Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 14 de 14
Filter
Add more filters










Publication year range
1.
JCI Insight ; 9(11)2024 May 16.
Article in English | MEDLINE | ID: mdl-38753465

ABSTRACT

Glycogen storage disease type III (GSDIII) is a rare metabolic disorder due to glycogen debranching enzyme (GDE) deficiency. Reduced GDE activity leads to pathological glycogen accumulation responsible for impaired hepatic metabolism and muscle weakness. To date, there is no curative treatment for GSDIII. We previously reported that 2 distinct dual AAV vectors encoding for GDE were needed to correct liver and muscle in a GSDIII mouse model. Here, we evaluated the efficacy of rapamycin in combination with AAV gene therapy. Simultaneous treatment with rapamycin and a potentially novel dual AAV vector expressing GDE in the liver and muscle resulted in a synergic effect demonstrated at biochemical and functional levels. Transcriptomic analysis confirmed synergy and suggested a putative mechanism based on the correction of lysosomal impairment. In GSDIII mice livers, dual AAV gene therapy combined with rapamycin reduced the effect of the immune response to AAV observed in this disease model. These data provide proof of concept of an approach exploiting the combination of gene therapy and rapamycin to improve efficacy and safety and to support clinical translation.


Subject(s)
Dependovirus , Disease Models, Animal , Genetic Therapy , Genetic Vectors , Liver , Sirolimus , Animals , Sirolimus/pharmacology , Sirolimus/therapeutic use , Dependovirus/genetics , Genetic Therapy/methods , Mice , Liver/metabolism , Genetic Vectors/genetics , Genetic Vectors/administration & dosage , Muscle, Skeletal/metabolism , Phenotype , Glycogen Debranching Enzyme System/genetics , Glycogen Debranching Enzyme System/metabolism , Humans , Male
2.
J Clin Invest ; 134(2)2024 Jan 16.
Article in English | MEDLINE | ID: mdl-38015640

ABSTRACT

Glycogen storage disease type III (GSDIII) is a rare inborn error of metabolism affecting liver, skeletal muscle, and heart due to mutations of the AGL gene encoding for the glycogen debranching enzyme (GDE). No curative treatment exists for GSDIII. The 4.6 kb GDE cDNA represents the major technical challenge toward the development of a single recombinant adeno-associated virus-derived (rAAV-derived) vector gene therapy strategy. Using information on GDE structure and molecular modeling, we generated multiple truncated GDEs. Among them, an N-terminal-truncated mutant, ΔNter2-GDE, had a similar efficacy in vivo compared with the full-size enzyme. A rAAV vector expressing ΔNter2-GDE allowed significant glycogen reduction in heart and muscle of Agl-/- mice 3 months after i.v. injection, as well as normalization of histology features and restoration of muscle strength. Similarly, glycogen accumulation and histological features were corrected in a recently generated Agl-/- rat model. Finally, transduction with rAAV vectors encoding ΔNter2-GDE corrected glycogen accumulation in an in vitro human skeletal muscle cellular model of GSDIII. In conclusion, our results demonstrated the ability of a single rAAV vector expressing a functional mini-GDE transgene to correct the muscle and heart phenotype in multiple models of GSDIII, supporting its clinical translation to patients with GSDIII.


Subject(s)
Glycogen Debranching Enzyme System , Glycogen Storage Disease Type III , Humans , Mice , Rats , Animals , Glycogen Storage Disease Type III/genetics , Glycogen Storage Disease Type III/therapy , Glycogen Debranching Enzyme System/genetics , Muscle, Skeletal/metabolism , Glycogen/metabolism , Transgenes
3.
Stem Cell Res ; 72: 103214, 2023 10.
Article in English | MEDLINE | ID: mdl-37769385

ABSTRACT

Glycogen storage disease type III (GSDIII) is an autosomal recessive disorder characterized by a deficiency of glycogen debranching enzyme (GDE) leading to cytosolic glycogen accumulation and inducing liver and muscle pathology. Skin fibroblasts from three GSDIII patients were reprogrammed into induced pluripotent stem cells (iPSCs) using non-integrated Sendai virus. All of the three lines exhibited normal morphology, expression of pluripotent markers, stable karyotype, potential of trilineage differentiation and absence of GDE expression, making them valuable tools for modeling GSDIII disease in vitro, studying pathological mechanisms and investigating potential treatments.


Subject(s)
Glycogen Debranching Enzyme System , Glycogen Storage Disease Type III , Induced Pluripotent Stem Cells , Humans , Glycogen Storage Disease Type III/metabolism , Glycogen Storage Disease Type III/pathology , Induced Pluripotent Stem Cells/metabolism , Liver/pathology , Muscles/metabolism , Muscles/pathology
4.
Front Cell Dev Biol ; 11: 1163427, 2023.
Article in English | MEDLINE | ID: mdl-37250895

ABSTRACT

Introduction: Glycogen storage disease type III (GSDIII) is a rare genetic disease caused by mutations in the AGL gene encoding the glycogen debranching enzyme (GDE). The deficiency of this enzyme, involved in cytosolic glycogen degradation, leads to pathological glycogen accumulation in liver, skeletal muscles and heart. Although the disease manifests with hypoglycemia and liver metabolism impairment, the progressive myopathy is the major disease burden in adult GSDIII patients, without any curative treatment currently available. Methods: Here, we combined the self-renewal and differentiation capabilities of human induced pluripotent stem cells (hiPSCs) with cutting edge CRISPR/Cas9 gene editing technology to establish a stable AGL knockout cell line and to explore glycogen metabolism in GSDIII. Results: Following skeletal muscle cells differentiation of the edited and control hiPSC lines, our study reports that the insertion of a frameshift mutation in AGL gene results in the loss of GDE expression and persistent glycogen accumulation under glucose starvation conditions. Phenotypically, we demonstrated that the edited skeletal muscle cells faithfully recapitulate the phenotype of differentiated skeletal muscle cells of hiPSCs derived from a GSDIII patient. We also demonstrated that treatment with recombinant AAV vectors expressing the human GDE cleared the accumulated glycogen. Discussion: This study describes the first skeletal muscle cell model of GSDIII derived from hiPSCs and establishes a platform to study the mechanisms that contribute to muscle impairments in GSDIII and to assess the therapeutic potential of pharmacological inducers of glycogen degradation or gene therapy approaches.

5.
Biomedicines ; 10(6)2022 Jun 16.
Article in English | MEDLINE | ID: mdl-35740450

ABSTRACT

Limb girdle muscular dystrophies (LGMD), caused by mutations in 29 different genes, are the fourth most prevalent group of genetic muscle diseases. Although the link between LGMD and its genetic origins has been determined, LGMD still represent an unmet medical need. Here, we describe a platform for modeling LGMD based on the use of human induced pluripotent stem cells (hiPSC). Thanks to the self-renewing and pluripotency properties of hiPSC, this platform provides a renewable and an alternative source of skeletal muscle cells (skMC) to primary, immortalized, or overexpressing cells. We report that skMC derived from hiPSC express the majority of the genes and proteins that cause LGMD. As a proof of concept, we demonstrate the importance of this cellular model for studying LGMDR9 by evaluating disease-specific phenotypes in skMC derived from hiPSC obtained from four patients.

6.
Front Pharmacol ; 13: 856804, 2022.
Article in English | MEDLINE | ID: mdl-35571097

ABSTRACT

Limb-girdle muscular dystrophy type R3 (LGMD R3) is a rare genetic disorder characterized by a progressive proximal muscle weakness and caused by mutations in the SGCA gene encoding alpha-sarcoglycan (α-SG). Here, we report the results of a mechanistic screening ascertaining the molecular mechanisms involved in the degradation of the most prevalent misfolded R77C-α-SG protein. We performed a combinatorial study to identify drugs potentializing the effect of a low dose of the proteasome inhibitor bortezomib on the R77C-α-SG degradation inhibition. Analysis of the screening associated to artificial intelligence-based predictive ADMET characterization of the hits led to identification of the HDAC inhibitor givinostat as potential therapeutical candidate. Functional characterization revealed that givinostat effect was related to autophagic pathway inhibition, unveiling new theories concerning degradation pathways of misfolded SG proteins. Beyond the identification of a new therapeutic option for LGMD R3 patients, our results shed light on the potential repurposing of givinostat for the treatment of other genetic diseases sharing similar protein degradation defects such as LGMD R5 and cystic fibrosis.

7.
Sci Rep ; 9(1): 6915, 2019 05 06.
Article in English | MEDLINE | ID: mdl-31061434

ABSTRACT

Limb-girdle muscular dystrophy type 2D (LGMD2D) is characterized by a progressive proximal muscle weakness. LGMD2D is caused by mutations in the gene encoding α-sarcoglycan (α-SG), a dystrophin-associated glycoprotein that plays a key role in the maintenance of sarcolemma integrity in striated muscles. We report here on the development of a new in vitro high-throughput screening assay that allows the monitoring of the proper localization of the most prevalent mutant form of α-SG (R77C substitution). Using this assay, we screened a library of 2560 FDA-approved drugs and bioactive compounds and identified thiostrepton, a cyclic antibiotic, as a potential drug to repurpose for LGMD2D treatment. Characterization of the thiostrepton effect revealed a positive impact on R77C-α-SG and other missense mutant protein localization (R34H, I124T, V247M) in fibroblasts overexpressing these proteins. Finally, further investigations of the molecular mechanisms of action of the compound revealed an inhibition of the chymotrypsin-like activity of the proteasome 24 h after thiostrepton treatment and a synergistic effect with bortezomib, an FDA-approved proteasome inhibitor. This study reports on the first in vitro model for LGMD2D that is compatible with high-throughput screening and proposes a new therapeutic option for LGMD2D caused by missense mutations of α-SG.


Subject(s)
Mutant Proteins/chemistry , Mutant Proteins/metabolism , Protein Folding/drug effects , Proteolysis/drug effects , Sarcoglycans/chemistry , Sarcoglycans/metabolism , Thiostrepton/pharmacology , Cell Line , Cell Membrane/drug effects , Cell Membrane/metabolism , Drug Evaluation, Preclinical , Humans , Induced Pluripotent Stem Cells/cytology , Mutant Proteins/genetics , Myoblasts/cytology , Myoblasts/drug effects , Sarcoglycans/genetics
8.
Sci Rep ; 8(1): 9112, 2018 06 14.
Article in English | MEDLINE | ID: mdl-29904107

ABSTRACT

Hutchinson-Gilford Progeria Syndrome (HGPS) is a rare genetic disorder that leads to premature aging. In this study, we used induced pluripotent stem cells to investigate the hypopigmentation phenotypes observed in patients with progeria. Accordingly, two iPS cell lines were derived from cells from HGPS patients and differentiated into melanocytes. Measurements of melanin content revealed a lower synthesis of melanin in HGPS melanocytes as compared to non-pathologic cells. Analysis of the melanosome maturation process by electron microscopy revealed a lower percentage of mature, fully pigmented melanosomes. Finally, a functional rescue experiment revealed the direct role of progerin in the regulation of melanogenesis. Overall, these results report a new dysregulated pathway in HGPS and open up novel perspectives in the study of pigmentation phenotypes that are associated with normal and pathological aging.


Subject(s)
Induced Pluripotent Stem Cells , Melanocytes , Melanosomes , Models, Biological , Pigmentation Disorders , Progeria , Humans , Induced Pluripotent Stem Cells/metabolism , Induced Pluripotent Stem Cells/pathology , Melanocytes/metabolism , Melanocytes/pathology , Melanosomes/metabolism , Melanosomes/pathology , Pigmentation Disorders/metabolism , Pigmentation Disorders/pathology , Progeria/metabolism , Progeria/pathology
9.
Eur J Med Chem ; 121: 747-757, 2016 Oct 04.
Article in English | MEDLINE | ID: mdl-27429255

ABSTRACT

Smoothened (Smo) is the signal transducer of the Hedgehog (Hh) pathway and its stimulation is considered a potential powerful tool in regenerative medicine to treat severe tissue injuries. Starting from GSA-10, a recently reported Hh activator acting on Smo, we have designed and synthesized a new class of quinolone-based compounds. Modification and decoration of three different portions of the original scaffold led to compounds able to induce differentiation of multipotent mesenchymal cells into osteoblasts. The submicromolar activity of several of these new quinolones (0.4-0.9 µM) is comparable to or better than that of SAG and purmorphamine, two reference Smo agonists. Structure-activity relationships allow identification of several molecular determinants important for the activity of these compounds.


Subject(s)
Drug Design , Osteogenesis/drug effects , Quinolones/chemistry , Quinolones/pharmacology , Animals , Chemistry Techniques, Synthetic , Drug Evaluation, Preclinical , Hedgehog Proteins/metabolism , Mice , Models, Molecular , NIH 3T3 Cells , Quinolones/chemical synthesis , Structure-Activity Relationship
10.
Sci Rep ; 6: 23479, 2016 Mar 24.
Article in English | MEDLINE | ID: mdl-27010359

ABSTRACT

Hedgehog (Hh) is a critical regulator of adipogenesis. Extracellular vesicles are natural Hh carriers, as illustrated by activated/apoptotic lymphocytes specifically shedding microparticles (MP) bearing the morphogen (MP(Hh+)). We show that MP(Hh+) inhibit adipocyte differentiation and orientate mesenchymal stem cells towards a pro-osteogenic program. Despite a Smoothened (Smo)-dependency, MP(Hh+) anti-adipogenic effects do not activate a canonical Hh signalling pathway in contrast to those elicited either by the Smo agonist SAG or recombinant Sonic Hedgehog. The Smo agonist GSA-10 recapitulates many of the hallmarks of MP(Hh+) anti-adipogenic effects. The adipogenesis blockade induced by MP(Hh+) and GSA-10 was abolished by the Smo antagonist LDE225. We further elucidate a Smo/Lkb1/Ampk axis as the non-canonical Hh pathway used by MP(Hh+) and GSA-10 to inhibit adipocyte differentiation. Our results highlight for the first time the ability of Hh-enriched MP to signal via a non-canonical pathway opening new perspectives to modulate fat development.


Subject(s)
Adipocytes/cytology , Cell Differentiation/physiology , Hedgehog Proteins/physiology , 3T3-L1 Cells , Animals , Cells, Cultured , Hedgehog Proteins/metabolism , Humans , Mesenchymal Stem Cells/cytology , Mice , Transcription Factors/metabolism
11.
FASEB J ; 29(5): 1817-29, 2015 May.
Article in English | MEDLINE | ID: mdl-25636740

ABSTRACT

The Smoothened (Smo) receptor, a member of class F G protein-coupled receptors, is the main transducer of the Hedgehog (Hh) signaling pathway implicated in a wide range of developmental and adult processes. Smo is the target of anticancer drugs that bind to a long and narrow cavity in the 7-transmembrane (7TM) domain. X-ray structures of human Smo (hSmo) bound to several ligands have revealed 2 types of 7TM-directed antagonists: those binding mostly to extracellular loops (site 1, e.g., LY2940680) and those penetrating deeply in the 7TM cavity (site 2, e.g., SANT-1). Here we report the development of the acylguanidine MRT-92, which displays subnanomolar antagonist activity against Smo in various Hh cell-based assays. MRT-92 inhibits rodent cerebellar granule cell proliferation induced by Hh pathway activation through pharmacologic (half maximal inhibitory concentration [IC50] = 0.4 nM) or genetic manipulation. Using [(3)H]MRT-92 (Kd = 0.3 nM for hSmo), we created a comprehensive framework for the interaction of small molecule modulators with hSmo and for understanding chemoresistance linked to hSmo mutations. Guided by molecular docking and site-directed mutagenesis data, our work convincingly confirms that MRT-92 simultaneously recognized and occupied both sites 1 and 2. Our data demonstrate the existence of a third type of Smo antagonists, those entirely filling the Smo binding cavity from the upper extracellular part to the lower cytoplasmic-proximal subpocket. Our studies should help design novel potent Smo antagonists and more effective therapeutic strategies for treating Hh-linked cancers and associated chemoresistance.


Subject(s)
Antineoplastic Agents/pharmacology , Cell Membrane/metabolism , Cerebellar Neoplasms/metabolism , Guanidines/pharmacology , Hedgehog Proteins/antagonists & inhibitors , Medulloblastoma/metabolism , Receptors, G-Protein-Coupled/antagonists & inhibitors , Small Molecule Libraries/pharmacology , Adult , Animals , Binding Sites , Blotting, Western , Cell Membrane/drug effects , Cell Proliferation/drug effects , Cells, Cultured , Cerebellar Neoplasms/drug therapy , Cerebellar Neoplasms/pathology , Drug Resistance, Neoplasm/drug effects , Drug Resistance, Neoplasm/genetics , Hedgehog Proteins/metabolism , Humans , Immunoenzyme Techniques , Medulloblastoma/drug therapy , Medulloblastoma/pathology , Mice , Molecular Docking Simulation , Mutagenesis, Site-Directed , Mutation/genetics , Protein Binding , Protein Conformation , Receptors, G-Protein-Coupled/metabolism , Signal Transduction/drug effects , Smoothened Receptor
12.
Trends Pharmacol Sci ; 35(5): 237-46, 2014 May.
Article in English | MEDLINE | ID: mdl-24703627

ABSTRACT

The Smoothened (Smo) receptor is a key transducer of the Hedgehog (Hh) signaling pathway, which plays a critical role in tissue maintenance and repair. Recent studies have highlighted the therapeutic value of Smo antagonists for treating Hh-linked cancers. Research on Smo agonists indicates that these molecules are important not only for delineating canonical versus noncanonical Hh signaling but also for understanding the role of Smo in physiological and pathological conditions. This review provides an update on the potential therapeutic importance of Smo modulators, and unravels the increasing complexity of its pharmacology with regard to clinical implications.


Subject(s)
Receptors, G-Protein-Coupled/agonists , Receptors, G-Protein-Coupled/antagonists & inhibitors , Animals , Hedgehog Proteins/metabolism , Humans , Ligands , Molecular Targeted Therapy , Receptors, G-Protein-Coupled/metabolism , Signal Transduction , Smoothened Receptor
13.
Med Sci (Paris) ; 29(10): 855-60, 2013 Oct.
Article in French | MEDLINE | ID: mdl-24148123

ABSTRACT

The Smoothened (Smo) receptor is a major component involved in signal transduction of the Hedgehog (Hh) morphogens both during embryogenesis and in the adult. Smo antagonists represent a promi-sing alternative for the treatment of cancers linked to abnormal Hh signalling. The crystal structure of the human Smo receptor bound to an antitumour agent demonstrates that this receptor belongs to the superfamily of G-protein coupled receptors. The antagonist binds to a pocket localized at the extracellular side formed by the seven transmembrane domains and the complex arrangement of the unusually long extracellular loops. The structure of the Smo receptor will promote the development of small molecules interacting with a key therapeutic target with interests in regenerative medicine and cancer.


Subject(s)
Receptors, G-Protein-Coupled/antagonists & inhibitors , Receptors, G-Protein-Coupled/chemistry , Adult , Antineoplastic Agents/therapeutic use , Clinical Trials as Topic , Crystallography , Humans , Models, Molecular , Molecular Targeted Therapy , Neoplasms/genetics , Neoplasms/therapy , Protein Structure, Tertiary , Receptors, G-Protein-Coupled/genetics , Smoothened Receptor
14.
Mol Pharmacol ; 83(5): 1020-9, 2013 May.
Article in English | MEDLINE | ID: mdl-23448715

ABSTRACT

Activation of the Smoothened (Smo) receptor mediates Hedgehog (Hh) signaling. Hh inhibitors are in clinical trials for cancer, and small-molecule Smo agonists may have therapeutic interests in regenerative medicine. Here, we have generated and validated a pharmacophoric model for Smo agonists and used this model for the virtual screening of a library of commercially available compounds. Among the 20 top-scoring ligands, we have identified and characterized a novel quinolinecarboxamide derivative, propyl 4-(1-hexyl-4-hydroxy-2-oxo-1,2-dihydroquinoline-3-carboxamido) benzoate, (GSA-10), as a Smo agonist. GSA-10 fits to the agonist pharmacophoric model with two hydrogen bond acceptor groups and four hydrophobic regions. Using pharmacological, biochemical, and molecular approaches, we provide compelling evidence that GSA-10 acts at Smo to promote the differentiation of multipotent mesenchymal progenitor cells into osteoblasts. However, this molecule does not display the hallmarks of reference Smo agonists. Remarkably, GSA-10 does not recognize the classic bodipy-cyclopamine binding site. Its effect on cell differentiation is inhibited by Smo antagonists, such as MRT-83, SANT-1, LDE225, and M25 in the nanomolar range, by GDC-0449 in the micromolar range, but not by cyclopamine and CUR61414. Thus, GSA-10 allows the pharmacological characterization of a novel Smo active site, which is notably not targeted to the primary cilium and strongly potentiated by forskolin and cholera toxin. GSA-10 belongs to a new class of Smo agonists and will be helpful for dissecting Hh mechanism of action, with important implications in physiology and in therapy.


Subject(s)
Quinolines/pharmacology , Receptors, G-Protein-Coupled/agonists , Receptors, G-Protein-Coupled/metabolism , Benzoates/pharmacology , Binding Sites/drug effects , Bone Morphogenetic Protein Receptors/metabolism , Cell Differentiation/drug effects , Cell Line , Cyclic AMP/metabolism , Cyclohexylamines/pharmacology , HEK293 Cells , Hedgehog Proteins/metabolism , Humans , Ligands , Receptors, G-Protein-Coupled/antagonists & inhibitors , Small Molecule Libraries , Smoothened Receptor , Thiophenes/pharmacology , Transcription Factors/metabolism , Wnt Proteins/metabolism , Zinc Finger Protein GLI1
SELECTION OF CITATIONS
SEARCH DETAIL
...