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1. Multiplex CRISPR/Cas9 system impairs HCMV replication by excising an essential viral gene

Author

Janina Gergen, Flora Coulon, Alison Creneguy, Nathan Elain-Duret, Alejandra Gutierrez, Olaf Pinkenburg, Els Verhoeyen, Ignacio Anegon, Tuan Huy Nguyen, Franck Albert Halary, Fabienne Haspot, Degauque, Nicolas, 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), Université de Nantes (UN)-Centre hospitalier universitaire de Nantes (CHU Nantes), Centre méditerranéen de médecine moléculaire (C3M), Université Nice Sophia Antipolis (1965 - 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), LabEX IGO Immunothérapie Grand Ouest, Nantes Université (Nantes Univ), Université Nice Sophia Antipolis (... - 2019) (UNS), and COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Université Côte d'Azur (UCA)-Institut National de la Santé et de la Recherche Médicale (INSERM)

Subjects
[SDV.IMM] Life Sciences [q-bio]/Immunology, Protein Expression, lcsh:Medicine, Cytomegalovirus, Artificial Gene Amplification and Extension, Microbial Genomics, Viral Structure, Virus Replication, Research and Analysis Methods, Polymerase Chain Reaction, Microbiology, Cell Line, Virions, Team3, Team2, Virology, Team1, Genetics, Gene Expression and Vector Techniques, Humans, Clustered Regularly Interspaced Short Palindromic Repeats, lcsh:Science, Molecular Biology Techniques, CRTI, Molecular Biology, ComputingMilieux_MISCELLANEOUS, Viral Genomics, Molecular Biology Assays and Analysis Techniques, lcsh:R, Microbial Mutation, Biology and Life Sciences, Genomics, Flow Cytometry, Viral Replication, Viral Persistence and Latency, Mutation, Viral Genome, [SDV.IMM]Life Sciences [q-bio]/Immunology, lcsh:Q, Research Article
Abstract

Anti-HCMV treatments used in immunosuppressed patients reduce viral replication, but resistant viral strains can emerge. Moreover, these drugs do not target latently infected cells. We designed two anti-viral CRISPR/Cas9 strategies to target the UL122/123 gene, a key regulator of lytic replication and reactivation from latency. The singleplex strategy contains one gRNA to target the start codon. The multiplex strategy contains three gRNAs to excise the complete UL122/123 gene. Primary fibroblasts and U-251 MG cells were transduced with lentiviral vectors encoding Cas9 and one or three gRNAs. Both strategies induced mutations in the target gene and a concomitant reduction of immediate early (IE) protein expression in primary fibroblasts. Further detailed analysis in U-251 MG cells showed that the singleplex strategy induced 50% of indels in the viral genome, leading to a reduction in IE protein expression. The multiplex strategy excised the IE gene in 90% of all viral genomes and thus led to the inhibition of IE protein expression. Consequently, viral genome replication and late protein expression were reduced by 90%. Finally, the production of new viral particles was nearly abrogated. In conclusion, the multiplex anti-UL122/123 CRISPR/Cas9 system can target the viral genome efficiently enough to significantly prevent viral replication.

Published
2018

2. Homology-directed repair in rodent zygotes using Cas9 and TALEN engineered proteins

Author

Laurent Tesson, Jean-Paul Concordet, Alison Creneguy, Dominique Gauguier, Carine Giovannangeli, Yacine Cherifi, Alexandre Fraichard, Claire Usal, Reynald Thinard, Ignacio Anegon, Séverine Remy, Jean-Baptiste Boulé, Charlotte Boix, Anne De Cian, Tuan H. Nguyen, Lucas Brusselle, Sandra Fontanière, Séverine Ménoret, Institut de transplantation urologie-néphrologie (ITUN), Université de Nantes (UN)-Centre hospitalier universitaire de Nantes (CHU Nantes), Santé - François Bonamy, Université de Nantes (UN)-Centre hospitalier universitaire de Nantes (CHU Nantes)-Institut de Recherche en Santé de l'Université de Nantes (IRS-UN)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), 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), 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), genOway, Lyon, Centre de Recherche des Cordeliers (CRC), Université Paris Diderot - Paris 7 (UPD7)-École pratique des hautes études (EPHE), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-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), Unité de Recherche sur les Maladies Cardiovasculaires, du Métabolisme et de la Nutrition = Institute of cardiometabolism and nutrition (ICAN), Université Pierre et Marie Curie - Paris 6 (UPMC)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-CHU Pitié-Salpêtrière [AP-HP], Sorbonne Université (SU)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU), Université de Nantes (UN)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche en Santé de l'Université de Nantes (IRS-UN)-Centre hospitalier universitaire de Nantes (CHU Nantes), Université Pierre et Marie Curie - Paris 6 (UPMC)-École Pratique des Hautes Études (EPHE), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Université Paris Diderot - Paris 7 (UPD7)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM), Unité de Recherche sur les Maladies Cardiovasculaires, du Métabolisme et de la Nutrition = Research Unit on Cardiovascular and Metabolic Diseases (ICAN), Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU)-Sorbonne Université (SU), HAL UPMC, Gestionnaire, Université Pierre et Marie Curie - Paris 6 (UPMC)-École pratique des hautes études (EPHE), Institut de transplantation urologie-néphrologie ( ITUN ), Université de Nantes ( UN ) -Centre hospitalier universitaire de Nantes ( CHU Nantes ), Université de Nantes ( UN ) -Institut de Recherche en Santé de l'Université de Nantes (IRSUN)-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 ), 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 ) -Centre National de la Recherche Scientifique ( CNRS ), Centre de Recherche des Cordeliers ( CRC ), Université Paris Diderot - Paris 7 ( UPD7 ) -École pratique des hautes études ( EPHE ) -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 ), Unité de Recherche sur les Maladies Cardiovasculaires, du Métabolisme et de la Nutrition = Institute of cardiometabolism and nutrition ( ICAN ), and Université Pierre et Marie Curie - Paris 6 ( UPMC ) -Assistance publique - Hôpitaux de Paris (AP-HP)-Institut National de la Santé et de la Recherche Médicale ( INSERM ) -CHU Pitié-Salpêtrière [APHP]

Subjects
Microinjections, Zygote, [ SDV.AEN ] Life Sciences [q-bio]/Food and Nutrition, [SDV.GEN] Life Sciences [q-bio]/Genetics, Biology, Protein Engineering, Article, Rats, Sprague-Dawley, Homology directed repair, Mice, 03 medical and health sciences, 0302 clinical medicine, Genome editing, [ SDV.MHEP ] Life Sciences [q-bio]/Human health and pathology, Animals, 030304 developmental biology, Genetics, 0303 health sciences, Messenger RNA, Transcription activator-like effector nuclease, [SDV.GEN]Life Sciences [q-bio]/Genetics, [SDV.MHEP] Life Sciences [q-bio]/Human health and pathology, Multidisciplinary, Cas9, Recombinational DNA Repair, Protein engineering, Rats, Cell biology, Mice, Inbred C57BL, [SDV.AEN] Life Sciences [q-bio]/Food and Nutrition, Nucleic acid, CRISPR-Cas Systems, [ SDV.GEN ] Life Sciences [q-bio]/Genetics, [SDV.AEN]Life Sciences [q-bio]/Food and Nutrition, 030217 neurology & neurosurgery, [SDV.MHEP]Life Sciences [q-bio]/Human health and pathology
Abstract

The generation of genetically-modified organisms has been revolutionized by the development of new genome editing technologies based on the use of gene-specific nucleases, such as meganucleases, ZFNs, TALENs and CRISPRs-Cas9 systems. The most rapid and cost-effective way to generate genetically-modified animals is by microinjection of the nucleic acids encoding gene-specific nucleases into zygotes. However, the efficiency of the procedure can still be improved. In this work we aim to increase the efficiency of CRISPRs-Cas9 and TALENs homology-directed repair by using TALENs and Cas9 proteins, instead of mRNA, microinjected into rat and mouse zygotes along with long or short donor DNAs. We observed that Cas9 protein was more efficient at homology-directed repair than mRNA, while TALEN protein was less efficient than mRNA at inducing homology-directed repair. Our results indicate that the use of Cas9 protein could represent a simple and practical methodological alternative to Cas9 mRNA in the generation of genetically-modified rats and mice as well as probably some other mammals.

Published
2015

3. Highly efficient in vitro and in vivo delivery of functional RNAs using new versatile MS2-chimeric retrovirus-like particles

Author

Prel, Anne, Caval, Vincent, Gayon, Régis, Ravassard, Philippe, Duthoit, Christine, Payen, Emmanuel, Maouche-Chretien, Leila, Creneguy, Alison, Nguyen, Tuan Huy, Martin, Nicolas, Piver, Eric, Sevrain, Raphaël, Lamouroux, Lucille, Leboulch, Philippe, Deschaseaux, Frédéric, Bouillé, Pascale, Sensébé, Luc, Pagès, Jean-Christophe, STROMALab, Université Toulouse III - Paul Sabatier (UT3), Université de Toulouse (UT)-Université de Toulouse (UT)-Ecole Nationale Vétérinaire de Toulouse (ENVT), Institut National Polytechnique (Toulouse) (Toulouse INP), Université de Toulouse (UT)-Université de Toulouse (UT)-Institut National Polytechnique (Toulouse) (Toulouse INP), Université de Toulouse (UT)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Etablissement Français du Sang-Centre National de la Recherche Scientifique (CNRS), Morphogénèse et antigénicité du VIH et du virus des Hépatites (MAVIVH - U1259 Inserm - CHRU Tours ), Centre Hospitalier Régional Universitaire de Tours (CHRU Tours)-Université de Tours (UT)-Institut National de la Santé et de la Recherche Médicale (INSERM), Vectalys SAS, Institut du Cerveau et de la Moëlle Epinière = Brain and Spine Institute (ICM), Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut National de la Santé et de la Recherche Médicale (INSERM)-CHU Pitié-Salpêtrière [AP-HP], 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), CEA [Fontenay-aux-Roses] (UGRA / SETA), Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Infectious Diseases Models for Innovative Therapies (IDMIT), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay, 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), Laboratoire de Biochimie et Biologie Moléculaire, Centre Hospitalier Régional Universitaire de Tours (CHRU Tours), Centre National de la Recherche Scientifique (CNRS)-Ecole Nationale Vétérinaire de Toulouse (ENVT), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Etablissement Français du Sang-Institut National de la Santé et de la Recherche Médicale (INSERM), HAL-UPMC, Gestionnaire, Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Ecole Nationale Vétérinaire de Toulouse (ENVT), Université Fédérale Toulouse Midi-Pyrénées-Institut National de la Santé et de la Recherche Médicale (INSERM)-Etablissement Français du Sang-Centre National de la Recherche Scientifique (CNRS), Morphogénèse et antigénicité du VIH et du virus des Hépatites (MAVIVH), Université de Tours-Centre Hospitalier Régional Universitaire de Tours (CHRU Tours)-Institut National de la Santé et de la Recherche Médicale (INSERM), Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut National de la Santé et de la Recherche Médicale (INSERM)-CHU Pitié-Salpêtrière [APHP]-Centre National de la Recherche Scientifique (CNRS), Centre Hospitalier Régional Universitaire de Tours (CHRU TOURS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Tours, 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)-CHU Pitié-Salpêtrière [AP-HP], and Sorbonne Université (SU)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)

Subjects
[SDV.MHEP.ME] Life Sciences [q-bio]/Human health and pathology/Emerging diseases, lcsh:Genetics, [SDV.MHEP.ME]Life Sciences [q-bio]/Human health and pathology/Emerging diseases, lcsh:QH426-470, lcsh:Cytology, Biological techniques, [SDV.BBM] Life Sciences [q-bio]/Biochemistry, Molecular Biology, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, lcsh:QH573-671, Article, Biotechnology
Abstract

International audience; RNA delivery is an attractive strategy to achieve transient gene expression in research projects and in cell- or gene-based therapies. Despite significant efforts investigating vector-directed RNA transfer, there is still a requirement for better efficiency of delivery to primary cells and in vivo. Retroviral platforms drive RNA delivery, yet retrovirus RNA-packaging constraints limit gene transfer to two genome-molecules per viral particle. To improve retroviral transfer, we designed a dimerization-independent MS2-driven RNA packaging system using MS2-Coat-retrovirus chimeras. The engineered chimeric particles promoted effective packaging of several types of RNAs and enabled efficient transfer of biologically active RNAs in various cell types, including human CD34+ and iPS cells. Systemic injection of high-titer particles led to gene expression in mouse liver and transferring Cre-recombinase mRNA in muscle permitted widespread editing at the ROSA26 locus. We could further show that the VLPs were able to activate an osteoblast differentiation pathway by delivering RUNX2- or DLX5-mRNA into primary human bone-marrow mesenchymal-stem cells. Thus, the novel chimeric MS2-lentiviral particles are a versatile tool for a wide range of applications including cellular-programming or genome-editing.

Published
2015

4. Efficient Generation of Myostatin Knock-Out Sheep Using CRISPR/Cas9 Technology and Microinjection into Zygotes

Author

Ignaco Anegón, F. Cuadro, A. P. Mulet, Laurent Tesson, N. Barrera, Martina Crispo, P.C. dos Santos-Neto, Alison Creneguy, Tuan H. Nguyen, Alejo Menchaca, Lucas Brusselle, Transgenic and Experimental Animal / Animales Transgénicos y de Experimentación [Montevideo], Institut Pasteur de Montevideo, Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP), 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), Instituto de Reproducción Animal Uruguay [Montevideo, Uruguay], Fundación IRAUy, Institut de transplantation urologie-néphrologie (ITUN), Université de Nantes (UN)-Centre hospitalier universitaire de Nantes (CHU Nantes), IHU-CESTI, French government, National Research Agency, Région Pays de la Loire through Biogenouest, IBiSA program, Fondation Progreffe and TEFOR, the integrative genomic facility of Nantes, FOCEM (MERCOSUR Structural Convergence Fund)COF 03/11(Uruguay), PEDECIBA (UdelaR, Uruguay)., and Le Bihan, Sylvie

Subjects
Microinjections, Zygote, lcsh:Medicine, Myostatin, Biology, Animals, Genetically Modified, symbols.namesake, Gene Knockout Techniques, Pregnancy, CRISPR, Animals, Genetically modified animal, lcsh:Science, Microinjection, Sheep, Domestic, 2. Zero hunger, Sanger sequencing, Multidisciplinary, [SDV.MHEP] Life Sciences [q-bio]/Human health and pathology, Cas9, lcsh:R, Embryo, Molecular biology, symbols, biology.protein, lcsh:Q, Female, CRISPR-Cas Systems, [SDV.MHEP]Life Sciences [q-bio]/Human health and pathology, Research Article
Abstract

International audience; While CRISPR/Cas9 technology has proven to be a valuable system to generate gene-targeted modified animals in several species, this tool has been scarcely reported in farm animals. Myostatin is encoded by MSTN gene involved in the inhibition of muscle differentiation and growth. We determined the efficiency of the CRISPR/Cas9 system to edit MSTN in sheep and generate knockout (KO) animals with the aim to promote muscle development and body growth. We generated CRISPR/Cas9 mRNAs specific for ovine MSTN and microinjected them into the cytoplasm of ovine zygotes. When embryo development of CRISPR/Cas9 microinjected zygotes (n = 216) was compared with buffer injected embryos (n = 183) and non microinjected embryos (n = 173), cleavage rate was lower for both micro-injected groups (P

Published
2015

5. Hepatic lentiviral gene transfer prevents the long-term onset of hepatic tumours of glycogen storage disease type 1a in mice

Author

Blandine Gri, Sophie Gaillard, Nicolas Ferry, Julie Clar, A. Stefanutti, Alison Creneguy, Tuan Huy Nguyen, Fabienne Rajas, Elodie Mutel, Gilles Mithieux, Olivier Beuf, Université de Lyon, Nutrition et cerveau (U1213, U855), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National de la Santé et de la Recherche Médicale (INSERM), 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), Université de Nantes (UN)-Centre hospitalier universitaire de Nantes (CHU Nantes), Plateforme d'Imagerie Multimodale LyonTech (PILoT), Centre de Recherche en Acquisition et Traitement de l'Image pour la Santé (CREATIS), Université de Lyon-Université de Lyon-Institut National des Sciences Appliquées de Lyon (INSA Lyon), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Hospices Civils de Lyon (HCL)-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 Claude Bernard Lyon 1 (UCBL), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Hospices Civils de Lyon (HCL)-Université Jean Monnet [Saint-Étienne] (UJM)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Biothérapies Hépatiques, Université de Nantes (UN)-Institut National de la Santé et de la Recherche Médicale (INSERM), Laboratoire d'Informatique, Signaux, et Systèmes de Sophia-Antipolis (I3S) / Equipe RAINBOW, Scalable and Pervasive softwARe and Knowledge Systems (Laboratoire I3S - SPARKS), Laboratoire d'Informatique, Signaux, et Systèmes de Sophia Antipolis (I3S), 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 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 Côte d'Azur (UCA)-Laboratoire d'Informatique, Signaux, et Systèmes de Sophia Antipolis (I3S), 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 Côte d'Azur (UCA), RMN et optique : De la mesure au biomarqueur, 'Association Française contre les Myopathies', and the 'Association Francophone des Glycogénoses'., ANR-07-MRAR-0011,GLUCOGENE THERAPY,Glycogénose de type Ia: de la physiopathologie à la thérapie génique(2007), ANR-11-BSV1-0009,GSD1,Glycogénose de type 1a : de la physiopathologie à la thérapie génique du foie(2011), Di Carlo, Marie-Ange, Maladies rares - Glycogénose de type Ia: de la physiopathologie à la thérapie génique - - GLUCOGENE THERAPY2007 - ANR-07-MRAR-0011 - MRAR - VALID, BLANC - Glycogénose de type 1a : de la physiopathologie à la thérapie génique du foie - - GSD12011 - ANR-11-BSV1-0009 - BLANC - VALID, Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Hospices Civils de Lyon (HCL)-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 Claude Bernard Lyon 1 (UCBL), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Hospices Civils de Lyon (HCL)-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), and 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 Côte d'Azur (UCA)-Université Nice Sophia Antipolis (1965 - 2019) (UNS)

Subjects
medicine.medical_specialty, G6PC, Transgene, Genetic enhancement, Genetic Vectors, Glycogen Storage Disease Type I, Biology, Malignant transformation, Mice, 03 medical and health sciences, chemistry.chemical_compound, 0302 clinical medicine, Internal medicine, Genetics, medicine, Animals, Humans, Glycogen storage disease, [SDV.NEU] Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC], Molecular Biology, Genetics (clinical), 030304 developmental biology, Mice, Knockout, 0303 health sciences, Glycogen, Lentivirus, Liver Neoplasms, Genetic Therapy, General Medicine, medicine.disease, biology.organism_classification, 3. Good health, Disease Models, Animal, Endocrinology, Liver, chemistry, 030220 oncology & carcinogenesis, Glucose-6-Phosphatase, [SDV.NEU]Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC], Steatosis
Abstract

Glycogen storage disease type 1a (GSD1a) is a rare disease due to the deficiency in the glucose-6-phosphatase (G6Pase) catalytic subunit (encoded by G6pc), which is essential for endogenous glucose production. Despite strict diet control to maintain blood glucose, patients with GSD1a develop hepatomegaly, steatosis and then hepatocellular adenomas (HCA), which can undergo malignant transformation. Recently, gene therapy has attracted attention as a potential treatment for GSD1a. In order to maintain long-term transgene expression, we developed an HIV-based vector, which allowed us to specifically express the human G6PC cDNA in the liver. We analysed the efficiency of this lentiviral vector in the prevention of the development of the hepatic disease in an original GSD1a mouse model, which exhibits G6Pase deficiency exclusively in the liver (L-G6pc(-/-) mice). Recombinant lentivirus were injected in B6.G6pc(ex3lox/ex3lox). SA(creERT2/w) neonates and G6pc deletion was induced by tamoxifen treatment at weaning. Magnetic resonance imaging was then performed to follow up the development of hepatic tumours. Lentiviral gene therapy restored glucose-6 phosphatase activity sufficient to correct fasting hypoglycaemia during 9 months. Moreover, lentivirus-treated L-G6pc(-/-) mice presented normal hepatic triglyceride levels, whereas untreated mice developed steatosis. Glycogen stores were also decreased although liver weight remained high. Interestingly, lentivirus-treated L-G6pc(-/-) mice were protected against the development of hepatic tumours after 9 months of gene therapy while most of untreated L-G6pc(-/-) mice developed millimetric HCA. Thus the treatment of newborns by recombinant lentivirus appears as an attractive approach to protect the liver from the development of steatosis and hepatic tumours associated to GSD1a pathology.

Published
2015

7. Surface expression of the hRSV nucleoprotein impairs immunological synapse formation with T cells

Author

Alison Creneguy, Catarina Sacristán, Susan M. Bueno, Jorge E. Mora, Michael Cammer, Tuan H. Nguyen, Alexis M. Kalergis, Juan Pablo Mackern-Oberti, David Depoil, Bruno A. Ramirez, Christian E. Palavecino, Michael L. Dustin, Roberto S. Gomez, Claudia A. Riedel, Sebastián A. Riquelme, Pablo F. Céspedes, Departamento de Microbiologıa y Genetica Molecular [Santiago, Chile] (Facultad de Ciencias Biologicas), Pontificia Universidad Católica de Chile (UC), 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), Helen and Martin Kimmel Center for Biology and Medicine [New York, NY, USA], New York University School of Medicine, NYU System (NYU)-NYU System (NYU)-New York University Langone Medical Center (NYU Langone Medical Center), NYU System (NYU), Kennedy Institute of Rheumatology [Oxford, UK], Imperial College London, Departamento de Ciencias Biologicas [Santiago, Chile] (Facultad de Ciencias Biologicas y Facultad de Medicina), Universidad Andrés Bello [Santiago] (UNAB), Departamento de Inmunologıa Clınica y Reumatologıa [Santiago, Chile] (Escuela de Medicina), Fondo Nacional de Desarrollo Científico y Tecnológico Grants 3100090, 1070352, and 3140455., Fondo de Fomento al Desarrollo Científico y Tecnológico D061008., Millennium Institute on Immunology and Immunotherapy (P07/088-F)., Grant 'Nouvelles Equipes-nouvelles thématiques' from the La Région Pays De La Loire., Evaluation-Orientation of Scientific Cooperation France-Chile Grant., Comisión Nacional de Investigación Científica y Tecnológica (CONICYT)., A Becas Chile scholarship., Proyecto de Inserción de Capital Humano en la Academia no. 79100015 from CONICYT., National Institutes of Health Grants AI043542 and AI080192., Wellcome Trust and Kennedy Trust., and Le Bihan, Sylvie

Subjects
CD4-Positive T-Lymphocytes, Cell signaling, Immunological Synapses, Lipid Bilayers, Receptors, Antigen, T-Cell, Golgi Apparatus, chemical and pharmacologic phenomena, Cell Communication, Respiratory Syncytial Virus Infections, Biology, Lymphocyte Activation, Virus Replication, Cell Line, Immunological synapse, Mice, Viral Proteins, 03 medical and health sciences, Histocompatibility Antigens, Animals, Humans, T lymphocyte priming, 030304 developmental biology, 0303 health sciences, [SDV.MHEP] Life Sciences [q-bio]/Human health and pathology, Brefeldin A, Multidisciplinary, Synapse assembly, Immunological synapse formation, 030306 microbiology, Cell Membrane, T-cell receptor, Dendritic Cells, Acquired immune system, Virology, 3. Good health, Transport protein, Cell biology, Mice, Inbred C57BL, Protein Transport, Nucleoproteins, PNAS Plus, Respiratory Syncytial Virus, Human, Signal transduction, Peptides, pSMAC, cSMAC, nucleocapsid protein, [SDV.MHEP]Life Sciences [q-bio]/Human health and pathology, Signal Transduction
Abstract

International audience; Human respiratory syncytial virus (hRSV) is the leading cause of bronchiolitis and pneumonia in young children worldwide. The recurrent hRSV outbreaks and reinfections are the cause of a significant public health burden and associate with an inefficient antiviral immunity, even after disease resolution. Although several mouse-and human cell-based studies have shown that hRSV infection prevents naïve T-cell activation by antigen-presenting cells, the mechanism underlying such inhibition remains unknown. Here, we show that the hRSV nucleoprotein (N) could be at least partially responsible for inhibiting T-cell activation during infection by this virus. Early after infection, the N protein was expressed on the surface of epithelial and dendritic cells, after interacting with trans-Golgi and lysosomal compartments. Further, experiments on supported lipid bilayers loaded with peptide-MHC (pMHC) complexes showed that surface anchored N protein prevented immunological synapse assembly by naive CD4 + T cells and, to a lesser extent, by antigen-experienced T-cell blasts. Synapse assembly inhibition was in part due to reduced T-cell receptor (TCR) signaling and pMHC clustering at the T-cell− bilayer interface, suggesting that N protein interferes with pMHC− TCR interactions. Moreover, N protein colocalized with the TCR independently of pMHC, consistent with a possible interaction with TCR complex components. Based on these data, we conclude that hRSV N protein expression at the surface of infected cells inhibits T-cell activation. Our study defines this protein as a major virulence factor that contributes to impairing acquired immunity and enhances susceptibility to reinfection by hRSV.

Published
2014

8. Codon swapping of zinc finger nucleases confers expression in primary cells and in vivo from a single lentiviral vector

Author

Ignacio Anegon, Cecilia Abarrategui-Pontes, Laurent Tesson, Tuan Huy Nguyen, Eli J. Fine, Thomas J. Cradick, Reynald Thinard, Alison Creneguy, Virginie Thepenier, Guillaume Podevin, Gang Bao, Laure Fournier Le Ray, 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), Université de Nantes (UN)-Centre hospitalier universitaire de Nantes (CHU Nantes), plate-forme Rat Transgenesis Immunophenomic (Inserm/CHU Nantes-IFR Santé François Bonamy), Structure fédérative de recherche François Bonamy (SFR François Bonamy), Université de Nantes (UN)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche en Santé de l'Université de Nantes (IRS-UN)-Université de Nantes (UN)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche en Santé de l'Université de Nantes (IRS-UN), Hémodynamique, Interaction Fibrose et Invasivité tumorales Hépatiques (HIFIH), and Université d'Angers (UA)

Subjects
Carcinoma, Hepatocellular, Transgene, [SDV]Life Sciences [q-bio], Genetic Vectors, lentiviral vectors, primary cells, Biology, Genome, Viral vector, 03 medical and health sciences, chemistry.chemical_compound, 0302 clinical medicine, Genome editing, Drug Discovery, Genetics, Animals, DNA Breaks, Double-Stranded, Glucuronosyltransferase, RNA, Small Interfering, Rats, Wistar, Codon, Molecular Biology, Genetics (clinical), Cells, Cultured, 030304 developmental biology, 0303 health sciences, fungi, Liver Neoplasms, Zinc Fingers, Glioma, Endonucleases, Molecular biology, Zinc finger nuclease, zinc finger nuclease, Cell biology, Rats, off-target sites, chemistry, Liver, Animals, Newborn, Cell culture, 030220 oncology & carcinogenesis, Hepatocytes, Molecular Medicine, Genetic Engineering, Ex vivo, DNA, Codon swapping
Abstract

International audience; BACKGROUND: Zinc finger nucleases (ZFNs) are promising tools for genome editing for biotechnological as well as therapeutic purposes. Delivery remains a major issue impeding targeted genome modification. Lentiviral vectors are highly efficient for delivering transgenes into cell lines, primary cells and into organs, such as the liver. However, the reverse transcription of lentiviral vectors leads to recombination of homologous sequences, as found between and within ZFN monomers.METHODS: We used a codon swapping strategy to both drastically disrupt sequence identity between ZFN monomers and to reduce sequence repeats within a monomer sequence. We constructed lentiviral vectors encoding codon-swapped ZFNs or unmodified ZFNs from a single mRNA transcript. Cell lines, primary hepatocytes and newborn rats were used to evaluate the efficacy of integrative-competent (ICLV) and integrative-deficient (IDLV) lentiviral vectors to deliver ZFNs into target cells.RESULTS: We reduced total identity between ZFN monomers from 90.9% to 61.4% and showed that a single ICLV allowed efficient expression of functional ZFNs targeting the rat UGT1A1 gene after codon-swapping, leading to much higher ZFN activity in cell lines (up to 7-fold increase compared to unmodified ZFNs and 60% activity in C6 cells), as compared to plasmid transfection or a single ICLV encoding unmodified ZFN monomers. Off-target analysis located several active sites for the 5-finger UGT1A1-ZFNs. Furthermore, we reported for the first time successful ZFN-induced targeted DNA double-strand breaks in primary cells (hepatocytes) and in vivo (liver) after delivery of a single IDLV encoding two ZFNs.CONCLUSION: These results demonstrate that a codon-swapping approach allowed a single lentiviral vector to efficiently express ZFNs and should stimulate the use of this viral platform for ZFN-mediated genome editing of primary cells, for both ex vivo or in vivo applications.

Published
2014

9. 768. The CRISPR/Cas9 System as an Anti-Viral Treatment to Prevent Primary Infection by HCMV Positive Hematopoietic Stem Cells

Author

Janina Gergen, Alison Creneguy, Fabienne Haspot, Franck Halary, Ignacio Anegon, Tuan Huy Nguyen, Flora Coulon, and Jean-Marie Heslan

Subjects
Pharmacology, viruses, medicine.medical_treatment, CD34, Hematopoietic stem cell transplantation, Biology, Virology, Viral vector, Haematopoiesis, Viral replication, Lytic cycle, Cell culture, Drug Discovery, Immunology, Genetics, medicine, Molecular Medicine, Stem cell, Molecular Biology
Abstract

HCMV is a beta-herpes virus, which induces a life long latency in hematopoietic stem cells after an asymptomatic primary infection. In hematopoietic stem cell transplantation, donor hematopoietic stem cells will engraft and differentiate in the recipient. This differentiation induces the reactivation of HCMV in the myeloid compartment and the release of new virions from the donor cells. The HCMV spread leads a primary infection of the immunocompromised HCMV negative recipient and can cause severe end organ diseases. The common treatments of HCMV in immunosuppressed patients are viral DNA replication inhibitors, which cause strong side effects, such as inhibition of hematopoiesis, kidney toxicity, and are likely to promote resistant strain emergence. As a consequence, none of the available drugs can target the HCMV in latent state.To circumvent this, we aim to directly alter the HCMV genome by using the CRIPSR/Cas9 system to knock-out the immediate early gene (IE) encoding essential viral proteins for lytic replication as well as the end of latency. We transduced the low HCMV-permissive U373-MG cells with a lentiviral vector encoding the Cas9 and tree different single gRNA targeting the IE gene. We FACS sorted those cell lines based on their Cas-9-GFP expression and then infected them with a HCMV laboratory strain. We detected mutations at the target site in up to 70% of the viral genomes. We observed a concomitant reduction of 50% less infected cell by FACS staining of the IE protein. Moreover, we set up an assay to analyze the virion release of infected U373-MG cells, which will allow us to address the decrease or inability to release new virions form U373-MG-gRNA-Cas9 HCMV infected cells. We are currently investigating a multiplex strategy with 3 gRNA targeting different parts of the IE gene which are delivered within the same lentiviral vector. The multiplex strategy is auspicious to show higher efficiency than a single gRNA. This strategy will be also tested on HCMV latently infected CD34+ hematopoietic stem cells to prevent viral reactivation.The CRISPR/Cas9 system anti-HCMV is a promising tool to block viral replication. With this anti-HCMV tool we will be able to treat HCMV positive donor cells in order to prevent the primary infection of the immunosuppressed recipient.

Published
2016

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