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1. Correction of the Exon 2 Duplication in DMD Myoblasts by a Single CRISPR/Cas9 System

Author

Stephanie Duguez, Annalisa Lattanzi, Kamel Mamchaoui, Araksya Izmiryan, Fulvio Mavilio, Samia Martin, Matteo Bovolenta, Francesco Bernardi, Arianna Moiani, Elena Barbon, Vincent Mouly, Généthon, Thérapie des maladies du muscle strié, 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), Sorbonne Université (SU), 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), Généthon, Evry, GENETHON, Genethon, Association française contre les myopathies (AFM-Téléthon), 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), Genethon - Inserm UMR_S951, Immunologie moléculaire et biothérapies innovantes (IMBI), GENETHON 3-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)-GENETHON 3-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)-Généthon, and Association française contre les myopathies (AFM-Téléthon)-Association française contre les myopathies (AFM-Téléthon)

Subjects
0301 basic medicine, [SDV]Life Sciences [q-bio], CRISPR/Cas9, duplication, dystrophin, gene editing, lentivirus, Socio-culturale, Biology, 03 medical and health sciences, Exon, 0302 clinical medicine, Economica, Genome editing, 030225 pediatrics, Drug Discovery, Gene duplication, dystrophin, CRISPR/Cas9, duplication, lentivirus, gene editing, medicine, Myocyte, CRISPR, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Guide RNA, Muscular dystrophy, Genetics (clinical), Genetics, [SDV.GEN]Life Sciences [q-bio]/Genetics, Cas9, lcsh:RM1-950, Ambientale, [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, medicine.disease, lcsh:Therapeutics. Pharmacology, 030104 developmental biology, Neurology, Pediatrics, Perinatology and Child Health, biology.protein, Molecular Medicine, Original Article, Neurology (clinical), Dystrophin, 030217 neurology & neurosurgery
Abstract

Exonic duplications account for 10%–15% of all mutations in Duchenne muscular dystrophy (DMD), a severe hereditary neuromuscular disorder. We report a CRISPR (clustered regularly interspaced short palindromic repeat)/Cas9-based strategy to correct the most frequent (exon 2) duplication in the DMD gene by targeted deletion, and tested the efficacy of such an approach in patient-derived myogenic cells. We demonstrate restoration of wild-type dystrophin expression at transcriptional and protein level in myotubes derived from genome-edited myoblasts in the absence of selection. Removal of the duplicated exon was achieved by the use of only one guide RNA (gRNA) directed against an intronic duplicated region, thereby increasing editing efficiency and reducing the risk of off-target effects. This study opens a novel therapeutic perspective for patients carrying disease-causing duplications., Graphical Abstract

Published
2017
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2. Constraints on Human CD34+ Cell Fate due to Lentiviral Vectors Can Be Relieved by Valproic Acid

Author

Guillaume Corre, Rudiyanto Gunawan, Alice Moussy, Saliha Majdoul, Andras Paldi, Nan Papili Gao, David Fenard, Valentina Poletti, Daniel Stockholm, Approches génétiques intégrées et nouvelles thérapies pour les maladies rares (INTEGRARE), 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), Università degli Studi di Modena e Reggio Emilia (UNIMORE), Immunologie moléculaire et biothérapies innovantes, École pratique des hautes études (EPHE)-Université d'Évry-Val-d'Essonne (UEVE)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Genethon, Genethon - Inserm UMR_S951, École pratique des hautes études (EPHE)-Université d'Évry-Val-d'Essonne (UEVE)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Genethon-École pratique des hautes études (EPHE)-Université d'Évry-Val-d'Essonne (UEVE)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Genethon-Généthon, Association française contre les myopathies (AFM-Téléthon)-Association française contre les myopathies (AFM-Téléthon), Généthon, Genethon, Institut des cellules souches pour le traitement et l'étude des maladies monogéniques (I-STEM), Université d'Évry-Val-d'Essonne (UEVE)-Institut National de la Santé et de la Recherche Médicale (INSERM), 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, Institut National de la Santé et de la Recherche Médicale (INSERM)-Généthon-Université d'Évry-Val-d'Essonne (UEVE), École Pratique des Hautes Études (EPHE), Università degli Studi di Modena e Reggio Emilia = University of Modena and Reggio Emilia (UNIMORE), Université d'Évry-Val-d'Essonne (UEVE)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Généthon, and ANR-17-CE12-0031,SinCity,Analyses transcriptomiques sur cellules uniques dont la généalogie est identifiée au cours d'un processus de différentiation(2017)

Subjects
Myeloid, [SDV.BIO]Life Sciences [q-bio]/Biotechnology, medicine.drug_class, Virus Integration, Genetic Vectors, CD34, Gene Expression, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, Viral vector, 03 medical and health sciences, Transduction (genetics), 0302 clinical medicine, Transduction, Genetic, Genetics, medicine, Humans, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Transgenes, Molecular Biology, ComputingMilieux_MISCELLANEOUS, 030304 developmental biology, Progenitor, 0303 health sciences, [SDV.GEN]Life Sciences [q-bio]/Genetics, Chemistry, Valproic Acid, Histone deacetylase inhibitor, Lentivirus, Gene Transfer Techniques, Hematopoietic stem cell, Cell Differentiation, Fetal Blood, Hematopoietic Stem Cells, [SDV.BIBS]Life Sciences [q-bio]/Quantitative Methods [q-bio.QM], Cell biology, Crosstalk (biology), medicine.anatomical_structure, Phenotype, 030220 oncology & carcinogenesis, Molecular Medicine, [SDV.IMM]Life Sciences [q-bio]/Immunology, Biomarkers
Abstract

The initial stages following the in vitro cytokine stimulation of human cord blood CD34+ cells overlap with the period when lentiviral gene transfer is typically performed. Single-cell transcriptional profiling and time-lapse microscopy were used to investigate how the vector-cell crosstalk impacts on the fate decision process. The single-cell transcription profiles were analyzed using a new algorithm, and it is shown that lentiviral transduction during the early stages of stimulation modifies the dynamics of the fate choice process of the CD34+ cells. The cells transduced with a lentiviral vector are biased toward the common myeloid progenitor lineage. Valproic acid, a histone deacetylase inhibitor known to increase the grafting potential of the CD34+ cells, improves the transduction efficiency to almost 100%. The cells transduced in the presence of valproic acid can subsequently undergo normal fate commitment. The higher gene transfer efficiency did not alter the genomic integration profile of the vector. These observations open the way to substantially improving lentiviral gene transfer protocols.

Published
2019
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3. Baboon envelope LVs efficiently transduced human adult, fetal, and progenitor T cells and corrected SCID-X1 T-cell deficiency

Author

David Fenard, Ornellie Bernadin, Anais Girard-Gagnepain, Camille Lévy, Chantal Lagresle-Peyrou, Kuiying Ma, Rana Mhaidly, Hanem Sadek, Marina Cavazzana, Fouzia Amirache, Caroline Costa, Ranjita Devi Moirangthem, Isabelle André, François-Loïc Cosset, Els Verhoeyen, Christian Reimann, Didier Nègre, Agata Cieslak, Vahid Asnafi, Virus enveloppés, vecteurs et immunothérapie – Enveloped viruses, Vectors and Immuno-therapy (EVIR), Centre International de Recherche en Infectiologie - UMR (CIRI), Institut National de la Santé et de la Recherche Médicale (INSERM)-École normale supérieure - Lyon (ENS Lyon)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-École normale supérieure - Lyon (ENS Lyon)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS), Human Lymphohematopoiesis Laboratory (Equipe Inserm U1163), Imagine - Institut des maladies génétiques (IMAGINE - U1163), Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM), Lucerne Cantonal Hospital [Luzern, Switzerland], Généthon, CHU Necker - Enfants Malades [AP-HP], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP), Institut Necker Enfants-Malades (INEM - UM 111 (UMR 8253 / U1151)), Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM), Clinical Investigation Center in Biotherapy, Institut National de la Santé et de la Recherche Médicale (INSERM), Centre méditerranéen de médecine moléculaire (C3M), Université Nice Sophia Antipolis (... - 2019) (UNS), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Côte d'Azur (UCA), Virus enveloppés, vecteurs et immunothérapie – Enveloped viruses, Vectors and Immuno-therapy (Equipe EVIR), École normale supérieure - Lyon (ENS Lyon)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-École normale supérieure - Lyon (ENS Lyon)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Genethon - Inserm UMR_S951, Immunologie moléculaire et biothérapies innovantes (IMBI), GENETHON 3-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)-GENETHON 3-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)-Généthon, Association française contre les myopathies (AFM-Téléthon)-Association française contre les myopathies (AFM-Téléthon), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Côte d'Azur (UCA)-Université Nice Sophia Antipolis (... - 2019) (UNS), COMUE Université Côte d'Azur (2015 - 2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015 - 2019) (COMUE UCA), Centre National de la Recherche Scientifique (CNRS)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM), Immunologie moléculaire et biothérapies innovantes, École pratique des hautes études (EPHE)-Université d'Évry-Val-d'Essonne (UEVE)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Genethon-École pratique des hautes études (EPHE)-Université d'Évry-Val-d'Essonne (UEVE)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Genethon-Généthon, Centre National de la Recherche Scientifique (CNRS)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM), Université Côte d'Azur (UCA)-Université Côte d'Azur (UCA)-Institut National de la Santé et de la Recherche Médicale (INSERM), Centre International de Recherche en Infectiologie (CIRI), École normale supérieure de Lyon (ENS de Lyon)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Université Jean Monnet - Saint-Étienne (UJM)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-École normale supérieure de Lyon (ENS de Lyon)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Université Jean Monnet - Saint-Étienne (UJM)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Université Nice Sophia Antipolis (1965 - 2019) (UNS), Human Virology Department, Virologie humaine, École normale supérieure - Lyon (ENS Lyon)-IFR128-Institut National de la Santé et de la Recherche Médicale (INSERM), Developpement Normal et Pathologique du Système Immunitaire, Université Nice Sophia Antipolis (UNS), Département de Biothérapie [CHU Necker], Assistance publique - Hôpitaux de Paris (AP-HP) (APHP)-Université Paris Descartes - Paris 5 (UPD5)-CHU Necker - Enfants Malades [AP-HP], Laboratoire d'Ingénierie des Systèmes Biologiques et des Procédés (LISBP), Centre National de la Recherche Scientifique (CNRS)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), and Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut National de la Recherche Agronomique (INRA)

Subjects
0301 basic medicine, medicine.medical_treatment, Genetic enhancement, [SDV]Life Sciences [q-bio], CD34, chemical and pharmacologic phenomena, Mice, SCID, Hematopoietic stem cell transplantation, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, Biology, complex mixtures, Mice, 03 medical and health sciences, 0302 clinical medicine, Mice, Inbred NOD, medicine, Animals, Progenitor cell, ComputingMilieux_MISCELLANEOUS, Severe combined immunodeficiency, integumentary system, Stem Cells, Lentivirus, hemic and immune systems, Gene Therapy, Hematology, bacterial infections and mycoses, medicine.disease, T cell deficiency, 3. Good health, Cell biology, Haematopoiesis, 030104 developmental biology, 030220 oncology & carcinogenesis, Stem cell, Papio
Abstract

T cells represent a valuable tool for treating cancers and infectious and inherited diseases; however, they are mainly short-lived in vivo. T-cell therapies would strongly benefit from gene transfer into long-lived persisting naive T cells or T-cell progenitors. Here we demonstrate that baboon envelope glycoprotein pseudotyped lentiviral vectors (BaEV-LVs) far outperformed other LV pseudotypes for transduction of naive adult and fetal interleukin-7–stimulated T cells. Remarkably, BaEV-LVs efficiently transduced thymocytes and T-cell progenitors generated by culture of CD34+ cells on Delta-like ligand 4 (Dll4). Upon NOD/SCIDγC−/− engraftment, high transduction levels (80%-90%) were maintained in all T-cell subpopulations. Moreover, T-cell lineage reconstitution was accelerated in NOD/SCIDγC−/− recipients after T-cell progenitor injection compared with hematopoietic stem cell transplantation. Furthermore, γC-encoding BaEV-LVs very efficiently transduced Dll4-generated T-cell precursors from a patient with X-linked severe combined immunodeficiency (SCID-X1), which fully rescued T-cell development in vitro. These results indicate that BaEV-LVs are valuable tools for the genetic modification of naive T cells, which are important targets for gene therapy. Moreover, they allowed for the generation of gene-corrected T-cell progenitors that rescued SCID-X1 T-cell development in vitro. Ultimately, the coinjection of LV-corrected T-cell progenitors and hematopoietic stem cells might accelerate T-cell reconstitution in immunodeficient patients.

Published
2019
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4. Dosage Thresholds and Influence of Transgene Cassette in Adeno-Associated Virus–Related Toxicity

Author

Hanen Khabou, Deniz Dalkara, Laure Pacot, Sylvain Fisson, Chloé Cordeau, Institut de la Vision, Centre National de la Recherche Scientifique (CNRS)-Sorbonne Université (SU)-Institut National de la Santé et de la Recherche Médicale (INSERM), Généthon, Genethon - Inserm UMR_S951, Immunologie moléculaire et biothérapies innovantes (IMBI), GENETHON 3-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)-GENETHON 3-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)-Généthon, and Association française contre les myopathies (AFM-Téléthon)-Association française contre les myopathies (AFM-Téléthon)

Subjects
0301 basic medicine, Retinal Disorder, MESH: gene therapy, retina, AAV, GFP, toxicity, promoter, Transgene, Genetic enhancement, viruses, Genetic Vectors, Green Fluorescent Proteins, Gene Dosage, medicine.disease_cause, Virus, Green fluorescent protein, 03 medical and health sciences, Capsid, 0302 clinical medicine, Genetics, medicine, Animals, Transgenes, Molecular Biology, Adeno-associated virus, Retina, [SDV.GEN]Life Sciences [q-bio]/Genetics, business.industry, Immunity, Genetic Therapy, Dependovirus, Virology, 3. Good health, Mice, Inbred C57BL, 030104 developmental biology, medicine.anatomical_structure, [SDV.TOX]Life Sciences [q-bio]/Toxicology, Toxicity, Molecular Medicine, business, 030217 neurology & neurosurgery
Abstract

International audience; Today, there are >500 published studies and 40 clinical trials to treat retinal disorders using gene therapy. The great majority of them rely on the use of adeno-associated virus vectors (AAV) for therapeutic gene delivery. Thus far, AAVs have an excellent safety profile in the clinic. Nevertheless, it is known that AAV-mediated gene delivery leads to toxicity at higher input doses in experimental gene therapy. This study reveals the factors that contribute to retinal toxicity after subretinal administration of AAV vectors in wild-type mice. The study shows that alongside the input dose, the nature of the transgene and the cells mediating the expression determine the extent of toxicity. Importantly, the study shows that AAV vectors encoding green fluorescent protein (GFP) used as controls in experimental gene therapy are toxic at doses as low as 5 × 109 vg, confounding the observed therapeutic effect in gene therapy paradigms. Altogether, the data show the importance of reducing input doses while increasing transgene expression levels via the use of more efficient capsids and promoters in order to avoid side effects in AAV-mediated gene therapy. Furthermore, the toxicity observed with AAV-GFP vectors imply a reinterpretation of previous gene therapy studies where the therapeutic effect was measured in relation to this control.

Published
2018
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5. Peptides derived from evolutionarily conserved domains in Beclin-1 and Beclin-2 enhance the entry of lentiviral vectors into human cells

Author

Sophie Frin, Nathalie Chazal, Saliha Majdoul, Lucile Espert, Laurence Briant, David Fenard, Nathalie Holic, Jérémie Cosette, Anne Galy, Ababacar K. Seye, Eric Bernard, 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, Institut de Recherche en Infectiologie de Montpellier (IRIM), Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS), Université d'Évry-Val-d'Essonne (UEVE)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris-Saclay-Généthon, Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM), École pratique des hautes études (EPHE), Immunologie moléculaire et biothérapies innovantes, École pratique des hautes études (EPHE)-Université d'Évry-Val-d'Essonne (UEVE)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Genethon, Centre de Recherche des Cordeliers (CRC (UMR_S 872)), Université Pierre et Marie Curie - Paris 6 (UPMC)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Infections rétrovirales et signalisation cellulaire (IRSC), Université Montpellier 1 (UM1)-Centre National de la Recherche Scientifique (CNRS), Groupe Immunologie et Thérapie Génique, Université Paris-Sud - Paris 11 (UP11)-Institut National de la Santé et de la Recherche Médicale (INSERM), Généthon, Evry, Genethon, Centre d’études d’Agents Pathogènes et Biotechologies pour la Santé (CPBS), Genethon - Inserm UMR_S951, École pratique des hautes études (EPHE)-Université d'Évry-Val-d'Essonne (UEVE)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Genethon-École pratique des hautes études (EPHE)-Université d'Évry-Val-d'Essonne (UEVE)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Genethon-Généthon, Association française contre les myopathies (AFM-Téléthon)-Association française contre les myopathies (AFM-Téléthon), Association française contre les myopathies (AFM-Téléthon), École pratique des hautes études ( EPHE ) -Université d'Évry-Val-d'Essonne ( UEVE ) -Institut National de la Santé et de la Recherche Médicale ( INSERM ) -Genethon, Centre de Recherche des Cordeliers ( CRC (UMR_S 872) ), Université Pierre et Marie Curie - Paris 6 ( UPMC ) -Université Paris Descartes - Paris 5 ( UPD5 ) -Institut National de la Santé et de la Recherche Médicale ( INSERM ) -Centre National de la Recherche Scientifique ( CNRS ), Institut de Recherche en Infectiologie de Montpellier ( IRIM ), Université de Montpellier ( UM ) -Centre National de la Recherche Scientifique ( CNRS ), Infections rétrovirales et signalisation cellulaire ( IRSC ), Université Montpellier 1 ( UM1 ) -Centre National de la Recherche Scientifique ( CNRS ), Université Paris-Sud - Paris 11 ( UP11 ) -Institut National de la Santé et de la Recherche Médicale ( INSERM ), and Chazal, Nathalie

Subjects
0301 basic medicine, [SDV.BIO]Life Sciences [q-bio]/Biotechnology, Genetic enhancement, [SDV]Life Sciences [q-bio], Biochemistry, Transduction (genetics), 0302 clinical medicine, Cells, Cultured, Conserved Sequence, ComputingMilieux_MISCELLANEOUS, Cell Line, Transformed, [SDV.MP.VIR] Life Sciences [q-bio]/Microbiology and Parasitology/Virology, Hematopoietic Stem Cell Transplantation, Intracellular Signaling Peptides and Proteins, Specific Pathogen-Free Organisms, Up-Regulation, 3. Good health, Cell biology, 030220 oncology & carcinogenesis, [SDV.MP.VIR]Life Sciences [q-bio]/Microbiology and Parasitology/Virology, Beclin-1, tat Gene Products, Human Immunodeficiency Virus, Recombinant Fusion Proteins, Protein domain, Mice, Transgenic, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, Biology, [ SDV.MP.VIR ] Life Sciences [q-bio]/Microbiology and Parasitology/Virology, Viral vector, 03 medical and health sciences, Viral entry, Cell Line, Tumor, Autophagy, Animals, Humans, Amino Acid Sequence, Progenitor cell, Enhancer, Molecular Biology, [SDV.BC] Life Sciences [q-bio]/Cellular Biology, Lentivirus, Genetic Therapy, Cell Biology, Virus Internalization, Hematopoietic Stem Cells, Virology, Peptide Fragments, [SDV.BIO] Life Sciences [q-bio]/Biotechnology, 030104 developmental biology, HIV-1
Abstract

International audience; Autophagy-related proteins such as Beclin-1 are involved in an array of complex processes, including antiviral responses, and may also modulate the efficiency of gene therapy viral vectors. The Tat-Beclin-1 (TB1) peptide has been reported as an autophagy-inducing factor inhibiting the replication of pathogens such as HIV, type 1 (HIV-1). However, autophagy-related proteins are also essential for the early steps of HIV-1 infection. Therefore, we examined the effects of the Beclin-1 evolution-arily conserved domain in TB1 on viral transduction and autophagy in single-round HIV infection or with nonreplicative HIV-1-derived lentiviral vectors. TB1 enhanced transduction with various pseudotypes but without inducing the autophagy process. TB1 augmented the transduction of human CD34؉ hematopoietic stem/progenitor cells while maintaining their capacity to engraft in vivo into humanized mice. TB1 was as effective as other transduction additives and functioned by enhancing the adhesion and fusion of viral particles with target cells but not their aggregation. We also found that the N-terminal L1 loop was critical for TB1 transduction-enhancing activity. Interestingly, the Tat-Beclin-2 (TB2) peptide, derived from the human Beclin-2 protein, was even more potent than TB1 in promoting viral transduction and infection. Taken together, our findings suggest that the TB1 and TB2 peptides enhance the viral entry step. Tat-Beclin peptides therefore represent a new family of viral transduction enhancers for potential use in gene therapy.

Published
2017
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6. Integrated time-lapse and single-cell transcription studies highlight the variable and dynamic nature of human hematopoietic cell fate commitment

Author

Moussy, Alice, Cosette, Jérémie, Parmentier, Romuald, da Silva, Cindy, Corre, Guillaume, Richard, Angélique, Gandrillon, Olivier, Stockholm, Daniel, Paldi, Andras, Huang, Sui, Centre de Recherche des Cordeliers (CRC (UMR_S 872)), Université Pierre et Marie Curie - Paris 6 (UPMC)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), GENETHON 3, Université d'Évry-Val-d'Essonne (UEVE), Genethon - Inserm UMR_S951, Immunologie moléculaire et biothérapies innovantes, École pratique des hautes études (EPHE)-Université d'Évry-Val-d'Essonne (UEVE)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Genethon-École pratique des hautes études (EPHE)-Université d'Évry-Val-d'Essonne (UEVE)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Genethon-Généthon, Association française contre les myopathies (AFM-Téléthon)-Association française contre les myopathies (AFM-Téléthon), Centre de génétique et de physiologie moléculaire et cellulaire (CGPhiMC), Centre National de la Recherche Scientifique (CNRS)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon, Laboratoire Analyse et Modélisation pour la Biologie et l'Environnement (LAMBE - UMR 8587), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université de Cergy Pontoise (UCP), Université Paris-Seine-Université Paris-Seine-Université d'Évry-Val-d'Essonne (UEVE)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Généthon, Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS), Laboratoire Analyse et Modélisation pour la Biologie et l'Environnement (LAMBE), Université Paris-Seine-Université Paris-Seine-Université d'Évry-Val-d'Essonne (UEVE)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Saclay (COmUE), and Université Paris Descartes - Paris 5 (UPD5)-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)

Subjects
[SDV.BIBS]Life Sciences [q-bio]/Quantitative Methods [q-bio.QM], ComputingMilieux_MISCELLANEOUS
Abstract

International audience

Published
2017
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7. 'RCL-Pooling Assay': A Simplified Method for the Detection of Replication-Competent Lentiviruses in Vector Batches Using Sequential Pooling

Author

Jean-Brice Marteau, Guillaume Corre, Anne Galy, Bruno Dalle, David Fenard, Michel Dessainte, Approches génétiques intégrées et nouvelles thérapies pour les maladies rares (INTEGRARE), 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), Laboratoire d'Onco-Hématologie (LOH), Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Departments of Hematology, Université Paris Diderot - Paris 7 (UPD7), Genethon - Inserm UMR_S951, Immunologie moléculaire et biothérapies innovantes, École pratique des hautes études (EPHE)-Université d'Évry-Val-d'Essonne (UEVE)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Genethon-École pratique des hautes études (EPHE)-Université d'Évry-Val-d'Essonne (UEVE)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Genethon-Généthon, Association française contre les myopathies (AFM-Téléthon)-Association française contre les myopathies (AFM-Téléthon), Généthon, Association française contre les myopathies (AFM-Téléthon), 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, École Pratique des Hautes Études (EPHE), GENOSAFE S.A, GENOSAFE, and Yposkesi

Subjects
0301 basic medicine, Virus inactivation, [SDV.BIO]Life Sciences [q-bio]/Biotechnology, Cost-Benefit Analysis, T-Lymphocytes, Genetic Vectors, Pooling, HIV Core Protein p24, Computational biology, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, Biology, Virus Replication, Sensitivity and Specificity, Cell Line, 03 medical and health sciences, Transduction (genetics), Transduction, Genetic, Genetics, Humans, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Purification methods, Molecular Biology, ComputingMilieux_MISCELLANEOUS, [SDV.GEN]Life Sciences [q-bio]/Genetics, Lentivirus, Virology, [SDV.BIBS]Life Sciences [q-bio]/Quantitative Methods [q-bio.QM], 3. Good health, 030104 developmental biology, HIV-1, Virus Inactivation, Molecular Medicine, [SDV.IMM]Life Sciences [q-bio]/Immunology, Biological Assay, Material handling
Abstract

Nonreplicative recombinant HIV-1-derived lentiviral vectors (LV) are increasingly used in gene therapy of various genetic diseases, infectious diseases, and cancer. Before they are used in humans, preparations of LV must undergo extensive quality control testing. In particular, testing of LV must demonstrate the absence of replication-competent lentiviruses (RCL) with suitable methods, on representative fractions of vector batches. Current methods based on cell culture are challenging because high titers of vector batches translate into high volumes of cell culture to be tested in RCL assays. As vector batch size and titers are continuously increasing because of the improvement of production and purification methods, it became necessary for us to modify the current RCL assay based on the detection of p24 in cultures of indicator cells. Here, we propose a practical optimization of this method using a pairwise pooling strategy enabling easier testing of higher vector inoculum volumes. These modifications significantly decrease material handling and operator time, leading to a cost-effective method, while maintaining optimal sensibility of the RCL testing. This optimized "RCL-pooling assay" ameliorates the feasibility of the quality control of large-scale batches of clinical-grade LV while maintaining the same sensitivity.

Published
2016
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8. Vpr expression abolishes the capacity of HIV-1 infected cells to repair uracilated DNA

Author

Nathalie Chazal, Eric Bernard, Patrick Eldin, Jean-François Guichou, Laurence Briant, David Fenard, Institut de Recherche en Infectiologie de Montpellier (IRIM), Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS), Genethon - Inserm UMR_S951, Immunologie moléculaire et biothérapies innovantes, École pratique des hautes études (EPHE)-Université d'Évry-Val-d'Essonne (UEVE)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Genethon-École pratique des hautes études (EPHE)-Université d'Évry-Val-d'Essonne (UEVE)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Genethon-Généthon, Association française contre les myopathies (AFM-Téléthon)-Association française contre les myopathies (AFM-Téléthon), Centre d’études d’Agents Pathogènes et Biotechologies pour la Santé (CPBS), Centre de Biochimie Structurale [Montpellier] (CBS), Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM)-Institut National de la Santé et de la Recherche Médicale (INSERM), Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM), and Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS)

Subjects
CD4-Positive T-Lymphocytes, DNA Repair, Cell Survival, DNA repair, Context (language use), Genome Integrity, Repair and Replication, Biology, Cell Line, DNA Glycosylases, 03 medical and health sciences, chemistry.chemical_compound, 0302 clinical medicine, Genetics, Humans, Uracil, ComputingMilieux_MISCELLANEOUS, Enzyme Assays, 030304 developmental biology, 0303 health sciences, Cell Cycle, [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, DNA, vpr Gene Products, Human Immunodeficiency Virus, Cell cycle, Molecular biology, 3. Good health, chemistry, DNA glycosylase, Cell culture, 030220 oncology & carcinogenesis, Ubiquitin ligase complex, [SDV.MP.VIR]Life Sciences [q-bio]/Microbiology and Parasitology/Virology, HIV-1
Abstract

International audience; The human immunodeficiency virus type 1 (HIV-1) Vpr protein binds to the cellular uracil-DNA glycosylase UNG2 and induces its degradation through the assembly with the DDB1-CUL4 ubiquitin ligase complex. This interaction counteracts the antiviral activity exerted by UNG2 on HIV-1 gene transcription, as previously reported by us. In this work, we show that Vpr expression in the context of HIV-1 infection markedly decreases UNG2 expression in transformed or primary CD4 + T lymphocytes. We demonstrate for the first time that Vpr-UNG2 interaction significantly impairs the uracil excision activity of infected cells. The loss of uracil excision activity coincides with a significant accumulation of uracilated bases in the genome of infected cells without changes in cell division. Although UNG2 expression and uracil-DNA glycosylase activity are recovered after the peak of retroviral replication, the mutagenic effect of transient DNA uracilation in cycling cells should be taken into account. Therefore, the possible consequences of Vpr-mediated temporary depletion of endo-genous nuclear UNG2 and subsequent alteration of the genomic integrity of infected cells need to be evaluated in the physiopathogenesis of HIV infection.

Published
2014
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9. Infectivity enhancement of different HIV-1-based lentiviral pseudotypes in presence of the cationic amphipathic peptide LAH4-L1

Author

Daniel Scherman, Sandrine Genries, David Fenard, Anne Galy, Samia Martin, Antoine Kichler, 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), Unité de pharmacologie chimique et génétique et d'imagerie (UPCGI - UMR 8151 / UMR_S 1022 ), Université Paris Descartes - Paris 5 (UPD5)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Ecole Nationale Supérieure de Chimie de Paris - Chimie ParisTech-PSL (ENSCP), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut de Chimie du CNRS (INC), Généthon, This work was supported by the Association Française contre les Myopathies., Fenard, David, 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, Ecole Nationale Supérieure de Chimie de Paris - Chimie ParisTech-PSL (ENSCP), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), École Pratique des Hautes Études (EPHE), Genethon - Inserm UMR_S951, Immunologie moléculaire et biothérapies innovantes, École pratique des hautes études ( EPHE ) -Université d'Évry-Val-d'Essonne ( UEVE ) -Institut National de la Santé et de la Recherche Médicale ( INSERM ) -Genethon-École pratique des hautes études ( EPHE ) -Université d'Évry-Val-d'Essonne ( UEVE ) -Institut National de la Santé et de la Recherche Médicale ( INSERM ) -Genethon-Généthon, Association française contre les myopathies ( AFM-Téléthon ) -Association française contre les myopathies ( AFM-Téléthon ), Association française contre les myopathies ( AFM-Téléthon ), École pratique des hautes études ( EPHE ) -Université d'Évry-Val-d'Essonne ( UEVE ) -Institut National de la Santé et de la Recherche Médicale ( INSERM ) -Genethon, Unité de pharmacologie chimique et génétique et d'imagerie ( UPCGI - UMR 8151 / UMR_S 1022 ), and Ecole Nationale Supérieure de Chimie de Paris- Chimie ParisTech-PSL ( ENSCP ) -Université Paris Descartes - Paris 5 ( UPD5 ) -Institut National de la Santé et de la Recherche Médicale ( INSERM ) -Centre National de la Recherche Scientifique ( CNRS )

Subjects
MESH : Hematopoietic stem cell, MESH : retrovirus, [SDV.BIO]Life Sciences [q-bio]/Biotechnology, Genetic enhancement, Virus Attachment, Biology, Hematopoietic stem cell, Viral vector, 03 medical and health sciences, Transduction (genetics), 0302 clinical medicine, Gene therapy, MESH: Gene therapy, Transduction, Genetic, MESH : Gene therapy, Virology, medicine, Humans, [INFO.INFO-BT]Computer Science [cs]/Biotechnology, MESH : cationic peptide, Cells, Cultured, 030304 developmental biology, Infectivity, 0303 health sciences, MESH: cationic peptide, Lentivirus, lentiviral vector, MESH: Hematopoietic stem cell, [ SDV.BIO ] Life Sciences [q-bio]/Biotechnology, Transfection, cationic amphipathic peptide, Recombinant Proteins, Fibronectins, [SDV.BIO] Life Sciences [q-bio]/Biotechnology, 3. Good health, [INFO.INFO-BT] Computer Science [cs]/Biotechnology, medicine.anatomical_structure, 030220 oncology & carcinogenesis, MESH: retrovirus, Stem cell, Peptides, [ INFO.INFO-BT ] Computer Science [cs]/Biotechnology, Ex vivo, Antimicrobial Cationic Peptides
Abstract

International audience; Lentiviral vectors (LVs) are promising delivery systems for gene therapy. To enhance the efficiency of target cell transduction by LVs, protocols often include the addition of culture additives. In this study, the cationic amphipathic peptide LAH4-L1 (KKALLAHALHLLALLALHLAHALKKA), a DNA transfection agent, was evaluated for its capacity to improve LV infectivity in cell lines and primary cells. Results show that LAH4-L1 enhances infectivity of all LV pseudotypes tested, particularly GALVTR-LVs. More importantly, LAH4-L1 promotes the transduction of CD34+ hematopoietic stem cells with GALVTR-LVs as efficiently as Retronectin, a culture additive used in ex vivo clinical protocols involving LVs. The action of LAH4-L1 relies both on the GALVTR-LV adhesion and post-adhesion steps. LAH4-L1 represents a new and attractive transduction enhancer for hematopoietic gene therapy protocols.

Published
2013
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10. 464. RCL-Pooling Assay: A Simplified Method for the Detection of Replication Competent Lentiviruses in Vector Batches Using Sequential Pooling

Author

Corre, Guillaume, Guillaume, Corre, Dessainte, Michel, Marteau, Jean-Brice, Dalle, Bruno, Fenard, David, Galy, Anne, 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, Laboratoire d'Onco-Hématologie (LOH), Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Departments of Hematology, Université Paris Diderot - Paris 7 (UPD7), Généthon, École pratique des hautes études (EPHE), 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), Genethon - Inserm UMR_S951, Immunologie moléculaire et biothérapies innovantes, École pratique des hautes études (EPHE)-Université d'Évry-Val-d'Essonne (UEVE)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Genethon-École pratique des hautes études (EPHE)-Université d'Évry-Val-d'Essonne (UEVE)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Genethon-Généthon, Association française contre les myopathies (AFM-Téléthon)-Association française contre les myopathies (AFM-Téléthon), and Association française contre les myopathies (AFM-Téléthon)

Subjects
[SDV.BIO]Life Sciences [q-bio]/Biotechnology, Current cell, Pooling, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, Computational biology, Biology, 03 medical and health sciences, 0302 clinical medicine, Drug Discovery, Replication (statistics), Genetics, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Vector (molecular biology), Purification methods, Molecular Biology, ComputingMilieux_MISCELLANEOUS, 030304 developmental biology, Pharmacology, [SDV.GEN]Life Sciences [q-bio]/Genetics, 0303 health sciences, [SDV.BIBS]Life Sciences [q-bio]/Quantitative Methods [q-bio.QM], Virology, Cell culture, 030220 oncology & carcinogenesis, [SDV.IMM]Life Sciences [q-bio]/Immunology, Molecular Medicine, Material handling
Abstract

Non-replicative recombinant HIV-1 derived lentiviral vectors (LV) are increasingly used in gene therapy clinical trials for various genetic diseases, infectious diseases or cancer. Before they are used in man, preparations of LV must undergo extensive biochemical and biological quality control testing. In particular, the legislation stipulates that the absence of replication-competent lentiviruses (RCL) must be demonstrated with suitable methods, on representative fractions of batches. Current standard and widely used methods based on cell culture are challenging because high titers of vector batches achieved translate into high volumes of cell culture that have to be tested. Since vector batch titers and sizes are continuously-increasing due to the improvement of production and purification methods, it is necessary to modify the current cell culture method. Here, we propose a practical optimization of the p24-decrease-based culture assay developed by Escarpe et al. (2003) using a pairwise pooling strategy enabling the test of higher vector inoculum volumes. These modifications significantly decrease material handling, operator time, leading to a cost effective method, while maintaining optimal sensibility of the RCL testing. This optimized RCL-pooling assay ameliorates the feasibility of the quality control of large-scale batches of clinical-grade LV while maintaining the same sensitivity.

Published
2016
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11. Phenotypic heterogeneity in clonal cell populations : origin and consequences

Author

corre, guillaume, Généthon, Ecole Doctorale 472 : Systèmes Intégrés, Environnement et Biodiversité (EPHE) (ED 472 SIEB), École pratique des hautes études (EPHE), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL), AXA research Funds, Ecole Pratique des Hautes Etudes, Andras Paldi, corre, guillaume, Genethon - Inserm UMR_S951, Immunologie moléculaire et biothérapies innovantes (IMBI), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Université d'Évry-Val-d'Essonne (UEVE)-GENETHON 3-Institut National de la Santé et de la Recherche Médicale (INSERM)-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)-GENETHON 3-Institut National de la Santé et de la Recherche Médicale (INSERM)-Généthon, and Association française contre les myopathies (AFM-Téléthon)-Association française contre les myopathies (AFM-Téléthon)

Subjects
Stochasticity, stochasticité, population clonale, cellules eucaryotes, Eukaryotic Cells, Phenotype, expression génique, variabilité, gene expression, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, Variability, clonal population, [SDV.BC] Life Sciences [q-bio]/Cellular Biology
Abstract

According to the actual paradigm, isogenic cells inthe same environment must display the same behavior as they share the same genetic program. In consequences, the clonal origin of cells is considered as the best guaranty of their homogeneity. Nevertheless, observing differences between isogenic cells, grown in the same environment is not a rare event as observed in different studies. The main objective of this work is to understand how those differences between genetically identical cells appear and to quantify the dynamics of these changes. The first part is dedicated to the study of long term variations in cells’ phenotype. Starting from single cells, we monitored the emergence of cell-to-cell variations in the resulting populations by weekly analysis during several weeks. We show that the establishment of cell-to-cell heterogeneity in our system is a long lasting process, suffering a memory effect which slowed the diversification of phenotype. Nevertheless, several weeks after subcloning individual cells, a relaxation process is observed in the resulting populations and their variability reached the same level as in the original population. In the second part, we quantified rapid changes in gene expression at the level of single cells. Based on video-microscopy experiments coupled with images analysis and signal processing, we highlight different single-cell dynamics that could explain the emergence of cell-to-cell differences in isogenic cells populations. We also show that a competition effect appears between similar transgenic constructs when they are close to each other. Finally, we highlight the limitations of the use oflong-life proteins for the study of gene expression dynamics., Le paradigme actuel de la biologie soutient que descellules possédant le même programme génétique doivent se comporter de façon similaire lorsqu’elles évoluent dans le même environnement. L'origine clonale de cellules est alors une garantie de grande homogénéité pour de nombreuses applications. Néanmoins, des observations montrent que malgré leur identité génétique, certaines cellules arborent des caractères différents dans le même environnement. L’objectif principal de ce travail est d’étudier la dynamique des fluctuations qui conduit à la diversification de ces cellules. Dans un premier temps, nous avons étudié la dynamique qui permet d’obtenir une population clonale hétérogène, à partir d’une cellule. En isolant des cellules et en analysant leurs descendants pendant plusieurs semaines, nous avons montré que ce processus est relativement long. Il semble subir uneffet mémoire à l’échelle cellulaire qui ralentie la diversification phénotypique. Néanmoins, après plusieurs semaines, un retour à l’équilibre s’opère et la variabilité des populations obtenues atteint celle de la population d’origine. Par la suite, nous avons quantifié les changements d’expression génique à l’échelle cellulaire. Cette seconde étape, basée sur la vidéo-microscopie couplée à l’analyse d’images et au traitement du signal, a permis d’estimer la dynamique qui aboutie à l’hétérogénéité observée dans la première partie. Nous avons aussi mis en évidence qu’une proximité spatiale entre deux constructions transgéniques pouvait entraîner une compétition entreelles. Enfin, nous avons identifié une limite à l’utilisation de certains gènes rapporteurs dans les études dynamiques de l’expression génique.

Published
2012

12. VSV-G pseudotyping rescues HIV-1 CA mutations that impair core assembly or stability

Author

Eric Bernard, Laurence Briant, Laurent Chaloin, David Fenard, Maxime Solignat, Nathalie Chazal, Christian Devaux, Sonia Brun, Institut de Recherche en Infectiologie de Montpellier (IRIM), Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS), Centre d’études d’Agents Pathogènes et Biotechologies pour la Santé (CPBS), Généthon, Genethon - Inserm UMR_S951, Immunologie moléculaire et biothérapies innovantes (IMBI), GENETHON 3-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)-GENETHON 3-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)-Généthon, and Association française contre les myopathies (AFM-Téléthon)-Association française contre les myopathies (AFM-Téléthon)

Subjects
lcsh:Immunologic diseases. Allergy, Alanine Substitution, Virus Integration, Endocytic cycle, Mutant, S178 Residue, medicine.disease_cause, 03 medical and health sciences, chemistry.chemical_compound, Capsid, Virology, medicine, Humans, Core Assembly, 030304 developmental biology, 0303 health sciences, Mutation, biology, Vesicular Stomatitis Virus Glycoprotein, Research, Viral Core Proteins, Virus Assembly, 030302 biochemistry & molecular biology, biology.organism_classification, Molecular biology, Reverse transcriptase, 3. Good health, Reverse Transcription Efficiency, Infectious Diseases, chemistry, Vesicular stomatitis virus, [SDV.MP.VIR]Life Sciences [q-bio]/Microbiology and Parasitology/Virology, DNA, Viral, Pseudotyping, HIV-1, lcsh:RC581-607, DNA
Abstract

Background The machinery of early HIV-1 replication still remains to be elucidated. Recently the viral core was reported to persist in the infected cell cytoplasm as an assembled particle, giving rise to the reverse transcription complex responsible for the synthesis of proviral DNA and its transport to the nucleus. Numerous studies have demonstrated that reverse transcription of the HIV-1 genome into proviral DNA is tightly dependent upon proper assembly of the capsid (CA) protein into mature cores that display appropriate stability. The functional impact of structural properties of the core in early replicative steps has yet to be determined. Results Here, we show that infectivity of HIV-1 mutants bearing S149A and S178A mutations in CA can be efficiently restored when pseudotyped with vesicular stomatitis virus envelope glycoprotein, that addresses the mutant cores through the endocytic pathway rather than by fusion at the plasma membrane. The mechanisms by which these mutations disrupt virus infectivity were investigated. S149A and S178A mutants were unable to complete reverse transcription and/or produce 2-LTR DNA. Morphological analysis of viral particles and in vitro uncoating assays of isolated cores demonstrated that infectivity defects resulted from disruption of the viral core assembly and stability for S149A and S178A mutants, respectively. Consistent with these results, both mutants failed to saturate TRIM-antiviral restriction activity. Conclusion Defects generated at the level of core assembly and stability by S149A and S178A mutations are sensitive to the way of delivery of viral nucleoprotein complexes into the target cell. Addressing CA mutants through the endocytic pathway may compensate for defects generated at the reverse transcription/nuclear import level subsequent to impairment of core assembly or stability.

Published
2008
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