Pathological evaluation of rats carrying in-frame mutations in the dystrophin gene: a new model of Becker muscular dystrophy

Teramoto, Naomi; Sugihara, Hidetoshi; Yamanouchi, Keitaro; Nakamura, Katsuyuki; Kimura, Kôichi; Okano, Tomoko; Shiga, Takanori; Shirakawa, Taku; Matsuo, Masafumi; Nagata, Tetsuya; Daimon, Masao; Matsuwaki, Takashi; Nishihara, Masugi

doi:10.1242/dmm.044701

Cited by 7 publications

(15 citation statements)

References 43 publications

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“…A previous report described generation of the first BMD rat model in which exons 3–16 of the rat Dmd gene were deleted using CRISPR/Cas9 38 . The BMD rat shows muscle inflammation, muscle fibrosis, heart fibrosis and reduced dystrophin protein, but not mRNA.…”

Section: Discussionmentioning

confidence: 99%

The X‐linked Becker muscular dystrophy (bmx) mouse models Becker muscular dystrophy via deletion of murine dystrophin exons 45–47

Heier

McCormack

Tully

et al. 2023

J cachexia sarcopenia muscle

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Background Becker muscular dystrophy (BMD) is a genetic neuromuscular disease of growing importance caused by in-frame, partial loss-of-function mutations in the dystrophin (DMD) gene. BMD presents with reduced severity compared with Duchenne muscular dystrophy (DMD), the allelic disorder of complete dystrophin deficiency. Significant therapeutic advancements have been made in DMD, including four FDA-approved drugs. BMD, however, is understudied and underserved-there are no drugs and few clinical trials. Discordance in therapeutic efforts is due in part to lack of a BMD mouse model which would enable greater understanding of disease and de-risk potential therapeutics before first-in-human trials. Importantly, a BMD mouse model is becoming increasingly critical as emerging DMD dystrophin restoration therapies aim to convert a DMD genotype into a BMD phenotype. Methods We use CRISPR/Cas9 technology to generate bmx (Becker muscular dystrophy, X-linked) mice, which express an in-frame ~40 000 BP deletion of exons 45-47 in the murine Dmd gene, reproducing the most common BMD patient mutation. Here, we characterize muscle pathogenesis using molecular and histological techniques and then test skeletal muscle and cardiac function using muscle function assays and echocardiography. Results Overall, bmx mice present with significant muscle weakness and heart dysfunction versus wild-type (WT) mice, despite a substantial improvement in pathology over dystrophin-null mdx52 mice. bmx mice show impaired motor function in grip strength (À39%, P < 0.0001), wire hang (P = 0.0025), and in vivo as well as ex vivo force assays. In aged bmx, echocardiography reveals decreased heart function through reduced fractional shortening (À25%, P = 0.0036). Additionally, muscle-specific serum CK is increased >60-fold (P < 0.0001), indicating increased muscle damage. Histologically, bmx muscles display increased myofibre size variability (minimal Feret's diameter: P = 0.0017) and centrally located nuclei indicating degeneration/regeneration (P < 0.0001). bmx muscles also display dystrophic pathology; however, levels of the following parameters are moderate in comparison with mdx52: inflammatory/necrotic foci (P < 0.0001), collagen deposition (+1.4-fold, P = 0.0217), and sarcolemmal damage measured by intracellular IgM (P = 0.0878). Like BMD patients, bmx muscles show reduced dystrophin protein levels (~20-50% of WT), whereas Dmd transcript levels are unchanged. At the molecular level, bmx muscles express increased levels of inflammatory genes, inflammatory miRNAs and fibrosis genes. Conclusions The bmx mouse recapitulates BMD disease phenotypes with histological, molecular and functional deficits. Importantly, it can inform both BMD pathology and DMD dystrophin restoration therapies. This novel model will enable further characterization of BMD disease progression, identification of biomarkers, identification of therapeutic targets and new preclinical drug studies aimed at developing therapies for BMD patients.

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Section: Discussionmentioning

confidence: 99%

The X‐linked Becker muscular dystrophy (bmx) mouse models Becker muscular dystrophy via deletion of murine dystrophin exons 45–47

Heier

McCormack

Tully

et al. 2023

J cachexia sarcopenia muscle

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“…Dystrophic mouse models, notably a series of mdx models, are valuable tools in the proof-of-concept stages of drug candidates because the strains have been established and well characterized. Novel dystrophic models with mice and rats are also further along in development utilizing their facility for genetic manipulation [ 45 , 46 , 47 ]. On the other hand, canine models of GRMD and CXMD J have played pivotal roles in DMD research, as they display many of the clinical manifestations seen in patients.…”

Section: Discussionmentioning

confidence: 99%

A Dystrophin Exon-52 Deleted Miniature Pig Model of Duchenne Muscular Dystrophy and Evaluation of Exon Skipping

Echigoya

Trieu

Duddy

et al. 2021

IJMS

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Duchenne muscular dystrophy (DMD) is a lethal X-linked recessive disorder caused by mutations in the DMD gene and the subsequent lack of dystrophin protein. Recently, phosphorodiamidate morpholino oligomer (PMO)-antisense oligonucleotides (ASOs) targeting exon 51 or 53 to reestablish the DMD reading frame have received regulatory approval as commercially available drugs. However, their applicability and efficacy remain limited to particular patients. Large animal models and exon skipping evaluation are essential to facilitate ASO development together with a deeper understanding of dystrophinopathies. Using recombinant adeno-associated virus-mediated gene targeting and somatic cell nuclear transfer, we generated a Yucatan miniature pig model of DMD with an exon 52 deletion mutation equivalent to one of the most common mutations seen in patients. Exon 52-deleted mRNA expression and dystrophin deficiency were confirmed in the skeletal and cardiac muscles of DMD pigs. Accordingly, dystrophin-associated proteins failed to be recruited to the sarcolemma. The DMD pigs manifested early disease onset with severe bodywide skeletal muscle degeneration and with poor growth accompanied by a physical abnormality, but with no obvious cardiac phenotype. We also demonstrated that in primary DMD pig skeletal muscle cells, the genetically engineered exon-52 deleted pig DMD gene enables the evaluation of exon 51 or 53 skipping with PMO and its advanced technology, peptide-conjugated PMO. The results show that the DMD pigs developed here can be an appropriate large animal model for evaluating in vivo exon skipping efficacy.

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“…Also these rats showed age-dependent decline of cardiac functions similar to DMD patients [72]. Later, based on this model, another rat model with an in-frame mutation in the dystrophin gene was generated [73]. New mutant rats had reduced expression of truncated dystrophin and mild phenotype similar to BMD patients.…”

Section: Animal Models To Study the Pathogenesis Of Dmdmentioning

confidence: 94%

Duchenne Muscular Dystrophy Animal Models

Егорова¹,

Galkin²,

Ivanova³

et al. 2022

Preclinical Animal Modeling in Medicine

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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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Pathological evaluation of rats carrying in-frame mutations in the dystrophin gene: a new model of Becker muscular dystrophy

Cited by 7 publications

References 43 publications

The X‐linked Becker muscular dystrophy (bmx) mouse models Becker muscular dystrophy via deletion of murine dystrophin exons 45–47

The X‐linked Becker muscular dystrophy (bmx) mouse models Becker muscular dystrophy via deletion of murine dystrophin exons 45–47

A Dystrophin Exon-52 Deleted Miniature Pig Model of Duchenne Muscular Dystrophy and Evaluation of Exon Skipping

Duchenne Muscular Dystrophy Animal Models

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