72 results on '"Torres-Torronteras J."'
Search Results
2. Hematopoietic gene therapy restores thymidine phosphorylase activity in a cell culture and a murine model of MNGIE
- Author
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Torres-Torronteras, J, Gómez, A, Eixarch, H, Palenzuela, L, Pizzorno, G, Hirano, M, Andreu, A L, Barquinero, J, and Martí, R
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- 2011
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3. Mitochondrial Neurogastrointestinal Encephalomyopathy (MNGIE): Biochemical Features and Therapeutic Approaches
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Lara, M. C., Valentino, M. L., Torres-Torronteras, J., Hirano, M., and Martí, R.
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- 2007
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4. Preclinical Efficacy and Safety Evaluation of Hematopoietic Stem Cell Gene Therapy in a Mouse Model of MNGIE
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Yadak, R.M.A. (Rana), Cabrera-Pérez, R. (Raquel), Torres-Torronteras, J., Bugiani, M. (Marianna), Haeck, J.C. (Joost), Huston, M.W. (Marshall W.), Bogaerts, E. (Elly), Goffart, S. (Steffi), Jacobs, E.H. (Edwin), Stok, M. (Merel), Leonardelli, L. (Lorena), Biasco, L. (Luca), Verdijk, R.M. (Robert), Bernsen, M.R. (Monique), Ruijter, G.J.G. (George), Martí, R. (Ramon), Wagemaker, G. (Gerard), Til, N.P. (Niek) van, Coo, I.F.M. (René) de, Yadak, R.M.A. (Rana), Cabrera-Pérez, R. (Raquel), Torres-Torronteras, J., Bugiani, M. (Marianna), Haeck, J.C. (Joost), Huston, M.W. (Marshall W.), Bogaerts, E. (Elly), Goffart, S. (Steffi), Jacobs, E.H. (Edwin), Stok, M. (Merel), Leonardelli, L. (Lorena), Biasco, L. (Luca), Verdijk, R.M. (Robert), Bernsen, M.R. (Monique), Ruijter, G.J.G. (George), Martí, R. (Ramon), Wagemaker, G. (Gerard), Til, N.P. (Niek) van, and Coo, I.F.M. (René) de
- Abstract
Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is an autosomal recessive disorder caused by thymidine phosphorylase (TP) deficiency resulting in systemic accumulation of thymidine (d-Thd) and deoxyuridine (d-Urd) and characterized by early-onset neurological and gastrointestinal symptoms. Long-term effective and safe treatment is not available. Allogeneic bone marrow transplantation may improve clinical manifestations but carries disease and transplant-related risks. In this study, lentiviral vector-based hematopoietic stem cell gene therapy (HSCGT) was performed in Tymp−/−Upp1−/− mice with the human phosphoglycerate kinase (PGK) promoter driving TYMP. Supranormal blood TP activity reduced intestinal nucleoside levels significantly at low vector copy number (median, 1.3; range, 0.2–3.6). Furthermore, we covered two major issues not addressed before. First, we demonstrate aberrant morphology of brain astrocytes in areas of spongy degeneration, which was reversed by HSCGT. Second, long-term follow-up and vector integration site analysis were performed to assess safety of the therapeutic LV vectors in depth. This report confirms and supplements previous work on the efficacy of HSCGT in reducing the toxic metabolites in Tymp−/−Upp1−/− mice, using a clinically applicable gene transfer vector and a highly efficient gene transfer method, and importantly demonstrates phenotypic correction with a favorable risk profile, warranting further development toward clinical implementation.
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- 2018
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5. Preclinical Efficacy and Safety Evaluation of Hematopoietic Stem Cell Gene Therapy in a Mouse Model of MNGIE
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Yadak, Rana, Cabrera-Perez, R, Torres-Torronteras, J, Bugiani, M, Haeck, Joost, Huston, MW, Taal, Elly, Goffart, S, Jacobs, Ed, Stok, M, Leonardelli, L, Biasco, L, Verdijk, Rob, Bernsen, Monique, Ruijter, GJG, Marti, R, Wagemaker, G, Til, NP, Coo, IFM, Yadak, Rana, Cabrera-Perez, R, Torres-Torronteras, J, Bugiani, M, Haeck, Joost, Huston, MW, Taal, Elly, Goffart, S, Jacobs, Ed, Stok, M, Leonardelli, L, Biasco, L, Verdijk, Rob, Bernsen, Monique, Ruijter, GJG, Marti, R, Wagemaker, G, Til, NP, and Coo, IFM
- Published
- 2018
6. Author Correction: Resveratrol Increases Hepatic SHBG Expression through Human Constitutive Androstane Receptor: a new Contribution to the French Paradox
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Saez-Lopez, Cristina, primary, Brianso-Llort, Laura, additional, Torres-Torronteras, J., additional, Simó, Rafael, additional, Hammond, Geoffrey L., additional, and Selva, David M., additional
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- 2018
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7. Resveratrol Increases Hepatic SHBG Expression through Human Constitutive Androstane Receptor: a new Contribution to the French Paradox
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Saez-Lopez, Cristina, primary, Brianso-Llort, Laura, additional, Torres-Torronteras, J., additional, Simó, Rafael, additional, Hammond, Geoffrey L., additional, and Selva, David M., additional
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- 2017
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8. Allogeneic haematopoietic stem cell transplantation for mitochondrial neurogastrointestinal encephalomyopathy
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Halter, J. P, Michael, W, Schüpbach, M, Mandel, H, Casali, C, Orchard, K, Collin, M, Valcarcel, D, Rovelli, A, Filosto, M, Dotti, M. T, Marotta, G, Pintos, G, Barba, P, Accarino, A, Ferra, C, Illa, I, Beguin, Y, Bakker, J. A, Boelens, J. J, De Coo, I. F. M, Fay, K, Sue, C. M, Nachbaur, D, Zoller, H, Sobreira, C, Pinto Simoes, B, Hammans, S. R, Savage, D, Martí, R, Chinnery, P. F, Elhasid, R, Gratwohl, A, Hirano, M, Barros Navarro, G, Benoist, J. F, Bierau, J, Bucalossi, A, Carluccio, M. A, Coll-Canti, J, Cotelli, M. S, Diesch, T, Di Fabio, R, Donati, M. A, Garvin, J. H, Hill, K, Kappeler, L, Ku Hne, T, Lara, M. C, Lenoci, M, Lucchini, G, Marques, W. Jr, Mattle, H. P, Meyer, A, Parini, R, Passweg, J. R, Pieroni, F, Rodriguez-Palmero, A, Santus, F, Scarpelli, M, Schlesser, P, Sicurelli, F, Stern, M, Stracieri, A. B, Tonin, P, Torres-Torronteras, J, Voltarelli, J. C, Zaidman, I., Radiotherapy, and Neurology
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Male ,DISORDER ,Pathology ,Neutrophils ,medicine.medical_treatment ,Neural Conduction ,Hematopoietic stem cell transplantation ,Gastroenterology ,Liver disease ,Fanconi anemia ,risk factors ,Non-U.S. Gov't ,Child ,610 Medicine & health ,Neurologic Examination ,OUTCOMES ,Ophthalmoplegia ,Research Support, Non-U.S. Gov't ,Hematopoietic Stem Cell Transplantation ,Brain ,THYMIDINE PHOSPHORYLASE-DEFICIENCY ,Magnetic Resonance Imaging ,Multicenter Study ,Haematopoiesis ,Treatment Outcome ,mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) ,MNGIE ,outcome ,Female ,Stem cell ,Adult ,medicine.medical_specialty ,Adolescent ,Thymidine phosphorylase activity ,Research Support ,thymidine phosphorylase ,PATIENT ,N.I.H ,Young Adult ,SDG 3 - Good Health and Well-being ,Muscular Dystrophy, Oculopharyngeal ,Research Support, N.I.H., Extramural ,Mitochondrial Encephalomyopathies ,Internal medicine ,medicine ,Journal Article ,Humans ,Transplantation, Homologous ,Thymidine phosphorylase ,Retrospective Studies ,business.industry ,MUTATIONS ,MUTAÇÃO GENÉTICA ,Body Weight ,Intestinal Pseudo-Obstruction ,Extramural ,allogeneic haematopoietic stem cell transplantation ,Original Articles ,medicine.disease ,Survival Analysis ,Transplantation ,DELETIONS ,FANCONI-ANEMIA ,Neurology (clinical) ,business ,Follow-Up Studies - Abstract
Haematopoietic stem cell transplantation has been proposed as treatment for mitochondrial neurogastrointestinal encephalomyopathy, a rare fatal autosomal recessive disease due to TYMP mutations that result in thymidine phosphorylase deficiency. We conducted a retrospective analysis of all known patients suffering from mitochondrial neurogastrointestinal encephalomyopathy who underwent allogeneic haematopoietic stem cell transplantation between 2005 and 2011. Twenty-four patients, 11 males and 13 females, median age 25 years (range 10-41 years) treated with haematopoietic stem cell transplantation from related (n = 9) or unrelated donors (n = 15) in 15 institutions worldwide were analysed for outcome and its associated factors. Overall, 9 of 24 patients (37.5%) were alive at last follow-up with a median follow-up of these surviving patients of 1430 days. Deaths were attributed to transplant in nine (including two after a second transplant due to graft failure), and to mitochondrial neurogastrointestinal encephalomyopathy in six patients. Thymidine phosphorylase activity rose from undetectable to normal levels (median 697 nmol/h/mg protein, range 262-1285) in all survivors. Seven patients (29%) who were engrafted and living more than 2 years after transplantation, showed improvement of body mass index, gastrointestinal manifestations, and peripheral neuropathy. Univariate statistical analysis demonstrated that survival was associated with two defined pre-transplant characteristics: human leukocyte antigen match (10/10 versus
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- 2015
9. Deoxyribonucleoside supply rescues mtDNA depletion in human POLG-deficient fibroblasts
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Blázquez-Bermejo, C., primary, Torres-Torronteras, J., additional, Cabrera, R., additional, Lombès, A., additional, Martí, R., additional, and Cámara, Y., additional
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- 2016
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10. OP45 – 3024: Efficient lentiviral vector-mediated hematopoietic stem cell gene therapy in MNGIE mice
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Yadak, R., primary, Torres-Torronteras, J., additional, Bogaerts, E., additional, de Ruijter, G., additional, Barquinero, J., additional, Marti, R., additional, Smeets, H.J., additional, Huston, M.W., additional, van Til, N.P., additional, Wagemaker, G., additional, and de Coo, I.F.M., additional
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- 2015
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11. P.302 - Deoxyribonucleoside supply rescues mtDNA depletion in human POLG-deficient fibroblasts
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Blázquez-Bermejo, C., Torres-Torronteras, J., Cabrera, R., Lombès, A., Martí, R., and Cámara, Y.
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- 2016
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12. RRM1 variants cause a mitochondrial DNA maintenance disorder via impaired de novo nucleotide synthesis
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Jonathan Shintaku, Wolfgang M. Pernice, Wafaa Eyaid, Jeevan B. GC, Zuben P. Brown, Marti Juanola-Falgarona, Javier Torres-Torronteras, Ewen W. Sommerville, Debby M.E.I. Hellebrekers, Emma L. Blakely, Alan Donaldson, Ingrid van de Laar, Cheng-Shiun Leu, Ramon Marti, Joachim Frank, Kurenai Tanji, David A. Koolen, Richard J. Rodenburg, Patrick F. Chinnery, H.J.M. Smeets, Gráinne S. Gorman, Penelope E. Bonnen, Robert W. Taylor, Michio Hirano, MUMC+: DA KG Lab Specialisten (9), MUMC+: DA KG Lab Centraal Lab (9), RS: MHeNs - R3 - Neuroscience, Klinische Genetica, Institut Català de la Salut, [Shintaku J, Pernice WM, Juanola-Falgarona M] Department of Neurology, H. Houston Merritt Neuromuscular Research Center, Columbia University Irving Medical Center, New York, New York, USA. [Eyaid W] Genetics Division, Department of Pediatrics, King Saud bin Abdulaziz University for Health Science, King Abdulaziz Medical City, Riyadh, Saudi Arabia. [GC JB, Brown ZP] Department of Biochemistry and Molecular Biophysics, Columbia University, New York, New York, USA. [Torres-Torronteras J, Marti R] Center for Biomedical Network Research on Rare Diseases, Instituto de Salud Carlos III, Madrid, Spain. Grups de Recerca en Malalties Neuromusculars i Mitocondrials, Vall d’Hebron Institut de Recerca (VHIR), Barcelona, Spain. Universitat Autònoma de Barcelona, Bellaterra, Spain, Vall d'Hebron Barcelona Hospital Campus, and Clinical Genetics
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DNA Replication ,Neurodevelopmental disorders Donders Center for Medical Neuroscience [Radboudumc 7] ,Mitochondrial Diseases ,Ribonucleoside Diphosphate Reductase ,Nucleotides ,Otros calificadores::Otros calificadores::/genética [Otros calificadores] ,Mitocondris - Malalties - Aspectes genètics ,Metabolic Disorders Radboud Institute for Molecular Life Sciences [Radboudumc 6] ,Nucleosides ,General Medicine ,ADN mitocondrial ,Nutritional and Metabolic Diseases::Metabolic Diseases::Mitochondrial Diseases [DISEASES] ,DNA, Mitochondrial ,DEFICIENCY ,All institutes and research themes of the Radboud University Medical Center ,PHOSPHORYLASE GENE-MUTATIONS ,Nucleic Acids, Nucleotides, and Nucleosides::Nucleic Acids::DNA::DNA, Circular::DNA, Mitochondrial [CHEMICALS AND DRUGS] ,ENCEPHALOMYOPATHY ,nucleótidos y nucleósidos de ácidos nucleicos::ácidos nucleicos::ADN::ADN circular::ADN mitocondrial [COMPUESTOS QUÍMICOS Y DROGAS] ,Ribonucleotide Reductases ,Other subheadings::Other subheadings::/genetics [Other subheadings] ,KINASE ,Humans ,RIBONUCLEOTIDE REDUCTASE ,enfermedades nutricionales y metabólicas::enfermedades metabólicas::enfermedades mitocondriales [ENFERMEDADES] - Abstract
Genetic diseases; Mitochondria; Molecular pathology Enfermedades genéticas; Mitocondrias; Patología molecular Malalties genètiques; Mitocondris; Patologia molecular Mitochondrial DNA (mtDNA) depletion/deletions syndromes (MDDS) encompass a clinically and etiologically heterogenous group of mitochondrial disorders caused by impaired mtDNA maintenance. Among the most frequent causes of MDDS are defects in nucleoside/nucleotide metabolism, which is critical for synthesis and homeostasis of the deoxynucleoside triphosphate (dNTP) substrates of mtDNA replication. A central enzyme for generating dNTPs is ribonucleotide reductase, a critical mediator of de novo nucleotide synthesis composed of catalytic RRM1 subunits in complex with RRM2 or p53R2. Here, we report 5 probands from 4 families who presented with ptosis and ophthalmoplegia as well as other clinical manifestations and multiple mtDNA deletions in muscle. We identified 3 RRM1 loss-of-function variants, including a dominant catalytic site variant (NP_001024.1: p.N427K) and 2 homozygous recessive variants at p.R381, which has evolutionarily conserved interactions with the specificity site. Atomistic molecular dynamics simulations indicate mechanisms by which RRM1 variants affect protein structure. Cultured primary skin fibroblasts of probands manifested mtDNA depletion under cycling conditions, indicating impaired de novo nucleotide synthesis. Fibroblasts also exhibited aberrant nucleoside diphosphate and dNTP pools and mtDNA ribonucleotide incorporation. Our data reveal that primary RRM1 deficiency and, by extension, impaired de novo nucleotide synthesis are causes of MDDS. This work was supported by Department of Defense Focused Program Award W81XWH2010807 (to MH), NIH research grant P01 HD32062 (to MH), and NIH grant 35 GM139453 (to JF). MH is supported by the Arturo Estopinan TK2 Research Fund, Nicholas Nunno Foundation, JDM Fund for Mitochondrial Research, Shuman Mitochondrial Disease Fund, the Marriott Mitochondrial Disease Clinic Research Fund from the J. Willard and Alice S. Marriott Foundation, and NIH grant U54 NS078059. Work in Newcastle upon Tyne was supported by the Wellcome Centre for Mitochondrial Research (203105/Z/16/Z), Medical Research Council International Centre for Genomic Medicine in Neuromuscular Disease (MR/S005021/1), UK NIHR Biomedical Research Centre in Age and Age Related Diseases award to the Newcastle upon Tyne Hospitals NHS Foundation, the Lily Foundation, and the UK National Health Service Highly Specialised Service for Rare Mitochondrial Disorders. RWT receives financial support from the Pathological Society. EWS was funded by a Medical Research Council PhD studentship. This work used the Extreme Science and Engineering Discovery Environment (XSEDE), which is supported by National Science Foundation grant ACI-1548562. JBGC is supported by grant BIO210070 from XSEDE. The authors thank the patients and their families for collaborating in this study and Saba Tadesse for technical support of mitochondrial respiratory chain enzyme activities. We also thank the Genome Technology Center at the Radboud University Medical Center and BGI Copenhagen for WES technical support.
- Published
- 2022
13. Most mitochondrial dGTP is tightly bound to respiratory complex I through the NDUFA10 subunit
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David Molina-Granada, Emiliano González-Vioque, Marris G. Dibley, Raquel Cabrera-Pérez, Antoni Vallbona-Garcia, Javier Torres-Torronteras, Leonid A. Sazanov, Michael T. Ryan, Yolanda Cámara, Ramon Martí, Institut Català de la Salut, [Molina-Granada D, Cabrera-Pérez R, Torres-Torronteras J, Cámara Y, Martí R] Grup de Recerca de Patologia Neuromuscular i Mitocondrial, Vall d’Hebron Institut de Recerca (VHIR), Barcelona, Spain. Universitat Autònoma de Barcelona, Bellaterra, Spain. Biomedical Network Research Centre on Rare Diseases (CIBERER), Instituto de Salud Carlos III, Madrid, Spain. [González-Vioque E] Grup de Recerca de Patologia Neuromuscular i Mitocondrial, Vall d’Hebron Institut de Recerca (VHIR), Barcelona, Spain. Universitat Autònoma de Barcelona, Bellaterra, Spain. Biomedical Network Research Centre on Rare Diseases (CIBERER), Instituto de Salud Carlos III, Madrid, Spain. Department of Clinical Biochemistry, Hospital Universitario Puerta del Hierro-Majadahonda, Madrid, Spain. [Dibley MG] Department of Biochemistry and Molecular Biology, Monash Biomedicine Discovery Institute, Monash University, Melbourne, Australia. [Vallbona-Garcia A] Grup de Recerca de Patologia Neuromuscular i Mitocondrial, Vall d’Hebron Institut de Recerca (VHIR), Barcelona, Spain. Universitat Autònoma de Barcelona, Bellaterra, Spain, and Vall d'Hebron Barcelona Hospital Campus
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Fisiologia cel·lular ,Electron Transport Complex I ,células::estructuras celulares::espacio intracelular::citoplasma::estructuras citoplasmáticas::orgánulos::mitocondrias [ANATOMÍA] ,Medicine (miscellaneous) ,Deoxyguanine Nucleotides ,NADH Dehydrogenase ,General Biochemistry, Genetics and Molecular Biology ,Mitocondris ,Mitochondria ,HEK293 Cells ,fenómenos fisiológicos celulares::respiración celular [FENÓMENOS Y PROCESOS] ,Humans ,Cells::Cellular Structures::Intracellular Space::Cytoplasm::Cytoplasmic Structures::Organelles::Mitochondria [ANATOMY] ,Ratolins ,General Agricultural and Biological Sciences ,Eukaryota::Animals::Chordata::Vertebrates::Mammals::Eutheria::Rodentia::Muridae::Murinae::Mice [ORGANISMS] ,Cell Physiological Phenomena::Cell Respiration [PHENOMENA AND PROCESSES] ,Eukaryota::animales::Chordata::vertebrados::mamíferos::Eutheria::Rodentia::Muridae::Murinae::ratones [ORGANISMOS] - Abstract
Biochemistry; Molecular medicine Bioquímica; Medicina molecular Bioquímica; Medicina molecular Imbalanced mitochondrial dNTP pools are known players in the pathogenesis of multiple human diseases. Here we show that, even under physiological conditions, dGTP is largely overrepresented among other dNTPs in mitochondria of mouse tissues and human cultured cells. In addition, a vast majority of mitochondrial dGTP is tightly bound to NDUFA10, an accessory subunit of complex I of the mitochondrial respiratory chain. NDUFA10 shares a deoxyribonucleoside kinase (dNK) domain with deoxyribonucleoside kinases in the nucleotide salvage pathway, though no specific function beyond stabilizing the complex I holoenzyme has been described for this subunit. We mutated the dNK domain of NDUFA10 in human HEK-293T cells while preserving complex I assembly and activity. The NDUFA10E160A/R161A shows reduced dGTP binding capacity in vitro and leads to a 50% reduction in mitochondrial dGTP content, proving that most dGTP is directly bound to the dNK domain of NDUFA10. This interaction may represent a hitherto unknown mechanism regulating mitochondrial dNTP availability and linking oxidative metabolism to DNA maintenance. We thank Dr, Luke Formosa (Department of Biochemistry and Molecular Biology, Monash Biomedicine Discovery Institute, Monash University, Melbourne, Australia) for his valuable advice and assistance on NDUFA10 molecular studies and Dr. Francesc Canals and his team (Proteomics Laboratory, Vall d’Hebron Institute of Oncology [VHIO], Universitat Autònoma de Barcelona, Barcelona, Spain) for their assistance with LC-MS/MS analyses. This work was supported by the Spanish Ministry of Industry, Economy and Competitiveness [grants BFU2014-52618-R, SAF2017-87506, and PID2020-112929RB-I00 to Y.C.], by the Spanish Instituto de Salud Carlos III [grants PI21/00554 and PMP15/00025 to R.M.], co-financed by the European Regional Development Fund (ERDF), and by an NHMRC Project grant to M.R. (GNT1164459).
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- 2022
14. Therapy Prospects for Mitochondrial DNA Maintenance Disorders
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Yolanda Cámara, Ferran Vila-Julià, David Molina-Granada, Maria Jesús Melià, Javier Torres-Torronteras, Miguel Molina-Berenguer, Ramon Martí, Elena García-Arumí, Javier Ramón, Institut Català de la Salut, [Ramón J, Vila-Julià F, Molina-Granada D, Molina-Berenguer M, Melià MJ, García-Arumí E, Torres-Torronteras J, Cámara Y, Martí R] Grup de Recerca en Malalties Neuromusculars i Mitocondrials, Vall d’Hebron Institut de Recerca, Barcelona, Spain. Universitat Autònoma de Barcelona, Bellaterra, Spain. Biomedical Network Research Centre on Rare Diseases (CIBERER), Instituto de Salud Carlos III, 28029 Madrid, Spain, and Vall d'Hebron Barcelona Hospital Campus
- Subjects
0301 basic medicine ,Mitochondrial Diseases ,Genetic enhancement ,medicine.medical_treatment ,Genetic Phenomena::DNA Replication [PHENOMENA AND PROCESSES] ,Multiple deletions ,Review ,Nutritional and Metabolic Diseases::Metabolic Diseases::Mitochondrial Diseases [DISEASES] ,Mitochondrion ,Liver transplantation ,Bioinformatics ,0302 clinical medicine ,Nucleoside ,Biology (General) ,Otros calificadores::/terapia [Otros calificadores] ,Spectroscopy ,depletion ,mtDNA ,Disease Management ,General Medicine ,multiple deletions ,Combined Modality Therapy ,gene therapy ,Computer Science Applications ,Mitochondria ,mitochondria ,Chemistry ,Mitocondris - Malalties - Tractament ,Disease Susceptibility ,Stem cell ,enfermedades nutricionales y metabólicas::enfermedades metabólicas::enfermedades mitocondriales [ENFERMEDADES] ,DNA Replication ,Mitochondrial DNA ,replication ,ADN - Duplicació ,QH301-705.5 ,Replication ,Dysfunctional family ,nucleoside ,DNA, Mitochondrial ,Catalysis ,Mitochondrial Proteins ,Inorganic Chemistry ,03 medical and health sciences ,Depletion ,fenómenos genéticos::replicación del ADN [FENÓMENOS Y PROCESOS] ,Gene therapy ,medicine ,Animals ,Humans ,Physical and Theoretical Chemistry ,QD1-999 ,Molecular Biology ,therapy ,business.industry ,Organic Chemistry ,Other subheadings::/therapy [Other subheadings] ,Mtdn ,Transplantation ,Clinical trial ,030104 developmental biology ,Gene Expression Regulation ,Mutation ,Therapy ,business ,030217 neurology & neurosurgery - Abstract
Esgotament; Teràpia gènica; Mitocondris Mitochondria; Depletion; Gene therapy Agotamiento; Terapia génica; Mitocondrias Mitochondrial DNA depletion and multiple deletions syndromes (MDDS) constitute a group of mitochondrial diseases defined by dysfunctional mitochondrial DNA (mtDNA) replication and maintenance. As is the case for many other mitochondrial diseases, the options for the treatment of these disorders are rather limited today. Some aggressive treatments such as liver transplantation or allogeneic stem cell transplantation are among the few available options for patients with some forms of MDDS. However, in recent years, significant advances in our knowledge of the biochemical pathomechanisms accounting for dysfunctional mtDNA replication have been achieved, which has opened new prospects for the treatment of these often fatal diseases. Current strategies under investigation to treat MDDS range from small molecule substrate enhancement approaches to more complex treatments, such as lentiviral or adenoassociated vector-mediated gene therapy. Some of these experimental therapies have already reached the clinical phase with very promising results, however, they are hampered by the fact that these are all rare disorders and so the patient recruitment potential for clinical trials is very limited.
- Published
- 2021
15. Identification and Characterization of New RNASEH1 Mutations Associated With PEO Syndrome and Multiple Mitochondrial DNA Deletions
- Author
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Institut Català de la Salut, [Carreño-Gago L, Blázquez-Bermejo C, Cámara Y, Martí R, Torres-Torronteras J] Departament de Patologia Mitocondrial i Neuromuscular, Vall d’Hebron Institut de Recerca (VHIR), Barcelona, Spain. Universitat Autònoma de Barcelona Barcelona, Spain. Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), Instituto de Salud Carlos III, Barcelona, Spain. [Díaz-Manera J, Gallardo E] Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), Instituto de Salud Carlos III, Barcelona, Spain. Servei de Neurologia, Malalties Neuromusculars, Hospital de la Santa Creu i Sant Pau, Barcelona, Spain. Institut de Recerca de HSCSP, Barcelona, Spain. Universitat Autònoma de Barcelona, Barcelona, Spain. [García-Arumí E] Departament de Patologia Mitocondrial i Neuromuscular, Vall d’Hebron Institut de Recerca (VHIR), Barcelona, Spain. Universitat Autònoma de Barcelona Barcelona, Spain. Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), Instituto de Salud Carlos III, Barcelona, Spain. Àrea de Genètica Clínica i Molecular, Hospital Universitari Vall d'Hebron, Barcelona, Spain, and Hospital Universitari Vall d'Hebron
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Ulls - Malalties - Aspectes genètics ,Ribonucleases ,Oftalmopatías::Trastornos de la Motilidad Ocular::Oftalmoplejía [ENFERMEDADES] ,fenómenos genéticos::variación genética::mutación::deleción de secuencias [FENÓMENOS Y PROCESOS] ,ADN mitocondrial - Malformacions ,Nucleic Acids, Nucleotides, and Nucleosides::Nucleic Acids::DNA::DNA, Circular::DNA, Mitochondrial [CHEMICALS AND DRUGS] ,Otros calificadores::Otros calificadores::/genética [Otros calificadores] ,nucleótidos y nucleósidos de ácidos nucleicos::ácidos nucleicos::ADN::ADN circular::ADN mitocondrial [COMPUESTOS QUÍMICOS Y DROGAS] ,Genetic Phenomena::Genetic Variation::Mutation::Sequence Deletion [PHENOMENA AND PROCESSES] ,Other subheadings::Other subheadings::/genetics [Other subheadings] ,Enzymes and Coenzymes::Enzymes::Hydrolases::Esterases::Endonucleases::Endoribonucleases::Ribonuclease H [CHEMICALS AND DRUGS] ,Enzimas y Coenzimas::Enzimas::Hidrolasas::Esterasas::Endonucleasas::Endorribonucleasas::Ribonucleasa H [COMPUESTOS QUÍMICOS Y DROGAS] ,Eye Diseases::Ocular Motility Disorders::Ophthalmoplegia [DISEASES] - Published
- 2021
16. Age-related metabolic changes limit efficacy of deoxynucleoside-based therapy in thymidine kinase 2-deficient mice
- Author
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[Blázquez-Bermejo C, Molina-Granada D, Vila-Julià F, Torres-Torronteras J, Martí R, Cámara Y] Grup de Recerca en Patologia Neuromuscular i Mitocondrial, Vall d'Hebron Institut de Recerca (VHIR), Universitat Autònoma de Barcelona (UAB), Barcelona, Spain. Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), Instituto de Salud Carlos III (ISCIII), Madrid, Spain. [Jiménez-Heis D] Grup de Recerca en Patologia Neuromuscular i Mitocondrial, Vall d'Hebron Institut de Recerca (VHIR), Universitat Autònoma de Barcelona (UAB), Barcelona, Spain. [Zhou X, Karlsson A] Division of Clinical Microbiology, Department of Laboratory Medicine, Karolinska Institute, Karolinska University Hospital, Stockholm, Sweden and Hospital Universitari Vall d'Hebron
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Fenómenos Fisiológicos::Crecimiento y Desarrollo::Envejecimiento::Longevidad [FENÓMENOS Y PROCESOS] ,Physiological Phenomena::Growth and Development::Aging::Longevity [PHENOMENA AND PROCESSES] ,Enzimas y Coenzimas::Enzimas::Transferasas::Fosfotransferasas::Fosfotransferasas (Aceptor de Grupo Alcohol)::Timidina Quinasa [COMPUESTOS QUÍMICOS Y DROGAS] ,Nucleic Acids, Nucleotides, and Nucleosides::Nucleic Acids::DNA::DNA, Circular::DNA, Mitochondrial [CHEMICALS AND DRUGS] ,nucleótidos y nucleósidos de ácidos nucleicos::ácidos nucleicos::ADN::ADN circular::ADN mitocondrial [COMPUESTOS QUÍMICOS Y DROGAS] ,Longevitat ,Enzymes and Coenzymes::Enzymes::Transferases::Phosphotransferases::Phosphotransferases (Alcohol Group Acceptor)::Thymidine Kinase [CHEMICALS AND DRUGS] ,ADN mitocondrial ,Timidina - Published
- 2021
17. Deoxynucleoside Therapy for Thymidine Kinase 2-Deficient Myopathy
- Author
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Francisco Javier Aguirre‐Rodríguez, Susana G. Kalko, Elena Martín-Hernández, Fabiola Mavillard, Michio Hirano, Javier Torres-Torronteras, Bruce Levin, Marcos Madruga-Garrido, Cecilia Jimenez-Mallebrera, Yuqi Tu, Juan P. Morealejo‐Aycinena, Yuelin Long, Karin Kleinsteuber, Ramon Martí, Itxaso Marti, Jasim Uddin, Olga Serrano, Caterina Garone, Concepcion Álvarez del Vayo, M. Alice Donati, Francina Munell, John L.P. Thompson, Carmen Paradas, Cristina Domínguez-González, Andrés Nascimento, M. Dolores Sardina, Kristen Engelstad, Dominguez-Gonzalez C., Madruga-Garrido M., Mavillard F., Garone C., Aguirre-Rodriguez F.J., Donati M.A., Kleinsteuber K., Marti I., Martin-Hernandez E., Morealejo-Aycinena J.P., Munell F., Nascimento A., Kalko S.G., Sardina M.D., Alvarez del Vayo C., Serrano O., Long Y., Tu Y., Levin B., Thompson J.L.P., Engelstad K., Uddin J., Torres-Torronteras J., Jimenez-Mallebrera C., Marti R., Paradas C., Hirano M., Instituto de Salud Carlos III, Generalitat de Catalunya, European Commission, Muscular Dystrophy Association (US), and Arturo Estopinan TK2 Research Fund
- Subjects
0301 basic medicine ,Adult ,Compassionate Use Trials ,Male ,medicine.medical_specialty ,Neurology ,Side effect ,medicine.medical_treatment ,Deoxyribonucleosides ,Walk Test ,Gastroenterology ,Thymidine Kinase ,Article ,03 medical and health sciences ,0302 clinical medicine ,Muscular Diseases ,Internal medicine ,tk2, myopathy, nucleosides, therapy ,medicine ,Humans ,Myopathy ,Child ,Feeding tube ,Mechanical ventilation ,business.industry ,Discontinuation ,Diarrhea ,030104 developmental biology ,Child, Preschool ,Female ,Neurology (clinical) ,GDF15 ,medicine.symptom ,business ,030217 neurology & neurosurgery - Abstract
[Objective] Thymidine kinase 2, encoded by the nuclear gene TK2, is required for mitochondrial DNA maintenance. Autosomal recessive TK2 mutations cause depletion and multiple deletions of mtDNA that manifest predominantly as a myopathy usually beginning in childhood and progressing relentlessly. We investigated the safety and efficacy of deoxynucleoside monophosphate and deoxynucleoside therapies., [Methods] We administered deoxynucleoside monophosphates and deoxynucleoside to 16 TK2‐deficient patients under a compassionate use program., [Results] In 5 patients with early onset and severe disease, survival and motor functions were better than historically untreated patients. In 11 childhood and adult onset patients, clinical measures stabilized or improved. Three of 8 patients who were nonambulatory at baseline gained the ability to walk on therapy; 4 of 5 patients who required enteric nutrition were able to discontinue feeding tube use; and 1 of 9 patients who required mechanical ventilation became able to breathe independently. In motor functional scales, improvements were observed in the 6‐minute walk test performance in 7 of 8 subjects, Egen Klassifikation in 2 of 3, and North Star Ambulatory Assessment in all 5 tested. Baseline elevated serum growth differentiation factor 15 levels decreased with treatment in all 7 patients tested. A side effect observed in 8 of the 16 patients was dose‐dependent diarrhea, which did not require withdrawal of treatment. Among 12 other TK2 patients treated with deoxynucleoside, 2 adults developed elevated liver enzymes that normalized following discontinuation of therapy., [Interpretation] This open‐label study indicates favorable side effect profiles and clinical efficacy of deoxynucleoside monophosphate and deoxynucleoside therapies for TK2 deficiency. ANN NEUROL 2019;86:293–303, This work was supported in part by grants from the Spanish Carlos III Health Institute (PMP15/00025 for C.P., F.Ma., and R.M.; PI16/00579 and CP09/00011 for C.J.‐M.), Muscular Dystrophy Association (577391), Arturo Estopinan TK2 Research Fund, Generalitat de Catalunya PERIS program (SLT002/16/00370 for J.T‐T.), and European Regional Development Fund.
- Published
- 2019
18. Identification and Characterization of New RNASEH1 Mutations Associated With PEO Syndrome and Multiple Mitochondrial DNA Deletions
- Author
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Lidia Carreño-Gago, Cora Blázquez-Bermejo, Jordi Díaz-Manera, Yolanda Cámara, Eduard Gallardo, Ramon Martí, Javier Torres-Torronteras, Elena García-Arumí, Institut Català de la Salut, [Carreño-Gago L, Blázquez-Bermejo C, Cámara Y, Martí R, Torres-Torronteras J] Departament de Patologia Mitocondrial i Neuromuscular, Vall d’Hebron Institut de Recerca (VHIR), Barcelona, Spain. Universitat Autònoma de Barcelona Barcelona, Spain. Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), Instituto de Salud Carlos III, Barcelona, Spain. [Díaz-Manera J, Gallardo E] Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), Instituto de Salud Carlos III, Barcelona, Spain. Servei de Neurologia, Malalties Neuromusculars, Hospital de la Santa Creu i Sant Pau, Barcelona, Spain. Institut de Recerca de HSCSP, Barcelona, Spain. Universitat Autònoma de Barcelona, Barcelona, Spain. [García-Arumí E] Departament de Patologia Mitocondrial i Neuromuscular, Vall d’Hebron Institut de Recerca (VHIR), Barcelona, Spain. Universitat Autònoma de Barcelona Barcelona, Spain. Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), Instituto de Salud Carlos III, Barcelona, Spain. Àrea de Genètica Clínica i Molecular, Hospital Universitari Vall d'Hebron, Barcelona, Spain, and Vall d'Hebron Barcelona Hospital Campus
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0301 basic medicine ,Mitochondrial DNA ,Ulls - Malalties - Aspectes genètics ,lcsh:QH426-470 ,Mitochondrial disease ,In silico ,medicine.disease_cause ,Eye Diseases::Ocular Motility Disorders::Ophthalmoplegia [DISEASES] ,enzimas y coenzimas::enzimas::hidrolasas::esterasas::endonucleasas::endorribonucleasas::ribonucleasa H [COMPUESTOS QUÍMICOS Y DROGAS] ,03 medical and health sciences ,0302 clinical medicine ,Ribonucleases ,Mitochondrial myopathy ,Nucleic Acids, Nucleotides, and Nucleosides::Nucleic Acids::DNA::DNA, Circular::DNA, Mitochondrial [CHEMICALS AND DRUGS] ,nucleótidos y nucleósidos de ácidos nucleicos::ácidos nucleicos::ADN::ADN circular::ADN mitocondrial [COMPUESTOS QUÍMICOS Y DROGAS] ,medicine ,Genetics ,Other subheadings::Other subheadings::/genetics [Other subheadings] ,Ribonuclease ,Gene ,Genetics (clinical) ,Original Research ,Mutation ,biology ,fenómenos genéticos::variación genética::mutación::deleción de secuencias [FENÓMENOS Y PROCESOS] ,mtDNA ,ADN mitocondrial - Malformacions ,Otros calificadores::Otros calificadores::/genética [Otros calificadores] ,Multiple mitochondrial DNA deletions ,multiple mtDNA deletions ,medicine.disease ,oftalmopatías::trastornos de la motilidad ocular::oftalmoplejía [ENFERMEDADES] ,lcsh:Genetics ,mitochondrial disease ,030104 developmental biology ,RNASEH1 ,030220 oncology & carcinogenesis ,PEO ,Genetic Phenomena::Genetic Variation::Mutation::Sequence Deletion [PHENOMENA AND PROCESSES] ,biology.protein ,Molecular Medicine ,Enzymes and Coenzymes::Enzymes::Hydrolases::Esterases::Endonucleases::Endoribonucleases::Ribonuclease H [CHEMICALS AND DRUGS] - Abstract
RNASEH1; Mitochondrial disease; MtDNA RNASEH1; Malaltia mitocondrial; ADN mitocondrial RNASEH1; Enfermedad mitocondrial; ADN mitocondrial Mitochondrial DNA (mtDNA) depletion and deletion syndrome encompasses a group of disorders caused by mutations in genes involved in mtDNA replication and maintenance. The clinical phenotype ranges from fatal infantile hepatocerebral forms to mild adult onset progressive external ophthalmoplegia (PEO). We report the case of a patient with PEO and multiple mtDNA deletions, with two new homozygous mutations in RNASEH1. The first mutation (c.487T>C) is located in the same catalytic domain as the four previously reported mutations, and the second (c.258_260del) is located in the connection domain, where no mutations have been reported. In silico study of the mutations predicted only the first mutation as pathogenic, but functional studies showed that both mutations cause loss of ribonuclease H1 activity. mtDNA replication dysfunction was demonstrated in patient fibroblasts, which were unable to recover normal mtDNA copy number after ethidium bromide-induced mtDNA depletion. Our results demonstrate the pathogenicity of two new RNASEH1 variants found in a patient with PEO syndrome, multiple deletions, and mild mitochondrial myopathy. This work was supported by the Spanish Instituto de Salud Carlos III, Fondo de Investigaciones Sanitarias and cofunded with ERDF funds (Grant No. FIS PI15/01428 to EG-A and FIS PI18/01574 to RM), the Spanish Ministry of Industry, Economy and Competitiveness (Grant No. SAF2017-87506-R to YC), and the Generalitat de Catalunya (a grant from the URDCat project PERIS to EG-A and RM). JT-T was funded by a fellowship granted by the Generalitat de Catalunya (PERIS program, SLT002/16/00370)
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- 2019
19. Age-related metabolic changes limit efficacy of deoxynucleoside-based therapy in thymidine kinase 2-deficient mice
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Anna Karlsson, Ramon Martí, Xiaoshan Zhou, Ferran Vila-Julià, David Molina-Granada, Javier Torres-Torronteras, Yolanda Cámara, Daniel Jiménez-Heis, Cora Blázquez-Bermejo, [Blázquez-Bermejo C, Molina-Granada D, Vila-Julià F, Torres-Torronteras J, Martí R, Cámara Y] Grup de Recerca en Patologia Neuromuscular i Mitocondrial, Vall d'Hebron Institut de Recerca (VHIR), Universitat Autònoma de Barcelona (UAB), Barcelona, Spain. Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), Instituto de Salud Carlos III (ISCIII), Madrid, Spain. [Jiménez-Heis D] Grup de Recerca en Patologia Neuromuscular i Mitocondrial, Vall d'Hebron Institut de Recerca (VHIR), Universitat Autònoma de Barcelona (UAB), Barcelona, Spain. [Zhou X, Karlsson A] Division of Clinical Microbiology, Department of Laboratory Medicine, Karolinska Institute, Karolinska University Hospital, Stockholm, Sweden, and Vall d'Hebron Barcelona Hospital Campus
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0301 basic medicine ,Mtdna, mitochondrial DNA ,dCK, deoxycytidine kinase ,Dtmp, deoxythymidine monophosphate ,Research paper ,Anabolism ,Dck, deoxycytidine kinase ,ADN mitocondrial ,Mitochondrion ,Deoxycytidine ,chemistry.chemical_compound ,dN, deoxynucleoside ,0302 clinical medicine ,nucleótidos y nucleósidos de ácidos nucleicos::ácidos nucleicos::ADN::ADN circular::ADN mitocondrial [COMPUESTOS QUÍMICOS Y DROGAS] ,Longevitat ,TK2 ,Dn, deoxynucleoside ,Deoxynucleoside therapy ,Mtdna depletion ,TK1, thymidine kinase 1 ,General Medicine ,Deoxycytidine kinase ,dCtd, deoxycytidine ,dThd, deoxythymidine ,030220 oncology & carcinogenesis ,Knockout mouse ,Enzymes and Coenzymes::Enzymes::Transferases::Phosphotransferases::Phosphotransferases (Alcohol Group Acceptor)::Thymidine Kinase [CHEMICALS AND DRUGS] ,medicine.medical_specialty ,Dnmp, deoxynucleoside monophosphate ,Tymp, thymidine phosphorylase gene ,Ndna, nuclear DNA ,Encephalomyopathy ,Timidina ,CNS, central nervous system ,General Biochemistry, Genetics and Molecular Biology ,TK2, thymidine kinase 2 ,03 medical and health sciences ,PBS, phosphate buffered saline ,Nucleic Acids, Nucleotides, and Nucleosides::Nucleic Acids::DNA::DNA, Circular::DNA, Mitochondrial [CHEMICALS AND DRUGS] ,Internal medicine ,medicine ,Dctd, deoxycytidine ,Dntp, deoxynucleoside triphosphate ,Thymidine phosphorylase ,Thymidine kinase 1 ,dTMP, deoxythymidine monophosphate ,Dthd, deoxythymidine ,Physiological Phenomena::Growth and Development::Aging::Longevity [PHENOMENA AND PROCESSES] ,dNMP, deoxynucleoside monophosphate ,KO, knockout ,Thy, thymine ,TP, thymidine phosphorylase ,Catabolism ,business.industry ,mtDNA depletion ,WT, wild-type ,mtDNA, mitochondrial DNA ,030104 developmental biology ,Endocrinology ,chemistry ,fenómenos fisiológicos::crecimiento y desarrollo::envejecimiento::longevidad [FENÓMENOS Y PROCESOS] ,enzimas y coenzimas::enzimas::transferasas::fosfotransferasas::fosfotransferasas (grupo alcohol aceptor)::timidina cinasa [COMPUESTOS QUÍMICOS Y DROGAS] ,dNTP, deoxynucleoside triphosphate ,Dcmp, deoxycytidine monophosphate ,nDNA, nuclear DNA ,Thymidine ,business ,dCMP, deoxycytidine monophosphate ,CDA, cytidine deaminase - Abstract
Deoxycytidine; Deoxynucleoside therapy; Encephalomyopathy Desoxicitidina; Terapia basada en desoxinucleósido; Encefalomiopatía Desoxicitidina; Teràpia basada en desoxinucleòsid; Encefalomiopatia BACKGROUND: Thymidine kinase 2 (TK2) catalyses the phosphorylation of deoxythymidine (dThd) and deoxycytidine (dCtd) within mitochondria. TK2 deficiency leads to mtDNA depletion or accumulation of multiple deletions. In patients, TK2 mutations typically manifest as a rapidly progressive myopathy with infantile onset, leading to respiratory insufficiency and encephalopathy in the most severe clinical presentations. TK2-deficient mice develop the most severe form of the disease and die at average postnatal day 16. dThd+dCtd administration delayed disease progression and expanded lifespan of a knockin murine model of the disease. METHODS: We daily administered TK2 knockout mice (Tk2KO) from postnatal day 4 with equimolar doses of dThd+dCtd, dTMP+dCMP, dThd alone or dCtd alone. We monitored body weight and survival and studied different variables at 12 or 29 days of age. We determined metabolite levels in plasma and target tissues, mtDNA copy number in tissues, and the expression and activities of enzymes with a relevant role in mitochondrial dNTP anabolism or catabolism. FINDINGS: dThd+dCtd treatment extended average lifespan of Tk2KO mice from 16 to 34 days, attenuated growth retardation, and rescued mtDNA depletion in skeletal muscle and other target tissues of 12-day-old mice, except in brain. However, the treatment was ineffective in 29-day-old mice that still died prematurely. Bioavailability of dThd and dCtd markedly decreased during mouse development. Activity of enzymes catabolizing dThd and dCtd increased with age in small intestine. Conversely, the activity of the anabolic enzymes decreased in target tissues during mouse development. We also found that administration of dThd alone had the same impact on survival to that of dThd+dCtd, whereas dCtd alone had no influence on lifespan. INTERPRETATION: dThd+dCtd treatment recruits alternative cytosolic salvage pathways for dNTP synthesis, suggesting that this therapy would be of benefit for any Tk2 mutation. dThd accounts for the therapeutic effect of the combined treatment in mice. During the first weeks after birth, mice experience marked tissue-specific metabolic regulations and ontogenetic changes in dNTP metabolism-related enzymes that limit therapeutic efficacy to early developmental stages. This study was funded by grants from the Spanish Ministry of Industry, Economy and Competitiveness [grant BFU2014-52618-R and SAF2017-87506-R to YC], the Spanish Instituto de Salud Carlos III [grant PI15/00465 and grant PMP 15/00025 to RM, co-financed with ERDF], the Fundacion Inocente, Inocente [grant 2017 to YC], and AFMTelethon [grant 19,965 to YC]. JT was funded by a fellowship granted by the Generalitat de Catalunya (PERIS program, SLT002/16/00370). The disclosed funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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- 2019
20. Mitochondrial Neurogastrointestinal Encephalomyopathy (MNGIE): Biochemical Features and Therapeutic Approaches
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Michio Hirano, Javier Torres-Torronteras, Maria Lucia Valentino, Ramon Martí, M. C. Lara, Lara, M.C., Valentino, M.L., Torres-Torronteras, J., Hirano, M., and Martí, R.
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TP ,Mitochondrial DNA ,Gastrointestinal Diseases ,Genetic enhancement ,Biophysics ,mitochondrial DNA ,Biology ,medicine.disease_cause ,DNA, Mitochondrial ,Biochemistry ,chemistry.chemical_compound ,Mitochondrial Encephalomyopathies ,medicine ,Humans ,Thymidine phosphorylase ,Molecular Biology ,Gene ,Thymidine Phosphorylase ,Mutation ,mitochondrial neurogastrointestinal encephalomyopathy ,mtDNA ,Cell Biology ,Molecular biology ,Deoxyuridine ,Human genetics ,Transplantation ,chemistry ,MNGIE ,Cancer research - Abstract
Over the last 15 years, important research has expanded our knowledge of the clinical, molecular genetic, and biochemical features of mitochondrial neurogastrointestinal encephalomyopathy (MNGIE). The characterization of mitochondrial involvement in this disorder and the seminal determination of its genetic cause, have opened new possibilities for more detailed and deeper studies on the pathomechanisms in this progressive and fatal disease. It has been established that MNGIE is caused by mutations in the gene encoding thymidine phosphorylase (TP), which lead to absolute or nearly complete loss of its catalytic activity, producing systemic accumulations of its substrates, thymidine (dThd) and deoxyuridine (dUrd). Findings obtained from in vitro and in vivo studies indicate that the biochemical imbalances specifically impair mitochondrial DNA (mtDNA) replication, repair, or both leading to mitochondrial dysfunction. We have proposed that therapy for MNGIE should be aimed at reducing the concentrations of these toxic nucleosides to normal or nearly normal levels. The first treatment, allogeneic stem-cell transplantation (alloSCT) reported in 2006, produced a nearly full biochemical correction of the dThd and dUrd imbalances in blood. Clinical follow-up of this and other patients receiving alloSCT is necessary to determine whether this and other therapies based on a permanent restoration of TP will be effective treatment for MNGIE. © 2007 The Biochemical Society.
- Published
- 2007
21. Gene Therapy Using a Liver-targeted AAV Vector Restores Nucleoside and Nucleotide Homeostasis in a Murine Model of MNGIE
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Alberto Auricchio, Michio Hirano, Jordi Barquinero, Yolanda Cámara, Ivano Di Meo, Raquel Cabrera-Pérez, Ramon Martí, Javier Torres-Torronteras, Massimo Zeviani, Carlo Viscomi, Giuseppe Pizzorno, Torres Torronteras, J, Viscomi, C, Cabrera P?rez, R, C?mara, Y, Di Meo, I, Barquinero, J, Auricchio, Alberto, Pizzorno, G, Hirano, M, Zeviani, M, and Mart?, R.
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Mitochondrial DNA ,Genetic enhancement ,Genetic Vectors ,Oculopharyngeal ,Biology ,medicine.disease_cause ,DNA, Mitochondrial ,chemistry.chemical_compound ,Mice ,Muscular Dystrophy, Oculopharyngeal ,Mitochondrial Encephalomyopathies ,Drug Discovery ,Genetics ,medicine ,Animals ,Dependovirus ,Disease Models, Animal ,Homeostasis ,Humans ,Intestinal Pseudo-Obstruction ,Liver ,Mutation ,Thymidine ,Thymidine Phosphorylase ,Genetic Therapy ,Muscular Dystrophy ,Thymidine phosphorylase ,Molecular Biology ,Pharmacology ,Ophthalmoplegia ,Deoxycytidine triphosphate ,Animal ,DNA ,Molecular biology ,Deoxyuridine ,3. Good health ,Mitochondrial ,chemistry ,Disease Models ,Molecular Medicine ,Original Article ,Nucleoside - Abstract
Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is an autosomal recessive disorder caused by mutations in TYMP , enconding thymidine phosphorylase (TP). TP deficiency results in systemic accumulation of thymidine and deoxyuridine, which interferes with mitochondrial DNA (mtDNA) replication and leads to mitochondrial dysfunction. To date, the only treatment available for MNGIE patients is allogeneic hematopoietic stem cell transplantation, which is associated with high morbidity and mortality. Here, we report that AAV2/8-mediated transfer of the human TYMP coding sequence (hcTYMP) under the control of a liver-specific promoter prevents the biochemical imbalances in a murine model of MNGIE. hcTYMP expression was restricted to liver, and a dose as low as 2 × 10 11 genome copies/kg led to a permanent reduction in systemic nucleoside levels to normal values in about 50% of treated mice. Higher doses resulted in reductions to normal or slightly below normal levels in virtually all mice treated. The nucleoside reduction achieved by this treatment prevented deoxycytidine triphosphate (dCTP) depletion, which is the limiting factor affecting mtDNA replication in this disease. These results demonstrate that the use of AAV to direct TYMP expression in liver is feasible as a potentially safe gene therapy strategy for MNGIE.
- Published
- 2014
22. Effect of Resveratrol Content in Red Wine on Circulating Sex Hormone-Binding Globulin: Lessons from a Pilot Clinical Trial.
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Briansó-Llort L, Simó-Servat O, Ramos-Perez L, Torres-Torronteras J, Hernandez C, Simó R, and Selva DM
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- Cholesterol, Female, Humans, Male, Pilot Projects, Resveratrol pharmacology, Sex Hormone-Binding Globulin, Cardiovascular Diseases prevention & control, Wine analysis
- Abstract
Scope: Low sex hormone-binding globulin (SHBG) levels are associated with higher risk of developing cardiovascular disease. Epidemiological studies have shown that red wine has beneficial effects on cardiovascular disease. In this work if resveratrol content in red wine increases SHBG levels is explored., Methods and Results: A pilot study aims at testing the effect of drinking for 14 days two types of red wine with different resveratrol content is conducted in 26 healthy volunteers. SHBG levels and several biochemical parameters are measured at the beginning and the end of every period. Results show that consumption of both wines does not change body mass index or biochemical markers of liver injury. The low resveratrol wine does not modify the lipid profile or SHBG levels. By contrast, red wine with high resveratrol content significantly reduces total cholesterol in both men and women. Finally, red wine with high resveratrol content increases circulating SHBG in women but not in men., Conclusions: Red wine rich in resveratrol reduces total cholesterol in men and women and increases SHBG only in women. Further research aims at investigating the potential SHBG role enhancement mediated by resveratrol regarding cardiovascular protection that presents women in comparison with men seems warranted., (© 2022 Wiley-VCH GmbH.)
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- 2022
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23. RRM1 variants cause a mitochondrial DNA maintenance disorder via impaired de novo nucleotide synthesis.
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Shintaku J, Pernice WM, Eyaid W, Gc JB, Brown ZP, Juanola-Falgarona M, Torres-Torronteras J, Sommerville EW, Hellebrekers DM, Blakely EL, Donaldson A, van de Laar I, Leu CS, Marti R, Frank J, Tanji K, Koolen DA, Rodenburg RJ, Chinnery PF, Smeets HJM, Gorman GS, Bonnen PE, Taylor RW, and Hirano M
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- DNA Replication, DNA, Mitochondrial genetics, DNA, Mitochondrial metabolism, Humans, Nucleosides, Nucleotides genetics, Ribonucleoside Diphosphate Reductase genetics, Ribonucleoside Diphosphate Reductase metabolism, Mitochondrial Diseases genetics, Ribonucleotide Reductases genetics, Ribonucleotide Reductases metabolism
- Abstract
Mitochondrial DNA (mtDNA) depletion/deletions syndromes (MDDS) encompass a clinically and etiologically heterogenous group of mitochondrial disorders caused by impaired mtDNA maintenance. Among the most frequent causes of MDDS are defects in nucleoside/nucleotide metabolism, which is critical for synthesis and homeostasis of the deoxynucleoside triphosphate (dNTP) substrates of mtDNA replication. A central enzyme for generating dNTPs is ribonucleotide reductase, a critical mediator of de novo nucleotide synthesis composed of catalytic RRM1 subunits in complex with RRM2 or p53R2. Here, we report 5 probands from 4 families who presented with ptosis and ophthalmoplegia as well as other clinical manifestations and multiple mtDNA deletions in muscle. We identified 3 RRM1 loss-of-function variants, including a dominant catalytic site variant (NP_001024.1: p.N427K) and 2 homozygous recessive variants at p.R381, which has evolutionarily conserved interactions with the specificity site. Atomistic molecular dynamics simulations indicate mechanisms by which RRM1 variants affect protein structure. Cultured primary skin fibroblasts of probands manifested mtDNA depletion under cycling conditions, indicating impaired de novo nucleotide synthesis. Fibroblasts also exhibited aberrant nucleoside diphosphate and dNTP pools and mtDNA ribonucleotide incorporation. Our data reveal that primary RRM1 deficiency and, by extension, impaired de novo nucleotide synthesis are causes of MDDS.
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- 2022
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24. Most mitochondrial dGTP is tightly bound to respiratory complex I through the NDUFA10 subunit.
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Molina-Granada D, González-Vioque E, Dibley MG, Cabrera-Pérez R, Vallbona-Garcia A, Torres-Torronteras J, Sazanov LA, Ryan MT, Cámara Y, and Martí R
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- Humans, HEK293 Cells, Mitochondria metabolism, Deoxyguanine Nucleotides metabolism, Electron Transport Complex I genetics, Electron Transport Complex I metabolism, NADH Dehydrogenase genetics, NADH Dehydrogenase metabolism
- Abstract
Imbalanced mitochondrial dNTP pools are known players in the pathogenesis of multiple human diseases. Here we show that, even under physiological conditions, dGTP is largely overrepresented among other dNTPs in mitochondria of mouse tissues and human cultured cells. In addition, a vast majority of mitochondrial dGTP is tightly bound to NDUFA10, an accessory subunit of complex I of the mitochondrial respiratory chain. NDUFA10 shares a deoxyribonucleoside kinase (dNK) domain with deoxyribonucleoside kinases in the nucleotide salvage pathway, though no specific function beyond stabilizing the complex I holoenzyme has been described for this subunit. We mutated the dNK domain of NDUFA10 in human HEK-293T cells while preserving complex I assembly and activity. The NDUFA10
E160A/R161A shows reduced dGTP binding capacity in vitro and leads to a 50% reduction in mitochondrial dGTP content, proving that most dGTP is directly bound to the dNK domain of NDUFA10. This interaction may represent a hitherto unknown mechanism regulating mitochondrial dNTP availability and linking oxidative metabolism to DNA maintenance., (© 2022. The Author(s).)- Published
- 2022
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25. Dysfunctional mitochondrial translation and combined oxidative phosphorylation deficiency in a mouse model of hepatoencephalopathy due to Gfm1 mutations.
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Molina-Berenguer M, Vila-Julià F, Pérez-Ramos S, Salcedo-Allende MT, Cámara Y, Torres-Torronteras J, and Martí R
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- Amino Acid Substitution, Animals, Disease Models, Animal, Electron Transport Complex IV genetics, Electron Transport Complex IV metabolism, Hepatic Encephalopathy genetics, Metabolism, Inborn Errors genetics, Mice, Mice, Knockout, Mitochondria, Liver genetics, Mitochondrial Proteins genetics, Peptide Elongation Factor G genetics, Hepatic Encephalopathy metabolism, Metabolism, Inborn Errors metabolism, Mitochondria, Liver metabolism, Mitochondrial Proteins metabolism, Mutation, Missense, Oxidative Phosphorylation, Peptide Elongation Factor G metabolism, Protein Biosynthesis
- Abstract
Hepatoencephalopathy due to combined oxidative phosphorylation deficiency type 1 (COXPD1) is a recessive mitochondrial translation disorder caused by mutations in GFM1, a nuclear gene encoding mitochondrial elongation factor G1 (EFG1). Patients with COXPD1 typically present hepatoencephalopathy early after birth with rapid disease progression, and usually die within the first few weeks or years of life. We have generated two different mouse models: a Gfm1 knock-in (KI) harboring the p.R671C missense mutation, found in at least 10 patients who survived more than 1 year, and a Gfm1 knock-out (KO) model. Homozygous KO mice (Gfm1
-/- ) were embryonically lethal, whereas homozygous KI (Gfm1R671C / R671C ) mice were viable and showed normal growth. R671C mutation in Gfm1 caused drastic reductions in the mitochondrial EFG1 protein content in different organs. Six- to eight-week-old Gfm1R671C / R671C mice showed partial reductions of in organello mitochondrial translation and respiratory complex IV enzyme activity in the liver. Compound heterozygous Gfm1R671C /- showed a more pronounced decrease of EFG1 protein in liver and brain mitochondria, as compared with Gfm1R671C / R671C mice. At 8 weeks of age, their mitochondrial translation rates were significantly reduced in both tissues. Additionally, Gfm1R671C /- mice showed combined oxidative phosphorylation deficiency (reduced complex I and IV enzyme activities in liver and brain), and blue native polyacrylamide gel electrophoresis analysis revealed lower amounts of both affected complexes. We conclude that the compound heterozygous Gfm1R671C /- mouse presents a clear dysfunctional molecular phenotype, showing impaired mitochondrial translation and combined respiratory chain dysfunction, making it a suitable animal model for the study of COXPD1., (© 2021 Federation of American Societies for Experimental Biology.)- Published
- 2022
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26. Therapy Prospects for Mitochondrial DNA Maintenance Disorders.
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Ramón J, Vila-Julià F, Molina-Granada D, Molina-Berenguer M, Melià MJ, García-Arumí E, Torres-Torronteras J, Cámara Y, and Martí R
- Subjects
- Animals, Combined Modality Therapy, DNA Replication, Disease Management, Disease Susceptibility, Gene Expression Regulation, Humans, Mitochondria metabolism, Mitochondrial Diseases diagnosis, Mitochondrial Diseases metabolism, Mitochondrial Proteins genetics, Mitochondrial Proteins metabolism, Mutation, DNA, Mitochondrial, Mitochondria genetics, Mitochondrial Diseases etiology, Mitochondrial Diseases therapy
- Abstract
Mitochondrial DNA depletion and multiple deletions syndromes (MDDS) constitute a group of mitochondrial diseases defined by dysfunctional mitochondrial DNA (mtDNA) replication and maintenance. As is the case for many other mitochondrial diseases, the options for the treatment of these disorders are rather limited today. Some aggressive treatments such as liver transplantation or allogeneic stem cell transplantation are among the few available options for patients with some forms of MDDS. However, in recent years, significant advances in our knowledge of the biochemical pathomechanisms accounting for dysfunctional mtDNA replication have been achieved, which has opened new prospects for the treatment of these often fatal diseases. Current strategies under investigation to treat MDDS range from small molecule substrate enhancement approaches to more complex treatments, such as lentiviral or adenoassociated vector-mediated gene therapy. Some of these experimental therapies have already reached the clinical phase with very promising results, however, they are hampered by the fact that these are all rare disorders and so the patient recruitment potential for clinical trials is very limited.
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- 2021
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27. Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE): Position paper on diagnosis, prognosis, and treatment by the MNGIE International Network.
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Hirano M, Carelli V, De Giorgio R, Pironi L, Accarino A, Cenacchi G, D'Alessandro R, Filosto M, Martí R, Nonino F, Pinna AD, Baldin E, Bax BE, Bolletta A, Bolletta R, Boschetti E, Cescon M, D'Angelo R, Dotti MT, Giordano C, Gramegna LL, Levene M, Lodi R, Mandel H, Morelli MC, Musumeci O, Pugliese A, Scarpelli M, Siniscalchi A, Spinazzola A, Tal G, Torres-Torronteras J, Vignatelli L, Zaidman I, Zoller H, Rinaldi R, and Zeviani M
- Subjects
- Consensus, DNA, Mitochondrial genetics, Gastrointestinal Diseases genetics, Gastrointestinal Diseases metabolism, Humans, International Cooperation, Mitochondrial Encephalomyopathies genetics, Mitochondrial Encephalomyopathies metabolism, Mutation, Thymidine Phosphorylase genetics, Thymidine Phosphorylase metabolism, Gastrointestinal Diseases diagnosis, Gastrointestinal Diseases therapy, Mitochondrial Encephalomyopathies diagnosis, Mitochondrial Encephalomyopathies therapy
- Abstract
Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is a rare autosomal recessive disease caused by TYMP mutations and thymidine phosphorylase (TP) deficiency. Thymidine and deoxyuridine accumulate impairing the mitochondrial DNA maintenance and integrity. Clinically, patients show severe and progressive gastrointestinal and neurological manifestations. The onset typically occurs in the second decade of life and mean age at death is 37 years. Signs and symptoms of MNGIE are heterogeneous and confirmatory diagnostic tests are not routinely performed by most laboratories, accounting for common misdiagnosis. Factors predictive of progression and appropriate tests for monitoring are still undefined. Several treatment options showed promising results in restoring the biochemical imbalance of MNGIE. The lack of controlled studies with appropriate follow-up accounts for the limited evidence informing diagnostic and therapeutic choices. The International Consensus Conference (ICC) on MNGIE, held in Bologna, Italy, on 30 March to 31 March 2019, aimed at an evidence-based consensus on diagnosis, prognosis, and treatment of MNGIE among experts, patients, caregivers and other stakeholders involved in caring the condition. The conference was conducted according to the National Institute of Health Consensus Conference methodology. A consensus development panel formulated a set of statements and proposed a research agenda. Specifically, the ICC produced recommendations on: (a) diagnostic pathway; (b) prognosis and the main predictors of disease progression; (c) efficacy and safety of treatments; and (f) research priorities on diagnosis, prognosis, and treatment. The Bologna ICC on diagnosis, management and treatment of MNGIE provided evidence-based guidance for clinicians incorporating patients' values and preferences., (© 2020 SSIEM.)
- Published
- 2021
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28. Efficacy of adeno-associated virus gene therapy in a MNGIE murine model enhanced by chronic exposure to nucleosides.
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Vila-Julià F, Cabrera-Pérez R, Cámara Y, Molina-Berenguer M, Lope-Piedrafita S, Hirano M, Mingozzi F, Torres-Torronteras J, and Martí R
- Subjects
- Animals, Combined Modality Therapy, Disease Models, Animal, Enzyme Activation, Gene Dosage, Gene Expression, Humans, Liver metabolism, Mice, Mice, Knockout, Mitochondrial Diseases genetics, Mitochondrial Diseases therapy, Ophthalmoplegia genetics, Ophthalmoplegia therapy, Phenotype, Thymidine Phosphorylase genetics, Treatment Outcome, Dependovirus genetics, Genetic Therapy methods, Genetic Vectors genetics, Intestinal Pseudo-Obstruction genetics, Intestinal Pseudo-Obstruction therapy, Muscular Dystrophy, Oculopharyngeal genetics, Muscular Dystrophy, Oculopharyngeal therapy, Nucleosides pharmacology, Ophthalmoplegia congenital
- Abstract
Background: Preclinical studies have shown that gene therapy is a feasible approach to treat mitochondrial neurogastrointestinal encephalomyopathy (MNGIE). However, the genetic murine model of the disease (Tymp/Upp1 double knockout, dKO) has a limited functional phenotype beyond the metabolic imbalances, and so the studies showing efficacy of gene therapy have relied almost exclusively on demonstrating correction of the biochemical phenotype. Chronic oral administration of thymidine (dThd) and deoxyuridine (dUrd) to dKO mice deteriorates the phenotype of the animals, providing a better model to test therapy approaches., Methods: dKO mice were treated with both dThd and dUrd in drinking water from weaning until the end of the study. At 8 - 11 weeks of age, mice were treated with several doses of adeno-associated virus (AAV) serotype 8 vector carrying the human TYMP coding sequence under the control of different liver-specific promoters (TBG, AAT, or HLP). The biochemical profile and functional phenotype were studied over the life of the animals., Findings: Nucleoside exposure resulted in 30-fold higher plasma nucleoside levels in dKO mice compared with non-exposed wild type mice. AAV-treatment provided elevated TP activity in liver and lowered systemic nucleoside levels in exposed dKO mice. Exposed dKO mice had enlarged brain ventricles (assessed by magnetic resonance imaging) and motor impairment (rotarod test); both were prevented by AAV treatment. Among all promoters tested, AAT showed the best efficacy., Interpretation: Our results show that AAV-mediated gene therapy restores the biochemical homeostasis in the murine model of MNGIE and, for the first time, demonstrate that this treatment improves the functional phenotype., Funding: This work was funded in part by the Spanish Instituto de Salud Carlos III, and the Generalitat de Catalunya. The disclosed funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript., Competing Interests: Declaration of Competing Interests RM, FV and MM report grants from the Instituto de Salud Carlos III during the conduct of the study; JT reports grants from Departament de Salut, Generalitat de Catalunya (PERIS program), during the conduct of the study; RM, MH, JT and YC report grants and non-financial support from Modis Therapeutics, personal fees and other from Modis Therapeutics, outside the submitted work; in addition, RM and MH have a patent “Deoxynucleoside therapy for diseases caused by unbalanced nucleotide pools including mitochondrial DNA depletion syndromes” (PCT/US16/038110) with royalties paid to Modis Therapeutics; MH reports grants and other support from Entrada Therapeutics, grants from Muscular Dystrophy Association and grants from NIH, outside the submitted work; FM is an employee of Spark Therapeutics; SL reports that the Nuclear Magnetic Resonance facility where she works charged the VHIR a fee for the MRI services; and RM, YC, JT and RC have a patent “Treatment of mitochondrial diseases” (PCT/EP2016/062636) with royalties paid to Modis Therapeutics., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)
- Published
- 2020
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29. Yeast Ppz1 protein phosphatase toxicity involves the alteration of multiple cellular targets.
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Velázquez D, Albacar M, Zhang C, Calafí C, López-Malo M, Torres-Torronteras J, Martí R, Kovalchuk SI, Pinson B, Jensen ON, Daignan-Fornier B, Casamayor A, and Ariño J
- Subjects
- Cell Cycle, DNA Damage, Phosphoprotein Phosphatases genetics, Phosphorylation, Reactive Oxygen Species, Saccharomyces cerevisiae genetics, Saccharomyces cerevisiae growth & development, Saccharomyces cerevisiae Proteins genetics, Gene Expression Regulation, Fungal, Metabolome, Phosphoprotein Phosphatases metabolism, Saccharomyces cerevisiae metabolism, Saccharomyces cerevisiae Proteins metabolism, Transcriptome
- Abstract
Control of the protein phosphorylation status is a major mechanism for regulation of cellular processes, and its alteration often lead to functional disorders. Ppz1, a protein phosphatase only found in fungi, is the most toxic protein when overexpressed in Saccharomyces cerevisiae. To investigate the molecular basis of this phenomenon, we carried out combined genome-wide transcriptomic and phosphoproteomic analyses. We have found that Ppz1 overexpression causes major changes in gene expression, affecting ~ 20% of the genome, together with oxidative stress and increase in total adenylate pools. Concurrently, we observe changes in the phosphorylation pattern of near 400 proteins (mainly dephosphorylated), including many proteins involved in mitotic cell cycle and bud emergence, rapid dephosphorylation of Snf1 and its downstream transcription factor Mig1, and phosphorylation of Hog1 and its downstream transcription factor Sko1. Deletion of HOG1 attenuates the growth defect of Ppz1-overexpressing cells, while that of SKO1 aggravates it. Our results demonstrate that Ppz1 overexpression has a widespread impact in the yeast cells and reveals new aspects of the regulation of the cell cycle.
- Published
- 2020
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30. Age-related metabolic changes limit efficacy of deoxynucleoside-based therapy in thymidine kinase 2-deficient mice.
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Blázquez-Bermejo C, Molina-Granada D, Vila-Julià F, Jiménez-Heis D, Zhou X, Torres-Torronteras J, Karlsson A, Martí R, and Cámara Y
- Subjects
- Age Factors, Animals, Biomarkers, Enzyme Activation, Gene Expression, Mice, Mice, Knockout, Mitochondria genetics, Mitochondria metabolism, Muscle, Skeletal metabolism, Thymidine Kinase genetics, Thymidine Kinase metabolism, Deoxyribonucleosides pharmacology, Energy Metabolism drug effects, Thymidine Kinase deficiency
- Abstract
Background: Thymidine kinase 2 (TK2) catalyses the phosphorylation of deoxythymidine (dThd) and deoxycytidine (dCtd) within mitochondria. TK2 deficiency leads to mtDNA depletion or accumulation of multiple deletions. In patients, TK2 mutations typically manifest as a rapidly progressive myopathy with infantile onset, leading to respiratory insufficiency and encephalopathy in the most severe clinical presentations. TK2-deficient mice develop the most severe form of the disease and die at average postnatal day 16. dThd+dCtd administration delayed disease progression and expanded lifespan of a knockin murine model of the disease., Methods: We daily administered TK2 knockout mice (Tk2
KO ) from postnatal day 4 with equimolar doses of dThd+dCtd, dTMP+dCMP, dThd alone or dCtd alone. We monitored body weight and survival and studied different variables at 12 or 29 days of age. We determined metabolite levels in plasma and target tissues, mtDNA copy number in tissues, and the expression and activities of enzymes with a relevant role in mitochondrial dNTP anabolism or catabolism., Findings: dThd+dCtd treatment extended average lifespan of Tk2KO mice from 16 to 34 days, attenuated growth retardation, and rescued mtDNA depletion in skeletal muscle and other target tissues of 12-day-old mice, except in brain. However, the treatment was ineffective in 29-day-old mice that still died prematurely. Bioavailability of dThd and dCtd markedly decreased during mouse development. Activity of enzymes catabolizing dThd and dCtd increased with age in small intestine. Conversely, the activity of the anabolic enzymes decreased in target tissues during mouse development. We also found that administration of dThd alone had the same impact on survival to that of dThd+dCtd, whereas dCtd alone had no influence on lifespan., Interpretation: dThd+dCtd treatment recruits alternative cytosolic salvage pathways for dNTP synthesis, suggesting that this therapy would be of benefit for any Tk2 mutation. dThd accounts for the therapeutic effect of the combined treatment in mice. During the first weeks after birth, mice experience marked tissue-specific metabolic regulations and ontogenetic changes in dNTP metabolism-related enzymes that limit therapeutic efficacy to early developmental stages. FUND: This study was funded by grants from the Spanish Ministry of Industry, Economy and Competitiveness, the Spanish Instituto de Salud Carlos III, the Fundación Inocente, Inocente, AFM Téléthon and the Generalitat de Catalunya. The disclosed funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript., (Copyright © 2019. Published by Elsevier B.V.)- Published
- 2019
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31. Alpha-1-Antitrypsin Promoter Improves the Efficacy of an Adeno-Associated Virus Vector for the Treatment of Mitochondrial Neurogastrointestinal Encephalomyopathy.
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Cabrera-Pérez R, Vila-Julià F, Hirano M, Mingozzi F, Torres-Torronteras J, and Martí R
- Subjects
- Animals, Disease Models, Animal, Gene Order, Genetic Vectors administration & dosage, Homeostasis, Liver metabolism, Liver pathology, Mice, Mice, Knockout, Mitochondria, Liver genetics, Mitochondria, Liver metabolism, Mitochondrial Encephalomyopathies metabolism, Thymidine Phosphorylase genetics, Transduction, Genetic, alpha 1-Antitrypsin metabolism, Dependovirus genetics, Gene Expression, Genetic Therapy methods, Genetic Vectors genetics, Mitochondrial Encephalomyopathies genetics, Mitochondrial Encephalomyopathies therapy, Promoter Regions, Genetic, alpha 1-Antitrypsin genetics
- Abstract
Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is a devastating disease caused by mutations in TYMP , which encodes thymidine phosphorylase (TP). In MNGIE patients, TP dysfunction results in systemic thymidine and deoxyuridine overload, which interferes with mitochondrial DNA replication. Preclinical studies have shown that gene therapy using a lentiviral vector targeted to hematopoietic stem cells or an adeno-associated virus (AAV) vector transcriptionally targeted to liver are feasible approaches to treat MNGIE. Here, we studied the effect of various promoters (thyroxine-binding globulin [TBG], phosphoglycerate kinase [PGK], hybrid liver-specific promoter [HLP], and alpha-1-antitrypsin [AAT]) and DNA configuration (single stranded or self complementary) on expression of the TYMP transgene in the AAV8 serotype in a murine model of MNGIE. All vectors restored liver TP activity and normalized nucleoside homeostasis in mice. However, the liver-specific promoters TBG, HLP, and AAT were more effective than the constitutive PGK promoter, and the self-complementary DNA configuration did not provide any therapeutic advantage over the single-stranded configuration. Among all constructs, only AAV-AAT was effective in all mice treated at the lowest dose (5 × 10
10 vector genomes/kg). As use of the AAT promoter will likely minimize the dose needed to achieve clinical efficacy as compared to the other promoters tested, we propose using the AAT promoter in the vector eventually designed for clinical use.- Published
- 2019
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32. Deoxynucleoside Therapy for Thymidine Kinase 2-Deficient Myopathy.
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Domínguez-González C, Madruga-Garrido M, Mavillard F, Garone C, Aguirre-Rodríguez FJ, Donati MA, Kleinsteuber K, Martí I, Martín-Hernández E, Morealejo-Aycinena JP, Munell F, Nascimento A, Kalko SG, Sardina MD, Álvarez Del Vayo C, Serrano O, Long Y, Tu Y, Levin B, Thompson JLP, Engelstad K, Uddin J, Torres-Torronteras J, Jimenez-Mallebrera C, Martí R, Paradas C, and Hirano M
- Subjects
- Adult, Child, Child, Preschool, Female, Humans, Male, Walk Test methods, Compassionate Use Trials methods, Deoxyribonucleosides therapeutic use, Muscular Diseases drug therapy, Muscular Diseases enzymology, Thymidine Kinase deficiency
- Abstract
Objective: Thymidine kinase 2, encoded by the nuclear gene TK2, is required for mitochondrial DNA maintenance. Autosomal recessive TK2 mutations cause depletion and multiple deletions of mtDNA that manifest predominantly as a myopathy usually beginning in childhood and progressing relentlessly. We investigated the safety and efficacy of deoxynucleoside monophosphate and deoxynucleoside therapies., Methods: We administered deoxynucleoside monophosphates and deoxynucleoside to 16 TK2-deficient patients under a compassionate use program., Results: In 5 patients with early onset and severe disease, survival and motor functions were better than historically untreated patients. In 11 childhood and adult onset patients, clinical measures stabilized or improved. Three of 8 patients who were nonambulatory at baseline gained the ability to walk on therapy; 4 of 5 patients who required enteric nutrition were able to discontinue feeding tube use; and 1 of 9 patients who required mechanical ventilation became able to breathe independently. In motor functional scales, improvements were observed in the 6-minute walk test performance in 7 of 8 subjects, Egen Klassifikation in 2 of 3, and North Star Ambulatory Assessment in all 5 tested. Baseline elevated serum growth differentiation factor 15 levels decreased with treatment in all 7 patients tested. A side effect observed in 8 of the 16 patients was dose-dependent diarrhea, which did not require withdrawal of treatment. Among 12 other TK2 patients treated with deoxynucleoside, 2 adults developed elevated liver enzymes that normalized following discontinuation of therapy., Interpretation: This open-label study indicates favorable side effect profiles and clinical efficacy of deoxynucleoside monophosphate and deoxynucleoside therapies for TK2 deficiency. ANN NEUROL 2019;86:293-303., (© 2019 American Neurological Association.)
- Published
- 2019
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33. Identification and Characterization of New RNASEH1 Mutations Associated With PEO Syndrome and Multiple Mitochondrial DNA Deletions.
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Carreño-Gago L, Blázquez-Bermejo C, Díaz-Manera J, Cámara Y, Gallardo E, Martí R, Torres-Torronteras J, and García-Arumí E
- Abstract
Mitochondrial DNA (mtDNA) depletion and deletion syndrome encompasses a group of disorders caused by mutations in genes involved in mtDNA replication and maintenance. The clinical phenotype ranges from fatal infantile hepatocerebral forms to mild adult onset progressive external ophthalmoplegia (PEO). We report the case of a patient with PEO and multiple mtDNA deletions, with two new homozygous mutations in RNASEH1 . The first mutation (c.487T>C) is located in the same catalytic domain as the four previously reported mutations, and the second (c.258_260del) is located in the connection domain, where no mutations have been reported. In silico study of the mutations predicted only the first mutation as pathogenic, but functional studies showed that both mutations cause loss of ribonuclease H1 activity. mtDNA replication dysfunction was demonstrated in patient fibroblasts, which were unable to recover normal mtDNA copy number after ethidium bromide-induced mtDNA depletion. Our results demonstrate the pathogenicity of two new RNASEH1 variants found in a patient with PEO syndrome, multiple deletions, and mild mitochondrial myopathy.
- Published
- 2019
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34. Increased dNTP pools rescue mtDNA depletion in human POLG-deficient fibroblasts.
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Blázquez-Bermejo C, Carreño-Gago L, Molina-Granada D, Aguirre J, Ramón J, Torres-Torronteras J, Cabrera-Pérez R, Martín MÁ, Domínguez-González C, de la Cruz X, Lombès A, García-Arumí E, Martí R, and Cámara Y
- Subjects
- Adenine analogs & derivatives, Adenine pharmacology, Adult, Catalytic Domain genetics, Cells, Cultured, DNA Polymerase gamma genetics, DNA Replication drug effects, DNA, Mitochondrial genetics, Deoxyribonucleotides metabolism, Ethidium pharmacology, Female, Fibroblasts drug effects, Genotype, Humans, Male, Mitochondria, Muscle genetics, Models, Molecular, Mutation, Missense, Phenotype, Point Mutation, Protein Conformation, Real-Time Polymerase Chain Reaction, Sequence Deletion, DNA Polymerase gamma deficiency, DNA, Mitochondrial metabolism, Deoxyribonucleotides pharmacology, Fibroblasts metabolism
- Abstract
Polymerase γ catalytic subunit ( POLG ) gene encodes the enzyme responsible for mitochondrial DNA (mtDNA) synthesis. Mutations affecting POLG are the most prevalent cause of mitochondrial disease because of defective mtDNA replication and lead to a wide spectrum of clinical phenotypes characterized by mtDNA deletions or depletion. Enhancing mitochondrial deoxyribonucleoside triphosphate (dNTP) synthesis effectively rescues mtDNA depletion in different models of defective mtDNA maintenance due to dNTP insufficiency. In this study, we studied mtDNA copy number recovery rates following ethidium bromide-forced depletion in quiescent fibroblasts from patients harboring mutations in different domains of POLG. Whereas control cells spontaneously recovered initial mtDNA levels, POLG-deficient cells experienced a more severe depletion and could not repopulate mtDNA. However, activation of deoxyribonucleoside (dN) salvage by supplementation with dNs plus erythro -9-(2-hydroxy-3-nonyl) adenine (inhibitor of deoxyadenosine degradation) led to increased mitochondrial dNTP pools and promoted mtDNA repopulation in all tested POLG -mutant cells independently of their specific genetic defect. The treatment did not compromise POLG fidelity because no increase in multiple deletions or point mutations was detected. Our study suggests that physiologic dNTP concentration limits the mtDNA replication rate. We thus propose that increasing mitochondrial dNTP availability could be of therapeutic interest for POLG deficiency and other conditions in which mtDNA maintenance is challenged.-Blázquez-Bermejo, C., Carreño-Gago, L., Molina-Granada, D., Aguirre, J., Ramón, J., Torres-Torronteras, J., Cabrera-Pérez, R., Martín, M. Á., Domínguez-González, C., de la Cruz, X., Lombès, A., García-Arumí, E., Martí, R., Cámara, Y. Increased dNTP pools rescue mtDNA depletion in human POLG-deficient fibroblasts.
- Published
- 2019
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35. Adipocyte MTERF4 regulates non-shivering adaptive thermogenesis and sympathetic-dependent glucose homeostasis.
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Castillo A, Vilà M, Pedriza I, Pardo R, Cámara Y, Martín E, Beiroa D, Torres-Torronteras J, Oteo M, Morcillo MA, Martí R, Simó R, Nogueiras R, and Villena JA
- Subjects
- Adipocytes drug effects, Adipocytes metabolism, Adipocytes pathology, Adipose Tissue, Brown drug effects, Adipose Tissue, Brown pathology, Adrenergic beta-Agonists pharmacology, Animals, Cold Temperature, Electron Transport Complex I genetics, Electron Transport Complex I metabolism, Electron Transport Complex III genetics, Electron Transport Complex III metabolism, Electron Transport Complex IV genetics, Electron Transport Complex IV metabolism, Gene Expression Regulation, Homeostasis genetics, Humans, Insulin metabolism, Insulin pharmacology, Insulin Resistance, Male, Mice, Mice, Knockout, Mitochondria drug effects, Mitochondria pathology, Mitochondrial Proteins deficiency, Organelle Biogenesis, Oxidative Phosphorylation drug effects, Signal Transduction, Transcription Factors deficiency, Adipose Tissue, Brown metabolism, Glucose metabolism, Mitochondria metabolism, Mitochondrial Proteins genetics, Thermogenesis genetics, Transcription Factors genetics
- Abstract
In humans, low brown adipose tissue (BAT) mass and activity have been associated with increased adiposity and fasting glucose levels, suggesting that defective BAT-dependent thermogenesis could contribute to the development of obesity and/or type 2 diabetes. The thermogenic function of BAT relies on a vast network of mitochondria exclusively equipped with UCP1. Mitochondrial biogenesis is exquisitely regulated by a well-defined network of transcription factors that coordinate the expression of nuclear genes required for the formation of functional mitochondria. However, less is known about the mitochondrial factors that control the expression of the genes encoded by the mitochondrial genome. Here, we have studied the role of mitochondrial transcription termination factor-4 (MTERF4) in BAT by using a new mouse model devoid of MTERF4 specifically in adipocytes (MTERF4-FAT-KO mice). Lack of MTERF4 in BAT leads to reduced OxPhos mitochondrial protein levels and impaired assembly of OxPhos complexes I, III and IV due to deficient translation of mtDNA-encoded proteins. As a result, brown adipocytes lacking MTERF4 exhibit impaired respiratory capacity. MTERF4-FAT-KO mice show a blunted thermogenic response and are unable to maintain body temperature when exposed to cold. Despite impaired BAT function, MTERF4-FAT-KO mice do not develop obesity or insulin resistance. Still, MTERF4-FAT-KO mice became resistant to the insulin-sensitizing effects of β
3 -specific adrenergic receptor agonists. Our results demonstrate that MTERF4 regulates mitochondrial protein translation and is essential for proper BAT thermogenic activity. Our study also supports the notion that pharmacological activation of BAT is a plausible therapeutic target for the treatment of insulin resistance., (Copyright © 2019 Elsevier B.V. All rights reserved.)- Published
- 2019
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36. Long-Term Sustained Effect of Liver-Targeted Adeno-Associated Virus Gene Therapy for Mitochondrial Neurogastrointestinal Encephalomyopathy.
- Author
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Torres-Torronteras J, Cabrera-Pérez R, Vila-Julià F, Viscomi C, Cámara Y, Hirano M, Zeviani M, and Martí R
- Subjects
- Animals, Carcinogenesis pathology, Deoxyuridine blood, Female, Gene Dosage, Genetic Vectors metabolism, Intestinal Pseudo-Obstruction blood, Kaplan-Meier Estimate, Male, Mice, Mitochondria, Liver metabolism, Muscular Dystrophy, Oculopharyngeal blood, Ophthalmoplegia congenital, Thymidine blood, Thymidine Phosphorylase genetics, Time Factors, Transgenes, Dependovirus genetics, Genetic Therapy, Intestinal Pseudo-Obstruction genetics, Intestinal Pseudo-Obstruction therapy, Liver pathology, Muscular Dystrophy, Oculopharyngeal genetics, Muscular Dystrophy, Oculopharyngeal therapy
- Abstract
Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is caused by mutations in TYMP, the gene encoding the enzyme thymidine phosphorylase (TP). TP dysfunction results in systemic accumulation of the noxious TP substrates thymidine and deoxyuridine. Gene therapy using either a lentiviral vector or adeno-associated vector (AAV) has proven to be a feasible strategy, as both vectors restore biochemical homeostasis in a murine model of the disease. This study shows that the effect of an AAV containing the TYMP coding sequence transcriptionally targeted to the liver persists long term in mice. Although the vector copy number was diluted and AAV-mediated liver TP activity eventually reduced or lost after 21 months at the lowest vector doses, the effect was sustained (with a negligible decrease in TP activity) and fully effective on nucleoside homeostasis for at least 21 months at a dose of 2 × 10
12 vg/kg. Macroscopic visual inspection of the animals' organs at completion of the study showed no adverse effects associated with the treatment. These results further support the feasibility of gene therapy for MNGIE.- Published
- 2018
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37. Preclinical Efficacy and Safety Evaluation of Hematopoietic Stem Cell Gene Therapy in a Mouse Model of MNGIE.
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Yadak R, Cabrera-Pérez R, Torres-Torronteras J, Bugiani M, Haeck JC, Huston MW, Bogaerts E, Goffart S, Jacobs EH, Stok M, Leonardelli L, Biasco L, Verdijk RM, Bernsen MR, Ruijter G, Martí R, Wagemaker G, van Til NP, and de Coo IFM
- Abstract
Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is an autosomal recessive disorder caused by thymidine phosphorylase (TP) deficiency resulting in systemic accumulation of thymidine (d-Thd) and deoxyuridine (d-Urd) and characterized by early-onset neurological and gastrointestinal symptoms. Long-term effective and safe treatment is not available. Allogeneic bone marrow transplantation may improve clinical manifestations but carries disease and transplant-related risks. In this study, lentiviral vector-based hematopoietic stem cell gene therapy (HSCGT) was performed in Tymp
-/- Upp1-/- mice with the human phosphoglycerate kinase (PGK) promoter driving TYMP . Supranormal blood TP activity reduced intestinal nucleoside levels significantly at low vector copy number (median, 1.3; range, 0.2-3.6). Furthermore, we covered two major issues not addressed before. First, we demonstrate aberrant morphology of brain astrocytes in areas of spongy degeneration, which was reversed by HSCGT. Second, long-term follow-up and vector integration site analysis were performed to assess safety of the therapeutic LV vectors in depth. This report confirms and supplements previous work on the efficacy of HSCGT in reducing the toxic metabolites in Tymp-/- Upp1-/- mice, using a clinically applicable gene transfer vector and a highly efficient gene transfer method, and importantly demonstrates phenotypic correction with a favorable risk profile, warranting further development toward clinical implementation.- Published
- 2018
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38. Polyphosphate is a key factor for cell survival after DNA damage in eukaryotic cells.
- Author
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Bru S, Samper-Martín B, Quandt E, Hernández-Ortega S, Martínez-Laínez JM, Garí E, Rafel M, Torres-Torronteras J, Martí R, Ribeiro MPC, Jiménez J, and Clotet J
- Subjects
- Deoxyribonucleotides metabolism, HEK293 Cells, Humans, Saccharomyces cerevisiae genetics, Saccharomyces cerevisiae metabolism, Saccharomyces cerevisiae physiology, Cell Survival, DNA metabolism, DNA Damage, DNA Repair, Polyphosphates metabolism
- Abstract
Cells require extra amounts of dNTPs to repair DNA after damage. Polyphosphate (polyP) is an evolutionary conserved linear polymer of up to several hundred inorganic phosphate (Pi) residues that is involved in many functions, including Pi storage. In the present article, we report on findings demonstrating that polyP functions as a source of Pi when required to sustain the dNTP increment essential for DNA repair after damage. We show that mutant yeast cells without polyP produce less dNTPs upon DNA damage and that their survival is compromised. In contrast, when polyP levels are ectopically increased, yeast cells become more resistant to DNA damage. More importantly, we show that when polyP is reduced in HEK293 mammalian cell line cells and in human dermal primary fibroblasts (HDFa), these cells become more sensitive to DNA damage, suggesting that the protective role of polyP against DNA damage is evolutionary conserved. In conclusion, we present polyP as a molecule involved in resistance to DNA damage and suggest that polyP may be a putative target for new approaches in cancer treatment or prevention., (Copyright © 2017 Elsevier B.V. All rights reserved.)
- Published
- 2017
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39. SAMHD1 is active in cycling cells permissive to HIV-1 infection.
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Badia R, Pujantell M, Torres-Torronteras J, Menéndez-Arias L, Martí R, Ruzo A, Pauls E, Clotet B, Ballana E, Esté JA, and Riveira-Muñoz E
- Subjects
- Cell Line, Clustered Regularly Interspaced Short Palindromic Repeats, DNA Replication drug effects, Gene Editing, Gene Expression, Gene Knockdown Techniques, HEK293 Cells, HIV Infections metabolism, HIV-1 pathogenicity, HIV-2 drug effects, Host-Pathogen Interactions, Humans, Phosphorylation, Reverse Transcription drug effects, SAM Domain and HD Domain-Containing Protein 1 genetics, SAM Domain and HD Domain-Containing Protein 1 metabolism, Viral Regulatory and Accessory Proteins drug effects, Zidovudine pharmacology, HIV-1 drug effects, SAM Domain and HD Domain-Containing Protein 1 pharmacology, Virus Replication drug effects
- Abstract
SAMHD1 is a triphosphohydrolase that restricts HIV-1 by limiting the intracellular dNTP pool required for reverse transcription. Although SAMHD1 is expressed and active/unphosphorylated in most cell lines, its restriction activity is thought to be relevant only in non-cycling cells. However, an in depth evaluation of SAMHD1 function and relevance in cycling cells is required. Here, we show that SAMHD1-induced degradation by HIV-2 Vpx affects the dNTP pool and HIV-1 replication capacity in the presence of the 3'-azido-3'-deoxythymidine (AZT) in cycling cells. Similarly, in SAMHD1 knockout cells, HIV-1 showed increased replicative capacity in the presence of nucleoside inhibitors, especially AZT, that was reverted by re-expression of wild type SAMHD1. Sensitivity to non-nucleoside inhibitors (nevirapine and efavirenz) or the integrase inhibitor raltegravir was not affected by SAMHD1. Combination of three mutations (S18A, T21A, T25A) significantly prevented SAMHD1 phosphorylation but did not significantly affect HIV-1 replication in the presence of AZT. Our results demonstrate that SAMHD1 is active in HIV-1 permissive cells, does not modify susceptibility to HIV-1 infection but strongly affects sensitivity to nucleoside inhibitors., (Copyright © 2017 Elsevier B.V. All rights reserved.)
- Published
- 2017
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40. Long-Term Restoration of Thymidine Phosphorylase Function and Nucleoside Homeostasis Using Hematopoietic Gene Therapy in a Murine Model of Mitochondrial Neurogastrointestinal Encephalomyopathy.
- Author
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Torres-Torronteras J, Cabrera-Pérez R, Barba I, Costa C, de Luna N, Andreu AL, Barquinero J, Hirano M, Cámara Y, and Martí R
- Subjects
- Animals, Combined Modality Therapy, Disease Models, Animal, Female, Homeostasis, Intestinal Pseudo-Obstruction genetics, Male, Mice, Mice, Inbred C57BL, Mice, Knockout, Mitochondrial Encephalomyopathies genetics, Muscular Dystrophy, Oculopharyngeal, Ophthalmoplegia congenital, Genetic Therapy, Genetic Vectors administration & dosage, Hematopoietic Stem Cell Transplantation, Intestinal Pseudo-Obstruction therapy, Lentivirus genetics, Mitochondrial Encephalomyopathies therapy, Nucleosides metabolism, Thymidine Phosphorylase genetics
- Abstract
Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is a metabolic disorder caused by mutations in TYMP, encoding thymidine phosphorylase (TP). In MNGIE patients, TP dysfunction produces systemic thymidine and deoxyuridine accumulation, which ultimately impairs mitochondrial DNA replication and results in mitochondrial dysfunction. To date, only allogeneic hematopoietic stem cell transplantation has demonstrated long-term clinical efficacy, but high morbidity and mortality associated with this procedure necessitate the search for safer alternatives. In a previous study, we demonstrated that hematopoietic stem cell gene therapy using a lentiviral vector containing the coding sequence of TYMP restored the biochemical homeostasis in an animal model of MNGIE. In the present follow-up study, we show that ectopic expression of TP in the hematopoietic system restores normal nucleoside levels in plasma, as well as in tissues affected in MNGIE such as small intestine, skeletal muscle, brain, and liver. Mitochondrial dNTP pool imbalances observed in liver of the animal model were also corrected by the treatment. The biochemical effects were maintained at least 20 months even with low levels of chimerism. No alterations in the blood cell counts or other toxic effects were observed in association with the lentiviral transduction or TP overexpression. These results further support the notion that gene therapy is a feasible treatment option for MNGIE., Competing Interests: Author Disclosure No competing financial interests exist.
- Published
- 2016
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41. The G1/S Specific Cyclin D2 Is a Regulator of HIV-1 Restriction in Non-proliferating Cells.
- Author
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Badia R, Pujantell M, Riveira-Muñoz E, Puig T, Torres-Torronteras J, Martí R, Clotet B, Ampudia RM, Vives-Pi M, Esté JA, and Ballana E
- Subjects
- Animals, Cell Proliferation, Cyclin-Dependent Kinase 4 immunology, Cyclin-Dependent Kinase Inhibitor p21 immunology, Granulocyte-Macrophage Colony-Stimulating Factor immunology, Humans, Macrophages virology, Mice, Monomeric GTP-Binding Proteins immunology, SAM Domain and HD Domain-Containing Protein 1, Cyclin D2 immunology, HIV Infections immunology, HIV-1 immunology, Macrophages immunology
- Abstract
Macrophages are a heterogeneous cell population strongly influenced by differentiation stimuli that become susceptible to HIV-1 infection after inactivation of the restriction factor SAMHD1 by cyclin-dependent kinases (CDK). Here, we have used primary human monocyte-derived macrophages differentiated through different stimuli to evaluate macrophage heterogeneity on cell activation and proliferation and susceptibility to HIV-1 infection. Stimulation of monocytes with GM-CSF induces a non-proliferating macrophage population highly restrictive to HIV-1 infection, characterized by the upregulation of the G1/S-specific cyclin D2, known to control early steps of cell cycle progression. Knockdown of cyclin D2, enhances HIV-1 replication in GM-CSF macrophages through inactivation of SAMHD1 restriction factor by phosphorylation. Co-immunoprecipitation experiments show that cyclin D2 forms a complex with CDK4 and p21, a factor known to restrict HIV-1 replication by affecting the function of the downstream cascade that leads to SAMHD1 deactivation. Thus, we demonstrate that cyclin D2 acts as regulator of cell cycle proteins affecting SAMHD1-mediated HIV-1 restriction in non-proliferating macrophages.
- Published
- 2016
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42. Polyphosphate is involved in cell cycle progression and genomic stability in Saccharomyces cerevisiae.
- Author
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Bru S, Martínez-Laínez JM, Hernández-Ortega S, Quandt E, Torres-Torronteras J, Martí R, Canadell D, Ariño J, Sharma S, Jiménez J, and Clotet J
- Subjects
- Cell Cycle Checkpoints physiology, Cell Division physiology, Organelles metabolism, Prokaryotic Cells metabolism, Saccharomyces cerevisiae cytology, Saccharomyces cerevisiae genetics, Genomic Instability, Polyphosphates metabolism, Saccharomyces cerevisiae metabolism
- Abstract
Polyphosphate (polyP) is a linear chain of up to hundreds of inorganic phosphate residues that is necessary for many physiological functions in all living organisms. In some bacteria, polyP supplies material to molecules such as DNA, thus playing an important role in biosynthetic processes in prokaryotes. In the present study, we set out to gain further insight into the role of polyP in eukaryotic cells. We observed that polyP amounts are cyclically regulated in Saccharomyces cerevisiae, and those mutants that cannot synthesise (vtc4Δ) or hydrolyse polyP (ppn1Δ, ppx1Δ) present impaired cell cycle progression. Further analysis revealed that polyP mutants show delayed nucleotide production and increased genomic instability. Based on these findings, we concluded that polyP not only maintains intracellular phosphate concentrations in response to fluctuations in extracellular phosphate levels, but also muffles internal cyclic phosphate fluctuations, such as those produced by the sudden demand of phosphate to synthetize deoxynucleotides just before and during DNA duplication. We propose that the presence of polyP in eukaryotic cells is required for the timely and accurate duplication of DNA., (© 2016 John Wiley & Sons Ltd.)
- Published
- 2016
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43. Inhibition of herpes simplex virus type 1 by the CDK6 inhibitor PD-0332991 (palbociclib) through the control of SAMHD1.
- Author
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Badia R, Angulo G, Riveira-Muñoz E, Pujantell M, Puig T, Ramirez C, Torres-Torronteras J, Martí R, Pauls E, Clotet B, Ballana E, and Esté JA
- Subjects
- Animals, Cells, Cultured, Herpesvirus 1, Human drug effects, Humans, SAM Domain and HD Domain-Containing Protein 1, Cyclin-Dependent Kinase 6 antagonists & inhibitors, Herpesvirus 1, Human physiology, Macrophages virology, Monomeric GTP-Binding Proteins metabolism, Piperazines pharmacology, Pyridines pharmacology, Virus Replication drug effects
- Abstract
Objectives: Sterile α motif and histidine-aspartate domain-containing protein 1 (SAMHD1) has been shown to restrict retroviruses and DNA viruses by decreasing the pool of intracellular deoxynucleotides. In turn, SAMHD1 is controlled by cyclin-dependent kinases (CDK) that regulate the cell cycle and cell proliferation. Here, we explore the effect of CDK6 inhibitors on the replication of herpes simplex virus type 1 (HSV-1) in primary monocyte-derived macrophages (MDM)., Methods: MDM were treated with palbociclib, a selective CDK4/6 inhibitor, and then infected with a GFP-expressing HSV-1. Intracellular deoxynucleotide triphosphate (dNTP) content was determined using a polymerase-based method., Results: CDK6 inhibitor palbociclib blocked SAMHD1 phosphorylation, intracellular dNTP levels and HSV-1 replication in MDM at subtoxic concentrations. Treatment of MDM with palbociclib reduced CDK2 activation, measured as the phosphorylation of the T-loop at Thr160. The antiviral activity of palbociclib was lost when SAMHD1 was degraded by viral protein X. Similarly, palbociclib did not block HSV-1 replication in SAMHD1-negative Vero cells at subtoxic concentrations, providing further evidence for a role of SAMHD1 in mediating the antiviral effect., Conclusions: SAMHD1-mediated HSV-1 restriction is controlled by CDK and points to a preferential role for CDK6 and CDK2 as mediators of SAMHD1 activation. Similarly, the restricting activity of SAMHD1 against DNA viruses suggests that control of dNTP availability is the major determinant of its antiviral activity. This is the first study describing the anti-HSV-1 activity of palbociclib., (© The Author 2015. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)
- Published
- 2016
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44. Cyclin D3-dependent control of the dNTP pool and HIV-1 replication in human macrophages.
- Author
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Ruiz A, Pauls E, Badia R, Torres-Torronteras J, Riveira-Muñoz E, Clotet B, Martí R, Ballana E, and Esté JA
- Subjects
- Cyclin D3 genetics, Gene Knockdown Techniques, Humans, Macrophages virology, Monomeric GTP-Binding Proteins metabolism, Phosphorylation, RNA Interference, SAM Domain and HD Domain-Containing Protein 1, Cyclin D3 metabolism, Cyclin-Dependent Kinase 6 metabolism, Deoxyribonucleotides metabolism, HIV-1 physiology, Macrophages physiology, Models, Biological, Virus Replication physiology
- Abstract
Cyclins control the activation of cyclin-dependent kinases (CDK), which in turn, control the cell cycle and cell division. Intracellular availability of deoxynucleotides (dNTP) plays a fundamental role in cell cycle progression. SAM domain and HD domain-containing protein 1 (SAMHD1) degrades nucleotide triphosphates and controls the size of the dNTP pool. SAMHD1 activity appears to be controlled by CDK. Here, we show that knockdown of cyclin D3 a partner of CDK6 and E2 a partner of CDK2 had a major impact in SAMHD1 phosphorylation and inactivation and led to decreased dNTP levels and inhibition of HIV-1 at the reverse transcription step in primary human macrophages. The effect of cyclin D3 RNA interference was lost after degradation of SAMHD1 by HIV-2 Vpx, demonstrating the specificity of the mechanism. Cyclin D3 inhibition correlated with decreased activation of CDK2. Our results confirm the fundamental role of the CDK6-cyclin D3 pair in controlling CDK2-dependent SAMHD1 phosphorylation and dNTP pool in primary macrophages.
- Published
- 2015
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45. Downregulation of duodenal SLC transporters and activation of proinflammatory signaling constitute the early response to high altitude in humans.
- Author
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Wojtal KA, Cee A, Lang S, Götze O, Frühauf H, Geier A, Pastor-Anglada M, Torres-Torronteras J, Martí R, Fried M, Lutz TA, Maggiorini M, Gassmann M, Rogler G, and Vavricka SR
- Subjects
- Biomarkers blood, Cell Hypoxia, Cell Line, Cytokines genetics, Down-Regulation, Duodenum physiopathology, Humans, Hypoxia blood, Hypoxia genetics, Hypoxia physiopathology, Intestinal Absorption, Intestinal Mucosa metabolism, Intestinal Mucosa physiopathology, Membrane Transport Proteins genetics, Oxygen metabolism, RNA, Messenger metabolism, Time Factors, Up-Regulation, Acclimatization, Altitude, Cytokines blood, Duodenum metabolism, Hypoxia metabolism, Inflammation Mediators blood, Membrane Transport Proteins metabolism, Signal Transduction
- Abstract
Solute carrier (SLC) transporters mediate the uptake of biologically active compounds in the intestine. Reduced oxygenation (hypoxia) is an important factor influencing intestinal homeostasis. The aim of this study was to investigate the pathophysiological consequences of hypoxia on the expression and function of SLCs in human intestine. Hypoxia was induced in human intestinal epithelial cells (IECs) in vitro (0.2; 1% O2 or CoCl2). For human in vivo studies, duodenal biopsies and serum samples were obtained from individuals (n = 16) acutely exposed to 4,554 meters above sea levels. Expression of relevant targets was analyzed by quantitative PCR, Western blotting, or immunofluorescence. Serum levels of inflammatory mediators and nucleosides were determined by ELISA and LC/MS-MS, respectively. In the duodenum of volunteers exposed to high altitude we observed decreased mRNA levels of apical sodium-dependent bile acid transporter (ASBT), concentrative nucleoside transporters 1/2 (CNT1/2), organic anion transporting polypeptide 2B1 (OATP2B1), organic cation transporter 2 (OCTN2), peptide transporter 1 (PEPT1), serotonin transporter (SERT), and higher levels of IFN-γ, IL-6, and IL-17A. Serum levels of IL-10, IFN-γ, matrix metalloproteinase-2 (MMP-2), and serotonin were elevated, whereas the levels of uridine decreased upon exposure to hypoxia. Hypoxic IECs showed reduced levels of equilibrative nucleoside transporter 2 (ENT2), OCTN2, and SERT mRNAs in vitro, which was confirmed on the protein level and was accompanied by activation of ERK1/2, increase of hypoxia-inducible factor (HIF) proteins, and production of IL-8 mRNA. Costimulation with IFN-γ and IL-6 during hypoxia further decreased the expression of SERT, ENT2, and CNT2 in vitro. Reduced oxygen supply affects the expression pattern of duodenal SLCs that is accompanied by changes in serum levels of proinflammatory cytokines and biologically active compounds demonstrating that intestinal transport is affected during systemic exposure to hypoxia in humans., (Copyright © 2014 the American Physiological Society.)
- Published
- 2014
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46. Palbociclib, a selective inhibitor of cyclin-dependent kinase4/6, blocks HIV-1 reverse transcription through the control of sterile α motif and HD domain-containing protein-1 (SAMHD1) activity.
- Author
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Pauls E, Badia R, Torres-Torronteras J, Ruiz A, Permanyer M, Riveira-Muñoz E, Clotet B, Marti R, Ballana E, and Esté JA
- Subjects
- Cells, Cultured, Cyclin-Dependent Kinase 2 metabolism, Cyclin-Dependent Kinase 6 metabolism, DNA, Viral analysis, Humans, Leukocytes, Mononuclear virology, Real-Time Polymerase Chain Reaction, SAM Domain and HD Domain-Containing Protein 1, Anti-HIV Agents metabolism, HIV-1 physiology, Monomeric GTP-Binding Proteins antagonists & inhibitors, Piperazines metabolism, Protein Kinase Inhibitors metabolism, Pyridines metabolism, Reverse Transcription
- Abstract
Background: Sterile α motif and HD domain-containing protein-1 (SAMHD1) inhibits HIV-1 reverse transcription by decreasing the pool of intracellular deoxynucleotides. SAMHD1 is controlled by cyclin-dependent kinase (CDK)-mediated phosphorylation. However, the exact mechanism of SAMHD1 regulation in primary cells is unclear. We explore the effect of palbociclib, a CDK6 inhibitor, in HIV-1 replication., Methods: Human primary monocytes were differentiated into macrophages with monocyte-colony stimulating factor and CD4 T lymphocytes stimulated with phytohaemagglutinin (PHA)/interleukin-2. Cells were treated with palbociclib and then infected with a Green fluorescent protein-expressing HIV-1 or R5 HIV-1 BaL. Viral DNA was measured by quantitative PCR and infection assessed by flow cytometry. Deoxynucleotide triphosphate (dNTP) content was determined using a polymerase-based method., Results: Pan-CDK inhibitors AT7519, roscovitine and purvalanol A reduced SAMHD1 phosphorylation. HIV-1 replication was blocked by AT7519 (66.4 ± 3.8%; n = 4), roscovitine (47.3 ± 3.9%; n = 4) and purvalanol A (55.7 ± 15.7%; n = 4) at subtoxic concentrations. Palbociclib, a potent and selective CDK6 inhibitor, blocked SAMHD1 phosphorylation, intracellular dNTP levels, HIV-1 reverse transcription and HIV-1 replication in primary macrophages and CD4 T lymphocytes. Notably, treatment of macrophages with palbociclib led to reduced CDK2 activation, measured as the phosphorylation of the T-loop at the Thr160. The antiviral effect was lost when SAMHD1 was degraded by Vpx, providing further evidence for a role of SAMHD1 in mediating the antiretroviral effect., Conclusions: Our results indicate that SAMHD1-mediated HIV-1 restriction is controlled by CDK as previously suggested but point to a preferential role for CDK2 and CDK6 as mediators of SAMHD1 activation. Our study provides a new signaling pathway susceptible for the development of new therapeutic approaches against HIV-1 infection.
- Published
- 2014
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47. Cell cycle control and HIV-1 susceptibility are linked by CDK6-dependent CDK2 phosphorylation of SAMHD1 in myeloid and lymphoid cells.
- Author
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Pauls E, Ruiz A, Badia R, Permanyer M, Gubern A, Riveira-Muñoz E, Torres-Torronteras J, Alvarez M, Mothe B, Brander C, Crespo M, Menéndez-Arias L, Clotet B, Keppler OT, Martí R, Posas F, Ballana E, and Esté JA
- Subjects
- Benzylamines, CD4-Positive T-Lymphocytes immunology, Cell Cycle immunology, Cells, Cultured, Cyclams, Cyclin-Dependent Kinase 2 antagonists & inhibitors, Cyclin-Dependent Kinase 2 genetics, Cyclin-Dependent Kinase 6 antagonists & inhibitors, Cyclin-Dependent Kinase 6 genetics, HEK293 Cells, HIV Infections virology, HIV-1 immunology, Heterocyclic Compounds pharmacology, Humans, Lymphocyte Activation immunology, Lymphocytes immunology, Macrophages immunology, Myeloid Cells immunology, Phosphorylation drug effects, Phosphorylation genetics, RNA Interference, RNA, Small Interfering, Receptors, CXCR4 antagonists & inhibitors, SAM Domain and HD Domain-Containing Protein 1, Cell Cycle Checkpoints immunology, Cyclin-Dependent Kinase 2 metabolism, Cyclin-Dependent Kinase 6 metabolism, HIV Infections immunology, Monomeric GTP-Binding Proteins metabolism
- Abstract
Proliferating cells are preferentially susceptible to infection by retroviruses. Sterile α motif and HD domain-containing protein-1 (SAMHD1) is a recently described deoxynucleotide phosphohydrolase controlling the size of the intracellular deoxynucleotide triphosphate (dNTP) pool, a limiting factor for retroviral reverse transcription in noncycling cells. Proliferating (Ki67(+)) primary CD4(+) T cells or macrophages express a phosphorylated form of SAMHD1 that corresponds with susceptibility to infection in cell culture. We identified cyclin-dependent kinase (CDK) 6 as an upstream regulator of CDK2 controlling SAMHD1 phosphorylation in primary T cells and macrophages susceptible to infection by HIV-1. In turn, CDK2 was strongly linked to cell cycle progression and coordinated SAMHD1 phosphorylation and inactivation. CDK inhibitors specifically blocked HIV-1 infection at the reverse transcription step in a SAMHD1-dependent manner, reducing the intracellular dNTP pool. Our findings identify a direct relationship between control of the cell cycle by CDK6 and SAMHD1 activity, which is important for replication of lentiviruses, as well as other viruses whose replication may be regulated by intracellular dNTP availability., (Copyright © 2014 by The American Association of Immunologists, Inc.)
- Published
- 2014
- Full Text
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48. SAMHD1 specifically affects the antiviral potency of thymidine analog HIV reverse transcriptase inhibitors.
- Author
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Ballana E, Badia R, Terradas G, Torres-Torronteras J, Ruiz A, Pauls E, Riveira-Muñoz E, Clotet B, Martí R, and Esté JA
- Subjects
- CD4-Positive T-Lymphocytes drug effects, CD4-Positive T-Lymphocytes metabolism, CD4-Positive T-Lymphocytes virology, Gene Expression, HIV Reverse Transcriptase genetics, HIV Reverse Transcriptase metabolism, HIV-2 enzymology, Host-Pathogen Interactions, Humans, Jurkat Cells, Macrophages drug effects, Macrophages metabolism, Macrophages virology, Monomeric GTP-Binding Proteins genetics, Primary Cell Culture, SAM Domain and HD Domain-Containing Protein 1, Thymidine metabolism, Viral Regulatory and Accessory Proteins genetics, Virus Replication drug effects, HIV Reverse Transcriptase antagonists & inhibitors, HIV-2 drug effects, Monomeric GTP-Binding Proteins metabolism, Reverse Transcriptase Inhibitors pharmacology, Stavudine pharmacology, Viral Regulatory and Accessory Proteins metabolism, Zidovudine pharmacology
- Abstract
Sterile alpha motif and histidine-aspartic domain-containing protein 1 (SAMHD1) is a deoxynucleoside triphosphate (dNTP) triphosphohydrolase recently recognized as an antiviral factor that acts by depleting dNTP availability for viral reverse transcriptase (RT). SAMHD1 restriction is counteracted by the human immunodeficiency virus type 2 (HIV-2) accessory protein Vpx, which targets SAMHD1 for proteosomal degradation, resulting in an increased availability of dNTPs and consequently enhanced viral replication. Nucleoside reverse transcriptase inhibitors (NRTI), one of the most common agents used in antiretroviral therapy, compete with intracellular dNTPs as the substrate for viral RT. Consequently, SAMHD1 activity may be influencing NRTI efficacy in inhibiting viral replication. Here, a panel of different RT inhibitors was analyzed for their different antiviral efficacy depending on SAMHD1. Antiviral potency was measured for all the inhibitors in transformed cell lines and primary monocyte-derived macrophages and CD4(+) T cells infected with HIV-1 with or without Vpx. No changes in sensitivity to non-NRTI or the integrase inhibitor raltegravir were observed, but for NRTI, sensitivity significantly changed only in the case of the thymidine analogs (AZT and d4T). The addition of exogenous thymidine mimicked the change in viral sensitivity observed after Vpx-mediated SAMHD1 degradation, pointing toward a differential effect of SAMHD1 activity on thymidine. Accordingly, sensitivity to AZT was also reduced in CD4(+) T cells infected with HIV-2 compared to infection with the HIV-2ΔVpx strain. In conclusion, reduction of SAMHD1 levels significantly decreases HIV sensitivity to thymidine but not other nucleotide RT analog inhibitors in both macrophages and lymphocytes., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)
- Published
- 2014
- Full Text
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49. Administration of deoxyribonucleosides or inhibition of their catabolism as a pharmacological approach for mitochondrial DNA depletion syndrome.
- Author
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Cámara Y, González-Vioque E, Scarpelli M, Torres-Torronteras J, Caballero A, Hirano M, and Martí R
- Subjects
- Animals, Cells, Cultured, DNA Copy Number Variations drug effects, DNA Copy Number Variations genetics, DNA, Mitochondrial metabolism, Humans, Intestinal Pseudo-Obstruction genetics, Male, Mice, Knockout, Mitochondria drug effects, Mitochondria metabolism, Mitochondrial Encephalomyopathies genetics, Muscular Dystrophy, Oculopharyngeal, Ophthalmoplegia congenital, DNA, Mitochondrial genetics, Deoxyribonucleosides therapeutic use, Intestinal Pseudo-Obstruction drug therapy, Intestinal Pseudo-Obstruction metabolism, Mitochondrial Encephalomyopathies drug therapy, Mitochondrial Encephalomyopathies metabolism
- Abstract
Mitochondrial DNA (mtDNA) depletion syndrome (MDS) is characterized by a reduction in mtDNA copy number and consequent mitochondrial dysfunction in affected tissues. A subgroup of MDS is caused by mutations in genes that disrupt deoxyribonucleotide metabolism, which ultimately leads to limited availability of one or several deoxyribonucleoside triphosphates (dNTPs), and subsequent mtDNA depletion. Here, using in vitro experimental approaches (primary cell culture of deoxyguanosine kinase-deficient cells and thymidine-induced mtDNA depletion in culture as a model of mitochondrial neurogastrointestinal encephalomyopathy, MNGIE), we show that supplements of those deoxyribonucleosides (dNs) involved in each biochemical defect (deoxyguanosine or deoxycytidine, dCtd) prevents mtDNA copy number reduction. Similar effects can be obtained by specific inhibition of dN catabolism using tetrahydrouridine (THU; inhibitor of cytidine deaminase) or immucillin H (inhibitor of purine nucleoside phosphorylase). In addition, using an MNGIE animal model, we provide evidence that mitochondrial dNTP content can be modulated in vivo by systemic administration of dCtd or THU. In spite of the severity associated with diseases due to defects in mtDNA replication, there are currently no effective therapeutic options available. Only in the case of MNGIE, allogeneic hematopoietic stem cell transplantation has proven efficient as a long-term therapeutic strategy. We propose increasing cellular availability of the deficient dNTP precursor by direct administration of the dN or inhibition of its catabolism, as a potential treatment for mtDNA depletion syndrome caused by defects in dNTP metabolism.
- Published
- 2014
- Full Text
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50. Gene therapy using a liver-targeted AAV vector restores nucleoside and nucleotide homeostasis in a murine model of MNGIE.
- Author
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Torres-Torronteras J, Viscomi C, Cabrera-Pérez R, Cámara Y, Di Meo I, Barquinero J, Auricchio A, Pizzorno G, Hirano M, Zeviani M, and Martí R
- Subjects
- Animals, DNA, Mitochondrial genetics, Dependovirus genetics, Disease Models, Animal, Genetic Vectors, Homeostasis genetics, Humans, Intestinal Pseudo-Obstruction pathology, Liver metabolism, Mice, Mitochondrial Encephalomyopathies pathology, Muscular Dystrophy, Oculopharyngeal, Mutation, Ophthalmoplegia congenital, Thymidine metabolism, Thymidine Phosphorylase biosynthesis, Genetic Therapy, Intestinal Pseudo-Obstruction genetics, Intestinal Pseudo-Obstruction therapy, Mitochondrial Encephalomyopathies genetics, Mitochondrial Encephalomyopathies therapy, Thymidine Phosphorylase genetics
- Abstract
Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is an autosomal recessive disorder caused by mutations in TYMP, enconding thymidine phosphorylase (TP). TP deficiency results in systemic accumulation of thymidine and deoxyuridine, which interferes with mitochondrial DNA (mtDNA) replication and leads to mitochondrial dysfunction. To date, the only treatment available for MNGIE patients is allogeneic hematopoietic stem cell transplantation, which is associated with high morbidity and mortality. Here, we report that AAV2/8-mediated transfer of the human TYMP coding sequence (hcTYMP) under the control of a liver-specific promoter prevents the biochemical imbalances in a murine model of MNGIE. hcTYMP expression was restricted to liver, and a dose as low as 2 × 10(11) genome copies/kg led to a permanent reduction in systemic nucleoside levels to normal values in about 50% of treated mice. Higher doses resulted in reductions to normal or slightly below normal levels in virtually all mice treated. The nucleoside reduction achieved by this treatment prevented deoxycytidine triphosphate (dCTP) depletion, which is the limiting factor affecting mtDNA replication in this disease. These results demonstrate that the use of AAV to direct TYMP expression in liver is feasible as a potentially safe gene therapy strategy for MNGIE.
- Published
- 2014
- Full Text
- View/download PDF
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