In vivo ARFI imaging of the mechanical properties of muscle in a dog model of Duchenne muscular dystrophy

Abstract Background Mutations in the DMD gene encoding dystrophin—a critical structural element in muscle cells—cause Duchenne muscular dystrophy (DMD), which is the most common fatal genetic disease. Clustered regularly interspaced short palindromic repeat (CRISPR)-mediated gene editing is a promising strategy for permanently curing DMD. Methods In this study, we developed a novel strategy for reframing DMD mutations via CRISPR-mediated large-scale excision of exons 46–54. We compared this approach with other DMD rescue strategies by using DMD patient-derived primary muscle-derived stem cells (DMD-MDSCs). Furthermore, a patient-derived xenograft (PDX) DMD mouse model was established by transplanting DMD-MDSCs into immunodeficient mice. CRISPR gene editing components were intramuscularly delivered into the mouse model by adeno-associated virus vectors. Results Results demonstrated that the large-scale excision of mutant DMD exons showed high efficiency in restoring dystrophin protein expression. We also confirmed that CRISPR from Prevotella and Francisella 1(Cas12a)-mediated genome editing could correct DMD mutation with the same efficiency as CRISPR-associated protein 9 (Cas9). In addition, more than 10% human DMD muscle fibers expressed dystrophin in the PDX DMD mouse model after treated by the large-scale excision strategies. The restored dystrophin in vivo was functional as demonstrated by the expression of the dystrophin glycoprotein complex member β-dystroglycan. Conclusions We demonstrated that the clinically relevant CRISPR/Cas9 could restore dystrophin in human muscle cells in vivo in the PDX DMD mouse model. This study demonstrated an approach for the application of gene therapy to other genetic diseases.

Download Full-text

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

Molecular Therapy ◽

10.1016/j.ymthe.2018.10.011 ◽

2019 ◽

Vol 27 (1) ◽

pp. 76-86 ◽

Cited By ~ 7

Author(s):

Kenji Rowel Q. Lim ◽

Yusuke Echigoya ◽

Tetsuya Nagata ◽

Mutsuki Kuraoka ◽

Masanori Kobayashi ◽

...

Keyword(s):

Duchenne Muscular Dystrophy ◽

Muscular Dystrophy ◽

Exon Skipping ◽

Dog Model

Download Full-text

Laminin-111 protein therapy enhances muscle regeneration and repair in the GRMD dog model of Duchenne muscular dystrophy

Human Molecular Genetics ◽

10.1093/hmg/ddz086 ◽

2019 ◽

Vol 28 (16) ◽

pp. 2686-2695 ◽

Cited By ~ 6

Author(s):

Pamela Barraza-Flores ◽

Tatiana M Fontelonga ◽

Ryan D Wuebbles ◽

Hailey J Hermann ◽

Andreia M Nunes ◽

...

Keyword(s):

Duchenne Muscular Dystrophy ◽

Muscular Dystrophy ◽

Muscle Strength ◽

Premature Death ◽

Muscle Disease ◽

Muscle Pathology ◽

Mdx Mouse ◽

Protein Therapy ◽

Muscle Fibrosis ◽

Dog Model

Abstract Duchenne muscular dystrophy (DMD) is a devastating X-linked disease affecting ~1 in 5000 males. DMD patients exhibit progressive muscle degeneration and weakness, leading to loss of ambulation and premature death from cardiopulmonary failure. We previously reported that mouse Laminin-111 (msLam-111) protein could reduce muscle pathology and improve muscle function in the mdx mouse model for DMD. In this study, we examined the ability of msLam-111 to prevent muscle disease progression in the golden retriever muscular dystrophy (GRMD) dog model of DMD. The msLam-111 protein was injected into the cranial tibial muscle compartment of GRMD dogs and muscle strength and pathology were assessed. The results showed that msLam-111 treatment increased muscle fiber regeneration and repair with improved muscle strength and reduced muscle fibrosis in the GRMD model. Together, these findings support the idea that Laminin-111 could serve as a novel protein therapy for the treatment of DMD.

Download Full-text

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.

Download Full-text

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

Journal of Personalized Medicine ◽

10.3390/jpm8040038 ◽

2018 ◽

Vol 8 (4) ◽

pp. 38 ◽

Cited By ~ 15

Author(s):

Kenji Lim ◽

Chantal Yoon ◽

Toshifumi Yokota

Keyword(s):

Duchenne Muscular Dystrophy ◽

Muscular Dystrophy ◽

Wide Spectrum ◽

Membrane Integrity ◽

Dystrophin Gene ◽

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 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.

Download Full-text