Mesoangioblasts from Facioscapulohumeral Muscular Dystrophy Display in Vivo a Variable Myogenic Ability Predictable by their in Vitro Behavior

Facioscapulohumeral muscular dystrophy (FSHD), characterized by progressive muscle weakness and deterioration, is genetically linked to aberrant expression ofDUX4in muscle. DUX4, in its full-length form, is cytotoxic in nongermline tissues. Here, we designed locked nucleic acid (LNA) gapmer antisense oligonucleotides (AOs) to knock downDUX4in immortalized FSHD myoblasts and theFLExDUX4FSHD mouse model. Using a screening method capable of reliably evaluating the knockdown efficiency of LNA gapmers against endogenousDUX4messenger RNA in vitro, we demonstrate that several designed LNA gapmers selectively and effectively reducedDUX4expression with nearly complete knockdown. We also found potential functional benefits of AOs on muscle fusion and structure in vitro. Finally, we show that one of the LNA gapmers was taken up and induced effective silencing ofDUX4upon local treatment in vivo. The LNA gapmers developed here will help facilitate the development of FSHD therapies.

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Cellular and animal models for facioscapulohumeral muscular dystrophy

Disease Models & Mechanisms ◽

10.1242/dmm.046904 ◽

2020 ◽

Vol 13 (10) ◽

pp. dmm046904

Author(s):

Alec M. DeSimone ◽

Justin Cohen ◽

Monkol Lek ◽

Angela Lek

Keyword(s):

Muscular Dystrophy ◽

Myogenic Differentiation ◽

Facioscapulohumeral Muscular Dystrophy ◽

Lower Limbs ◽

Repeat Array ◽

Drug Candidates ◽

Cellular Models ◽

Wide Range

ABSTRACTFacioscapulohumeral muscular dystrophy (FSHD) is one of the most common forms of muscular dystrophy and presents with weakness of the facial, scapular and humeral muscles, which frequently progresses to the lower limbs and truncal areas, causing profound disability. Myopathy results from epigenetic de-repression of the D4Z4 microsatellite repeat array on chromosome 4, which allows misexpression of the developmentally regulated DUX4 gene. DUX4 is toxic when misexpressed in skeletal muscle and disrupts several cellular pathways, including myogenic differentiation and fusion, which likely underpins pathology. DUX4 and the D4Z4 array are strongly conserved only in primates, making FSHD modeling in non-primate animals difficult. Additionally, its cytotoxicity and unusual mosaic expression pattern further complicate the generation of in vitro and in vivo models of FSHD. However, the pressing need to develop systems to test therapeutic approaches has led to the creation of multiple engineered FSHD models. Owing to the complex genetic, epigenetic and molecular factors underlying FSHD, it is difficult to engineer a system that accurately recapitulates every aspect of the human disease. Nevertheless, the past several years have seen the development of many new disease models, each with their own associated strengths that emphasize different aspects of the disease. Here, we review the wide range of FSHD models, including several in vitro cellular models, and an array of transgenic and xenograft in vivo models, with particular attention to newly developed systems and how they are being used to deepen our understanding of FSHD pathology and to test the efficacy of drug candidates.

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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.

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BIOCHEMICAL CHANGES IN PROGRESSIVE MUSCULAR DYSTROPHY: VI. INCORPORATION OF URIDINE-2-14C INTO RNA OF VARIOUS TISSUE OF NORMAL AND DYSTROPHIC MICE

Canadian Journal of Biochemistry ◽

10.1139/o67-167 ◽

1967 ◽

Vol 45 (9) ◽

pp. 1419-1425 ◽

Cited By ~ 14

Author(s):

Uma Srivastava

Keyword(s):

Muscular Dystrophy ◽

Soluble Fraction ◽

Normal Liver ◽

Biochemical Changes ◽

Normal Muscle ◽

Dystrophic Muscle ◽

Progressive Muscular Dystrophy ◽

Dystrophic Mice

Normal and dystrophic mice were injected intravenously with uridine-2-14C at various stages of the disease. Radioactivity in the acid-soluble fraction of most of the tissues studied was unchanged or not significantly different in dystrophic animals. In vivo incorporation of uridine-2-14C into RNA increased in dystrophic muscle as compared to normal muscle at 30 days, remained the same at 60 days, and was reduced at 90 days. Similar results were also observed on the in vitro incorporation of uridine-2-14C catalyzed by homogenates of normal and dystrophic muscle. Dystrophic brain and pancreas showed a decrease in the incorporation at each stage investigated as compared to controls. No change in the incorporation was noted in dystrophic and normal liver, kidney, spleen, and heart. The decrease in uridine-2-14C incorporation in dystrophic muscle at 90 days could be due to an increased RNA content. Such a phenomenon was explained as due to infiltration of dystrophic muscle by invading macrophages.It is concluded that the metabolism of RNA is not decreased in the dystrophic muscle in preliminary stages of the disease as compared to the control.

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Nintedanib as a new therapeutic agent for Duchenne muscular dystrophy: preclinical in vitro and in vivo studies

Neuromuscular Disorders ◽

10.1016/j.nmd.2017.06.354 ◽

2017 ◽

Vol 27 ◽

pp. S191

Author(s):

P. Piñol ◽

E. Fernández-Simón ◽

X. Suárez ◽

N. de Luna ◽

A. Molins ◽

...

Keyword(s):

Duchenne Muscular Dystrophy ◽

Muscular Dystrophy ◽

Therapeutic Agent ◽

In Vivo Studies

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Collagen-VI supplementation by cell transplantation improves muscle regeneration in Ullrich congenital muscular dystrophy model mice

Stem Cell Research & Therapy ◽

10.1186/s13287-021-02514-3 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Nana Takenaka-Ninagawa ◽

Jinsol Kim ◽

Mingming Zhao ◽

Masae Sato ◽

Tatsuya Jonouchi ◽

...

Keyword(s):

Skeletal Muscle ◽

Muscular Dystrophy ◽

Cell Transplantation ◽

Muscle Regeneration ◽

Congenital Muscular Dystrophy ◽

Induced Pluripotent Stem Cell ◽

Skeletal Muscle Regeneration ◽

Ullrich Congenital Muscular Dystrophy

Abstract Background Mesenchymal stromal cells (MSCs) function as supportive cells on skeletal muscle homeostasis through several secretory factors including type 6 collagen (COL6). Several mutations of COL6A1, 2, and 3 genes cause Ullrich congenital muscular dystrophy (UCMD). Skeletal muscle regeneration deficiency has been reported as a characteristic phenotype in muscle biopsy samples of human UCMD patients and UCMD model mice. However, little is known about the COL6-dependent mechanism for the occurrence and progression of the deficiency. The purpose of this study was to clarify the pathological mechanism of UCMD by supplementing COL6 through cell transplantation. Methods To test whether COL6 supplementation has a therapeutic effect for UCMD, in vivo and in vitro experiments were conducted using four types of MSCs: (1) healthy donors derived-primary MSCs (pMSCs), (2) MSCs derived from healthy donor induced pluripotent stem cell (iMSCs), (3) COL6-knockout iMSCs (COL6KO-iMSCs), and (4) UCMD patient-derived iMSCs (UCMD-iMSCs). Results All four MSC types could engraft for at least 12 weeks when transplanted into the tibialis anterior muscles of immunodeficient UCMD model (Col6a1KO) mice. COL6 protein was restored by the MSC transplantation if the MSCs were not COL6-deficient (types 1 and 2). Moreover, muscle regeneration and maturation in Col6a1KO mice were promoted with the transplantation of the COL6-producing MSCs only in the region supplemented with COL6. Skeletal muscle satellite cells derived from UCMD model mice (Col6a1KO-MuSCs) co-cultured with type 1 or 2 MSCs showed improved proliferation, differentiation, and maturation, whereas those co-cultured with type 3 or 4 MSCs did not. Conclusions These findings indicate that COL6 supplementation improves muscle regeneration and maturation in UCMD model mice.

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A Comment on “Muscle Xenografts Reproduce Key Molecular Features of Facioscapulohumeral Muscular Dystrophy”: What Is New and What Has Already been Done and Reported but Was Not Quoted?

Cell Transplantation ◽

10.1177/0963689720939120 ◽

2020 ◽

Vol 29 ◽

pp. 096368972093912

Author(s):

Daniel Skuk ◽

Jacques P Tremblay

Keyword(s):

Muscular Dystrophy ◽

Scientific Work ◽

Facioscapulohumeral Muscular Dystrophy ◽

In Vivo Model ◽

Immunodeficient Mice ◽

Molecular Features ◽

Bibliographic Search ◽

Scientific Context ◽

Muscle Precursor Cells

A study was recently published that sought to develop an in vivo model of facioscapulohumeral muscular dystrophy by transplanting muscle precursor cells from a patient into immunodeficient mice. The study largely applied the methodology used by our team in a study published more than two decades ago with a similar objective, albeit for another muscular dystrophy. However, our study is not cited, leaving the wrong idea that the concept, methodology, and part of the results are original to this recent study. Although the recent study is of interest, the omission of our publication, as well as other relevant references, deprives it of an adequate scientific context. We, therefore, want to point out the importance of a careful bibliographic search in any scientific work.

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Role of proteoglycans and glycosaminoglycans in Duchenne muscular dystrophy

Glycobiology ◽

10.1093/glycob/cwy058 ◽

2018 ◽

Vol 29 (2) ◽

pp. 110-123 ◽

Cited By ~ 8

Author(s):

Laurino Carmen ◽

Vadala’ Maria ◽

Julio Cesar Morales-Medina ◽

Annamaria Vallelunga ◽

Beniamino Palmieri ◽

...

Keyword(s):

Duchenne Muscular Dystrophy ◽

Muscular Dystrophy ◽

Mammalian Cells ◽

Muscle Development ◽

Glycoprotein Complex ◽

Experimental Therapies ◽

Dystrophin Protein

Abstract Duchenne muscular dystrophy (DMD) is an inherited fatal X-linked myogenic disorder with a prevalence of 1 in 3500 male live births. It affects voluntary muscles, and heart and breathing muscles. DMD is characterized by continuous degeneration and regeneration cycles resulting in extensive fibrosis and a progressive reduction in muscle mass. Since the identification of a reduction in dystrophin protein as the cause of this disorder, numerous innovative and experimental therapies, focusing on increasing the levels of dystrophin, have been proposed, but the clinical improvement has been unsatisfactory. Dystrophin forms the dystrophin-associated glycoprotein complex and its proteins have been studied as a promising novel therapeutic target to treat DMD. Among these proteins, cell surface glycosaminoglycans (GAGs) are found almost ubiquitously on the surface and in the extracellular matrix (ECM) of mammalian cells. These macromolecules interact with numerous ligands, including ECM constituents, adhesion molecules and growth factors that play a crucial role in muscle development and maintenance. In this article, we have reviewed in vitro, in vivo and clinical studies focused on the functional role of GAGs in the pathophysiology of DMD with the final aim of summarizing the state of the art of GAG dysregulation within the ECM in DMD and discussing future therapeutic perspectives.

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Phosphorylation of the carboxyl-terminal region of dystrophin

Biochemistry and Cell Biology ◽

10.1139/o96-047 ◽

1996 ◽

Vol 74 (4) ◽

pp. 431-437 ◽

Cited By ~ 21

Author(s):

Marek Michalak ◽

Susan Y. Fu ◽

Rachel E. Milner ◽

Jody L. Busaan ◽

Jacqueline E. Hance

Keyword(s):

Extracellular Matrix ◽

Muscular Dystrophy ◽

Protein Kinases ◽

Protein Interactions ◽

Matrix Protein ◽

Casein Kinase ◽

Terminal Region ◽

Extracellular Matrix Protein

Dystrophin is a protein product of the gene responsible for Duchenne and Becker muscular dystrophy. The protein is localized to the inner surface of sarcolemma and is associated with a group of membrane (glyco)proteins. Dystrophin links cytoskeletal actins via the dystrophin-associated protein complex to extracellular matrix protein, laminin. This structural organization implicates the role of dystrophin in stabilizing the sarcolemma of muscle fibers. Precisely how dystrophin functions is far from clear. The presence of an array of isoforms of the C-terminal region of dystrophin suggests that dystrophin may have functions other than structural. In agreement, many potential phosphorylation sites are found in the C-terminal region of dystrophin, and the C-terminal region of dystrophin is phosphorylated both in vitro and in vivo by many protein kinases, including MAP kinase, p34cdc2 kinase, CaM kinase, and casein kinase, and is dephosphorylated by calcineurin. The C-terminal domain of dystrophin is also a substrate for hierarchical phosporylation by casein kinase-2 and GSK-3. These observations, in accordance with the finding that the cysteine-rich region binds to Ca2+, Zn2+, and calmodulin, suggest an active involvement of dystrophin in transducing signals across muscle sarcolemma. Phosphorylation–dephosphorylation of the C-terminal region of dystrophin may play a role in regulating dystrophin–protein interactions and (or) transducing signal from the extracellular matrix via the dystrophin molecule to the cytoskeleton.Key words: Duchenne muscular dystrophy, protein phosphorylation, protein kinases, calcineurin, cytoskeleton.

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