toward the correction of muscular dystrophy by gene editing

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2004840117 ◽

2021 ◽

Vol 118 (22) ◽

pp. e2004840117

Author(s):

Eric N. Olson

Keyword(s):

Muscular Dystrophy ◽

Gene Editing ◽

Genetic Disorder ◽

Premature Death ◽

Lessons Learned ◽

Large Mammals ◽

Gene Encoding ◽

Monogenic Disorders ◽

Dystrophin Expression ◽

And Function

Recent advances in gene editing technologies are enabling the potential correction of devastating monogenic disorders through elimination of underlying genetic mutations. Duchenne muscular dystrophy (DMD) is an especially severe genetic disorder caused by mutations in the gene encoding dystrophin, a membrane-associated protein required for maintenance of muscle structure and function. Patients with DMD succumb to loss of mobility early in life, culminating in premature death from cardiac and respiratory failure. The disease has thus far defied all curative strategies. CRISPR gene editing has provided new opportunities to ameliorate the disease by eliminating DMD mutations and thereby restore dystrophin expression throughout skeletal and cardiac muscle. Proof-of-concept studies in rodents, large mammals, and human cells have validated the potential of this approach, but numerous challenges remain to be addressed, including optimization of gene editing, delivery of gene editing components throughout the musculature, and mitigation of possible immune responses. This paper provides an overview of recent work from our laboratory and others toward the genetic correction of DMD and considers the opportunities and challenges in the path to clinical translation. Lessons learned from these studies will undoubtedly enable further applications of gene editing to numerous other diseases of muscle and other tissues.

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Restoring Dystrophin Expression in Duchenne Muscular Dystrophy Muscle

American Journal Of Pathology ◽

10.1016/j.ajpath.2011.03.050 ◽

2011 ◽

Vol 179 (1) ◽

pp. 12-22 ◽

Cited By ~ 61

Author(s):

Eric P. Hoffman ◽

Abby Bronson ◽

Arthur A. Levin ◽

Shin'ichi Takeda ◽

Toshifumi Yokota ◽

...

Keyword(s):

Duchenne Muscular Dystrophy ◽

Muscular Dystrophy ◽

Dystrophin Expression

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Correction of Dystrophin Expression in Cells From Duchenne Muscular Dystrophy Patients Through Genomic Excision of Exon 51 by Zinc Finger Nucleases

Molecular Therapy ◽

10.1038/mt.2014.234 ◽

2015 ◽

Vol 23 (3) ◽

pp. 523-532 ◽

Cited By ~ 72

Author(s):

David G Ousterout ◽

Ami M Kabadi ◽

Pratiksha I Thakore ◽

Pablo Perez-Pinera ◽

Matthew T Brown ◽

...

Keyword(s):

Duchenne Muscular Dystrophy ◽

Muscular Dystrophy ◽

Zinc Finger ◽

Zinc Finger Nucleases ◽

Dystrophin Expression ◽

In Cells

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The Long Journey from Diagnosis to Therapy

Annual Review of Genomics and Human Genetics ◽

10.1146/annurev-genom-112019-083518 ◽

2020 ◽

Vol 21 (1) ◽

pp. 1-13

Author(s):

Kay E. Davies

Keyword(s):

Duchenne Muscular Dystrophy ◽

Muscular Dystrophy ◽

Somatic Cell ◽

Human Genetics ◽

Annual Review ◽

Editorial Committee ◽

Single Cell Sequencing ◽

Monogenic Disorders ◽

Mendelian Disorders ◽

Opening Up

I was honored to be asked by the Editorial Committee of the Annual Review of Genomics and Genetics to write an autobiographical account of my life in science and in genetics in particular. The field has moved from mapping Mendelian disorders 40 years ago to the delivery of effective therapies for some monogenic disorders today. My 40-year journey from diagnosis to therapy for Duchenne muscular dystrophy has depended on collaborations among basic scientists, clinicians, medical charities, genetic counselors, biotech companies, and affected families. The future of human genetics looks even more exciting, with techniques such as single-cell sequencing and somatic cell CRISPR editing opening up opportunities for precision medicine and accelerating progress.

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Therapeutic restoration of dystrophin expression in Duchenne muscular dystrophy

Journal of Muscle Research and Cell Motility ◽

10.1007/s10974-006-9081-6 ◽

2006 ◽

Vol 27 (5-7) ◽

pp. 387-398 ◽

Cited By ~ 19

Author(s):

Dominic J. Wells

Keyword(s):

Duchenne Muscular Dystrophy ◽

Muscular Dystrophy ◽

Dystrophin Expression

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Eteplirsen treatment for Duchenne muscular dystrophy

Neurology ◽

10.1212/wnl.0000000000005680 ◽

2018 ◽

Vol 90 (24) ◽

pp. e2146-e2154 ◽

Cited By ~ 67

Author(s):

Jay S. Charleston ◽

Frederick J. Schnell ◽

Johannes Dworzak ◽

Cas Donoghue ◽

Sarah Lewis ◽

...

Keyword(s):

Duchenne Muscular Dystrophy ◽

Muscular Dystrophy ◽

Western Blot ◽

Reverse Transcription ◽

Exon Skipping ◽

Fold Increase ◽

Cell Penetration ◽

Reverse Transcription Pcr ◽

Dystrophin Expression ◽

Immunofluorescence Intensity

ObjectiveTo describe the quantification of novel dystrophin production in patients with Duchenne muscular dystrophy (DMD) after long-term treatment with eteplirsen.MethodsClinical study 202 was an observational, open-label extension of the randomized, controlled study 201 assessing the safety and efficacy of eteplirsen in patients with DMD with a confirmed mutation in the DMD gene amenable to correction by skipping of exon 51. Patients received once-weekly IV doses of eteplirsen 30 or 50 mg/kg. Upper extremity muscle biopsy samples were collected at combined study week 180, blinded, and assessed for dystrophin-related content by Western blot, Bioquant software measurement of dystrophin-associated immunofluorescence intensity, and percent dystrophin-positive fibers (PDPF). Results were contrasted with matched untreated biopsies from patients with DMD. Reverse transcription PCR followed by Sanger sequencing of newly formed slice junctions was used to confirm the mechanism of action of eteplirsen.ResultsReverse transcription PCR analysis and sequencing of the newly formed splice junction confirmed that 100% of treated patients displayed the expected skipped exon 51 sequence. In treated patients vs untreated controls, Western blot analysis of dystrophin content demonstrated an 11.6-fold increase (p = 0.007), and PDPF analysis demonstrated a 7.4-fold increase (p < 0.001). The PDPF findings were confirmed in a re-examination of the sample (15.5-fold increase, p < 0.001). Dystrophin immunofluorescence intensity was 2.4-fold greater in treated patients than in untreated controls (p < 0.001).ConclusionTaken together, the 4 assays, each based on unique evaluation mechanisms, provided evidence of eteplirsen muscle cell penetration, exon skipping, and induction of novel dystrophin expression.Classification of evidenceThis study provides Class II evidence of the muscle cell penetration, exon skipping, and induction of novel dystrophin expression by eteplirsen, as confirmed by 4 assays.

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A Simplified Immune Suppression Scheme Leads to Persistent Micro-dystrophin Expression in Duchenne Muscular Dystrophy Dogs

Human Gene Therapy ◽

10.1089/hum.2011.147 ◽

2012 ◽

Vol 23 (2) ◽

pp. 202-209 ◽

Cited By ~ 35

Author(s):

Jin-Hong Shin ◽

Yongping Yue ◽

Arun Srivastava ◽

Bruce Smith ◽

Yi Lai ◽

...

Keyword(s):

Duchenne Muscular Dystrophy ◽

Muscular Dystrophy ◽

Immune Suppression ◽

Dystrophin Expression

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Function and Genetics of Dystrophin and Dystrophin-Related Proteins in Muscle

Physiological Reviews ◽

10.1152/physrev.00028.2001 ◽

2002 ◽

Vol 82 (2) ◽

pp. 291-329 ◽

Cited By ~ 721

Author(s):

Derek J. Blake ◽

Andrew Weir ◽

Sarah E. Newey ◽

Kay E. Davies

Keyword(s):

Duchenne Muscular Dystrophy ◽

Muscular Dystrophy ◽

Protein Complex ◽

Muscle Disease ◽

Calcium Handling ◽

Dystrophic Muscle ◽

Gene Encoding ◽

Muscle Wasting Disease ◽

Novel Therapeutic Strategies ◽

Related Proteins

The X-linked muscle-wasting disease Duchenne muscular dystrophy is caused by mutations in the gene encoding dystrophin. There is currently no effective treatment for the disease; however, the complex molecular pathology of this disorder is now being unravelled. Dystrophin is located at the muscle sarcolemma in a membrane-spanning protein complex that connects the cytoskeleton to the basal lamina. Mutations in many components of the dystrophin protein complex cause other forms of autosomally inherited muscular dystrophy, indicating the importance of this complex in normal muscle function. Although the precise function of dystrophin is unknown, the lack of protein causes membrane destabilization and the activation of multiple pathophysiological processes, many of which converge on alterations in intracellular calcium handling. Dystrophin is also the prototype of a family of dystrophin-related proteins, many of which are found in muscle. This family includes utrophin and α-dystrobrevin, which are involved in the maintenance of the neuromuscular junction architecture and in muscle homeostasis. New insights into the pathophysiology of dystrophic muscle, the identification of compensating proteins, and the discovery of new binding partners are paving the way for novel therapeutic strategies to treat this fatal muscle disease. This review discusses the role of the dystrophin complex and protein family in muscle and describes the physiological processes that are affected in Duchenne muscular dystrophy.

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