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1. Characterization of brain dystrophins absence and impact in dystrophin-deficient Dmdmdx rat model

Author

Virginie François, Ignacio Anegon, Mireille Ledevin, Caroline Le Guiner, Thibaut Larcher, D. Caudal, Corinne Huchet, A. Lafoux, Therassay Platform, CAPACITES, Université de Nantes (UN), UMR1089, Nantes Gene Therapy Laboratory, Institut National de la Santé et de la Recherche Médicale (INSERM), Développement et Pathologie du Tissu Musculaire (DPTM), Institut National de la Recherche Agronomique (INRA)-Ecole Nationale Vétérinaire de Nantes, UMR 1064-CRTI, UMR 1089, Nantes Gene Therapy Laboratory, UMR 1089,Nantes Gene Therapy Laboratory, 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), Physiopathologie Animale et bioThérapie du muscle et du système nerveux (PAnTher), Ecole Nationale Vétérinaire, Agroalimentaire et de l'alimentation Nantes-Atlantique (ONIRIS)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Expertise en Anatomie Pathologique (APEX), Ecole Nationale Vétérinaire, Agroalimentaire et de l'alimentation Nantes-Atlantique (ONIRIS)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Ecole Nationale Vétérinaire, Agroalimentaire et de l'alimentation Nantes-Atlantique (ONIRIS)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), ProdInra, Archive Ouverte, École nationale vétérinaire, agroalimentaire et de l'alimentation Nantes-Atlantique (ONIRIS)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), and École nationale vétérinaire, agroalimentaire et de l'alimentation Nantes-Atlantique (ONIRIS)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-École nationale vétérinaire, agroalimentaire et de l'alimentation Nantes-Atlantique (ONIRIS)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)

Subjects
Central Nervous System, 0301 basic medicine, Sucrose, Heredity, Genetic Linkage, Physiology, Duchenne muscular dystrophy, [SDV]Life Sciences [q-bio], Synaptogenesis, Hippocampus, Duchenne Muscular Dystrophy, Anxiety, Disaccharides, Biochemistry, Nervous System, Muscular Dystrophies, Dystrophin, 0302 clinical medicine, Medicine and Health Sciences, Protein Isoforms, Muscular dystrophy, health care economics and organizations, Mammals, Multidisciplinary, biology, Organic Compounds, Physics, Eukaryota, Classical Mechanics, Brain, Animal Models, musculoskeletal system, Phenotype, [SDV] Life Sciences [q-bio], Chemistry, medicine.anatomical_structure, Experimental Organism Systems, Neurology, X-Linked Traits, Sex Linkage, Vertebrates, Physical Sciences, Mechanical Stress, Medicine, Anatomy, Rats, Transgenic, Locomotion, Research Article, musculoskeletal diseases, medicine.medical_specialty, congenital, hereditary, and neonatal diseases and abnormalities, Transgene, Science, Central nervous system, education, Carbohydrates, Research and Analysis Methods, Rodents, 03 medical and health sciences, Model Organisms, Internal medicine, Genetics, medicine, Animals, Maze Learning, Muscle, Skeletal, Clinical Genetics, Biological Locomotion, Myocardium, Organic Chemistry, Organisms, Chemical Compounds, Biology and Life Sciences, Proteins, Muscular Dystrophy, Animal, medicine.disease, Rats, Cytoskeletal Proteins, Thermal Stresses, 030104 developmental biology, Endocrinology, Amniotes, Animal Studies, Mice, Inbred mdx, biology.protein, Stress, Psychological, 030217 neurology & neurosurgery
Abstract

Duchenne Muscular Dystrophy (DMD) is a severe muscle-wasting disease caused by mutations in the DMD gene encoding dystrophin, expressed mainly in muscles but also in other tissues like retina and brain. Non-progressing cognitive dysfunction occurs in 20 to 50% of DMD patients. Furthermore, loss of expression of the Dp427 dystrophin isoform in the brain of mdx mice, the most used animal model of DMD, leads to behavioral deficits thought to be linked to insufficiencies in synaptogenesis and channel clustering at synapses. Mdx mice where the locomotor phenotype is mild also display a high and maladaptive response to stress. Recently, we generated Dmdmdx rats carrying an out-of frame mutation in exon 23 of the DMD gene and exhibiting a skeletal and cardiac muscle phenotype similar to DMD patients. In order to evaluate the impact of dystrophin loss on behavior, we explored locomotion parameters as well as anhedonia, anxiety and response to stress, in Dmdmdx rats aged from 1.5 to 7 months, in comparison to wild-type (WT) littermates. Pattern of dystrophin expression in the brain of WT and Dmdmdx rats was characterized by western-blot analyses and immunohistochemistry. We showed that dystrophin-deficient Dmdmdx rats displayed motor deficits in the beam test, without association with depressive or anxiety-like phenotype. However, Dmdmdx rats exhibited a strong response to restraint-induced stress, with a large increase in freezings frequency and duration, suggesting an alteration in a functional circuit including the amygdala. In brain, large dystrophin isoform Dp427 was not expressed in mutant animals. Dmdmdx rat is therefore a good animal model for preclinical evaluations of new treatments for DMD but care must be taken with their responses to mild stress.

Published
2020
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2. 5 years of successful inducible transgene expression following locoregional AAV delivery in nonhuman primates with no detectable immunity

Author

Mickaël Guilbaud, Marie Devaux, Célia Couzinié, Johanne Le Duff, Alice Toromanoff, Céline Vandamme, Nicolas Jaulin, Gwladys Gernoux, Thibaut Larcher, Philippe Moullier, Caroline Le Guiner, Oumeya Adjali, UMR 1089, Institut National de la Santé et de la Recherche Médicale (INSERM), UMR1089, Department of Clinical Microbiology, Institue of Clinical Medicine, University of Eastern Finland, Gene Therapy Center, University of Massachusetts Medical School [Worcester] (UMASS), University of Massachusetts System (UMASS), Physiopathologie Animale et bioThérapie du muscle et du système nerveux (PAnTher), Institut National de la Recherche Agronomique (INRA)-Ecole Nationale Vétérinaire, Agroalimentaire et de l'alimentation Nantes-Atlantique (ONIRIS), 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), Department of Clinical Microbiology [Kuopio, Finland], University of Eastern Finland- Institute of Clinical Medicine [Kuopio, Finland], Développement et Pathologie du Tissu Musculaire (DPTM), Institut National de la Recherche Agronomique (INRA)-Ecole Nationale Vétérinaire de Nantes, JAULIN, Nicolas, 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), Institut National de la Recherche Agronomique (INRA)-École nationale vétérinaire, agroalimentaire et de l'alimentation Nantes-Atlantique (ONIRIS), ProdInra, Archive Ouverte, Ecole Nationale Vétérinaire de Nantes-Institut National de la Recherche Agronomique (INRA), and Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Nantes (UN)

Subjects
[SDV] Life Sciences [q-bio], [SDV.MHEP] Life Sciences [q-bio]/Human health and pathology, viruses, [SDV]Life Sciences [q-bio], biochemical phenomena, metabolism, and nutrition, ComputingMilieux_MISCELLANEOUS, [SDV.MHEP]Life Sciences [q-bio]/Human health and pathology
Abstract

5 years of successful inducible transgene expression following locoregional AAV delivery in nonhuman primates with no detectable immunity

Published
2019
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3. RNA-Seq Analysis of an Antisense Sequence Optimized for Exon Skipping in Duchenne Patients Reveals No Off-Target Effect

Author

Thomas Voit, M. Allais, Marie Montus, Caroline Le Guiner, Yann Audic, Adrien Leger, Emilie Lecomte, Laurent Servais, Philippe Moullier, Claire Domenger, Virginie François, 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), Généthon, CHU Trousseau [APHP], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU), NIHR Biomedical Research Centre [London], Guy's and St Thomas' NHS Foundation Trust-King‘s College London, Institut de Génétique et Développement de Rennes (IGDR), Structure Fédérative de Recherche en Biologie et Santé de Rennes ( Biosit : Biologie - Santé - Innovation Technologique )-Centre National de la Recherche Scientifique (CNRS)-Université de Rennes 1 (UR1), Université de Rennes (UNIV-RENNES)-Université de Rennes (UNIV-RENNES), This project was supported by FRM (Fondation pour la Recherche Médicale, grant FDT20140931046), by AFM-Téléthon (Association Française contre les Myopathies), and by ADNA (Advanced Diagnostics for New Therapeutic Approaches), a program dedicated to personalized medicine, coordinated by Institut Mérieux, and supported by research and innovation aid from the French public agency, Bpifrance., JAULIN, Nicolas, and Université de Rennes (UR)-Centre National de la Recherche Scientifique (CNRS)-Structure Fédérative de Recherche en Biologie et Santé de Rennes ( Biosit : Biologie - Santé - Innovation Technologique )

Subjects
0301 basic medicine, congenital, hereditary, and neonatal diseases and abnormalities, Duchenne muscular dystrophy, [SDV]Life Sciences [q-bio], RNA-Seq, Computational biology, Biology, Article, 03 medical and health sciences, Exon, Drug Discovery, Gene expression, medicine, Messenger RNA, [SDV.MHEP] Life Sciences [q-bio]/Human health and pathology, lcsh:RM1-950, RNA, RNA sequencing, medicine.disease, Duchenne, Exon skipping, 3. Good health, [SDV] Life Sciences [q-bio], lcsh:Therapeutics. Pharmacology, 030104 developmental biology, RNA splicing, Molecular Medicine, U7snRNA, off-target, [SDV.MHEP]Life Sciences [q-bio]/Human health and pathology, exon skipping
Abstract

International audience; Non-coding uridine-rich small nuclear RNAs (UsnRNAs) have emerged in recent years as effective tools for exon skipping for the treatment of Duchenne muscular dystrophy (DMD), a degenerative muscular genetic disorder. We recently showed the high capacity of a recombinant adeno-associated virus (rAAV)-U7snRNA vector to restore the reading frame of the DMD mRNA in the muscles of DMD dogs. We are now moving toward a phase I/II clinical trial with an rAAV-U7snRNA-E53, carrying an antisense sequence designed to hybridize exon 53 of the human DMD messenger. As observed for genome-editing tools, antisense sequences present a risk of off-target effects, reflecting partial hybridization onto unintended transcripts. To characterize the clinical antisense sequence, we studied its expression and explored the occurrence of its off-target effects in human in vitro models of skeletal muscle and liver. We presented a comprehensive methodology combining RNA sequencing and in silico filtering to analyze off-targets. We showed that U7snRNA-E53 induced the effective exon skipping of the DMD transcript without inducing the notable deregulation of transcripts in human cells, neither at gene expression nor at the mRNA splicing level. Altogether, these results suggest that the use of the rAAV-U7snRNA-E53 vector for exon skipping could be safe in eligible DMD patients.

Published
2018
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4. Gait characterization in golden retriever muscular dystrophy dogs using linear discriminant analysis

Author

Stéphane Blot, Jean-Yves Hogrel, El Mostafa Qannari, Chantal Thorin, Caroline Le Guiner, Inès Barthélémy, Yan Cherel, Karl Rouger, Bodvael Fraysse, UMR 1089, Atlantic Gene Therapies, Institut National de la Santé et de la Recherche Médicale (INSERM), UPR de neurobiologie, École nationale vétérinaire d'Alfort (ENVA), Chercheur indépendant, Physiopathologie Animale et bioThérapie du muscle et du système nerveux (PAnTher), Institut National de la Recherche Agronomique (INRA)-Ecole Nationale Vétérinaire, Agroalimentaire et de l'alimentation Nantes-Atlantique (ONIRIS), UMR 1089, Atlantic Gene Therapies & Genethon, Neuromuscular Physiology and Evaluation Laboratory, Institut de Myologie, 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), Développement et Pathologie du Tissu Musculaire (DPTM), Ecole Nationale Vétérinaire de Nantes-Institut National de la Recherche Agronomique (INRA), Université de Nantes (UN)-Institut National de la Santé et de la Recherche Médicale (INSERM), and Institut National de la Recherche Agronomique (INRA)-Ecole Nationale Vétérinaire de Nantes

Subjects
Male, 0301 basic medicine, lcsh:Diseases of the musculoskeletal system, [SDV]Life Sciences [q-bio], 0302 clinical medicine, Gait (human), Accelerometry, Treatment evaluation, Orthopedics and Sports Medicine, Statistical analysis, Muscular dystrophy, Gait, ComputingMilieux_MISCELLANEOUS, Age Factors, Discriminant analysis, Phenotype, Treatment Outcome, Technical Advance, Disease Progression, GRMD, Immunosuppressive Agents, Golden retriever muscular dystrophy, medicine.medical_specialty, Genotype, Clinical Decision-Making, Sensitivity and Specificity, Gait assessment, 03 medical and health sciences, Dogs, Animal model, Physical medicine and rehabilitation, Rheumatology, medicine, Animals, [SDV.BA.MVSA]Life Sciences [q-bio]/Animal biology/Veterinary medicine and animal Health, business.industry, Disease progression, Muscular Dystrophy, Animal, medicine.disease, Linear discriminant analysis, Muscular Dystrophy, Duchenne, Disease Models, Animal, 030104 developmental biology, Gait analysis, Linear Models, [SDV.SP.PHARMA]Life Sciences [q-bio]/Pharmaceutical sciences/Pharmacology, lcsh:RC925-935, business, [SDV.AEN]Life Sciences [q-bio]/Food and Nutrition, 030217 neurology & neurosurgery
Abstract

Background Accelerometric analysis of gait abnormalities in golden retriever muscular dystrophy (GRMD) dogs is of limited sensitivity, and produces highly complex data. The use of discriminant analysis may enable simpler and more sensitive evaluation of treatment benefits in this important preclinical model. Methods Accelerometry was performed twice monthly between the ages of 2 and 12 months on 8 healthy and 20 GRMD dogs. Seven accelerometric parameters were analysed using linear discriminant analysis (LDA). Manipulation of the dependent and independent variables produced three distinct models. The ability of each model to detect gait alterations and their pattern change with age was tested using a leave-one-out cross-validation approach. Results Selecting genotype (healthy or GRMD) as the dependent variable resulted in a model (Model 1) allowing a good discrimination between the gait phenotype of GRMD and healthy dogs. However, this model was not sufficiently representative of the disease progression. In Model 2, age in months was added as a supplementary dependent variable (GRMD_2 to GRMD_12 and Healthy_2 to Healthy_9.5), resulting in a high overall misclassification rate (83.2%). To improve accuracy, a third model (Model 3) was created in which age was also included as an explanatory variable. This resulted in an overall misclassification rate lower than 12%. Model 3 was evaluated using blinded data pertaining to 81 healthy and GRMD dogs. In all but one case, the model correctly matched gait phenotype to the actual genotype. Finally, we used Model 3 to reanalyse data from a previous study regarding the effects of immunosuppressive treatments on muscular dystrophy in GRMD dogs. Our model identified significant effect of immunosuppressive treatments on gait quality, corroborating the original findings, with the added advantages of direct statistical analysis with greater sensitivity and more comprehensible data representation. Conclusions Gait analysis using LDA allows for improved analysis of accelerometry data by applying a decision-making analysis approach to the evaluation of preclinical treatment benefits in GRMD dogs.

Published
2017
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5. Long-term microdystrophin gene therapy is effective in a canine model of Duchenne muscular dystrophy

Author

Virginie François, Oumeya Adjali, Jean-Yves Hogrel, George Dickson, Thibaut Larcher, Maeva Dutilleul, Taeyoung Koo, Marie Montus, Stéphane Ederhy, Stéphane Blot, M. Allais, Carole Masurier, Alberto Malerba, Marie Devaux, B. Matot, Karim Wahbi, Sophie Moullec, Bernard Gjata, Bodvael Fraysse, Laurent Servais, Fulvio Mavilio, Inès Barthélémy, Pierre G. Carlier, Takis Athanasopoulos, Isabelle Testault, Samia Martin, Jack-Yves Deschamps, Philippe Moullier, Federico Mingozzi, Philippe Veron, Christophe Georger, Johanne Le Duff, Caroline Le Guiner, Thomas Voit, Jean-Laurent Thibaut, U1089, Institut National de la Santé et de la Recherche Médicale (INSERM), Service of Clinical Trials and Databases, Institut de Myologie, Université Pierre et Marie Curie - Paris 6 (UPMC)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Association française contre les myopathies (AFM-Téléthon)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Association française contre les myopathies (AFM-Téléthon)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Généthon, Centre de recherche en Myologie – U974 SU-INSERM, Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU), 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), Centre de Boisbonne, Atlantic Gene Therapies, Ecole Nationale Vétérinaire Agroalimentaire et de l'Alimentation Nantes Atlantique (ONIRIS), Thérapie génique translationnelle pour les maladies neuromusculaires et de la rétine, Université de Nantes (UN)-Institut National de la Santé et de la Recherche Médicale (INSERM), Physiopathologie Animale et bioThérapie du muscle et du système nerveux (PAnTher), Institut National de la Recherche Agronomique (INRA)-École nationale vétérinaire, agroalimentaire et de l'alimentation Nantes-Atlantique (ONIRIS), Royal Holloway, University of London, Laboratoire de Résonance Magnétique Nucléaire (LRMN), Modélisation thérapeutique en neurologie: myologie et biothérapies des myopathies canines, Université Paris-Est Créteil Val-de-Marne - Paris 12 (UPEC UP12), Chercheur indépendant, Centre Hospitalier Vétérinaire Atlantia, Service de Cardiologie [CHU Cochin], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Hôpital Cochin [AP-HP], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP), CHU Saint-Antoine [AP-HP], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU), Laboratoire des Sciences de l'Information et des Systèmes (LSIS), Aix Marseille Université (AMU)-Université de Toulon (UTLN)-Arts et Métiers Paristech ENSAM Aix-en-Provence-Centre National de la Recherche Scientifique (CNRS), Approches génétiques intégrées et nouvelles thérapies pour les maladies rares (INTEGRARE), École Pratique des Hautes Études (EPHE), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Université d'Évry-Val-d'Essonne (UEVE)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Généthon, University of Wolverhampton, Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Association française contre les myopathies (AFM-Téléthon)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS), Thérapie des maladies du muscle strié, Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), UMR1089, Laboratoire de Thérapie Génique, Université de Nantes (UN), Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Association française contre les myopathies (AFM-Téléthon)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Association française contre les myopathies (AFM-Téléthon)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Pierre et Marie Curie - Paris 6 (UPMC), Institut National de la Recherche Agronomique (INRA)-Ecole Nationale Vétérinaire, Agroalimentaire et de l'alimentation Nantes-Atlantique (ONIRIS), Service de Cardiologie [CHU Saint-Antoine], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU), Centre National de la Recherche Scientifique (CNRS)-Arts et Métiers Paristech ENSAM Aix-en-Provence-Université de Toulon (UTLN)-Aix Marseille Université (AMU), École pratique des hautes études (EPHE), Laboratoire RMN, Institut de Myologie, Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Pierre et Marie Curie - Paris 6 (UPMC), Centre de recherche en myologie, Université Pierre et Marie Curie - Paris 6 (UPMC)-Association française contre les myopathies (AFM-Téléthon)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Université Pierre et Marie Curie - Paris 6 (UPMC)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Assistance publique - Hôpitaux de Paris (AP-HP) (APHP)-Association française contre les myopathies (AFM-Téléthon)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Assistance publique - Hôpitaux de Paris (AP-HP) (APHP)-Association française contre les myopathies (AFM-Téléthon)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), UMR 1089, Atlantic Gene Therapies, Développement et Pathologie du Tissu Musculaire (DPTM), Institut National de la Recherche Agronomique (INRA)-Ecole Nationale Vétérinaire de Nantes, Assistance publique - Hôpitaux de Paris (AP-HP) (APHP)-CHU Cochin [AP-HP], Assistance publique - Hôpitaux de Paris (AP-HP) (APHP)-CHU Saint-Antoine [APHP], Association française contre les myopathies (AFM-Téléthon), École pratique des hautes études (EPHE)-Université d'Évry-Val-d'Essonne (UEVE)-GENETHON 3-Institut National de la Santé et de la Recherche Médicale (INSERM), UMR 1089, Atlantic Gene Therapies & Genethon, Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Association française contre les myopathies (AFM-Téléthon)-Université Pierre et Marie Curie - Paris 6 (UPMC), Recherche et développement [Généthon, Evry] (R & D Généthon), Généthon [Evry], Hôpital Cochin [AP-HP], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP), Université d'Évry-Val-d'Essonne (UEVE)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Généthon-École pratique des hautes études (EPHE), and Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)

Subjects
0301 basic medicine, Male, myopathie de duchenne, Duchenne muscular dystrophy, Genetic enhancement, medicine.medical_treatment, General Physics and Astronomy, Bioinformatics, Dystrophin, Muscular Dystrophy, Vector (molecular biology), Transgenes, Muscular dystrophy, Multidisciplinary, biology, Gene Transfer Techniques, Immunosuppression, Skeletal, Dependovirus, 3. Good health, thérapie génique, Administration, Muscle, Administration, Intravenous, Intravenous, ITGA7, musculoskeletal diseases, congenital, hereditary, and neonatal diseases and abnormalities, Neuromuscular disease, Science, Genetic Vectors, Animals, Disease Models, Animal, Dogs, Genetic Therapy, Muscle, Skeletal, Muscular Dystrophy, Animal, Muscular Dystrophy, Duchenne, General Biochemistry, Genetics and Molecular Biology, Article, 03 medical and health sciences, medicine, Animal, business.industry, [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, General Chemistry, Duchenne, medicine.disease, 030104 developmental biology, Disease Models, biology.protein, business
Abstract

Duchenne muscular dystrophy (DMD) is an incurable X-linked muscle-wasting disease caused by mutations in the dystrophin gene. Gene therapy using highly functional microdystrophin genes and recombinant adeno-associated virus (rAAV) vectors is an attractive strategy to treat DMD. Here we show that locoregional and systemic delivery of a rAAV2/8 vector expressing a canine microdystrophin (cMD1) is effective in restoring dystrophin expression and stabilizing clinical symptoms in studies performed on a total of 12 treated golden retriever muscular dystrophy (GRMD) dogs. Locoregional delivery induces high levels of microdystrophin expression in limb musculature and significant amelioration of histological and functional parameters. Systemic intravenous administration without immunosuppression results in significant and sustained levels of microdystrophin in skeletal muscles and reduces dystrophic symptoms for over 2 years. No toxicity or adverse immune consequences of vector administration are observed. These studies indicate safety and efficacy of systemic rAAV-cMD1 delivery in a large animal model of DMD, and pave the way towards clinical trials of rAAV–microdystrophin gene therapy in DMD patients., Duchenne muscular dystrophy is a progressive degenerative disease of muscles caused by mutations in the dystrophin gene. Here the authors use AAV vectors to deliver microdystrophin to dogs with muscular dystrophy, and show restoration of dystrophin expression and reduction of symptoms up to 26 months of age.

Published
2017
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6. Non-ambulant Duchenne patients theoretically treatable by Exon 53 skipping have severe phenotype

Author

Laurent Servais, Marie Montus, Caroline Le Guiner, Rabah Ben Yaou, Mélanie Annoussamy, Amélie Moraux, Jean-Yves Hogrel, Andreea M. Seferian, Karima Zehrouni, Anne-Gaëlle Le Moing, Teresa Gidaro, Catherine Vanhulle, Vincent Laugel, Nina Butoianu, Jean-Marie Cuisset, Pascal Sabouraud, Claude Cances, Andrea Klein, France Leturcq, Philippe Moullier, Thomas Voit, Institut de Myologie, Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Association française contre les myopathies (AFM-Téléthon)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS), Approches génétiques intégrées et nouvelles thérapies pour les maladies rares (INTEGRARE), École Pratique des Hautes Études (EPHE), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Université d'Évry-Val-d'Essonne (UEVE)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Généthon, Généthon, 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), Service de pédiatrie néonatale et réanimation - neuropédiatrie [CHU Rouen], Hôpital Charles Nicolle [Rouen], CHU Rouen, Normandie Université (NU)-Normandie Université (NU)-CHU Rouen, Normandie Université (NU)-Normandie Université (NU)-Université de Rouen Normandie (UNIROUEN), Normandie Université (NU), Laboratoire de Génétique Médicale (LGM), Université de Strasbourg (UNISTRA)-Institut National de la Santé et de la Recherche Médicale (INSERM), Centre Hospitalier Universitaire de Toulouse (CHU Toulouse), University Children’s Hospital Zurich, Service de biochimie et de génétique moléculaire [CHU Cochin], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Hôpital Cochin [AP-HP], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP), Centre National de la Recherche Scientifique (CNRS)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Association française contre les myopathies (AFM-Téléthon)-Sorbonne Université (SU), École pratique des hautes études (EPHE), Hôpital Charles Nicolle [Rouen]-CHU Rouen, Hôpital Purpan [Toulouse], CHU Toulouse [Toulouse], University of Zurich, and Servais, L

Subjects
Research Report, musculoskeletal diseases, Duchenne muscular dystrophy, Pediatrics, medicine.medical_specialty, congenital, hereditary, and neonatal diseases and abnormalities, [SDV]Life Sciences [q-bio], Population, phenotype-genotype correlation, 610 Medicine & health, 03 medical and health sciences, Exon, outcome measures, 0302 clinical medicine, medicine, education, 030304 developmental biology, Muscle contracture, 0303 health sciences, education.field_of_study, Ejection fraction, business.industry, medicine.disease, Exon skipping, nervous system diseases, Clinical trial, 2728 Neurology (clinical), Neurology, 10036 Medical Clinic, 2808 Neurology, Cohort, Cancer research, Neurology (clinical), business, 030217 neurology & neurosurgery, exon skipping
Abstract

Background: Exon skipping therapy is an emerging approach in Duchenne Muscular Dystrophy (DMD). Antisense oligonucleotides that induce skipping of exon 51, 44, 45, or 53 are currently being evaluated in clinical trials. These trials were designed on the basis of data available in general DMD population. Objectives: Our objective was to compare the clinical and functional statuses of non-ambulant DMD patients theoretically treatable by exon 53 skipping and of DMD patients with other mutations. Methods: We first compared fifteen non-ambulant DMD patients carrying deletions theoretically treatable by exon 53 skipping (DMD-53) with fifteen closely age-matched DMD patients with mutations not treatable by exon 53 skipping (DMD-all-non-53) then with fifteen DMD patients carrying deletions not treatable by exon 53 skipping (DMD-del-non-53). Results: We found that DMD-53 patients had a lower left ventricular ejection fraction, more contractures and they tend to have weaker grips and pinch strengths than other DMD patients. DMD-53 patients lost ambulation significantly younger than other DMD patients. This result was confirmed by comparing ages at loss of ambulation in all non-ambulant DMD patients of the DMD cohort identified in a molecular diagnostic lab. Conclusions: These prospective and retrospective data demonstrate that DMD-53 patients have clinically more severe phenotypes than other DMD patients.

Published
2015
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7. Liver-specific transcriptional modules identified by genome-wide in silico analysis enable efficient gene therapy in mice and non-human primates

Author

Olivier D. Christophe, Janka Matrai, Marinee Chuah, Jessica Willems, Pieter De Bleser, Melvin Y Rincon, Bing Yan, Caroline Le Guiner, Véronique Blouin, Mario Di Matteo, Gwladys Gernoux, Philippe Moullier, Oumeya Adjali, Inge Petrus, Hanneke Evens, Nisha Nair, Ermira Samara-Kuko, Thierry VandenDriessche, Abel Acosta-Sanchez, Amine Meliani, Federico Mingozzi, Ghislaine Cherel, 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), Université libre de Bruxelles (ULB), Max Planck Institute for Chemistry (MPIC), Max-Planck-Gesellschaft, Immunologie moléculaire et biothérapies innovantes (IMBI), Généthon-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université d'Évry-Val-d'Essonne (UEVE)-École pratique des hautes études (EPHE), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL), Hémostase et biologie vasculaire, Université Paris-Sud - Paris 11 (UP11)-IFR93-Institut National de la Santé et de la Recherche Médicale (INSERM), Center for Molecular and Vascular Biology, Catholic University of Leuven - Katholieke Universiteit Leuven (KU Leuven), École Pratique des Hautes Études (EPHE), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Université d'Évry-Val-d'Essonne (UEVE)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Généthon, Division of Gene Therapy & Regenerative Medicine, Cell Biology and Histology, and Basic (bio-) Medical Sciences

Subjects
EXPRESSION, mice, DATABASE, Transgene, Genetic enhancement, In silico, [SDV]Life Sciences [q-bio], Computational biology, Biology, TRANSDUCTION, Conserved sequence, VIVO, 03 medical and health sciences, Transduction (genetics), 0302 clinical medicine, Gene expression, Drug Discovery, Medicine and Health Sciences, Genetics, Molecular Biology, 030304 developmental biology, Pharmacology, 0303 health sciences, SEQUENCES, Robustness (evolution), Gene Therapy, FACTOR-IX, EFFICACY, DNA binding site, MODEL, HEMOPHILIA-B, 030220 oncology & carcinogenesis, VECTORS, Molecular Medicine, Original Article, non-human primates, Liver-Specific Transcriptional Modules
Abstract

International audience; The robustness and safety of liver-directed gene therapy can be substantially improved by enhancing expression of the therapeutic transgene in the liver. To achieve this, we developed a new approach of rational in silico vector design. This approach relies on a genome-wide bio-informatics strategy to identify cis-acting regulatory modules (CRMs) containing evolutionary conserved clusters of transcription factor binding site motifs that determine high tissue-specific gene expression. Incorporation of these CRMs into adeno-associated viral (AAV) and non-viral vectors enhanced gene expression in mice liver 10 to 100-fold, depending on the promoter used. Furthermore, these CRMs resulted in robust and sustained liver-specific expression of coagulation factor IX (FIX), validating their immediate therapeutic and translational relevance. Subsequent translational studies indicated that therapeutic FIX expression levels could be attained reaching 20-35% of normal levels after AAV-based liver-directed gene therapy in cynomolgus macaques. This study underscores the potential of rational vector design using computational approaches to improve their robustness and therefore allows for the use of lower and thus safer vector doses for gene therapy, while maximizing therapeutic efficacy.

Published
2014
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8. Transgene regulation using the tetracycline-inducible TetR-KRAB system after AAV-mediated gene transfer in rodents and nonhuman primates

Author

Fabienne Rolling, Lydie Guigand, Alexandra Mendes-Madeira, Knut Stieger, Oumeya Adjali, Yan Cherel, Alice Toromanoff, Philippe Moullier, Caroline Le Guiner, Marie Devaux, Mickaël Guilbaud, Laboratoire de Thérapie Génique Translationnelle des Maladies Génétiques (Inserm UMR 1089), Université de Nantes (UN)-Institut National de la Santé et de la Recherche Médicale (INSERM), Department of Ophtalmology, Justus-Liebig-Universität Gießen (JLU), Développement et Pathologie du Tissu Musculaire (DPTM), Institut National de la Recherche Agronomique (INRA)-Ecole Nationale Vétérinaire de Nantes, 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), Physiopathologie Animale et bioThérapie du muscle et du système nerveux (PAnTher), Institut National de la Recherche Agronomique (INRA)-Ecole Nationale Vétérinaire, Agroalimentaire et de l'alimentation Nantes-Atlantique (ONIRIS), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Nantes (UN), and Ecole Nationale Vétérinaire de Nantes-Institut National de la Recherche Agronomique (INRA)

Subjects
Male, lcsh:Medicine, Transactivation, chemistry.chemical_compound, Mice, 0302 clinical medicine, Gene expression, Medicine and Health Sciences, Vector (molecular biology), Transgenes, lcsh:Science, Immune Response, Regulation of gene expression, Genetics, 0303 health sciences, Immunity, Cellular, Multidisciplinary, Gene Transfer Techniques, Genomics, Gene Therapy, Dependovirus, Cell biology, 030220 oncology & carcinogenesis, Doxycycline, Genetic Engineering, Research Article, Biotechnology, Transgene, Genetic Vectors, Immunology, Kruppel-Like Transcription Factors, Biology, Gene delivery, Retina, 03 medical and health sciences, Immune system, Genomic Medicine, Animals, Humans, TetR, Rats, Wistar, Muscle, Skeletal, Molecular Biology Techniques, Molecular Biology, 030304 developmental biology, Clinical Genetics, lcsh:R, Biology and Life Sciences, Tetracycline, Rats, Mice, Inbred C57BL, chemistry, Gene Expression Regulation, Macaca, lcsh:Q, [SDV.MHEP]Life Sciences [q-bio]/Human health and pathology
Abstract

Numerous studies have demonstrated the efficacy of the Adeno-Associated Virus (AAV)-based gene delivery platform in vivo. The control of transgene expression in many protocols is highly desirable for therapeutic applications and/or safety reasons. To date, the tetracycline and the rapamycin dependent regulatory systems have been the most widely evaluated. While the long-term regulation of the transgene has been obtained in rodent models, the translation of these studies to larger animals, especially to nonhuman primates (NHP), has often resulted in an immune response against the recombinant regulator protein involved in transgene expression regulation. These immune responses were dependent on the target tissue and vector delivery route. Here, using AAV vectors, we evaluated a doxycyclin-inducible system in rodents and macaques in which the TetR protein is fused to the human Kru ̈ ppel associated box (KRAB) protein. We demonstrated long term gene regulation efficiency in rodents after subretinal and intramuscular administration of AAV5 and AAV1 vectors, respectively. However, as previously described for other chimeric transactivators, the TetR-KRAB-based system failed to achieve long term regulation in the macaque after intramuscular vector delivery because of the development of an immune response. Thus, immunity against the chimeric transactivator TetR-KRAB emerged as the primary limitation for the clinical translation of the system when targeting the skeletal muscle, as previously described for other regulatory proteins. New developments in the field of chimeric drug-sensitive transactivators with the potential to not trigger the host immune system are still needed.

Published
2014
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9. Characterization of Dystrophin Deficient Rats: A New Model for Duchenne Muscular Dystrophy

Author

Laurent Tesson, Helicia Goubin, Jean-Baptiste Renaud, Thibaut Larcher, Caroline Le Guiner, Yan Cherel, Carine Giovannangeli, Corinne Huchet, Maeva Dutilleul, Ignacio Anegon, Gilles Toumaniantz, Anne De Cian, Lydie Guigand, Jean-Paul Concordet, Séverine Remy, Charlotte Boix, A. Lafoux, Virginie François, Virginie Thepenier, Physiopathologie Animale et bioThérapie du muscle et du système nerveux (PAnTher), Institut National de la Recherche Agronomique (INRA)-Ecole Nationale Vétérinaire, Agroalimentaire et de l'alimentation Nantes-Atlantique (ONIRIS), Atlantic Gene Therapies, 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 hospitalier universitaire de Nantes (CHU Nantes), Centre de Recherche en Transplantation et Immunologie (U1064 Inserm - CRTI), 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), Institut de Transplantation Urologie-Néphrologie (ITUN), Institut de Transplantation Urologie-Néphrologie ( ITUN), Laboratoire de Thérapie Génique Translationnelle des Maladies Génétiques (Inserm UMR 1089), Généthon, Structure et Instabilité des Génomes (STRING), Muséum national d'Histoire naturelle (MNHN)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Région Pays de la Loire (Biogenouest), IBiSA program, Fondation Progreffe, TEFOR (Infrastructures d'Avenir du Gouvernement Français), Développement et Pathologie du Tissu Musculaire (DPTM), Institut National de la Recherche Agronomique (INRA)-Ecole Nationale Vétérinaire de Nantes, 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)-Institut National de la Santé et de la Recherche Médicale (INSERM), Muséum national d'Histoire naturelle (MNHN)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Ecole Nationale Vétérinaire de Nantes-Institut National de la Recherche Agronomique (INRA), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Nantes (UN), APEX - Plate-forme d'expertise en anatomie pathologique pour la Recherche [Nantes] (PAnTher), Ecole Nationale Vétérinaire, Agroalimentaire et de l'alimentation Nantes-Atlantique (ONIRIS)-INRA - Oniris (INRA UMR0703), Institut National de la Recherche Agronomique (INRA)-Institut National de la Recherche Agronomique (INRA), Institut de transplantation urologie-néphrologie (ITUN), Université de Nantes (UN)-Centre hospitalier universitaire de Nantes (CHU Nantes), Thérapie génique translationnelle pour les maladies neuromusculaires et de la rétine, Recherche et développement [Généthon, Evry] (R & D Généthon), Généthon [Evry], Muséum national d'Histoire naturelle (MNHN)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS), Région Pays de la Loire through Biogenouest, IBiSA program, Fondation Progreffe and TEFOR (Infrastructures d’Avenir of the French goverment), and the integrative genomic facility of Nantes for sequencing experiments., Muséum national d'Histoire naturelle (MNHN)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut de Chimie du CNRS (INC)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS), Institut National de la Recherche Agronomique (INRA)-École nationale vétérinaire, agroalimentaire et de l'alimentation Nantes-Atlantique (ONIRIS), Unité de recherche de l'institut du thorax (ITX-lab), Le Bihan, Sylvie, and Lafoux, Aude

Subjects
Male, Pathology, Necrosis, myopathie de duchenne, Physiology, Duchenne muscular dystrophy, Gene Expression, Adipose tissue, Dystrophin, 0302 clinical medicine, Medicine and Health Sciences, Medicine, rat, Muscular dystrophy, Creatine Kinase, 0303 health sciences, Muscle Weakness, [SDV.MHEP] Life Sciences [q-bio]/Human health and pathology, Multidisciplinary, Ventricular Remodeling, biology, Dilated cardiomyopathy, Exons, Diaphragm (structural system), Neurology, Gene Targeting, Female, medicine.symptom, Research Article, Biotechnology, musculoskeletal diseases, medicine.medical_specialty, congenital, hereditary, and neonatal diseases and abnormalities, Science, Médecine humaine et pathologie, modèle d'analyse, 03 medical and health sciences, Genetics, Congenital Disorders, Animals, RNA, Messenger, Muscle, Skeletal, Molecular Biology, 030304 developmental biology, Base Sequence, business.industry, Myocardium, Biology and Life Sciences, Muscle weakness, Cell Biology, Muscular Dystrophy, Animal, medicine.disease, Fibrosis, Rats, Muscular Dystrophy, Duchenne, Disease Models, Animal, Mutation, biology.protein, Human health and pathology, business, mutant déficient, Gene Deletion, 030217 neurology & neurosurgery, [SDV.MHEP]Life Sciences [q-bio]/Human health and pathology
Abstract

International audience; A few animal models of Duchenne muscular dystrophy (DMD) are available, large ones such as pigs or dogs being expensive and difficult to handle. Mdx (X-linked muscular dystrophy) mice only partially mimic the human disease, with limited chronic muscular lesions and muscle weakness. Their small size also imposes limitations on analyses. A rat model could represent a useful alternative since rats are small animals but 10 times bigger than mice and could better reflect the lesions and functional abnormalities observed in DMD patients. Two lines of Dmd mutated-rats (Dmd[mdx]) were generated using TALENs targeting exon 23. Muscles of animals of both lines showed undetectable levels of dystrophin by western blot and less than 5% of dystrophin positive fibers by immunohistochemistry. At 3 months, limb and diaphragm muscles from Dmd[mdx] rats displayed severe necrosis and regeneration. At 7 months, these muscles also showed severe fibrosis and some adipose tissue infiltration. Dmd[mdx] rats showed significant reduction in muscle strength and a decrease in spontaneous motor activity. Furthermore, heart morphology was indicative of dilated cardiomyopathy associated histologically with necrotic and fibrotic changes. Echocardiography showed significant concentric remodeling and alteration of diastolic function. In conclusion, Dmd[mdx] rats represent a new faithful small animal model of DMD.

Published
2014
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10. Gene transfer of human CD40Ig does not prevent rejection in a non-human primate kidney allotransplantation model

Author

Antoine Blancher, Nahzli Dilek, Jeremy Hervouet, Yan Cherel, Jack-Yves Deschamps, Alice Toromanoff, Bernard Vanhove, Caroline Le Guiner, Gilles Blancho, David Minault, Xavier Tillou, Brigitte Le Mauff, Karine Renaudin, Nicolas Poirier, Ignacio Anegon, Mathieu Angin, ProdInra, Migration, Institut National de la Santé et de la Recherche Médicale (INSERM), UMR1089, Laboratoire d'Immunogénétique Moléculaire (LIMT), Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées, Physiopathologie Animale et bioThérapie du muscle et du système nerveux (PAnTher), Institut National de la Recherche Agronomique (INRA)-École nationale vétérinaire, agroalimentaire et de l'alimentation Nantes-Atlantique (ONIRIS), Service d'Anatomie Pathologique, Centre Hospitalier Universitaire G. R. Laennec (HGRL Saint-Herblain)-Centre hospitalier universitaire de Nantes (CHU Nantes), CHU Caen, Normandie Université (NU)-Tumorothèque de Caen Basse-Normandie (TCBN), Institut National de la Recherche Agronomique (INRA)-Ecole Nationale Vétérinaire, Agroalimentaire et de l'alimentation Nantes-Atlantique (ONIRIS), and Centre hospitalier universitaire de Nantes (CHU Nantes)-Centre Hospitalier Universitaire G. R. Laennec (HGRL Saint-Herblain)

Subjects
Graft Rejection, Male, medicine.medical_treatment, primate, 0302 clinical medicine, Immunology and Allergy, 0303 health sciences, [SDV.MHEP] Life Sciences [q-bio]/Human health and pathology, biology, Graft Survival, Gene Transfer Techniques, CD28, hemic and immune systems, Dependovirus, Mixed lymphocyte reaction, 3. Good health, Models, Animal, Veterinary medicine and animal Health, Antibody, Recombinant Fusion Proteins, Immunology, CD40 Ligand, Genetic Vectors, Médecine humaine et pathologie, Immunoglobulins, 03 medical and health sciences, Immune system, Transplantation Immunology, medicine, Animals, Humans, Transplantation, Homologous, CD40 Antigens, 030304 developmental biology, Transplantation, [SDV.BA.MVSA]Life Sciences [q-bio]/Animal biology/Veterinary medicine and animal Health, tolérance, [SDV.BA.MVSA] Life Sciences [q-bio]/Animal biology/Veterinary medicine and animal Health, Fusion protein, Kidney Transplantation, Blockade, Immunity, Humoral, Macaca fascicularis, Médecine vétérinaire et santé animal, biology.protein, Human health and pathology, [SDV.MHEP]Life Sciences [q-bio]/Human health and pathology, 030215 immunology, Allotransplantation
Abstract

Background Blockade of costimulation signaling required for immune response, such as CD40/CD40L and CD28/B7, is a reasonable strategy to prevent rejection and in defined combinations may allow donor specific tolerance. Indeed, in rodents, costimulation blockade with CD28/B7 antagonists or with CD40Ig was able to induce regulatory T cells and transplant tolerance whereas in primates, anti-CD40 antibodies, anti-CD40L antibodies or CTLA4Ig, used as monotherapy, significantly delayed graft rejection. Methods Using an adeno-associated virus (AAV) vector mediated gene transfer of a human CD40Ig fusion protein (hCD40Ig) in primates, we evaluated the capacity of this costimulation blockade molecule interfering with CD40/CD40L signaling in prolonging kidney transplants in cynomolgus monkeys. Results This gene transfer strategy allowed for maintaining a plateau of hCD40Ig production within two months and avoided a high-scale production phase of this molecule. Although the hCD40Ig was able to bind efficiently to human and macaque CD40L and high (> 200 μg/ml) transgene expression was obtained, no effect on graft survival was observed. In addition, there was no inhibition of humoral response to vaccination. In vitro , hCD40Ig strongly increased mixed lymphocyte reaction, and when compared to the anti-CD40L antibody h5C8, was not as potent to induce complement-dependent cytotoxicity. Conclusion These data suggest that CD40/CD40L blockade using a non-depleting CD40Ig fusion protein, a therapeutic strategy that showed efficacy in rodents, is not able to modulate the immune response in primates. These data highlight important biological differences between rodent and primate models to evaluate therapeutic strategies at the preclinical level.

Published
2012

11. Lack of Immunotoxicity After Regional Intravenous (RI) Delivery of rAAV to Nonhuman Primate Skeletal Muscle

Author

Fabienne Rolling, Gilles Blancho, Jack-Yves Deschamps, Oumeya Adjali, Olivier Gauthier, Lydie Guigand, Thibaut Larcher, Yan Cherel, Alice Toromanoff, Marcelo Hill, Philippe Moullier, Pierre Chenuaud, Bernard Vanhove, Caroline Le Guiner, Ignacio Anegon, ProdInra, Migration, UMR649, 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], Développement et Pathologie du Tissu Musculaire (DPTM), Institut National de la Recherche Agronomique (INRA)-Ecole Nationale Vétérinaire de Nantes, UMR643, École nationale vétérinaire, agroalimentaire et de l'alimentation Nantes-Atlantique (ONIRIS), Department of Molecular Genetics and Microbiology [Gainesville] (UF|MGM), Department of Medicine [Gainesville] (UF|Medicine), University of Florida [Gainesville] (UF)-University of Florida [Gainesville] (UF), and Ecole Nationale Vétérinaire, Agroalimentaire et de l'alimentation Nantes-Atlantique (ONIRIS)

Subjects
Male, CANINE HEMOPHILIA-B, viruses, [SDV]Life Sciences [q-bio], Muscle Fibers, Skeletal, T-CELL, law.invention, Transduction (genetics), 0302 clinical medicine, Transduction, Genetic, law, Drug Discovery, MEDIATED GENE-TRANSFER, IMMUNE-RESPONSE, In Situ Hybridization, 0303 health sciences, Reverse Transcriptase Polymerase Chain Reaction, Dependovirus, Immunohistochemistry, 3. Good health, Genetically modified organism, [SDV] Life Sciences [q-bio], medicine.anatomical_structure, 030220 oncology & carcinogenesis, Injections, Intravenous, Recombinant DNA, Molecular Medicine, REGULATED TRANSGENE EXPRESSION, COAGULATION FACTOR-IX, RECOMBINANT-ADENOASSOCIATED-VIRUS, INTRAMUSCULAR INJECTION, Transgene, Genetic Vectors, Enzyme-Linked Immunosorbent Assay, In situ hybridization, Biology, Injections, Intramuscular, Virus, LIPOPROTEIN-LIPASE DEFICIENCY, 03 medical and health sciences, Immune system, medicine, Genetics, Animals, Muscle, Skeletal, Molecular Biology, 030304 developmental biology, Pharmacology, Skeletal muscle, Original Articles, LONG-TERM CORRECTION, Immunology, Macaca
Abstract

International audience; In the absence of an immune response from the host, intramuscular (IM) injection of recombinant adeno-associated virus (rAAV) results in the permanent expression of the transgene from mouse to primate models. However, recent gene transfer studies into animal models and humans indicate that the risk of transgene and/or capsid-specific immune responses occurs and depends on multiple factors. Among these factors, the route of delivery is important, although poorly addressed in large animal models. Here, we compare the IM and the drug-free regional intravenous (RI) deliveries of rAAV in nonhuman primate (NHP) skeletal muscle monitoring the host immune response toward the transgene. We show that IM is consistently associated with immunotoxicity and the destruction of the genetically modified myofibers, whereas RI allows the stable expression of the transgene. This has important implications for the design of clinical trials for gene transfer in skeletal muscle.

Published
2010
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12. Safety and efficacy of regional intravenous (RI) versus intramuscular (IM) delivery of rAAV1 and rAAV8 to nonhuman primate skeletal muscle

Author

Fabienne Rolling, Katherine A. High, Valder R. Arruda, Magalie Penaud-Budloo, Yan Cherel, Richard O. Snyder, Alice Toromanoff, Mickaël Guilbaud, Ignacio Anegon, Philippe Moullier, Jack-Yves Deschamps, Hansell H. Stedman, Guillaume Podevin, Lydie Guigand, Mark E. Haskins, Caroline Le Guiner, UMR649, Institut National de la Santé et de la Recherche Médicale (INSERM), Centre hospitalier universitaire de Nantes (CHU Nantes), Université de Nantes (UN), Développement et Pathologie du Tissu Musculaire (DPTM), Institut National de la Recherche Agronomique (INRA)-Ecole Nationale Vétérinaire de Nantes, Etablissement Français du Sang, Department of Molecular Genetics and Microbiology [Gainesville] (UF|MGM), Department of Medicine [Gainesville] (UF|Medicine), University of Florida [Gainesville] (UF)-University of Florida [Gainesville] (UF), University of Pennsylvania, Ecole Nationale Vétérinaire de Nantes, Howard Hughes Medical Institute (HHMI), and UMR643

Subjects
Male, Biodistribution, RECOMBINANT-ADENOASSOCIATED-VIRUS, Transgene, [SDV]Life Sciences [q-bio], Genetic Vectors, Cell, Gene Expression, Biology, Injections, Intramuscular, Virus, law.invention, DUCHENNE MUSCULAR-DYSTROPHY, 03 medical and health sciences, Transduction (genetics), 0302 clinical medicine, Transduction, Genetic, law, Drug Discovery, MEDIATED GENE-TRANSFER, medicine, Genetics, Animals, VECTOR GENOMES, Transgenes, Muscle, Skeletal, Molecular Biology, 030304 developmental biology, Southern blot, Pharmacology, 0303 health sciences, AUTOIMMUNE ANEMIA, IMMUNE-RESPONSES, Skeletal muscle, Dependovirus, SEVERE HEMOPHILIA-B, FACTOR-IX, Virology, 3. Good health, Macaca fascicularis, medicine.anatomical_structure, 030220 oncology & carcinogenesis, DNA, Viral, Injections, Intravenous, Recombinant DNA, T-CELLS, Molecular Medicine, REGULATED TRANSGENE EXPRESSION
Abstract

International audience; We developed a drug-free regional intravenous (RI) delivery protocol of recombinant adeno-associated virus (rAAV) 1 and 8 to an entire limb in the nonhuman primate (NHP), and compared the results with those produced by intramuscular (IM) delivery of the same dose of vector. We show that RI delivery of both serotypes was remarkably well tolerated with no adverse side-effects. After IM, muscle transduction was restricted to the site of injection with a high number of vector copies per cell for rAAV1. In contrast, although RI delivery resulted in a lower vector copy per cell, it was detectable in the vast majority of muscles of the injected limb. The amounts of circulating infectious rAAV were similar for both serotypes and modes of delivery. At autopsy at up to 34 months after vector administration, similar biodistribution patterns were found for both vectors and for both modes of delivery, with numerous organs found to be positive for vector sequence when assayed using PCR and Southern blot. Altogether, we demonstrated that RI is a simple and efficient transduction protocol in NHPs, resulting in higher expression of the transgene with a lower number of vector genomes per cell. However, regardless of the mode of delivery, concerns continue to be raised by the presence of vector sequences detected at distant sites.

Published
2008
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