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1. Circulating PCSK9 levels are not associated with the severity of hepatic steatosis and NASH in a high-risk population

Author

Bertrand Cariou, Odile Blanchet, Matthieu Wargny, Sarra Smati, L. Arnaud, Matthieu Pichelin, Benjamin Bouillet, Luc Van Gaal, Sven Francque, Philippe Lefebvre, Jean-Michel Petit, Jérôme Boursier, Pierre-Henri Ducluzeau, Cédric Le May, Bart Staels, Adrien Lannes, Bruno Vergès, unité de recherche de l'institut du thorax UMR1087 UMR6291 (ITX), Université de Nantes - UFR de Médecine et des Techniques Médicales (UFR MEDECINE), Université de Nantes (UN)-Université de Nantes (UN)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Centre d’Investigation Clinique de Nantes (CIC Nantes), Université de Nantes (UN)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre hospitalier universitaire de Nantes (CHU Nantes), Service d'Endocrinologie (CHRU Tours), Centre Hospitalier Régional Universitaire de Tours (CHRU Tours), Université Bourgogne Franche-Comté [COMUE] (UBFC), Lipides - Nutrition - Cancer [Dijon - U1231] (LNC), Université de Bourgogne (UB)-Institut National de la Santé et de la Recherche Médicale (INSERM)-AgroSup Dijon - Institut National Supérieur des Sciences Agronomiques, de l'Alimentation et de l'Environnement, Service d'Endocrinologie, Diabétologie et Maladies Métaboliques (CHU de Dijon), Centre Hospitalier Universitaire de Dijon - Hôpital François Mitterrand (CHU Dijon), Département d'hépatologie (CHU Angers), Centre Hospitalier Universitaire d'Angers (CHU Angers), PRES Université Nantes Angers Le Mans (UNAM)-PRES Université Nantes Angers Le Mans (UNAM), Centre de Ressources Biologiques [CHU d'Angers] (CRB CHU d'Angers BB-0033-00038), Récepteurs nucléaires, maladies cardiovasculaires et diabète - U 1011 (RNMCD), Institut Pasteur de Lille, Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP)-Université de Lille-Centre Hospitalier Régional Universitaire [Lille] (CHRU Lille)-Institut National de la Santé et de la Recherche Médicale (INSERM), Department of Gastroenterology and Hepatology (Antwerp University Hospital), Antwerp University Hospital [Edegem] (UZA), Department of Endocrinology, Diabetology and Metabolism (Antwerp University Hospital), Hémodynamique, Interaction Fibrose et Invasivité tumorales Hépatiques (HIFIH), Université d'Angers (UA), LUNAM Université, F-49933 Angers, France, Plateforme PACeM (Plateforme d'Analyse Cellulaire et Moléculaire), SFR ICAT4208, F-49933 Angers, France., PACeM Angers, Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université de Nantes - UFR de Médecine et des Techniques Médicales (UFR MEDECINE), Université de Nantes (UN)-Université de Nantes (UN), Unité de recherche de l'institut du thorax (ITX-lab), Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Lille-Centre Hospitalier Régional Universitaire [Lille] (CHRU Lille), Plateforme d'Analyse Cellulaire et Moléculaire [SFR ICAT - UA] (PACEM), SFR UA 4208 Interactions Cellulaires et Applications Thérapeutiques (ICAT), Université d'Angers (UA)-Université d'Angers (UA), ANR-16-RHUS-0007,CHOPIN,CHOPIN(2016), and Université de Bourgogne (UB)-AgroSup Dijon - Institut National Supérieur des Sciences Agronomiques, de l'Alimentation et de l'Environnement-Institut National de la Santé et de la Recherche Médicale (INSERM)

Subjects
0301 basic medicine, Male, Biopsy, Liver steatosis, Type 2 diabetes, 030204 cardiovascular system & hematology, Gastroenterology, Severity of Illness Index, PCSK9, 0302 clinical medicine, Fibrosis, Non-alcoholic Fatty Liver Disease, LDL-cholesterol, ComputingMilieux_MISCELLANEOUS, education.field_of_study, medicine.diagnostic_test, Middle Aged, Liver biopsy, [SDV.MHEP.EM]Life Sciences [q-bio]/Human health and pathology/Endocrinology and metabolism, Magnetic Resonance Imaging, 3. Good health, Liver, Female, France, Proprotein Convertase 9, Cardiology and Cardiovascular Medicine, MRI, Adult, Risk, medicine.medical_specialty, Population, Liver fibrosis, 03 medical and health sciences, [SDV.MHEP.CSC]Life Sciences [q-bio]/Human health and pathology/Cardiology and cardiovascular system, Internal medicine, medicine, [SDV.MHEP.PHY]Life Sciences [q-bio]/Human health and pathology/Tissues and Organs [q-bio.TO], Humans, education, Aged, business.industry, [SDV.MHEP.HEG]Life Sciences [q-bio]/Human health and pathology/Hépatology and Gastroenterology, Hepatology, medicine.disease, Fatty Liver, 030104 developmental biology, Cross-Sectional Studies, Diabetes Mellitus, Type 2, Linear Models, Human medicine, Steatosis, Steatohepatitis, business
Abstract

Background and aims: Some studies suggested that proprotein convertase subtilisin kexin type 9 (PCSK9) is linked to liver steatosis severity and non-alcoholic steatohepatitis (NASH). We aimed to assess whether circulating PCSK9 levels are associated with either liver fat content (LFC) or histological markers of NASH in high-risk patients. Methods: We present results from three cross-sectional studies from two French Hospitals: Dijon and Numevox (departments of Endocrinology) and Angers (department of Hepatology). Only patients without lipid-lowering therapy were included. All 132 patients had type 2 diabetes in Dijon, compared to 55/224 in Numevox (25%) and 39/122 in Angers (32%). LFC was assessed on MRI (Dijon and Numevox), and NASH lesion on liver biopsy (Angers). Additionally, we included mRNA results from 138 overweight patients of a Belgian Hospital (Antwerp). Results: While circulating levels of PCSK9 were positively correlated with total cholesterol, LDL-C, triglycerides and non-HDL-C in all 3 cohorts, no significant association was found between PCSK9 and transaminases. Furthermore, no association was found between plasma PCSK9 levels and LFC in both Numevox (beta(adjusted) = 0.71 +/- 1.33, p = 0.60) and Dijon (beta(adjusted) = 1.03 +/- 0.90, p = 0.25). There was no correlation between circulating PCSK9 and histological liver lesions: steatosis severity (beta(adjusted) = -3.95 +/- 2.75, p = 0.15), NASH activity score (beta(adjusted) = -0.31 +/- 0.17, p = 0.082), lobular (beta = -0.067 +/- 0.055, p = 0.22) or portal inflammation (beta = -0.088 +/- 0.079, p = 0.27), ballooning (beta = -0.025 +/- 0.065, p = 0.70) and fibrosis (beta = -0.17 +/- 0.11, p = 0.12). Finally, hepatic PCSK9 mRNA levels were not correlated with NASH histological severity. Conclusions: Circulating PCSK9 concentrations are not associated with the severity of liver steatosis or histological markers of NASH. These data are reassuring regarding the clinical use of PCSK9 inhibitors in cardiovascular diseases.

Published
2018
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2. A novel ex vivo Huntington’s disease model for studying GABAergic neurons and cell grafts by laser microdissection

Author

André, E, Daviaud, N., Sindji, Laurence, Cayon, J., Perrot, R., Montero-Menei, Claudia, Design and Application of Innovative Local Treatments in Glioblastoma (CRCINA-ÉQUIPE 17), Centre de Recherche en Cancérologie et Immunologie Nantes-Angers (CRCINA), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Nantes - UFR de Médecine et des Techniques Médicales (UFR MEDECINE), Université de Nantes (UN)-Université de Nantes (UN)-Centre hospitalier universitaire de Nantes (CHU Nantes)-Centre National de la Recherche Scientifique (CNRS)-Université d'Angers (UA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Nantes - UFR de Médecine et des Techniques Médicales (UFR MEDECINE), Université de Nantes (UN)-Université de Nantes (UN)-Centre hospitalier universitaire de Nantes (CHU Nantes)-Centre National de la Recherche Scientifique (CNRS)-Université d'Angers (UA), Fishberg Department of Neuroscience [New York, NY, USA] (Friedman Brain Institute), Icahn School of Medicine at Mount Sinai [New York] (MSSM), Plateforme d’Analyses Cellulaires et Moléculaires - PACeM [Angers], Université d'Angers (UA), Service Commun d'Imagerie et d'Analyse Microscopique - SCIAM [Angers], This project is supported by the 'Education Audiovisual' and the cultural executive agency of the European Union through the NanoFar Erasmus Mundus joint Doctoral program, by Angers Loire MeÂtropole and by the 'Fondation de l'Avenir' & 'Inserm', France., Design and Application of Innovative Local Treatments in Glioblastoma ( CRCINA - Département NOHMAD - Equipe 17 ), Centre de recherche de Cancérologie et d'Immunologie / Nantes - Angers ( CRCINA ), Université d'Angers ( UA ) -Université de Nantes ( UN ) -Institut National de la Santé et de la Recherche Médicale ( INSERM ) -Centre National de la Recherche Scientifique ( CNRS ) -Institut de Recherche en Santé de l'Université de Nantes ( IRS-UN ) -Centre hospitalier universitaire de Nantes ( CHU Nantes ) -Université d'Angers ( UA ) -Université de Nantes ( UN ) -Institut National de la Santé et de la Recherche Médicale ( INSERM ) -Centre National de la Recherche Scientifique ( CNRS ) -Institut de Recherche en Santé de l'Université de Nantes ( IRS-UN ) -Centre hospitalier universitaire de Nantes ( CHU Nantes ), Fishberg Department of Neuroscience [New York, NY, USA] ( Friedman Brain Institute ), Icahn School of Medicine at Mount Sinai [New York], Université d'Angers ( UA ), Bernardo, Elizabeth, Université d'Angers (UA)-Université de Nantes (UN)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Centre hospitalier universitaire de Nantes (CHU Nantes)-Université d'Angers (UA)-Université de Nantes (UN)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Centre hospitalier universitaire de Nantes (CHU Nantes), Service Commun d'Imagerie et d'Analyse Microscopique [SFR ICAT - UA] (SCIAM), SFR UA 4208 Interactions Cellulaires et Applications Thérapeutiques (ICAT), and Université d'Angers (UA)-Université d'Angers (UA)

Subjects
Cell Transplantation, lcsh:Medicine, Pathology and Laboratory Medicine, Nervous System, [ SDV.CAN ] Life Sciences [q-bio]/Cancer, Rats, Sprague-Dawley, Animal Cells, Neural Pathways, Medicine and Health Sciences, GABAergic Neurons, lcsh:Science, Dehydration (Medicine), Neurons, Physics, Brain, Neurodegenerative Diseases, Condensed Matter Physics, Huntington Disease, Neurology, Optical Equipment, Genetic Diseases, Physical Sciences, Engineering and Technology, Anatomy, Cellular Types, Phase Transitions, Research Article, Cell Survival, Sublimation, Equipment, [SDV.CAN]Life Sciences [q-bio]/Cancer, Laser Capture Microdissection, Extraction techniques, Signs and Symptoms, [SDV.CAN] Life Sciences [q-bio]/Cancer, Diagnostic Medicine, Animals, RNA, Messenger, Clinical Genetics, Lasers, lcsh:R, Autosomal Dominant Diseases, Biology and Life Sciences, Cell Biology, RNA extraction, Rats, Neostriatum, Research and analysis methods, Disease Models, Animal, Neuroanatomy, Cellular Neuroscience, lcsh:Q, Neuroscience
Abstract

International audience; Organotypic brain slice cultures have been recently used to study neurodegenerative disorders such as Parkinson's disease and Huntington's disease (HD). They preserve brain three-dimensional architecture, synaptic connectivity and brain cells microenvironment. Here, we developed an innovative model of Huntington's disease from coronal rat brain slices, that include all the areas involved in the pathology. HD-like neurodegeneration was obtained in only one week, in a single step, during organotypic slice preparation, without the use of neurotoxins. HD-like histopathology was analysed and after one week, a reduction of 40% of medium spiny neurons was observed. To analyse new therapeutic approaches in this innovative HD model, we developed a novel protocol of laser microdissection to isolate and analyse by RT-qPCR, grafted cells as well as surrounding tissue of fresh organotypic slices. We determined that laser microdissection could be performed on a 400μm organoty-pic slice after alcohol dehydration protocol, allowing the analysis of mRNA expression in the rat tissue as well as in grafted cells. In conclusion, we developed a new approach for modeling Huntington's disease ex vivo, and provided a useful innovative method for screening new potential therapies for neurodegenerative diseases especially when associated with laser microdissection.

Published
2018
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3. Locoregional Confinement and Major Clinical Benefit of 188 Re-Loaded CXCR4-Targeted Nanocarriers in an Orthotopic Human to Mouse Model of Glioblastoma

Author

Séhédic, Delphine, Chourpa, Igor, Tétaud, Clément, Griveau, Audrey, Loussouarn, Claire, Avril, Sylvie, Legendre, Claire, Lepareur, Nicolas, Wion, Didier, Hindré, François, Davodeau, François, Garcion, Emmanuel, Design and Application of Innovative Local Treatments in Glioblastoma (CRCINA-ÉQUIPE 17), Centre de Recherche en Cancérologie et Immunologie Nantes-Angers (CRCINA), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Nantes - UFR de Médecine et des Techniques Médicales (UFR MEDECINE), Université de Nantes (UN)-Université de Nantes (UN)-Centre hospitalier universitaire de Nantes (CHU Nantes)-Centre National de la Recherche Scientifique (CNRS)-Université d'Angers (UA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Nantes - UFR de Médecine et des Techniques Médicales (UFR MEDECINE), Université de Nantes (UN)-Université de Nantes (UN)-Centre hospitalier universitaire de Nantes (CHU Nantes)-Centre National de la Recherche Scientifique (CNRS)-Université d'Angers (UA), Nanomédicaments et Nanosondes, EA 6295 (NMNS), Université de Tours, Plateforme de Radiobiologie et d’Imagerie EXperimentale - PRIMEX [Angers], Université d'Angers (UA), Département d'oncologie médicale [Rennes], CRLCC Eugène Marquis (CRLCC), BrainTech Laboratory [CHU Grenoble Alpes - Inserm U1205] (Brain Tech Lab ), CHU Grenoble-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), Nuclear Oncology (CRCINA-ÉQUIPE 13), Plateforme d’Analyses Cellulaires et Moléculaires - PACeM [Angers], This work was supported by 'La Région Pays-de-la-Loire' through the Nuclear Technology for Health project (NucSan), by the 'Institut National de la Santé et de la Recherche Médicale' (INSERM), by 'La Ligue Nationale Contre le Cancer' through an 'Equipe Labellisée 2012' grant and by the 'Axe Vectorisation and Radiothérapies of the Cancéropôle Grand-Ouest'. Delphine Séhédic was a Ph.D. student from the NucSan project and received a fellowship from 'La Région Pays-de-la-Loire'. N.L., F.H., F.D. and E.G. are also member of the LabEx IRON 'Innovative Radiopharmaceuticals in Oncology and Neurology' as part of the French government 'Investissements d’Avenir' program. The presented work is also related to the PL-BIO 2014-2020 INCA (Institut National du Cancer) project MARENGO - 'MicroRNA agonist and antagonist Nanomedicines for GliOblastoma treatment: from molecular programmation to preclinical validation', to the ANR (French National Research Agency) program RADIOHEAD 'Nanocapsules for Selective Internal RadioTherapy of glioblastoma, and to the 'Région Pays-de-la-Loire' project IRAD' Pre-clinical validation of an innovative Internal RADiotherapy of glioma in a large spontaneous animal model'. C.Lo., and C.Le. were posdoctoral fellows Inca and Fondation ARC, respectively., Université de Tours (UT), Bernardo, Elizabeth, Université d'Angers (UA)-Université de Nantes (UN)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Centre hospitalier universitaire de Nantes (CHU Nantes)-Université d'Angers (UA)-Université de Nantes (UN)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Centre hospitalier universitaire de Nantes (CHU Nantes), Plateforme de Radiobiologie et d’Imageries EXpérimentale [SFR ICAT - UA] (PRIMEX), SFR UA 4208 Interactions Cellulaires et Applications Thérapeutiques (ICAT), and Université d'Angers (UA)-Université d'Angers (UA)

Subjects
CXCR4, nanoparticle, [SDV.CAN] Life Sciences [q-bio]/Cancer, glioblastoma, immuno-targeting, spectral imaging, [SDV.CAN]Life Sciences [q-bio]/Cancer, macrophage, radiation therapy
Abstract

International audience; Purpose: Gold standard beam radiation for glioblastoma (GBM) treatment is challenged by resistance phenomena occurring in cellular populations well prepared to survive or to repair damage caused by radiation. Among signals that have been linked with radio-resistance, the SDF1/CXCR4 axis, associated with cancer stem-like cell, may be an opportune target. To avoid the problem of systemic toxicity and blood-brain barrier crossing, the relevance and efficacy of an original system of local brain internal radiation therapy combining a radiopharmaceutical with an immuno-nanoparticle was investigated.Experiment design: The nanocarrier combined lipophilic thiobenzoate complexes of rhenium-188 loaded in the core of a lipid nanocapsule (LNC188Re) with a function-blocking antibody, 12G5 directed at the CXCR4, on its surface. The efficiency of 12G5-LNC188Re was investigated in an orthotopic and xenogenic GBM model of CXCR4-positive U87MG cells implanted in the striatum of Scid mice.Results: We demonstrated that 12G5-LNC188Re single infusion treatment by convection-enhanced delivery resulted in a major clinical improvement in median survival that was accompanied by locoregional effects on tumor development including hypovascularization and stimulation of the recruitment of bone marrow derived CD11b- or CD68-positive cells as confirmed by immunohistochemistry analysis. Interestingly, thorough analysis by spectral imaging in a chimeric U87MG GBM model containing CXCR4-positive/red fluorescent protein (RFP)-positive- and CXCR4-negative/RFP-negative-GBM cells revealed greater confinement of DiD-labeled 12G5-LNCs than control IgG2a-LNCs in RFP compartments. Main conclusion: These findings on locoregional impact and targeting of disseminated cancer cells in tumor margins suggest that intracerebral active targeting of nanocarriers loaded with radiopharmaceuticals may have considerable benefits in clinical applications.

Published
2017
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4. Effects of mesenchymal stem cell therapy, in association with pharmacologically active microcarriers releasing VEGF, in an ischaemic stroke model in the rat

Author

Jérôme Cayon, Didier Divoux, Simon Roussel, Omar Touzani, Léna Marteau, Myriam Lecocq, Jérôme Toutain, Claudia N. Montero-Menei, Marie-Sophie Quittet, Fabien Fillesoye, Laurence Sindji, Myriam Bernaudin, Hypoxie, physiopathologies cérébrovasculaire et tumorale (CERVOxy), Imagerie et Stratégies Thérapeutiques des pathologies Cérébrales et Tumorales (ISTCT), Université de Caen Normandie (UNICAEN), Normandie Université (NU)-Normandie Université (NU)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)-Université de Caen Normandie (UNICAEN), Normandie Université (NU)-Normandie Université (NU)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), Micro et Nanomédecines Biomimétiques (MINT), Université d'Angers (UA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Bretagne Loire (UBL), Plateforme d'Analyse Cellulaire et Moléculaire [SFR ICAT - UA] (PACEM), SFR UA 4208 Interactions Cellulaires et Applications Thérapeutiques (ICAT), Université d'Angers (UA)-Université d'Angers (UA), LUNAM Université, F-49933 Angers, France, Plateforme PACeM (Plateforme d'Analyse Cellulaire et Moléculaire), SFR ICAT4208, F-49933 Angers, France., and PACeM Angers

Subjects
Doublecortin Domain Proteins, Male, Vascular Endothelial Growth Factor A, Angiogenesis, [SDV]Life Sciences [q-bio], Biochemistry, Brain Ischemia, Rats, Sprague-Dawley, chemistry.chemical_compound, 0302 clinical medicine, Medicine, Stroke, 0303 health sciences, Drug Carriers, Behavior, Animal, Infarction, Middle Cerebral Artery, General Medicine, VEGF, Magnetic Resonance Imaging, Vascular endothelial growth factor, Pharmacologically active microcarriers, Treatment Outcome, medicine.symptom, Microtubule-Associated Proteins, Biotechnology, medicine.medical_specialty, Biomedical Engineering, Context (language use), Brain damage, Mesenchymal Stem Cell Transplantation, Biomaterials, 03 medical and health sciences, In vivo, parasitic diseases, Animals, Molecular Biology, 030304 developmental biology, business.industry, Mesenchymal stem cell, Neuropeptides, Mesenchymal Stem Cells, Recovery of Function, Hypoxia (medical), medicine.disease, Surgery, Disease Models, Animal, chemistry, [SDV.SP.PHARMA]Life Sciences [q-bio]/Pharmaceutical sciences/Pharmacology, Cancer research, Blood Vessels, Laminin, business, 030217 neurology & neurosurgery
Abstract

CERVOXY; International audience; Few effective therapeutic interventions are available to limit brain damage and functional deficits after ischaemic stroke. Within this context, mesenchymal stem cell (MSC) therapy carries minimal risks while remaining efficacious through the secretion of trophic, protective, neurogenic and angiogenic factors. The limited survival rate of MSCs restricts their beneficial effects. The usefulness of a three-dimensional support, such as a pharmacologically active microcarrier (PAM), on the survival of MSCs during hypoxia has been shown in vitro, especially when the PAMs were loaded with vascular endothelial growth factor (VEGF). In the present study, the effect of MSCs attached to laminin-PAMs (LM-PAMs), releasing VEGF or not, was evaluated in vivo in a model of transient stroke. The parameters assessed were infarct volume, functional recovery and endogenous cellular reactions. LM-PAMs induced the expression of neuronal markers by MSCs both in vitro and in vivo. Moreover, the prolonged release of VEGF increased angiogenesis around the site of implantation of the LM-PAMs and facilitated the migration of immature neurons towards the ischaemic tissue. Nonetheless, MSCs/LM-PAMs-VEGF failed to improve sensorimotor functions. The use of LM-PAMs to convey MSCs and to deliver growth factors could be an effective strategy to repair the brain damage caused by a stroke.

Published
2014
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5. Lentiviral Vectors That Express UGT1A1 in Liver and Contain Mir-142 Target Sequences Normalize Hyperbilirubinemia in Gunn Rats

Author

Anne Dariel, Séverine Remy, Tuan Huy Nguyen, Nicolas Ferry, Ignacio Anegon, Sébastien Boni, Guillaume Podevin, Claire Usal, Sophie Laplanche, Françoise Schmitt, Virginie Pichard, Anne Myara, Maude Flageul, Philippe Labrune, Hémodynamique, Interaction Fibrose et Invasivité tumorales Hépatiques (HIFIH), Université d'Angers (UA), Institut National de la Santé et de la Recherche Médicale (INSERM), Centre hospitalier universitaire de Nantes (CHU Nantes), Faculté de Médecine, Université Francois Rabelais [Tours], Laboratoire de Thérapie Génique Translationnelle des Maladies Génétiques (Inserm UMR 1089), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Nantes - UFR de Médecine et des Techniques Médicales (UFR MEDECINE), Université de Nantes (UN)-Université de Nantes (UN), Faculté de Médecine - Université de Nantes, Institut de transplantation urologie-néphrologie (ITUN), Université de Nantes (UN)-Centre hospitalier universitaire de Nantes (CHU Nantes), Plateforme d’Analyses Cellulaires et Moléculaires - PACeM [Angers], Immunointervention dans les allo et xénotransplantations, Université de Nantes (UN)-IFR26-Institut National de la Santé et de la Recherche Médicale (INSERM)-ITUN, Service de Biologie [GH Saint-Joseph - APHP], Centre hospitalier Saint-Joseph [Paris], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP), UMR643, Service de pédiatrie [Béclère], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-AP-HP - Hôpital Antoine Béclère [Clamart], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP), UMR S948 Biothérapies hépatiques, and Neurology

Subjects
Male, Time Factors, Crigler–Najjar syndrome, RNA Stability, Genetic enhancement, [SDV]Life Sciences [q-bio], Genetic Vectors, Rats, Gunn, Gene Transfer, Antigen-Presenting Cells, Biology, digestive system, Antibodies, 03 medical and health sciences, 0302 clinical medicine, Multiplicity of infection, Immune system, Transduction, Genetic, medicine, Animals, Humans, Prealbumin, RNA, Messenger, Vector (molecular biology), Crigler–Najjar Syndrome, Glucuronosyltransferase, Promoter Regions, Genetic, Antigen-presenting cell, Crigler-Najjar Syndrome, 030304 developmental biology, 0303 health sciences, Hepatology, Lentivirus, Gastroenterology, Bilirubin, MicroRNA, Genetic Therapy, medicine.disease, Molecular biology, Rats, 3. Good health, Disease Models, Animal, MicroRNAs, Haematopoiesis, Real-time polymerase chain reaction, Liver, 030220 oncology & carcinogenesis, Biomarkers, HeLa Cells
Abstract

International audience; Background & AimsCrigler–Najjar type 1 (CN-I) is an inherited liver disease caused by an absence of bilirubin–uridine 5′-diphosphate–glucuronosyltransferase (UGT1A1) activity. It results in life-threatening levels of unconjugated bilirubin, and therapeutic options are limited. We used adult Gunn rats (an animal model of the disease) to evaluate the efficiency of lentiviral-based gene therapy to express UGT1A1 in liver. Methods Gunn rats were given intraportal injections of VSVG-pseudotyped lentiviral vectors that encode UGT1A1 under the control of a liver-specific transthyretin promoter (mTTR.hUGT1A1); this vector does not contain target sequences for miR-142, a microRNA that is expressed specifically in hematopoietic cells. Rats were also injected with the vector mTTR.hUGT1A1.142T, which contains 4 copies of the miR-142 target sequences; its messenger RNA should be degraded in antigen-presenting cells. Bilirubinemia was monitored, and the presence of transduced hepatocytes was analyzed by quantitative polymerase chain reaction. Vector expression was tested in vitro in rat hematopoietic cells. Results In Gunn rats, bilirubin levels normalized 2 weeks after administration of mTTR.hUGT1A1. However, hyperbilirubinemia resumed 8 weeks after vector administration, concomitant with the induction of an immune response. In contrast, in rats injected with mTTR-UGT1A1.142T, bilirubin levels normalized for up to 6 months and transduced cells were not eliminated. Conclusions Lentiviral vectors that express UGT1A1 reduce hyperbilirubinemia in immunocompetent Gunn rats for at least 6 months. The immune response against virally expressed UGT1A1 can be circumvented by inclusion of miR-142 target sequences, which reduce vector expression in antigen-presenting cells. This lentiviral-based gene therapy approach might be developed to treat patients with CN-I.

Published
2010
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6. Transient Increase in Intrahepatic Pressure Mediates Successful Treatment of the Gunn Rat with Reduced Doses of Lentiviral Vector

Author

Françoise Schmitt, Maude Flageul, Anne Dariel, Anne Myara, Sébastien Boni, Virginie Pichard, Nicolas Ferry, Tuan Huy Nguyen, Guillaume Podevin, Cecilia Abarrategui Pontes, Hémodynamique, Interaction Fibrose et Invasivité tumorales Hépatiques (HIFIH), Université d'Angers (UA), Laboratoire de Thérapie Génique Translationnelle des Maladies Génétiques (Inserm UMR 1089), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Nantes - UFR de Médecine et des Techniques Médicales (UFR MEDECINE), Université de Nantes (UN)-Université de Nantes (UN), Faculté de Médecine - Université de Nantes, Institut de transplantation urologie-néphrologie (ITUN), Université de Nantes (UN)-Centre hospitalier universitaire de Nantes (CHU Nantes), Plateforme d’Analyses Cellulaires et Moléculaires - PACeM [Angers], Service de Biologie [GH Saint-Joseph - APHP], Centre hospitalier Saint-Joseph [Paris], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP), and Neurology

Subjects
Endothelium, Genetic enhancement, [SDV]Life Sciences [q-bio], Genetic Vectors, Rats, Gunn, Blood Pressure, Pharmacology, Biology, Polymerase Chain Reaction, Viral vector, 03 medical and health sciences, Transduction (genetics), 0302 clinical medicine, Multiplicity of infection, Liver Function Tests, In vivo, Transduction, Genetic, Genetics, medicine, Pressure, Animals, Molecular Biology, 030304 developmental biology, Crigler-Najjar Syndrome, Hyperbilirubinemia, 0303 health sciences, Lentivirus, Bilirubin, Blood flow, Genetic Therapy, Gunn rat, 3. Good health, Rats, Disease Models, Animal, medicine.anatomical_structure, Liver, 030220 oncology & carcinogenesis, Immunology, Hepatocytes, Molecular Medicine, Biomarkers
Abstract

International audience; Lentiviral vectors can stably transduce hepatocytes and are promising tools for gene therapy of hepatic diseases. Although hepatocytes are accessible to blood-borne viral vectors through fenestrations of the hepatic endothelium, improved liver transduction after delivery of vectors to the blood stream is needed. As the normal endothelial fenestration and lentiviral vectors are similar in size (150 nm), we hypothesized that a transient increase in hepatic blood pressure may enhance in vivo gene transfer to hepatocytes. We designed a simple surgical procedure, by which the liver is temporarily excluded from blood flow. Lentiviral vectors were injected in a large volume to increase intrahepatic pressure. We demonstrated that in the Gunn rat, a model of Crigler–Najjar disease, the administration of low vector doses (corresponding to a multiplicity of infection of 0.2) by this procedure resulted in therapeutic correction of hyperbilirubinemia, without toxicity. The correction was sustained for 10 months (end of study). The same vector amounts yielded only partial correction after intraportal delivery. We believe that this new and clinically applicable strategy may broaden the range of genetic liver diseases accessible to gene therapy.

Published
2010
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7. A novel ex vivo Huntington's disease model for studying GABAergic neurons and cell grafts by laser microdissection.

Author

André EM, Daviaud N, Sindji L, Cayon J, Perrot R, and Montero-Menei CN

Subjects
Animals, Brain pathology, Cell Survival, Disease Models, Animal, Huntington Disease metabolism, Huntington Disease surgery, RNA, Messenger genetics, RNA, Messenger metabolism, Rats, Rats, Sprague-Dawley, Cell Transplantation, GABAergic Neurons pathology, Huntington Disease pathology, Laser Capture Microdissection
Abstract

Organotypic brain slice cultures have been recently used to study neurodegenerative disorders such as Parkinson's disease and Huntington's disease (HD). They preserve brain three-dimensional architecture, synaptic connectivity and brain cells microenvironment. Here, we developed an innovative model of Huntington's disease from coronal rat brain slices, that include all the areas involved in the pathology. HD-like neurodegeneration was obtained in only one week, in a single step, during organotypic slice preparation, without the use of neurotoxins. HD-like histopathology was analysed and after one week, a reduction of 40% of medium spiny neurons was observed. To analyse new therapeutic approaches in this innovative HD model, we developed a novel protocol of laser microdissection to isolate and analyse by RT-qPCR, grafted cells as well as surrounding tissue of fresh organotypic slices. We determined that laser microdissection could be performed on a 400μm organotypic slice after alcohol dehydration protocol, allowing the analysis of mRNA expression in the rat tissue as well as in grafted cells. In conclusion, we developed a new approach for modeling Huntington's disease ex vivo, and provided a useful innovative method for screening new potential therapies for neurodegenerative diseases especially when associated with laser microdissection.

Published
2018
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