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1.
Front Mol Neurosci ; 13: 3, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32116541

RESUMO

LAMA2-related congenital muscular dystrophy, also known as MDC1A, is caused by loss-of-function mutations in the alpha2 chain of Laminin-211. Loss of this protein interrupts the connection between the muscle cell and its extracellular environment and results in an aggressive, congenital-onset muscular dystrophy characterized by severe hypotonia, lack of independent ambulation, and early mortality driven by respiratory complications and/or failure to thrive. Of the pathomechanisms of MDC1A, the earliest and most prominent is widespread and rampant fibrosis. Here, we will discuss some of the key drivers of fibrosis including TGF-beta and renin-angiotensin system signaling and consequences of these pathways including myofibroblast transdifferentiation and matrix remodeling. We will also highlight some of the differences in fibrogenesis in congenital muscular dystrophy (CMD) with that seen in Duchenne muscular dystrophy (DMD). Finally, we will connect the key signaling pathways in the pathogenesis of MDC1A to the current status of the therapeutic approaches that have been tested in the preclinical models of MDC1A to treat fibrosis.

2.
Mol Cell Biol ; 39(14)2019 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-31036568

RESUMO

GALGT2 (also B4GALNT2) encodes a glycosyltransferase that is normally confined to the neuromuscular and myotendinous junction in adult skeletal muscle. GALGT2 overexpression in muscle can inhibit muscular dystrophy in mouse models of the disease by inducing the overexpression of surrogate muscle proteins, including utrophin, agrin, laminins, and integrins. Despite its well-documented biological properties, little is known about the endogenous regulation of muscle GALGT2 expression. Here, we demonstrate that epidermal growth factor receptor (EGFR) ligands can activate the human GALGT2 promoter. Overexpression of one such ligand, soluble heparin-binding EGF-like growth factor (sHB-EGF), also stimulated mouse muscle Galgt2 gene expression and expression of GALGT2-inducible surrogate muscle genes. Deletion analysis of the GALGT2 promoter identified a 45-bp region containing a TFAP4-binding site that was required for sHB-EGF activation. sHB-EGF increased TFAP4 binding to this site in muscle cells and increased endogenous Tfap4 gene expression. sHB-EGF also increased muscle EGFR protein expression and activated EGFR-Akt signaling. sHB-EGF expression was concentrated at the neuromuscular junction, and Hbegf deletion reduced Galgt2-dependent synaptic glycosylation. Hbegf deletion also mimicked Galgt2-dependent neuromuscular and muscular dystrophy phenotypes. These data demonstrate that sHB-EGF is an endogenous regulator of muscle Galgt2 gene expression and can mimic Galgt2-dependent muscle phenotypes.


Assuntos
Fator de Crescimento Semelhante a EGF de Ligação à Heparina/genética , Fator de Crescimento Semelhante a EGF de Ligação à Heparina/metabolismo , Músculo Esquelético/metabolismo , N-Acetilgalactosaminiltransferases/genética , Junção Neuromuscular/metabolismo , Animais , Sítios de Ligação , Células CHO , Linhagem Celular , Cricetulus , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Glicosilação , Células HEK293 , Humanos , Masculino , Camundongos , N-Acetilgalactosaminiltransferases/química , N-Acetilgalactosaminiltransferases/metabolismo , Regiões Promotoras Genéticas , Transdução de Sinais , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
3.
Mol Ther Methods Clin Dev ; 10: 89-104, 2018 Sep 21.
Artigo em Inglês | MEDLINE | ID: mdl-30073180

RESUMO

Recombinant adeno-associated virus (rAAV)rh74.MCK.GALGT2 is a muscle-specific gene therapy that is being developed to treat forms of muscular dystrophy. Here we report on an isolated limb infusion technique in a non-human primate model, where hindlimb blood flow is transiently isolated using balloon catheters to concentrate vector in targeted leg muscles. A bilateral dose of 2.5 × 1013 vector genomes (vg)/kg/limb was sufficient to induce GALGT2-induced glycosylation in 10%-60% of skeletal myofibers in all leg muscles examined. There was a 19-fold ± 6-fold average limb-wide increase in vector genomes per microgram genomic DNA at a bilateral dose of 2.5 × 1013 vg/kg/limb compared with a bilateral dose of 6 × 1012 vg/kg/limb. A unilateral dose of 6 × 1013 vg/kg/limb showed a 12- ± 3-fold increase in treated limb muscles compared to contralateral untreated limb muscles, which received vector only after release into the systemic circulation from the treated limb. Variability in AAV biodistribution between different segments of the same muscle was 125% ± 18% for any given dose, while variability between the same muscle for any given treatment dose was 45% ± 7%. These experiments demonstrate that treatment of muscles throughout the leg with rAAVrh74.MCK.GALGT2 can be accomplished safely using an isolated limb infusion technique, where balloon catheters transiently isolate the limb vasculature, but that intra- and inter-muscle transduction variability is a significant issue.

4.
Mol Cell Biol ; 37(10)2017 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-28265002

RESUMO

Sarcopenia, the loss of muscle mass and strength during normal aging, involves coordinate changes in skeletal myofibers and the cells that contact them, including satellite cells and motor neurons. Here we show that the protein O-fucosyltransferase 1 gene (Pofut1), which encodes a glycosyltransferase required for NotchR-mediated cell-cell signaling, has reduced expression in aging skeletal muscle. Moreover, premature postnatal deletion of Pofut1 in skeletal myofibers can induce aging-related phenotypes in cis within skeletal myofibers and in trans within satellite cells and within motor neurons via the neuromuscular junction. Changed phenotypes include reduced skeletal muscle size and strength, decreased myofiber size, increased slow fiber (type 1) density, increased muscle degeneration and regeneration in aged muscles, decreased satellite cell self-renewal and regenerative potential, and increased neuromuscular fragmentation and occasional denervation. Pofut1 deletion in skeletal myofibers reduced NotchR signaling in young adult muscles, but this effect was lost with age. Increasing muscle NotchR signaling also reduced muscle size. Gene expression studies point to regulation of cell cycle genes, muscle myosins, NotchR and Wnt pathway genes, and connective tissue growth factor by Pofut1 in skeletal muscle, with additional effects on α dystroglycan glycosylation.


Assuntos
Envelhecimento/fisiologia , Fucosiltransferases/fisiologia , Neurônios Motores/fisiologia , Fibras Musculares Esqueléticas/fisiologia , Junção Neuromuscular/fisiologia , Células Satélites de Músculo Esquelético/fisiologia , Animais , Proteínas de Ciclo Celular/metabolismo , Células Cultivadas , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neurônios Motores/citologia , Fibras Musculares Esqueléticas/citologia , Junção Neuromuscular/patologia , Fenótipo , Receptores Notch/metabolismo , Sarcopenia/etiologia , Sarcopenia/metabolismo , Sarcopenia/patologia , Células Satélites de Músculo Esquelético/citologia , Transdução de Sinais
5.
Hum Gene Ther ; 28(6): 493-509, 2017 06.
Artigo em Inglês | MEDLINE | ID: mdl-28345428

RESUMO

Use of adeno-associated virus (AAV) to transduce genes into skeletal muscles can be associated with T-cell responses to viral capsid and/or to transgenic protein. Intramuscular mononuclear cell infiltrates primarily consisting of CD8+ T cells and also containing FOXP3+ regulatory T cells were present in rhesus macaque skeletal muscle treated with rAAVrh74.MCK.GALGT2 by vascular delivery. Administration of oral prednisone prior to AAV gene delivery and throughout the study reduced such infiltrates by 60% at 24 weeks post AAV delivery compared with AAV-treated animals not receiving prednisone, regardless of the presence of pre-existing AAV serum antibodies at the time of treatment. The majority of CD8+ T cells in AAV-treated muscles expressed activated caspase 3 and programmed cell death protein 1 (PD1), suggesting ongoing programmed cell death. AAV-transduced skeletal muscles also had elevated expression of programmed death ligand 2 (PDL2) on skeletal myofibers, and this increase in expression extended to muscles where transgene was not overexpressed. These data demonstrate that prednisone can reduce the extent of intramuscular T-cell infiltrates in AAV-treated muscles, which may aid in achieving long-term transgene expression, as may the induction of PDL2 expression on skeletal myofibers to promote PD1-mediated programmed T-cell death.


Assuntos
Dependovirus/genética , Vetores Genéticos/imunologia , Imunossupressores/farmacologia , Distrofias Musculares/terapia , Prednisona/farmacologia , Proteína 2 Ligante de Morte Celular Programada 1/genética , Receptor de Morte Celular Programada 1/genética , Administração Oral , Animais , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/patologia , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/imunologia , Morte Celular , Dependovirus/imunologia , Expressão Gênica , Terapia Genética/métodos , Vetores Genéticos/administração & dosagem , Vetores Genéticos/química , Imunidade Celular/efeitos dos fármacos , Injeções Intra-Arteriais , Injeções Intramusculares , Macaca mulatta , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/imunologia , Músculo Esquelético/patologia , Distrofias Musculares/genética , Distrofias Musculares/imunologia , Distrofias Musculares/patologia , N-Acetilgalactosaminiltransferases/genética , N-Acetilgalactosaminiltransferases/imunologia , Proteína 2 Ligante de Morte Celular Programada 1/agonistas , Proteína 2 Ligante de Morte Celular Programada 1/imunologia , Receptor de Morte Celular Programada 1/agonistas , Receptor de Morte Celular Programada 1/imunologia , Linfócitos T Reguladores/efeitos dos fármacos , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/patologia , Transgenes
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