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1. Prevalence Study of Cellular Capsid-Specific Immune Responses to AAV2, 4, 5, 8, 9, and rh10 in Healthy Donors.

Author: Xicluna, Rebecca, Avenel, Allan, Vandamme, Céline, Devaux, Marie, Jaulin, Nicolas, Couzinié, Célia, Le Duff, Johanne, Charrier, Alicia, Guilbaud, Mickaël, Adjali, Oumeya, and Gernoux, Gwladys
Published: 2024
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2. Prevalence study of cellular capsid-specific immune responses to AAV1, 2, 4, 5, 8, 9 and rh10 reveals particular features for AAV9

Author: Xicluna, Rebecca, primary, Avenel, Allan, additional, Vandamme, Celine, additional, Devaux, Marie, additional, Jaulin, Nicolas, additional, Couzinie, Celia, additional, Le Duff, Johanne, additional, Charrier, Alicia, additional, Guilbaud, Mickael, additional, Adjali, Oumeya, additional, and Gernoux, Gwladys, additional
Published: 2023
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3. Transcriptomic Analysis Reveals the Inability of Recombinant AAV8 to Activate Human Monocyte-Derived Dendritic Cells

Author: Masri, Samer, primary, Carré, Laure, additional, Jaulin, Nicolas, additional, Vandamme, Céline, additional, Couzinié, Célia, additional, Guy-Duché, Aurélien, additional, Dupont, Jean-Baptiste, additional, Pereira, Allwyn, additional, Charpentier, Eric, additional, David, Laurent, additional, Gernoux, Gwladys, additional, Guilbaud, Mickaël, additional, and Adjali, Oumeya, additional
Published: 2023
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4. AAV8 locoregional delivery induces long-term expression of an immunogenic transgene in macaques despite persisting local inflammation

Author: Gernoux, Gwladys, primary, Guilbaud, Mickaël, additional, Devaux, Marie, additional, Journou, Malo, additional, Pichard, Virginie, additional, Jaulin, Nicolas, additional, Léger, Adrien, additional, Le Duff, Johanne, additional, Deschamps, Jack-Yves, additional, Le Guiner, Caroline, additional, Moullier, Philippe, additional, Cherel, Yan, additional, and Adjali, Oumeya, additional
Published: 2021
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5. Cross-Packaging and Capsid Mosaic Formation in Multiplexed AAV Libraries

Author: Schmit, Pauline F., primary, Pacouret, Simon, additional, Zinn, Eric, additional, Telford, Elizabeth, additional, Nicolaou, Fotini, additional, Broucque, Frédéric, additional, Andres-Mateos, Eva, additional, Xiao, Ru, additional, Penaud-Budloo, Magalie, additional, Bouzelha, Mohammed, additional, Jaulin, Nicolas, additional, Adjali, Oumeya, additional, Ayuso, Eduard, additional, and Vandenberghe, Luk H., additional
Published: 2020
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6. Tetramer-Based Enrichment of Preexisting Anti-AAV8 CD8+ T Cells in Human Donors Allows the Detection of a TEMRA Subpopulation

Author: Vandamme, Céline, primary, Xicluna, Rebecca, additional, Hesnard, Leslie, additional, Devaux, Marie, additional, Jaulin, Nicolas, additional, Guilbaud, Mickaël, additional, Le Duff, Johanne, additional, Couzinié, Célia, additional, Moullier, Philippe, additional, Saulquin, Xavier, additional, and Adjali, Oumeya, additional
Published: 2020
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7. Supplemental Material, Lorant_et_al_CT-1923_R1_Table_S1 - Vascular Delivery of Allogeneic MuStem Cells in Dystrophic Dogs Requires Only Short-Term Immunosuppression to Avoid Host Immunity and Generate Clinical/Tissue Benefits

Author: Lorant, Judith, Larcher, Thibaut, Jaulin, Nicolas, Hedan, Benoît, Lardenois, Aurélie, Leroux, Isabelle, Dubreil, Laurence, Ledevin, Mireille, Hélicia Goubin, Moullec, Sophie, Jack-Yves Deschamps, Thorin, Chantal, André, Catherine, Oumeya Adjali, and Rouger, Karl
Subjects: Medicine, Cell Biology
Abstract: Supplemental Material, Lorant_et_al_CT-1923_R1_Table_S1 for Vascular Delivery of Allogeneic MuStem Cells in Dystrophic Dogs Requires Only Short-Term Immunosuppression to Avoid Host Immunity and Generate Clinical/Tissue Benefits by Judith Lorant, Thibaut Larcher, Nicolas Jaulin, Benoît Hedan, Aurélie Lardenois, Isabelle Leroux, Laurence Dubreil, Mireille Ledevin, Hélicia Goubin, Sophie Moullec, Jack-Yves Deschamps, Chantal Thorin, Catherine André, Oumeya Adjali, and Karl Rouger in Cell Transplantation
Published: 2018
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8. Five Years of Successful Inducible Transgene Expression Following Locoregional Adeno-Associated Virus Delivery in Nonhuman Primates with No Detectable Immunity

Author: Guilbaud, Mickaël, primary, Devaux, Marie, additional, Couzinié, Celia, additional, Le Duff, Johanne, additional, Toromanoff, Alice, additional, Vandamme, Céline, additional, Jaulin, Nicolas, additional, Gernoux, Gwladys, additional, Larcher, Thibaut, additional, Moullier, Philippe, additional, Le Guiner, Caroline, additional, and Adjali, Oumeya, additional
Published: 2019
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9. Vascular Delivery of Allogeneic MuStem Cells in Dystrophic Dogs Requires Only Short-Term Immunosuppression to Avoid Host Immunity and Generate Clinical/Tissue Benefits

Author: Lorant, Judith, primary, Larcher, Thibaut, additional, Jaulin, Nicolas, additional, Hedan, Benoît, additional, Lardenois, Aurélie, additional, Leroux, Isabelle, additional, Dubreil, Laurence, additional, Ledevin, Mireille, additional, Goubin, Hélicia, additional, Moullec, Sophie, additional, Deschamps, Jack-Yves, additional, Thorin, Chantal, additional, André, Catherine, additional, Adjali, Oumeya, additional, and Rouger, Karl, additional
Published: 2018
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10. Human MuStem cells, a promising therapeutic candidate for muscular dystrophies with immunomodulatory properties

Author: Lorant, Judith, Jaulin, Nicolas, Charrier, Marine, Lieubeau-Teillet, Blandine, Leroux, Isabelle, Schleder, Cindy, Magot, Armelle, Hamel, Antoine, Pereon, Yann, Adjali, Oumeya, Rouger, Karl, Physiopathologie Animale et bioThérapie du muscle et du système nerveux (PAnTher), Institut National de la Recherche Agronomique (INRA)-Ecole Nationale Vétérinaire, Agroalimentaire et de l'alimentation Nantes-Atlantique (ONIRIS), Laboratoire de Thérapie Génique Translationnelle des Maladies Génétiques (Inserm UMR 1089), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Nantes - UFR de Médecine et des Techniques Médicales (UFR MEDECINE), Université de Nantes (UN)-Université de Nantes (UN), Institut National de la Recherche Agronomique (INRA), Immuno-Endocrinologie Cellulaire et Moléculaire (IECM), Ecole Nationale Vétérinaire de Nantes-Université de Nantes (UN)-Institut National de la Recherche Agronomique (INRA), Centre de Référence des maladies neuromusculaires rares Nantes-Angers, Service des Explorations Fonctionnelles, Centre hospitalier universitaire de Nantes (CHU Nantes), Service de Chirurgie Infantile, Développement et Pathologie du Tissu Musculaire (DPTM), Institut National de la Recherche Agronomique (INRA)-Ecole Nationale Vétérinaire de Nantes, Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Nantes (UN), Institut National de la Recherche Agronomique (INRA)-Université de Nantes (UN)-Ecole Nationale Vétérinaire de Nantes, and Université de Nantes (UN)-Institut National de la Santé et de la Recherche Médicale (INSERM)
Subjects: pseudohypertrophic childhood muscular dystrophy, myopathie de duchenne, [SDV.IMM]Life Sciences [q-bio]/Immunology, thérapie cellulaire, cell therapy, immunomodulation, [SDV.MHEP]Life Sciences [q-bio]/Human health and pathology
Abstract: Nowadays, allogeneic cell-based therapeutic approaches for regenerative medecine are limited by graft rejection. To counteract this major deleterious effect, immunosuppressive regimens are developed and given to patients, improving their lifespan but causing in return severe adverse effects. Over the last years, a number of adult stem cell populations including mesenchymal stem cells (MSC) and vessel-associated stem cells were described for the treatment of genetic muscle diseases. Those cells were shown to display immunomodulatory properties by acting, directly or through the secretion of soluble factors, on a large number of immune cell partners (Cossu et al., 2012; English et al., 2013). Duchenne Muscular Dystrophy (DMD) is a degenerative muscle disease characterized notably by an inflammatory component that negatively impacts on muscle regeneration activity. In this context, these original immune features attributed to stem cells could be a great advantage to improve cell engraftment and efficiency. In the laboratory, we have isolated a population of muscle-derived stem cells from healthy dog muscle tissue, called cMuStem cells, and made the proof of concept of their systemic delivery efficiency in the preclinical GRMD canine model of DMD (Rouger et al, 2011; Robriquet et al, 2015). Recently, we managed to isolate the same population from Paravertebralis muscle of 9 to 15-years old patients free of known muscle disease (hMuStem cells). It was defined as a mixed population composed of both myogenic progenitors and mesenchymal perivascular cells, and characterized by a large proliferation rate, an oligopotency as well as an in vivo myogenic regenerative potential. The aim of the study was to determine whether hMuStem cells also exhibit immunomodulatory properties. Interestingly, our preliminary data show the ability of hMuStem cells from different donors to modulate allogeneic T cell proliferation and to secrete various immunomodulatory molecules (prostaglandin-E2, indoleamin-2,3-deoxygenase-1, heme oxygenase-1 and inducible nitric-oxide synthase-1). Our data also suggest that hMuStem cells are able to interact with the complement system by inhibiting complement-mediated lysis of erythrocytes. This effect seems to be mediated by factor H, an alternative inhibitory complement pathway. Overall, our study is critical for the understanding of the interaction between MuStem cells and the immune system, as well as the design of safe and efficient allogeneic stem cell-based therapy for the treatment of muscular dystrophies.
Published: 2016

11. Human MuStem cells: a cell-based therapy candidate for Duchenne Muscular Dystrophy, with immunomodulatory properties

Author: Lorant, Judith, Jaulin, Nicolas, Leroux, Isabelle, Zuber, Céline, Schleder, Cindy, Charrier, Marine, Lieubeau-Teillet, Blandine, Pereon, Yann, Magot, A, Hamel, A, Adjali, Oumeya, Rouger, Karl, Physiopathologie Animale et bioThérapie du muscle et du système nerveux (PAnTher), Institut National de la Recherche Agronomique (INRA)-Ecole Nationale Vétérinaire, Agroalimentaire et de l'alimentation Nantes-Atlantique (ONIRIS), Immuno-Endocrinologie Cellulaire et Moléculaire (IECM), Institut National de la Recherche Agronomique (INRA)-Université de Nantes (UN)-Ecole Nationale Vétérinaire de Nantes, Centre Hospitalier Universiatire Hôtel-Dieu de Nantes (CHU Hôtel-Dieu), UMR1089, and Institut National de la Santé et de la Recherche Médicale (INSERM)
Subjects: ComputingMilieux_MISCELLANEOUS, [SDV.MHEP]Life Sciences [q-bio]/Human health and pathology
Abstract: National audience
Published: 2016

12. Immunomodulatory properties of human MuStem cells: assessing their impact on adaptive and innate immunity

Author: Lorant, Judith, Jaulin, Nicolas, Leroux, Isabelle, Schleder, Cindy, Zuber, Céline, Charrier, Marine, Lieubeau-Teillet, Blandine, Péréon, Yann, Magot, Armelle, Hamel, Antoine, Adjali, Oumeya, Rouger, Karl, Physiopathologie Animale et bioThérapie du muscle et du système nerveux (PAnTher), Institut National de la Recherche Agronomique (INRA)-Ecole Nationale Vétérinaire, Agroalimentaire et de l'alimentation Nantes-Atlantique (ONIRIS), Laboratoire de Thérapie Génique Translationnelle des Maladies Génétiques (Inserm UMR 1089), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Nantes - UFR de Médecine et des Techniques Médicales (UFR MEDECINE), Université de Nantes (UN)-Université de Nantes (UN), Ecole Nationale Vétérinaire, Agroalimentaire et de l'alimentation Nantes-Atlantique (ONIRIS), Immuno-Endocrinologie Cellulaire et Moléculaire (IECM), Ecole Nationale Vétérinaire de Nantes-Université de Nantes (UN)-Institut National de la Recherche Agronomique (INRA), Immuno-Endocrinologie Cellulaire (IECM), Service des Explorations Fonctionnelles - Centre de Référence des Maladies Neuromusculaires Rares, Centre hospitalier universitaire de Nantes (CHU Nantes), Centre Hospitalier Universitaire de l'Hôtel Dieu (CHU Hôtel Dieu), Service de Chirurgie Infantile, Développement et Pathologie du Tissu Musculaire (DPTM), Ecole Nationale Vétérinaire de Nantes-Institut National de la Recherche Agronomique (INRA), Université de Nantes (UN)-Institut National de la Santé et de la Recherche Médicale (INSERM), Institut National de la Recherche Agronomique (INRA)-Université de Nantes (UN)-Ecole Nationale Vétérinaire de Nantes, Institut National de la Recherche Agronomique (INRA)-Ecole Nationale Vétérinaire de Nantes, Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Nantes (UN), Institut National de la Recherche Agronomique (INRA)-École nationale vétérinaire, agroalimentaire et de l'alimentation Nantes-Atlantique (ONIRIS), École nationale vétérinaire, agroalimentaire et de l'alimentation Nantes-Atlantique (ONIRIS), Institut National de la Recherche Agronomique (INRA)-Université de Nantes (UN)-Ecole Nationale Vétérinaire de Nantes-École nationale vétérinaire, agroalimentaire et de l'alimentation Nantes-Atlantique (ONIRIS), and ProdInra, Archive Ouverte
Subjects: [SDV.MHEP] Life Sciences [q-bio]/Human health and pathology, [SDV.IMM] Life Sciences [q-bio]/Immunology, myopathie de duchenne, Immunologie, Immunology, Médecine humaine et pathologie, [SDV.IMM]Life Sciences [q-bio]/Immunology, Human health and pathology, thérapie cellulaire, immunomodulation, [SDV.MHEP]Life Sciences [q-bio]/Human health and pathology
Abstract: Several preclinical approaches based on allogeneic stem cell delivery were shown to be attractive for the treatment of genetic muscular dystrophies. Nevertheless, a significant hurdle for their clinical translation is the immune rejection of donor cells. Immunosuppressive regimens are generally used to overcome host immunity and can allow the improvement of graft survival. Nevertheless, they are associated with a number of side effects, limiting their long term use. Recently, some tissue-specific adult stem cell populations were described to exhibit immunomodulatory properties that could increase their ability to engraft in an allogeneic recipient and improve their regenerative potential. We have previously demonstrated that allogeneic muscle-derived delayed adherent stem cells (that we called MuStem cells) are able to phenotypically and clin- ically correct the Duchenne dystrophic canine model (Rouger et al., 2011; Robriquet et al., 2015). Recently, we isolated human MuStem cells and assessed their immunomodulatory potential. We evaluated their ability to inhibit T cell prolif- eration and to modulate the complement pathway. Interest- ingly, our preliminary data showed that human MuStem cells were able to modulate allogeneic T cell proliferation and to express immunomodulatory molecules such as prostaglandin- E2, indoleamin-2,3-deoxygenase-1 and TGFb2. Moreover, MuStem cells were also able to secrete Factor H molecule suggesting a potential effect on the alternate pathway of the complement system. Overall, our study is critical for the un- derstanding of the crosstalk between MuStem cells and the im- mune system, as well as the design of safe and efficient allogeneic stem cell-based therapy for the treatment of muscle dystrophies
Published: 2015

13. Early interaction of AAV8 with the host immune system following intramuscular delivery results in weak but detectable lymphocyte and dendritic cell transduction

Author: Gernoux, Gwaldys, Guilbaud, Mickaël, Dubreil, Laurence, Larcher, Thibaut, Babarit, Candice, Ledevin, Mireille, Jaulin, Nicolas, Planel, Pierre, Moullier, Philippe, Adjali, Oumeya, UMR1089, Institut National de la Santé et de la Recherche Médicale (INSERM), Atlantic Gene Therapies, Centre hospitalier universitaire de Nantes (CHU Nantes), Physiopathologie Animale et bioThérapie du muscle et du système nerveux (PAnTher), Institut National de la Recherche Agronomique (INRA)-Ecole Nationale Vétérinaire, Agroalimentaire et de l'alimentation Nantes-Atlantique (ONIRIS), and University of Florida [Gainesville]
Subjects: viruses, [SDV.IMM]Life Sciences [q-bio]/Immunology, [SDV.MHEP]Life Sciences [q-bio]/Human health and pathology
Abstract: Following in vivo recombinant Adeno-Associated-Virus (rAAV)-based gene transfer, adaptive immune responses specific to the vector or the transgene product have emerged as a potential roadblock to successful clinical translation. The occurrence of such responses depends on several parameters including the route of vector administration as well as the viral serotype and the genome configuration, either self complementary (sc) or single stranded (ss). These parameters influence rAAV vector-associated immunity by modulating the crosstalk between the vector and the host immune system including vector ability to interact or even transduce lymphoid tissues in general and antigen-presenting cells (APCs) in particular. Little is known about immune cell populations that are targeted in vivo by rAAV vectors. Moreover, the transduction of dendritic cells (DC) is still controversial and not directly demonstrated. Here, we show that intramuscular administration of a sc rAAV8 vector in the mouse leads to a rapid distribution of viral genomes in the lymphoid tissues which is associated to transgene expression. Transduced cells were detected in follicular areas of the spleen and the draining lymph nodes. In addition to B and T lymphocytes, transduced professional APC were detected although at very low frequency. In addition, viral genomes and transgene transcripts were also detected in these cell populations after ss rAAV8 vector administration. Although the functional significance of those observations needs further explorations, our results highlight an early and intricate interaction between the rAAV vector upon its in vivo delivery and the host immune system.
Published: 2015

14. In vitro and in vivo characterization of immunomodulatory properties of MuStem cells

Author: Lorant, Judith, Jaulin, Nicolas, Larcher, Thibaut, Hédan, Benoît, Deschamps, Jack-Yves, Leroux, Isabelle, Zuber, Céline, Schleder, Cindy, Ledevin, Mireille, Dutilleul, Maeva, Goubin, Hélicia, Jounier, Corinne, André, Catherine, Cherel, Yan, Adjali, Oumeya, Rouger, Karl, Physiopathologie Animale et bioThérapie du muscle et du système nerveux (PAnTher), Institut National de la Recherche Agronomique (INRA)-Ecole Nationale Vétérinaire, Agroalimentaire et de l'alimentation Nantes-Atlantique (ONIRIS), LUNAM Université [Nantes Angers Le Mans], Atlantic Gene Therapies, UMR1089, Institut National de la Santé et de la Recherche Médicale (INSERM), Institut de Génétique et Développement de Rennes (IGDR), Structure Fédérative de Recherche en Biologie et Santé de Rennes ( Biosit : Biologie - Santé - Innovation Technologique )-Centre National de la Recherche Scientifique (CNRS)-Université de Rennes 1 (UR1), Université de Rennes (UNIV-RENNES)-Université de Rennes (UNIV-RENNES), Boisbonne Gene and Cell Therapy Center, Partenaires INRAE, ESGCT., Institut National de la Recherche Agronomique (INRA)-École nationale vétérinaire, agroalimentaire et de l'alimentation Nantes-Atlantique (ONIRIS), and Université de Rennes (UR)-Centre National de la Recherche Scientifique (CNRS)-Structure Fédérative de Recherche en Biologie et Santé de Rennes ( Biosit : Biologie - Santé - Innovation Technologique )
Subjects: [SDV.IMM]Life Sciences [q-bio]/Immunology, ComputingMilieux_MISCELLANEOUS, [SDV.MHEP]Life Sciences [q-bio]/Human health and pathology
Abstract: International audience
Published: 2014

15. 697. Phenotypic and Functional Characterisation of Human Anti-AAV CD8+ T Cells Using MHC Class I Tetramer-Associated Magnetic Enrichment

Author: Vandamme, Celine, primary, Devaux, Marie, additional, Jaulin, Nicolas, additional, Guilbaud, Mickaël, additional, Duff, Johanne Le, additional, Hesnard, Leslie, additional, Djamai, Hanane, additional, Bonneville, Marc, additional, Moullier, Philippe, additional, Saulquin, Xavier, additional, and Adjali, Oumeya, additional
Published: 2016
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16. Early Interaction of Adeno-Associated Virus Serotype 8 Vector with the Host Immune System Following Intramuscular Delivery Results in Weak but Detectable Lymphocyte and Dendritic Cell Transduction

Author: Gernoux, Gwladys, primary, Guilbaud, Mickaël, additional, Dubreil, Laurence, additional, Larcher, Thibaut, additional, Babarit, Candice, additional, Ledevin, Mireille, additional, Jaulin, Nicolas, additional, Planel, Pierre, additional, Moullier, Philippe, additional, and Adjali, Oumeya, additional
Published: 2015
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17. Forelimb Treatment in a Large Cohort of Dystrophic Dogs Supports Delivery of a Recombinant AAV for Exon Skipping in Duchenne Patients

Author: Le Guiner, Caroline, primary, Montus, Marie, additional, Servais, Laurent, additional, Cherel, Yan, additional, Francois, Virginie, additional, Thibaud, Jean-Laurent, additional, Wary, Claire, additional, Matot, Béatrice, additional, Larcher, Thibaut, additional, Guigand, Lydie, additional, Dutilleul, Maeva, additional, Domenger, Claire, additional, Allais, Marine, additional, Beuvin, Maud, additional, Moraux, Amélie, additional, Le Duff, Johanne, additional, Devaux, Marie, additional, Jaulin, Nicolas, additional, Guilbaud, Mickaël, additional, Latournerie, Virginie, additional, Veron, Philippe, additional, Boutin, Sylvie, additional, Leborgne, Christian, additional, Desgue, Diana, additional, Deschamps, Jack-Yves, additional, Moullec, Sophie, additional, Fromes, Yves, additional, Vulin, Adeline, additional, Smith, Richard H, additional, Laroudie, Nicolas, additional, Barnay-Toutain, Frédéric, additional, Rivière, Christel, additional, Bucher, Stéphanie, additional, Le, Thanh-Hoa, additional, Delaunay, Nicolas, additional, Gasmi, Mehdi, additional, Kotin, Robert M, additional, Bonne, Gisèle, additional, Adjali, Oumeya, additional, Masurier, Carole, additional, Hogrel, Jean-Yves, additional, Carlier, Pierre, additional, Moullier, Philippe, additional, and Voit, Thomas, additional
Published: 2014
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18. Generation and in vivo evaluation of IL10-treated dendritic cells in a nonhuman primate model of AAV-based gene transfer

Author: Moreau, Aurélie, primary, Vandamme, Céline, additional, Segovia, Mercedes, additional, Devaux, Marie, additional, Guilbaud, Mickaël, additional, Tilly, Gaëlle, additional, Jaulin, Nicolas, additional, Le Duff, Johanne, additional, Cherel, Yan, additional, Deschamps, Jack-Yves, additional, Anegon, Ignacio, additional, Moullier, Philippe, additional, Cuturi, Maria Cristina, additional, and Adjali, Oumeya, additional
Published: 2014
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19. Tetramer-Based Enrichment of Preexisting Anti-AAV8 CD8+ T Cells in Human Donors Allows the Detection of a TEMRA Subpopulation

Author: Vandamme, Céline, Xicluna, Rebecca, Hesnard, Leslie, Devaux, Marie, Jaulin, Nicolas, Guilbaud, Mickaël, Le Duff, Johanne, Couzinié, Célia, Moullier, Philippe, Saulquin, Xavier, Adjali, Oumeya, Laboratoire de Thérapie Génique Translationnelle des Maladies Génétiques (Inserm UMR 1089), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Nantes - UFR de Médecine et des Techniques Médicales (UFR MEDECINE), Université de Nantes (UN)-Université de Nantes (UN), Immunobiology of Human αβ and γδ T Cells and Immunotherapeutic Applications (CRCINA-ÉQUIPE 1), Centre de Recherche en Cancérologie et Immunologie Nantes-Angers (CRCINA), Université de Nantes (UN)-Université de Nantes (UN)-Centre hospitalier universitaire de Nantes (CHU Nantes)-Centre National de la Recherche Scientifique (CNRS)-Université d'Angers (UA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Nantes - UFR de Médecine et des Techniques Médicales (UFR MEDECINE), Université de Nantes (UN)-Université de Nantes (UN)-Centre hospitalier universitaire de Nantes (CHU Nantes)-Centre National de la Recherche Scientifique (CNRS)-Université d'Angers (UA), JAULIN, Nicolas, and Université d'Angers (UA)-Université de Nantes (UN)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Centre hospitalier universitaire de Nantes (CHU Nantes)-Université d'Angers (UA)-Université de Nantes (UN)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Centre hospitalier universitaire de Nantes (CHU Nantes)
Subjects: Adult, Male, lcsh:Immunologic diseases. Allergy, Receptors, CCR7, tetramer-associated magnetic enrichment, [SDV]Life Sciences [q-bio], viruses, Genetic Vectors, Immunology, chemical and pharmacologic phenomena, CD8-Positive T-Lymphocytes, immune response, Interferon-gamma, Young Adult, Immunology and Allergy, Humans, CD8 + T lymphocytes, Aged, Original Research, AAV, Genetic Therapy, Dependovirus, Middle Aged, CD8+ T lymphocytes, gene therapy, [SDV] Life Sciences [q-bio], Capsid Proteins, Female, lcsh:RC581-607, Immunologic Memory
Abstract: Pre-existing immunity to AAV capsid may compromise the safety and efficiency of rAAV-mediated gene transfer in patients. Anti-capsid cytotoxic immune responses have proven to be a challenge to characterize because of the scarcity of circulating AAV-specific CD8+T lymphocytes which can seldom be detected with conventional flow cytometry or ELISpot assays. Here, we used fluorescent MHC class I tetramers combined with magnetic enrichment to detect and phenotype AAV8-specific CD8+T cells in human PBMCs without prior amplification. We showed that all healthy individuals tested carried a pool of AAV8-specific CD8+T cells with a CD45RA+CCR7−terminally-differentiated effector memory cell (TEMRA) fraction.Ex vivofrequencies of total AAV-specific CD8+T cells were not predictive of IFNγ ELISpot responses but interestingly we evidenced a correlation between the proportion of TEMRAcells and IFNγ ELISpot positive responses. TEMRAcells may then play a role in recombinant AAV-mediated cytotoxicity in patients with preexisting immunity. Overall, our results encourage the development of new methods combining increased detection sensitivity of AAV-specific T cells and their poly-functional assessment to better characterize and monitor AAV capsid-specific cellular immune responses in the perspective of rAAV-mediated clinical trials.
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20. AAV8 locoregional delivery induces long-term expression of an immunogenic transgene in macaques despite persisting local inflammation

Author: Virginie Pichard, Oumeya Adjali, Nicolas Jaulin, Malo Journou, Johanne Le Duff, Gwladys Gernoux, Adrien Leger, Caroline Le Guiner, Yan Cherel, Marie Devaux, Mickaël Guilbaud, Jack-Yves Deschamps, Philippe Moullier, Laboratoire de Thérapie Génique Translationnelle des Maladies Génétiques (Inserm UMR 1089), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Nantes - UFR de Médecine et des Techniques Médicales (UFR MEDECINE), Université de Nantes (UN)-Université de Nantes (UN), Centre de Boisbonne [Nantes], Ecole Nationale Vétérinaire, Agroalimentaire et de l'alimentation Nantes-Atlantique (ONIRIS), Physiopathologie Animale et bioThérapie du muscle et du système nerveux (PAnTher), Ecole Nationale Vétérinaire, Agroalimentaire et de l'alimentation Nantes-Atlantique (ONIRIS)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Institut National de la Sante et de la Recherche Medicale (Inserm)European CommissionCHU de NantesEuropean CommissionAFM-Telethon (Association Francaise contre les Myopathies)Appeared in source as:National Research AgencyRegion Pays de la LoireRegion Pays de la Loire, ANR-09-BLAN-0265,GENETOL,INDUCTION DE TOLERANCE SPECIFIQUE DU TRANSGENE APRES THERAPIE GENIQUE(2009), ANR-10-IBHU-0005,CESTI (TSI-IHU),Centre Européen des Sciences de la Transplantation et de l'Immunothérapie (TSI-IHU)(2010), ANR-17-CE17-0007,TOLGEN,Transfert de gène dans le muscle squelettique à l'aide d'AAV: Décryptage de la réponse immune de l'hôte(2017), JAULIN, Nicolas, Blanc - INDUCTION DE TOLERANCE SPECIFIQUE DU TRANSGENE APRES THERAPIE GENIQUE - - GENETOL2009 - ANR-09-BLAN-0265 - Blanc - VALID, Instituts Hospitalo-Universitaires B - Centre Européen des Sciences de la Transplantation et de l'Immunothérapie (TSI-IHU) - - CESTI (TSI-IHU)2010 - ANR-10-IBHU-0005 - IBHU - VALID, Transfert de gène dans le muscle squelettique à l'aide d'AAV: Décryptage de la réponse immune de l'hôte - - TOLGEN2017 - ANR-17-CE17-0007 - AAPG2017 - VALID, Université de Nantes - UFR de Médecine et des Techniques Médicales (UFR MEDECINE), Université de Nantes (UN)-Université de Nantes (UN)-Institut National de la Santé et de la Recherche Médicale (INSERM), École nationale vétérinaire, agroalimentaire et de l'alimentation Nantes-Atlantique (ONIRIS), and École nationale vétérinaire, agroalimentaire et de l'alimentation Nantes-Atlantique (ONIRIS)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)
Subjects: 0301 basic medicine, regulatory T cell, lcsh:QH426-470, macaques, Regulatory T cell, [SDV]Life Sciences [q-bio], Transgene, locoregional delivery, Inflammation, immune response, Virus, Green fluorescent protein, 03 medical and health sciences, Transduction (genetics), 0302 clinical medicine, Immune system, Genetics, medicine, Vector (molecular biology), lcsh:QH573-671, tolerance, gene transfer, Molecular Biology, lcsh:Cytology, business.industry, AAV, 3. Good health, [SDV] Life Sciences [q-bio], Treg, lcsh:Genetics, 030104 developmental biology, medicine.anatomical_structure, inflammation, 030220 oncology & carcinogenesis, Immunology, Molecular Medicine, Original Article, medicine.symptom, business
Abstract: Adeno-associated virus (AAV) vectors are considered efficient vectors for gene transfer, as illustrated by recent successful clinical trials targeting retinal or neurodegenerative disorders. However, limitations as host immune responses to AAV capsid or transduction of limited regions must still be overcome. Here, we focused on locoregional (LR) intravenous perfusion vector delivery that allows transduction of large muscular areas and is considered to be less immunogenic than intramuscular (IM) injection. To confirm this hypothesis, we injected 6 cynomolgus monkeys with an AAV serotype 8 (AAV8) vector encoding for the highly immunogenic GFP driven by either a muscle-specific promoter (n = 3) or a cytomegalovirus (CMV) promoter (n = 3). We report that LR delivery allows long-term GFP expression in the perfused limb (up to 1 year) despite the initiation of a peripheral transgene-specific immune response. The analysis of the immune status of the perfused limb shows that LR delivery induces persisting inflammation. However, this inflammation is not sufficient to result in transgene clearance and is balanced by resident regulatory T cells. Overall, our results suggest that LR delivery promotes persisting transgene expression by induction of Treg cells in situ and might be a safe alternative to IM route to target large muscle territories for the expression of secreted therapeutic factors., Graphical Abstract, Immune responses can be a major limit to rAAV gene transfer efficiency. Gernoux et al. show that locoregional intravenous perfusion (LR) of an AAV8 vector encoding for the highly immunogenic GFP protein in monkeys leads to persisting transgene expression. The inflammation observed in situ is balanced by regulatory T cells.
Published: 2021

21. Intrinsic Differential Scanning Fluorimetry for Fast and Easy Identification of Adeno-Associated Virus Serotypes

Author: Martin Biel, Stylianos Michalakis, Gerhard Winter, Tim Menzen, Axel Rossi, Ruth Rieser, Eduard Ayuso, Hildegard Büning, Magalie Penaud-Budloo, Mohammed Bouzelha, Department of Pharmacy [Munich, Germany] (Center for Drug Research), Ludwig-Maximilians-Universität München (LMU), Laboratoire de Thérapie Génique Translationnelle des Maladies Génétiques (Inserm UMR 1089), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Nantes - UFR de Médecine et des Techniques Médicales (UFR MEDECINE), Université de Nantes (UN)-Université de Nantes (UN), Institute of Experimental Hematology [Hannover, Germany], Hannover Medical School [Hannover] (MHH), Coriolis Pharma [Martinsried, Germany], Center for Integrated Protein Science (CIPSM), Technische Universität Munchen - Université Technique de Munich [Munich, Allemagne] (TUM)-Helmholtz-Zentrum München (HZM)-Ludwig Maximilian University of Munich [Germany] (LMU München), German Center for Infection Research - partner site Hannover-Braunschweig (DZIF), Department of Ophthalmology [Munich, Germany], This work was supported by the Deutsche Forschungsgemeinschaft (EXC114)., JAULIN, Nicolas, and Technische Universität Munchen - Université Technique de Munich [Munich, Allemagne] (TUM)-Ludwig-Maximilians-Universität München (LMU)-Helmholtz Zentrum München = German Research Center for Environmental Health
Subjects: Protein Denaturation, Time Factors, [SDV]Life Sciences [q-bio], viruses, Genetic enhancement, Pharmaceutical Science, adeno-associated virus, medicine.disease_cause, melting temperature, 030226 pharmacology & pharmacy, Virus, Workflow, law.invention, high-throughput technology, 03 medical and health sciences, Transduction (genetics), capsid thermal stability, 0302 clinical medicine, law, Gene expression, medicine, Transition Temperature, High throughput technology, Fluorometry, Adeno-associated virus, Fluorescent Dyes, Protein Unfolding, 030304 developmental biology, 0303 health sciences, Protein Stability, Chemistry, intrinsic fluorescence, Dependovirus, High-Throughput Screening Assays, Cell biology, [SDV] Life Sciences [q-bio], AAV vector, Capsid, Recombinant DNA, Capsid Proteins, Hydrophobic and Hydrophilic Interactions
Abstract: International audience; Recombinant adeno-associated virus (AAV) vectors have evolved as the most promising technology for gene therapy due to their good safety profile, high transduction efficacy, and long-term gene expression in non-dividing cells. AAV-based gene therapy holds great promise for treating genetic disorders like inherited blindness, muscular atrophy, or bleeding disorders. Multiple naturally occurring and engineered AAV serotypes exist, which differ in capsid sequence and as a consequence in cellular tropism. Individual AAV capsids differ in thermal stability and have a characteristic melting temperature (Tm), which enables serotype-specific discrimination of AAV vectors. Differential scanning fluorimetry (DSF) combined with a dye-like SYPRO Orange (SO-DSF), which binds to hydrophobic regions of unfolded proteins, has been successfully applied to determine the Tm of AAV capsids. Here, we present DSF measurement of intrinsic fluorescence signal (iDSF) as a simple alternative method for determination of AAV capsid Tm. The study demonstrates that DSF measurement of intrinsic fluorescence signal is a simple, accurate, and rapid alternative to SO-DSF, which enables characterization of AAV capsid stability with excellent precision and without the need of SO or any other dye.
Published: 2020

22. Genetic diseases in the omics era

Author: Jean-Baptiste Dupont, Laboratoire de Thérapie Génique Translationnelle des Maladies Génétiques (Inserm UMR 1089), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Nantes - UFR de Médecine et des Techniques Médicales (UFR MEDECINE), Université de Nantes (UN)-Université de Nantes (UN), Université de Nantes - UFR de Médecine et des Techniques Médicales (UFR MEDECINE), Université de Nantes (UN)-Université de Nantes (UN)-Institut National de la Santé et de la Recherche Médicale (INSERM), and JAULIN, Nicolas
Subjects: Proteomics, [SDV]Life Sciences [q-bio], MEDLINE, Nerve Tissue Proteins, Computational biology, Mass Spectrometry, Dynamin II, Mice, 03 medical and health sciences, 0302 clinical medicine, Drug Discovery, Genetics, Animals, Humans, Medicine, Longitudinal Studies, Molecular Biology, ComputingMilieux_MISCELLANEOUS, Adaptor Proteins, Signal Transducing, 030304 developmental biology, Pharmacology, 0303 health sciences, Sequence Analysis, RNA, business.industry, Gene Expression Profiling, Tumor Suppressor Proteins, Oligonucleotides, Antisense, Protein Tyrosine Phosphatases, Non-Receptor, Omics, [SDV] Life Sciences [q-bio], Disease Models, Animal, Tamoxifen, Commentary, Molecular Medicine, business, 030217 neurology & neurosurgery, Myopathies, Structural, Congenital
Abstract: Omics analyses are powerful methods to obtain an integrated view of complex biological processes, disease progression, or therapy efficiency. However, few studies have compared different disease forms and different therapy strategies to define the common molecular signatures representing the most significant implicated pathways. In this study, we used RNA sequencing and mass spectrometry to profile the transcriptomes and proteomes of mouse models for three forms of centronuclear myopathies (CNMs), untreated or treated with either a drug (tamoxifen), antisense oligonucleotides reducing the level of dynamin 2 (DNM2), or following modulation of DNM2 or amphiphysin 2 (BIN1) through genetic crosses. Unsupervised analysis and differential gene and protein expression were performed to retrieve CNM molecular signatures. Longitudinal studies before, at, and after disease onset highlighted potential disease causes and consequences. Main pathways in the common CNM disease signature include muscle contraction, regeneration and inflammation. The common therapy signature revealed novel potential therapeutic targets, including the calcium regulator sarcolipin. We identified several novel biomarkers validated in muscle and/or plasma through RNA quantification, western blotting, and enzyme-linked immunosorbent assay (ELISA) assays, including ANXA2 and IGFBP2. This study validates the concept of using multi-omics approaches to identify molecular signatures common to different disease forms and therapeutic strategies.
Published: 2021

23. Chemical modification of the adeno-associated virus capsid to improve gene delivery

Author: Mirja Hommel, Dimitri Alvarez-Dorta, Oumeya Adjali, Aurélien Leray, Eduard Ayuso, Gloria González-Aseguinolaza, Simon Pacouret, Mickaël Guilbaud, Mohammed Bouzelha, Philippe Moullier, Sébastien G. Gouin, Laurence Dubreil, Mathieu Mével, Véronique Blouin, Jean Philippe Combal, David Deniaud, Magalie Penaud-Budloo, INSERM, UMR 1089, Centre hospitalier universitaire de Nantes (CHU Nantes), Chimie Et Interdisciplinarité : Synthèse, Analyse, Modélisation (CEISAM), Université de Nantes - UFR des Sciences et des Techniques (UN UFR ST), Université de Nantes (UN)-Université de Nantes (UN)-Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie du CNRS (INC), Physiopathologie Animale et bioThérapie du muscle et du système nerveux (PAnTher), Ecole Nationale Vétérinaire, Agroalimentaire et de l'alimentation Nantes-Atlantique (ONIRIS)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Expertise en Anatomie Pathologique (APEX), Ecole Nationale Vétérinaire, Agroalimentaire et de l'alimentation Nantes-Atlantique (ONIRIS)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Ecole Nationale Vétérinaire, Agroalimentaire et de l'alimentation Nantes-Atlantique (ONIRIS)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Vivet Therapeutics SAS, Universidad de Navarra [Pamplona] (UNAV), Université de Nantes (UN)-Université de Nantes (UN)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), École nationale vétérinaire, agroalimentaire et de l'alimentation Nantes-Atlantique (ONIRIS)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), École nationale vétérinaire, agroalimentaire et de l'alimentation Nantes-Atlantique (ONIRIS)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-École nationale vétérinaire, agroalimentaire et de l'alimentation Nantes-Atlantique (ONIRIS)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), and JAULIN, Nicolas
Subjects: 0303 health sciences, biology, Chemistry, [SDV]Life Sciences [q-bio], viruses, Asialoglycoprotein, General Chemistry, Gene delivery, medicine.disease_cause, Genome, Virus, 3. Good health, Cell biology, [SDV] Life Sciences [q-bio], 03 medical and health sciences, Transduction (genetics), 0302 clinical medicine, Capsid, 030220 oncology & carcinogenesis, medicine, biology.protein, Antibody, Adeno-associated virus, 030304 developmental biology
Abstract: Gene delivery vectors based on adeno-associated virus (AAV) are highly promising due to several desirable features of this parent virus, including a lack of pathogenicity, efficient infection of dividing and non-dividing cells and sustained maintenance of the viral genome. However, the conclusion from clinical data using these vectors is that there is a need to develop new AAVs with a higher transduction efficiency and specificity for relevant target tissues. To overcome these limitations, we chemically modified the surface of the capsid of AAV vectors. These modifications were achieved by chemical coupling of a ligand by the formation of a thiourea functionality between the amino group of the capsid proteins and the reactive isothiocyanate motif incorporated into the ligand. This strategy does not require genetic engineering of the capsid sequence. The proof of concept was first evidenced using a fluorophore (FITC). Next, we coupled the N-acetylgalactosamine ligand onto the surface of the AAV capsid for asialoglycoprotein receptor-mediated hepatocyte-targeted delivery. Chemically-modified capsids also showed reduced interactions with neutralizing antibodies. Taken together, our findings reveal the possibility of creating a specific engineered platform for targeting AAVs via chemical coupling., Bioconjugated AAV vectors, achieved by coupling of ligands on amino groups of the capsid, are of great interest for gene delivery. Chemical modifications can be used to enhance cell tropism and to decrease interactions with neutralizing antibodies.
Published: 2021

24. Homologous Recombination Offers Advantages over Transposition‐Based Systems to Generate Recombinant Baculovirus for Adeno‐Associated Viral Vector Production

Author: Cécile Robin, Laurie Brun, Frédéric Broucque, Luk H. Vandenberghe, Magalie Penaud-Budloo, Lucie Ménard, Aurélien Jacob, Oumeya Adjali, Eduard Ayuso, Achille François, Mohammed Bouzelha, Aline Roblin, Véronique Blouin, Paloma Jiménez Gil, Laboratoire de Thérapie Génique Translationnelle des Maladies Génétiques (Inserm UMR 1089), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Nantes - UFR de Médecine et des Techniques Médicales (UFR MEDECINE), Université de Nantes (UN)-Université de Nantes (UN), Department of Ophthalmology [Boston, MA, USA] (Ocular Genomics Institute), Harvard Medical School [Boston] (HMS), Grousbeck Gene Therapy Center [Boston, MA, USA], Broad Institute of MIT and Harvard (BROAD INSTITUTE), Harvard Medical School [Boston] (HMS)-Massachusetts Institute of Technology (MIT)-Massachusetts General Hospital [Boston], Harvard Stem Cell Institute [Cambridge, USA] (HSCI), Harvard University [Cambridge], JAULIN, Nicolas, Université de Nantes - UFR de Médecine et des Techniques Médicales (UFR MEDECINE), and Université de Nantes (UN)-Université de Nantes (UN)-Institut National de la Santé et de la Recherche Médicale (INSERM)
Subjects: 0106 biological sciences, baculovirus expression vector, viruses, [SDV]Life Sciences [q-bio], Genetic Vectors, homologous recombination, Computational biology, Gene delivery, Biology, 01 natural sciences, Applied Microbiology and Biotechnology, Genome, Viral vector, law.invention, Transposition (music), Tn7 transposition, law, 010608 biotechnology, Vector (molecular biology), Gene, 010401 analytical chemistry, Gene Transfer Techniques, General Medicine, Dependovirus, gene therapy, 0104 chemical sciences, [SDV] Life Sciences [q-bio], manufacturing, rAAV vectors, Recombinant DNA, Molecular Medicine, Homologous recombination, Baculoviridae
Abstract: International audience; Viral vectors have a great potential for gene delivery, but manufacturing is a big challenge for the industry. The baculovirus-insect cell is one of the most scalable platforms to produce recombinant adeno-associated virus (rAAV) vectors. The standard procedure to generate recombinant baculovirus is based on Tn7 transposition which is time-consuming and suffers technical constraints. Moreover, baculoviral sequences adjacent to the AAV ITRs are preferentially encapsidated into the rAAV vector particles. This observation raises concerns about safety due to the presence of bacterial and antibiotic resistance coding sequences with a Tn7-mediated system for the construction of baculoviruses reagents. Here, a faster and safer method based on homologous recombination (HR) is investigated. First, the functionality of the inserted cassette and the absence of undesirable genes into HR-derived baculoviral genomes are confirmed. Strikingly, it is found that the exogenous cassette showed increased stability over passages when using the HR system. Finally, both materials generated high rAAV vector genome titers, with the advantage of the HR system being exempted from undesirable bacterial genes which provides an additional level of safety for its manufacturing. Overall, this study highlights the importance of the upstream process and starting biologic materials to generate safer rAAV biotherapeutic products.
Published: 2020

25. Tyrosine conjugation methods for protein labelling

Author: David Deniaud, Mathieu Mével, Sébastien G. Gouin, Dimitri Alvarez Dorta, Chimie Et Interdisciplinarité : Synthèse, Analyse, Modélisation (CEISAM), Université de Nantes - UFR des Sciences et des Techniques (UN UFR ST), Université de Nantes (UN)-Université de Nantes (UN)-Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie du CNRS (INC), Laboratoire de Thérapie Génique Translationnelle des Maladies Génétiques (Inserm UMR 1089), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Nantes - UFR de Médecine et des Techniques Médicales (UFR MEDECINE), Université de Nantes (UN)-Université de Nantes (UN), JAULIN, Nicolas, and Université de Nantes (UN)-Université de Nantes (UN)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)
Subjects: Immunoconjugates, [SDV]Life Sciences [q-bio], Lysine, bioorthogonality, Chemical biology, Protein aggregation, 010402 general chemistry, 01 natural sciences, Catalysis, site-specificity, Labelling, Cysteine, chemistry.chemical_classification, Bioconjugation, Staining and Labeling, 010405 organic chemistry, Biomolecule, Organic Chemistry, Proteins, General Chemistry, bioconjugates, Combinatorial chemistry, 0104 chemical sciences, [SDV] Life Sciences [q-bio], chemistry, click chemistry, Click chemistry, tyrosine
Abstract: International audience; Over the last two decades, the development of chemical biology and the need for more defined protein conjugates have fostered active research on new bioconjugation techniques. In particular, a wide range of biorthogonal labelling strategies have been reported to functionalize the phenol side chain of tyrosines (Tyr). Tyr occur at medium frequency and are partially buried at the protein surface, offering interesting opportunities for site-selective labelling of the most reactive residues. Tyr-targeting has proved effective for designing a wide range of important biomolecules including antibody-drug conjugates, fluorescent or radioactive protein probes, glycovaccines, protein aggregates and PEG-conjugates. Innovative methods have also been reported for site-specific labelling with ligand-directed anchors and for specific affinity capture of proteins. This review will present and discuss these promising alternatives to the conventional labelling of the nucleophilic lysine and cysteine residues.
Published: 2020

26. The Contractile Phenotype of Skeletal Muscle in TRPV1 Knockout Mice Is Gender-Specific and Exercise-Dependent

Author: Sylvie Ducreux, Sabine Lotteau, A. Lafoux, Corinne Huchet, JAULIN, Nicolas, Therassay Platform, CAPACITES, Université de Nantes (UN), Cardiovasculaire, métabolisme, diabétologie et nutrition (CarMeN), Université Claude Bernard Lyon 1 (UCBL), 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)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Laboratoire de Thérapie Génique Translationnelle des Maladies Génétiques (Inserm UMR 1089), Université de Nantes - UFR de Médecine et des Techniques Médicales (UFR MEDECINE), Université de Nantes (UN)-Université de Nantes (UN)-Institut National de la Santé et de la Recherche Médicale (INSERM), Hospices Civils de Lyon (HCL), Universite Claude Bernard Lyon 1, Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Hospices Civils de Lyon (HCL), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Nantes - UFR de Médecine et des Techniques Médicales (UFR MEDECINE), and Université de Nantes (UN)-Université de Nantes (UN)
Subjects: medicine.medical_specialty, Contraction (grammar), [SDV]Life Sciences [q-bio], TRPV1, Muscle disorder, General Biochemistry, Genetics and Molecular Biology, Article, 03 medical and health sciences, Transient receptor potential channel, 0302 clinical medicine, Internal medicine, Medicine, skeletal muscle, 10. No inequality, Receptor, lcsh:Science, Ecology, Evolution, Behavior and Systematics, 030304 developmental biology, 0303 health sciences, exercise, business.industry, musculoskeletal, neural, and ocular physiology, Malignant hyperthermia, Paleontology, Skeletal muscle, medicine.disease, [SDV] Life Sciences [q-bio], medicine.anatomical_structure, Endocrinology, nervous system, Space and Planetary Science, Knockout mouse, lcsh:Q, lipids (amino acids, peptides, and proteins), business, 030217 neurology & neurosurgery, psychological phenomena and processes
Abstract: The transient receptor potential vanilloid 1 (TRPV1) belongs to the transient receptor potential superfamily of sensory receptors. TRPV1 is a non-selective cation channel permeable to Ca2+ that is capable of detecting noxious heat temperature and acidosis. In skeletal muscles, TRPV1 operates as a reticular Ca2+-leak channel and several TRPV1 mutations have been associated with two muscle disorders: malignant hyperthermia (MH) and exertional heat stroke (EHS). Although TRPV1&minus, /&minus, mice have been available since the 2000s, TRPV1&rsquo, s role in muscle physiology has not been thoroughly studied. Therefore, the focus of this work was to characterize the contractile phenotype of skeletal muscles of TRPV1-deficient mice at rest and after four weeks of exercise. As MS and EHS have a higher incidence in men than in women, we also investigated sex-related phenotype differences. Our results indicated that, without exercise, TRPV1&minus, mice improved in vivo muscle strength with an impairment of skeletal muscle in vitro twitch features, i.e., delayed contraction and relaxation. Additionally, exercise appeared detrimental to TRPV1&minus, slow-twitch muscles, especially in female animals.
Published: 2020

27. The Assembly-Activating Protein Promotes Stability and Interactions between AAV’s Viral Proteins to Nucleate Capsid Assembly

Author: Ana Karla Cepeda Diaz, Luk H. Vandenberghe, Anna C. Maurer, Jessica Blake, Eva Andres-Mateos, Simon Pacouret, Grousbeck Gene Therapy Center [Boston, MA, USA], Harvard Medical School [Boston] (HMS), Department of Ophthalmology [Boston, MA, USA] (Ocular Genomics Institute), Laboratoire de Thérapie Génique Translationnelle des Maladies Génétiques (Inserm UMR 1089), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Nantes - UFR de Médecine et des Techniques Médicales (UFR MEDECINE), Université de Nantes (UN)-Université de Nantes (UN), Harvard Stem Cell Institute [Cambridge, USA] (HSCI), Harvard University [Cambridge], Massachusetts Institute of Technology (MIT), Broad Institute of MIT and Harvard (BROAD INSTITUTE), Harvard Medical School [Boston] (HMS)-Massachusetts Institute of Technology (MIT)-Massachusetts General Hospital [Boston], This work was supported by NIH National Eye InstituteCore Grant P30EY003790, the NIH Common Fund (5DP1EY023177-03), Giving/Grousbeck, and Lonza Houston (to L.H.V.)., JAULIN, Nicolas, Université de Nantes - UFR de Médecine et des Techniques Médicales (UFR MEDECINE), and Université de Nantes (UN)-Université de Nantes (UN)-Institut National de la Santé et de la Recherche Médicale (INSERM)
Subjects: 0301 basic medicine, Models, Molecular, Genetic enhancement, engineering, [SDV]Life Sciences [q-bio], viruses, Amino Acid Motifs, Host tropism, medicine.disease_cause, structure-function, 0302 clinical medicine, capsid, Adeno-associated virus, lcsh:QH301-705.5, Phylogeny, [SDV.MHEP] Life Sciences [q-bio]/Human health and pathology, AAP, Chemistry, Protein Stability, Structure function, AAV, Dependovirus, Phenotype, gene therapy, Cell biology, [SDV] Life Sciences [q-bio], Capsid, 030220 oncology & carcinogenesis, Gain of Function Mutation, Protein Binding, adeno-associated virus, General Biochemistry, Genetics and Molecular Biology, Virus, Article, 03 medical and health sciences, medicine, Humans, Serotyping, capsid assembly, Virus Assembly, Virion, manufacturing, 030104 developmental biology, HEK293 Cells, lcsh:Biology (General), Capsid Proteins, Protein Multimerization, vector, [SDV.MHEP]Life Sciences [q-bio]/Human health and pathology
Abstract: Summary: The adeno-associated virus (AAV) vector is a preferred delivery platform for in vivo gene therapy. Natural and engineered variations of the AAV capsid affect a plurality of phenotypes relevant to gene therapy, including vector production and host tropism. Fundamental to these aspects is the mechanism of AAV capsid assembly. Here, the role of the viral co-factor assembly-activating protein (AAP) was evaluated in 12 naturally occurring AAVs and 9 putative ancestral capsid intermediates. The results demonstrate increased capsid protein stability and VP-VP interactions in the presence of AAP. The capsid’s dependence on AAP can be partly overcome by strengthening interactions between monomers within the assembly, as illustrated by the transfer of a minimal motif defined by a phenotype-to-phylogeny mapping method. These findings suggest that the emergence of AAP within the Dependovirus genus relaxes structural constraints on AAV assembly in favor of increasing the degrees of freedom for the capsid to evolve. : Maurer et al. describe a phenotype-to-phylogeny mapping strategy correlating phenotypic variation in AAVs to a reconstructed phylogeny, revealing capsid structure-function relationships relevant to that phenotype. Dependence on the viral co-factor AAP for capsid assembly is examined, and capsid functional motifs, in addition to mechanistic roles of AAP, are elucidated. Keywords: AAV, AAP, adeno-associated virus, capsid assembly, manufacturing, capsid, vector engineering, structure-function, gene therapy
Published: 2018

28. Pharmacology of Recombinant Adeno-associated Virus Production

Author: Magalie Penaud-Budloo, Nathalie Clement, Achille François, Eduard Ayuso, JAULIN, Nicolas, Démonstrateurs - Consortium préindustriel des vecteurs de thérapie génique - - PGT2010 - ANR-10-DPBS-0001 - DPBS - VALID, Laboratoire de Thérapie Génique Translationnelle des Maladies Génétiques (Inserm UMR 1089), Université de Nantes - UFR de Médecine et des Techniques Médicales (UFR MEDECINE), Université de Nantes (UN)-Université de Nantes (UN)-Institut National de la Santé et de la Recherche Médicale (INSERM), Powell Gene Therapy [Gainesville, FL, USA], Department of Pediatrics [Gainesville, FL, USA], University of Florida [Gainesville] (UF)-University of Florida [Gainesville] (UF), Financial support to E.A.’s laboratory was provided by INSERM, University of Nantes, CHU of Nantes, Fondation d’Entreprises pour la Thérapie Génique en Pays de la Loire, and Région Pays de la Loire and Pre-industrial gene therapy consortium (Agence Nationale de la Recherche-investissements d’avenir, grant number 10-DPBS-0001)., ANR-10-DPBS-0001,PGT,Consortium préindustriel des vecteurs de thérapie génique(2010), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Nantes - UFR de Médecine et des Techniques Médicales (UFR MEDECINE), and Université de Nantes (UN)-Université de Nantes (UN)
Subjects: 0301 basic medicine, lcsh:QH426-470, viral vectors, [SDV]Life Sciences [q-bio], Genetic enhancement, viruses, quality controls, Biology, medicine.disease_cause, impurities, Article, law.invention, Viral vector, 03 medical and health sciences, Plasmid, law, Genetics, medicine, lcsh:QH573-671, Molecular Biology, Adeno-associated virus, lcsh:Cytology, AAV, Virology, gene therapy, [SDV] Life Sciences [q-bio], lcsh:Genetics, manufacturing, 030104 developmental biology, Herpes simplex virus, Helper virus, Recombinant DNA, Molecular Medicine, Homologous recombination
Abstract: International audience; Recombinant adeno-associated viral (rAAV) vectors have been used in more than 150 clinical trials with a good safety profile and significant clinical benefit in many genetic diseases. In addition, due to their ability to infect non-dividing and dividing cells and to serve as efficient substrate for homologous recombination, rAAVs are being used as a tool for gene-editing approaches. However, manufacturing of these vectors at high quantities and fulfilling current good manufacturing practices (GMP) is still a challenge, and several technological platforms are competing for this niche. Herein, we will describe the most commonly used upstream methods to produce rAAVs, paying particular attention to the starting materials (input) used in each platform and which related impurities can be expected in final products (output). The most commonly found impurities in rAAV stocks include defective particles (i.e., AAV capsids that do contain the therapeutic gene or are not infectious), residual proteins from host cells and helper viruses (adenovirus, herpes simplex virus, or baculoviruses), and illegitimate DNA from plasmids, cells, or helper viruses that may be encapsidated into rAAV particles. Given the role that impurities may play in immunotoxicity, this article reviews the impurities inherently associated with each manufacturing platform.
Published: 2018

29. Unraveling the Complex Story of Immune Responses to AAV Vectors Trial After Trial

Author: Vandamme, Céline, Adjali, Oumeya, Mingozzi, Federico, Department of Clinical Microbiology [Kuopio, Finland], University of Eastern Finland- Institute of Clinical Medicine [Kuopio, Finland], Laboratoire de Thérapie Génique Translationnelle des Maladies Génétiques (Inserm UMR 1089), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Nantes - UFR de Médecine et des Techniques Médicales (UFR MEDECINE), Université de Nantes (UN)-Université de Nantes (UN), 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, 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), F.M.’s work was supported by Genethon. It was also supported by the European Union’s research and innovation program under Grant Agreement No. 667751 (MYOCURE to F.M.), the European Research Council Consolidator Grant under Grant Agreement No. 617432 (MoMAAV to F.M.), and the R-Rare2 consortium grant SMART-HaemoCare. O.A. and C.V. were supported by the Inserm, the French Ministry of Research, the F.R.M. (Fondation pour la Recherche Me´dicale), the University Hospital of Nantes, the Fondation pour la Thérapie Génique en Pays de Loire, the AFM-Téléthon (Association Française contre les Myopathies), the Région Pays de la Loire (IMBIO-DC consortium), and the IHU-CESTI, which is supported by the French National Research Agency (ANR) via the ‘‘Investment Into The Future’’ program ANR-10-IBHU-005., ANR-10-IBHU-0005,CESTI (TSI-IHU),Centre Européen des Sciences de la Transplantation et de l'Immunothérapie (TSI-IHU)(2010), European Project: 667751,H2020,H2020-PHC-2015-two-stage,MYOCURE(2016), European Project: 617432,EC:FP7:ERC,ERC-2013-CoG,MOMAAV(2014), École Pratique des Hautes Études (EPHE), Centre de recherche en Myologie – U974 SU-INSERM, Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU), JAULIN, Nicolas, Instituts Hospitalo-Universitaires B - Centre Européen des Sciences de la Transplantation et de l'Immunothérapie (TSI-IHU) - - CESTI (TSI-IHU)2010 - ANR-10-IBHU-0005 - IBHU - VALID, Development of an innovative gene therapy platform to cure rare hereditary muscle disorders - MYOCURE - - H20202016-01-01 - 2019-12-31 - 667751 - VALID, and Molecular signatures and Modulation of immunity to Adeno-Associated Virus vectors - MOMAAV - - EC:FP7:ERC2014-07-01 - 2019-06-30 - 617432 - VALID
Subjects: Clinical Trials as Topic, Immunity, Cellular, [SDV.MHEP] Life Sciences [q-bio]/Human health and pathology, [SDV]Life Sciences [q-bio], viruses, Genetic Vectors, T cells, Genetic Therapy, CD8-Positive T-Lymphocytes, Dependovirus, gene therapy, immune responses, AAV vectors, [SDV] Life Sciences [q-bio], Humans, Capsid Proteins, antibody responses, Review Articles, [SDV.MHEP]Life Sciences [q-bio]/Human health and pathology
Abstract: International audience; Over the past decade, vectors derived from adeno-associated virus (AAV) have established themselves as apowerful tool for in vivo gene transfer, allowing long-lasting and safe transgene expression in a variety of humantissues. Nevertheless, clinical trials demonstrated how B and T cell immune responses directed against the AAVcapsid, likely arising after natural infection with wild-type AAV, might potentially impact gene transfer safetyand efficacy in patients. Seroprevalence studies have evidenced that most individuals carry anti-AAV neutralizingantibodies that can inhibit recombinant AAV transduction of target cells following in vivo administration ofvector particles. Likewise, liver- and muscle-directed clinical trials have shown that capsid-reactive memoryCD8+T cells could be reactivated and expanded upon presentation of capsid-derived antigens on transduced cells,potentially leading to loss of transgene expression and immune-mediated toxicities. In celebration of the 25thanniversary of the European Society of Gene and Cell Therapy, this review article summarizes progress madeduring the past decade in understanding and modulating AAV vector immunogenicity. While the knowledgegenerated has contributed to yield impressive clinical results, several important questions remain unanswered,making the study of immune responses to AAV a priority for the field of in vivo transfer.
Published: 2017

30. Phagocytosis-shielded lentiviral vectors improve liver gene therapy in nonhuman primates

Author: Mauro Biffi, Daniela Cesana, Federica Moalli, Tongyao Liu, Matteo Iannacone, Douglas Drager, Patrizia Cristofori, Sara Bartolaccini, Fabio Russo, Andrea Raimondi, Alessio Cantore, Ilaria Visigalli, Robert T. Peters, Eugenio Montini, Andrea Calabria, Michela Milani, Susannah Patarroyo-White, Andrea Annoni, Eduard Ayuso, Luigi Naldini, San Raffaele Telethon Institute for Gene Therapy [Milan, Italy] (SR-Tiget), Vita-Salute San Raffaele University and Center for Translational Genomics and Bioinformatics, IRCCS Ospedale San Raffaele [Milan, Italy], Bioverativ [Waltham, MA, USA], GlaxoSmithKline R&D UK [Ware, UK], Laboratoire de Thérapie Génique Translationnelle des Maladies Génétiques (Inserm UMR 1089), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Nantes - UFR de Médecine et des Techniques Médicales (UFR MEDECINE), Université de Nantes (UN)-Université de Nantes (UN), This work was supported by Telethon (SR-Tiget Core Grant 2011–2016) and Bioverativ sponsored research agreement., JAULIN, Nicolas, Université de Nantes - UFR de Médecine et des Techniques Médicales (UFR MEDECINE), Université de Nantes (UN)-Université de Nantes (UN)-Institut National de la Santé et de la Recherche Médicale (INSERM), Milani, M., Annoni, A., Moalli, F., Liu, T., Cesana, D., Calabria, A., Bartolaccini, S., Biffi, M., Russo, F., Visigalli, I., Raimondi, A., Patarroyo-White, S., Drager, D., Cristofori, P., Ayuso, E., Montini, E., Peters, R., Iannacone, M., Cantore, A., and Naldini, L.
Subjects: Kupffer Cells, [SDV]Life Sciences [q-bio], Phagocytosis, Genetic enhancement, Transgene, Genetic Vectors, CD47 Antigen, Article, Immune tolerance, Viral vector, 03 medical and health sciences, 0302 clinical medicine, Mice, Inbred NOD, Immunity, Immune Tolerance, Animals, Humans, Medicine, Tissue Distribution, 030304 developmental biology, Phagocytes, 0303 health sciences, Innate immune system, business.industry, Lentivirus, Gene Transfer Techniques, Genetic Therapy, General Medicine, Immunity, Innate, 3. Good health, Mice, Inbred C57BL, [SDV] Life Sciences [q-bio], Liver, 030220 oncology & carcinogenesis, Immunology, Hepatocytes, Systemic administration, Macaca, business
Abstract: International audience; Liver-directed gene therapy for the coagulation disorder hemophilia showed safe and effective results in clinical trials using adeno-associated viral vectors to replace a functional coagulation factor, although some unmet needs remain. Lentiviral vectors (LVs) may address some of these hurdles because of their potential for stable expression and the low prevalence of preexisting viral immunity in humans. However, systemic LV administration to hemophilic dogs was associated to mild acute toxicity and low efficacy at the administered doses. Here, exploiting intravital microscopy and LV surface engineering, we report a major role of the human phagocytosis inhibitor CD47, incorporated into LV cell membrane, in protecting LVs from uptake by professional phagocytes and innate immune sensing, thus favoring biodistribution to hepatocytes after systemic administration. By enforcing high CD47 surface content, we generated phagocytosis-shielded LVs which, upon intravenous administration to nonhuman primates, showed selective liver and spleen targeting and enhanced hepatocyte gene transfer compared to parental LV, reaching supraphysiological activity of human coagulation factor IX, the protein encoded by the transgene, without signs of toxicity or clonal expansion of transduced cells.
Published: 2019

31. O-GlcNAc stimulation: A new metabolic approach to treat septic shock

Author: Ferron, Marine, Cadiet, julien, Persello, Antoine, Prat, Valentine, Denis, Manon, Erraud, Angélique, Aillerie, Virginie, Mével, Mathieu, Bigot, Edith, Chatham, John, Gauthier, Chantal, Rozec, Bertrand, Lauzier, Benjamin, JAULIN, Nicolas, Unité de recherche de l'institut du thorax (ITX-lab), Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université de Nantes - UFR de Médecine et des Techniques Médicales (UFR MEDECINE), Université de Nantes (UN)-Université de Nantes (UN), Laboratoire de Thérapie Génique Translationnelle des Maladies Génétiques (Inserm UMR 1089), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Nantes - UFR de Médecine et des Techniques Médicales (UFR MEDECINE), Centre hospitalier universitaire de Nantes (CHU Nantes), Division of Molecular and Cellular Pathology [Birmingham], and unité de recherche de l'institut du thorax UMR1087 UMR6291 (ITX)
Subjects: Lipopolysaccharides, Male, [SDV]Life Sciences [q-bio], lcsh:R, lcsh:Medicine, Bridged Bicyclo Compounds, Heterocyclic, Shock, Septic, beta-N-Acetylhexosaminidases, Article, Acetylglucosamine, Rats, Sarcoplasmic Reticulum Calcium-Transporting ATPases, [SDV] Life Sciences [q-bio], Disease Models, Animal, Animals, Fluid Therapy, Humans, lcsh:Q, Bacterial infection, lcsh:Science, Protein Processing, Post-Translational, Cardiac device therapy
Abstract: International audience; Septic shock is a systemic inflammation associated with cell metabolism disorders and cardiovascular dysfunction. Increases in O-GlcNAcylation have shown beneficial cardiovascular effects in acute pathologies. We used two different rat models to evaluate the beneficial effects of O-GlcNAc stimulation at the early phase of septic shock. Rats received lipopolysaccharide (LPS) to induce endotoxemic shock or saline (control) and fluid resuscitation (R) with or without O-GlcNAc stimulation (NButGT-10 mg/kg) 1 hour after shock induction. For the second model, rats received cecal ligature and puncture (CLP) surgery and fluid therapy with or without NButGT. Cardiovascular function was evaluated and heart and blood samples were collected and analysed. NButGT treatment efficiently increased total O-GlcNAc without modification of HBP enzyme expression.Treatment improved circulating parameters and cardiovascular function in both models, and restored SERCA2a expression levels. NButGT treatment also reduced animal mortality. In this study, we demonstrate that in septic shock O-GlcNAc stimulation improves global animal and cardiovascular function outcomes associated with a restoration of SERCA2a levels. This pre-clinical study opens avenues for a potential therapy of early-stage septic shock.
Published: 2019

32. RAAV WITH CHEMICALLY MODIFIED CAPSID

Author: Mével, Mathieu, Deniaud, David, Ayuso, Eduard, Laboratoire de Thérapie Génique Translationnelle des Maladies Génétiques (Inserm UMR 1089), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Nantes - UFR de Médecine et des Techniques Médicales (UFR MEDECINE), Université de Nantes (UN)-Université de Nantes (UN), Chimie Et Interdisciplinarité : Synthèse, Analyse, Modélisation (CEISAM), Université de Nantes - UFR des Sciences et des Techniques (UN UFR ST), Université de Nantes (UN)-Université de Nantes (UN)-Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie du CNRS (INC), JAULIN, Nicolas, and Université de Nantes (UN)-Université de Nantes (UN)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)
Subjects: [SDV] Life Sciences [q-bio], viruses, [SDV]Life Sciences [q-bio]
Abstract: The invention is directed to the field of gene therapy, i.e. gene delivery into target cells, tissue, organ and organism, and more particularly to gene delivery via viral vectors. The inventors showed that it is possible by chemical coupling to modulate the coupling of a ligand in the surface of the capsid of AAV, for example AAV2 and AAV3b. In particular, the present invention relates to a recombinant Adeno-Associated Virus (rAAV) vector particle having at least one primary amino group contained in the capsid proteins, chemically coupled with at least one ligand L, wherein coupling of said ligand L is implemented through a bond comprising a -CSNH- bond and an optionally substituted aromatic moiety. Particularly, the inventors tested the chemical coupling of mannose ligand on AAV2 for subretinally injection to rats. The present invention further relates to a method for chemically coupling an Adeno-Associated Virus (AAV) vector particle with at least one ligand L and to a Recombinant Adeno-Associated Virus (rAAV) vector particle obtained by said method as well as a pharmaceutical composition comprising it and their corresponding medical use.
Published: 2019

33. Single-Stranded DNA Virus Sequencing (SSV-Seq) for Characterization of Residual DNA and AAV Vector Genomes

Author: Lecomte, Emilie, Leger, Adrien, Penaud-Budloo, Magalie, Ayuso, Eduard, Laboratoire de Thérapie Génique Translationnelle des Maladies Génétiques (Inserm UMR 1089), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Nantes - UFR de Médecine et des Techniques Médicales (UFR MEDECINE), Université de Nantes (UN)-Université de Nantes (UN), JAULIN, Nicolas, Université de Nantes - UFR de Médecine et des Techniques Médicales (UFR MEDECINE), and Université de Nantes (UN)-Université de Nantes (UN)-Institut National de la Santé et de la Recherche Médicale (INSERM)
Subjects: [SDV] Life Sciences [q-bio], [SDV.MHEP] Life Sciences [q-bio]/Human health and pathology, [SDV]Life Sciences [q-bio], ComputingMilieux_MISCELLANEOUS, [SDV.MHEP]Life Sciences [q-bio]/Human health and pathology
Abstract: International audience
Published: 2019

34. 5 years of successful inducible transgene expression following locoregional AAV delivery in nonhuman primates with no detectable immunity

Author: Mickaël Guilbaud, Marie Devaux, Célia Couzinié, Johanne Le Duff, Alice Toromanoff, Céline Vandamme, Nicolas Jaulin, Gwladys Gernoux, Thibaut Larcher, Philippe Moullier, Caroline Le Guiner, Oumeya Adjali, UMR 1089, Institut National de la Santé et de la Recherche Médicale (INSERM), UMR1089, Department of Clinical Microbiology, Institue of Clinical Medicine, University of Eastern Finland, Gene Therapy Center, University of Massachusetts Medical School [Worcester] (UMASS), University of Massachusetts System (UMASS), Physiopathologie Animale et bioThérapie du muscle et du système nerveux (PAnTher), Institut National de la Recherche Agronomique (INRA)-Ecole Nationale Vétérinaire, Agroalimentaire et de l'alimentation Nantes-Atlantique (ONIRIS), Laboratoire de Thérapie Génique Translationnelle des Maladies Génétiques (Inserm UMR 1089), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Nantes - UFR de Médecine et des Techniques Médicales (UFR MEDECINE), Université de Nantes (UN)-Université de Nantes (UN), Department of Clinical Microbiology [Kuopio, Finland], University of Eastern Finland- Institute of Clinical Medicine [Kuopio, Finland], Développement et Pathologie du Tissu Musculaire (DPTM), Institut National de la Recherche Agronomique (INRA)-Ecole Nationale Vétérinaire de Nantes, JAULIN, Nicolas, Université de Nantes - UFR de Médecine et des Techniques Médicales (UFR MEDECINE), Université de Nantes (UN)-Université de Nantes (UN)-Institut National de la Santé et de la Recherche Médicale (INSERM), Institut National de la Recherche Agronomique (INRA)-École nationale vétérinaire, agroalimentaire et de l'alimentation Nantes-Atlantique (ONIRIS), ProdInra, Archive Ouverte, Ecole Nationale Vétérinaire de Nantes-Institut National de la Recherche Agronomique (INRA), and Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Nantes (UN)
Subjects: [SDV] Life Sciences [q-bio], [SDV.MHEP] Life Sciences [q-bio]/Human health and pathology, viruses, [SDV]Life Sciences [q-bio], biochemical phenomena, metabolism, and nutrition, ComputingMilieux_MISCELLANEOUS, [SDV.MHEP]Life Sciences [q-bio]/Human health and pathology
Abstract: 5 years of successful inducible transgene expression following locoregional AAV delivery in nonhuman primates with no detectable immunity
Published: 2019

35. Stability of the adeno-associated virus 8 reference standard material

Author: Frédéric Broucque, Magalie Penaud-Budloo, Katell Harrouet, Susan M. D'Costa, Mohammed Bouzelha, Martin Lock, Richard O. Snyder, Véronique Blouin, Sandy Douthe, Eduard Ayuso, Sylvie Saleun, Oumeya Adjali, Sushma Ogram, Laboratoire de Thérapie Génique Translationnelle des Maladies Génétiques (Inserm UMR 1089), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Nantes - UFR de Médecine et des Techniques Médicales (UFR MEDECINE), Université de Nantes (UN)-Université de Nantes (UN), Brammer Bio [Alachua, FL, USA], Department of Pathology and Laboratory Medicine [Philadelphia, PA, USA] (Perelman School of Medicine), University of Pennsylvania [Philadelphia], Financial support to E.A.’s laboratory was provided by INSERM, University of Nantes, CHU of Nantes, Fondation d’Entreprises pour la Thérapie Génique en Pays de la Loire, and Région Pays de la Loire and Pre-industrial gene therapy consortium (Agence Nationale de la Recherche - Investissements d’Avenir, grant number 10-DPBS-0001)., ANR-10-DPBS-0001,PGT,Consortium préindustriel des vecteurs de thérapie génique(2010), University of Pennsylvania, JAULIN, Nicolas, and Démonstrateurs - Consortium préindustriel des vecteurs de thérapie génique - - PGT2010 - ANR-10-DPBS-0001 - DPBS - VALID
Subjects: 0301 basic medicine, Serotype, [SDV]Life Sciences [q-bio], Genetic Vectors, Biology, medicine.disease_cause, Virus Replication, Virus, 03 medical and health sciences, 0302 clinical medicine, Genetics, medicine, Humans, Dosing, Vector (molecular biology), Molecular Biology, Reference standards, Adeno-associated virus, Cryopreservation, Protein Stability, Dependovirus, Reference Standards, Virology, 3. Good health, [SDV] Life Sciences [q-bio], Titer, 030104 developmental biology, HEK293 Cells, Capsid, 030220 oncology & carcinogenesis, Practice Guidelines as Topic, Molecular Medicine, Capsid Proteins
Abstract: International audience; Adeno-associated virus (AAV) vectors are extensively used for gene therapy clinical trials. Accurate and standardized titration methods are essential for characterizing and dosing AAV-based drugs and thus to assess their safety and efficacy. To this end, the Reference Standard Materials (RSM) working group generated standards for AAV serotype 2 and serotype 8. The AAV8RSM (ATCC® VR-1816™) was deposited to the American Type Culture Collection in 2014 and is available to the scientific community. Here, three independent laboratories of the RSM working group provide stability data of the AAV8RSM 2 years after the initial characterization and after container relabeling performed at the ATCC. The AAV8RSM showed constant titers across experimental conditions: 1.48 ± 0.62 × 1012 vector genome (vg)/ml, 9.38 ± 11.4 × 108 infectious units (IU)/ml and 5.76 ± 2.39 × 1011 total particles (p)/ml as determined by qPCR, TCID50 and ELISA, respectively. Additionally, the AAV8RSM capsid protein integrity assessed by SDS-PAGE was equivalent to the original analyses. In conclusion, the AAV8RSM titers remained stable for two years under appropriate storage conditions (
Published: 2018

36. The SSV‐Seq 2.0 PCR‐Free Method Improves the Sequencing of Adeno‐Associated Viral Vector Genomes Containing GC‐Rich Regions and Homopolymers

Author: Magalie Penaud-Budloo, Eduard Ayuso, Véronique Blouin, Emilie Lecomte, Mathieu Bolteau, Oumeya Adjali, Sylvie Saleun, Aurélien Guy-Duché, Laboratoire de Thérapie Génique Translationnelle des Maladies Génétiques (Inserm UMR 1089), Université de Nantes - UFR de Médecine et des Techniques Médicales (UFR MEDECINE), Université de Nantes (UN)-Université de Nantes (UN)-Institut National de la Santé et de la Recherche Médicale (INSERM), JAULIN, Nicolas, Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Nantes - UFR de Médecine et des Techniques Médicales (UFR MEDECINE), and Université de Nantes (UN)-Université de Nantes (UN)
Subjects: [SDV]Life Sciences [q-bio], viruses, Genetic Vectors, Genome, Viral, Computational biology, Biology, Polymerase Chain Reaction, Applied Microbiology and Biotechnology, Genome, DNA sequencing, AAV vectors, PCR-free library, law.invention, Viral vector, 03 medical and health sciences, chemistry.chemical_compound, 0302 clinical medicine, law, Vector (molecular biology), GC-content, Polymerase chain reaction, 030304 developmental biology, homopolymers, 0303 health sciences, High-Throughput Nucleotide Sequencing, high-throughput sequencing, General Medicine, Dependovirus, [SDV] Life Sciences [q-bio], chemistry, 030220 oncology & carcinogenesis, DNA, Viral, Recombinant DNA, Molecular Medicine, DNA
Abstract: International audience; Adeno-associated viral vectors (AAV) are efficient engineered tools for delivering genetic material into host cells. The commercialization of AAV-based drugs must be accompanied by the development of appropriate quality control (QC) assays. Given the potential risk of co-transfer of oncogenic or immunogenic sequences with therapeutic vectors, accurate methods to assess the level of residual DNA in AAV vector stocks are particularly important. An assay based on high-throughput sequencing (HTS) to identify and quantify DNA species in recombinant AAV batches is developed. Here, it is shown that PCR amplification of regions that have a local GC content >90% and include successive mononucleotide stretches, such as the CAG promoter, can introduce bias during DNA library preparation, leading to drops in sequencing coverage. To circumvent this problem, SSV-Seq 2.0, a PCR-free protocol for sequencing AAV vector genomes containing such sequences, is developed. The PCR-free protocol improves the evenness of the rAAV genome coverage and consequently leads to a more accurate relative quantification of residual DNA. HTS-based assays provide a more comprehensive assessment of DNA impurities and AAV vector genome integrity than conventional QC tests based on real-time PCR and are useful methods to improve the safety and efficacy of these viral vectors.
Published: 2020

37. Accurate Titration of Infectious AAV Particles Requires Measurement of Biologically Active Vector Genomes and Suitable Controls

Author: Eduard Ayuso, Véronique Blouin, Philippe Moullier, Achille François, Magalie Penaud-Budloo, Oumeya Adjali, Emilie Lecomte, Mohammed Bouzelha, Frédéric Broucque, Laboratoire de Thérapie Génique Translationnelle des Maladies Génétiques (Inserm UMR 1089), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Nantes - UFR de Médecine et des Techniques Médicales (UFR MEDECINE), Université de Nantes (UN)-Université de Nantes (UN), This research was supported by the Fondation d’Entreprise Thérapie Génique en Pays de Loire, the Commissariat Général à l’Investissement (ANR Program, Investissements d’Avenir, Preindustrial Gene Therapy vector consortium), the Association Française contre les Myopathies (AFM), the Agence Nationale de Sécurité des Médicaments (ANSM), the Centre Hospitalier Universitaire (CHU) of Nantes, and the INSERM., JAULIN, Nicolas, Université de Nantes - UFR de Médecine et des Techniques Médicales (UFR MEDECINE), and Université de Nantes (UN)-Université de Nantes (UN)-Institut National de la Santé et de la Recherche Médicale (INSERM)
Subjects: 0301 basic medicine, titration, [SDV.BIO]Life Sciences [q-bio]/Biotechnology, lcsh:QH426-470, Transgene, Genetic enhancement, viruses, Computational biology, Biology, Genome, Virus, Article, law.invention, AAV vectors, 03 medical and health sciences, law, Genetics, Vector (molecular biology), lcsh:QH573-671, quality control, Molecular Biology, Infectivity, lcsh:Cytology, infectivity, [SDV.SP]Life Sciences [q-bio]/Pharmaceutical sciences, gene therapy, [SDV.BIO] Life Sciences [q-bio]/Biotechnology, [SDV.SP] Life Sciences [q-bio]/Pharmaceutical sciences, lcsh:Genetics, 030104 developmental biology, Recombinant DNA, Molecular Medicine, Cell fractionation
Abstract: Although the clinical use of recombinant adeno-associated virus (rAAV) vectors is constantly increasing, the development of suitable quality control methods is still needed for accurate vector characterization. Among the quality criteria, the titration of infectious particles is critical to determine vector efficacy. Different methods have been developed for the measurement of rAAV infectivity in vitro, based on detection of vector genome replication in trans-complementing cells infected with adenovirus, detection of transgene expression in permissive cells, or simply detection of intracellular vector genomes following the infection of indicator cells. In the present study, we have compared these methods for the titration of infectious rAAV8 vector particles, and, to assess their ability to discriminate infectious and non-infectious rAAV serotype 8 particles, we have generated a VP1-defective AAV8-GFP vector. Since VP1 is required to enter the cell nucleus, the lack of VP1 should drastically reduce the infectivity of rAAV particles. The AAV8 reference standard material was used as a positive control. Our results demonstrated that methods based on measurement of rAAV biological activity (i.e., vector genome replication or transgene expression) were able to accurately discriminate infectious versus non-infectious particles, whereas methods simply measuring intracellular vector genomes were not. Several cell fractionation protocols were tested in an attempt to specifically measure vector genomes that had reached the nucleus, but genomes from wild-type and VP1-defective AAV8 particles were equally detected in the nuclear fraction by qPCR. These data highlight the importance of using suitable controls, including a negative control, for the development of biological assays such as infectious unit titration. Keywords: AAV vectors, gene therapy, quality control, titration, infectivity
Published: 2018

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

Author: Thomas Voit, M. Allais, Marie Montus, Caroline Le Guiner, Yann Audic, Adrien Leger, Emilie Lecomte, Laurent Servais, Philippe Moullier, Claire Domenger, Virginie François, Laboratoire de Thérapie Génique Translationnelle des Maladies Génétiques (Inserm UMR 1089), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Nantes - UFR de Médecine et des Techniques Médicales (UFR MEDECINE), Université de Nantes (UN)-Université de Nantes (UN), Généthon, CHU Trousseau [APHP], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU), NIHR Biomedical Research Centre [London], Guy's and St Thomas' NHS Foundation Trust-King‘s College London, Institut de Génétique et Développement de Rennes (IGDR), Structure Fédérative de Recherche en Biologie et Santé de Rennes ( Biosit : Biologie - Santé - Innovation Technologique )-Centre National de la Recherche Scientifique (CNRS)-Université de Rennes 1 (UR1), Université de Rennes (UNIV-RENNES)-Université de Rennes (UNIV-RENNES), This project was supported by FRM (Fondation pour la Recherche Médicale, grant FDT20140931046), by AFM-Téléthon (Association Française contre les Myopathies), and by ADNA (Advanced Diagnostics for New Therapeutic Approaches), a program dedicated to personalized medicine, coordinated by Institut Mérieux, and supported by research and innovation aid from the French public agency, Bpifrance., JAULIN, Nicolas, and Université de Rennes (UR)-Centre National de la Recherche Scientifique (CNRS)-Structure Fédérative de Recherche en Biologie et Santé de Rennes ( Biosit : Biologie - Santé - Innovation Technologique )
Subjects: 0301 basic medicine, congenital, hereditary, and neonatal diseases and abnormalities, Duchenne muscular dystrophy, [SDV]Life Sciences [q-bio], RNA-Seq, Computational biology, Biology, Article, 03 medical and health sciences, Exon, Drug Discovery, Gene expression, medicine, Messenger RNA, [SDV.MHEP] Life Sciences [q-bio]/Human health and pathology, lcsh:RM1-950, RNA, RNA sequencing, medicine.disease, Duchenne, Exon skipping, 3. Good health, [SDV] Life Sciences [q-bio], lcsh:Therapeutics. Pharmacology, 030104 developmental biology, RNA splicing, Molecular Medicine, U7snRNA, off-target, [SDV.MHEP]Life Sciences [q-bio]/Human health and pathology, exon skipping
Abstract: International audience; Non-coding uridine-rich small nuclear RNAs (UsnRNAs) have emerged in recent years as effective tools for exon skipping for the treatment of Duchenne muscular dystrophy (DMD), a degenerative muscular genetic disorder. We recently showed the high capacity of a recombinant adeno-associated virus (rAAV)-U7snRNA vector to restore the reading frame of the DMD mRNA in the muscles of DMD dogs. We are now moving toward a phase I/II clinical trial with an rAAV-U7snRNA-E53, carrying an antisense sequence designed to hybridize exon 53 of the human DMD messenger. As observed for genome-editing tools, antisense sequences present a risk of off-target effects, reflecting partial hybridization onto unintended transcripts. To characterize the clinical antisense sequence, we studied its expression and explored the occurrence of its off-target effects in human in vitro models of skeletal muscle and liver. We presented a comprehensive methodology combining RNA sequencing and in silico filtering to analyze off-targets. We showed that U7snRNA-E53 induced the effective exon skipping of the DMD transcript without inducing the notable deregulation of transcripts in human cells, neither at gene expression nor at the mRNA splicing level. Altogether, these results suggest that the use of the rAAV-U7snRNA-E53 vector for exon skipping could be safe in eligible DMD patients.
Published: 2018

39. Safety and Long-Term Efficacy of AAV4 Gene Therapy in Patients with RPE65 Leber Congenital Amaurosis

Author: Michel Weber, Oumeya Adjali, Guylène Le Meur, Sébastien Schmitt, Catherine Ivan, Romain Valabregue, Christophe Darmon, Fabienne Rolling, Stéphane Bézieau, Fanny Billaud, Philippe Moullier, Yann Péréon, Pierre Lebranchu, Service d'ophtalmologie [CHU Nantes], Centre hospitalier universitaire de Nantes (CHU Nantes), Laboratoire de Thérapie Génique Translationnelle des Maladies Génétiques (Inserm UMR 1089), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Nantes - UFR de Médecine et des Techniques Médicales (UFR MEDECINE), Université de Nantes (UN)-Université de Nantes (UN), Image Perception Interaction (IPI), Laboratoire des Sciences du Numérique de Nantes (LS2N), Université de Nantes - UFR des Sciences et des Techniques (UN UFR ST), Université de Nantes (UN)-Université de Nantes (UN)-École Centrale de Nantes (ECN)-Centre National de la Recherche Scientifique (CNRS)-IMT Atlantique Bretagne-Pays de la Loire (IMT Atlantique), Institut Mines-Télécom [Paris] (IMT)-Institut Mines-Télécom [Paris] (IMT)-Université de Nantes - UFR des Sciences et des Techniques (UN UFR ST), Institut Mines-Télécom [Paris] (IMT)-Institut Mines-Télécom [Paris] (IMT), Service de génétique médicale [CHU Nantes], Reference Centre for Neuromuscular Disorders ( FILNEMUS), Centre de Neuro-Imagerie de Recherche (CENIR), Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-CHU Pitié-Salpêtrière [AP-HP], Sorbonne Université (SU)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU), JAULIN, Nicolas, Image Perception Interaction (LS2N - équipe IPI), Université de Nantes (UN)-Université de Nantes (UN)-École Centrale de Nantes (ECN)-Centre National de la Recherche Scientifique (CNRS)-IMT Atlantique (IMT Atlantique), Center for NeuroImaging Research-Human MRI Neuroimaging core facility for clinical research [ICM Paris] (CENIR), Institut du Cerveau = Paris Brain Institute (ICM), 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], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU)-Sorbonne Université (SU)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)-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], and Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU)-Sorbonne Université (SU)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)
Subjects: 0301 basic medicine, Retinal degeneration, Visual acuity, genetic structures, Genetic enhancement, [SDV]Life Sciences [q-bio], Leber Congenital Amaurosis, Nystagmus, RPE65, 0302 clinical medicine, Drug Discovery, Child, [SDV.MHEP] Life Sciences [q-bio]/Human health and pathology, clinical trial, Dependovirus, Magnetic Resonance Imaging, gene therapy, 3. Good health, Visual field, [SDV] Life Sciences [q-bio], AAV vector, Molecular Medicine, Original Article, medicine.symptom, Tomography, Optical Coherence, Adult, cis-trans-Isomerases, medicine.medical_specialty, Adolescent, Genetic Vectors, inherited retinal dystrophies, Young Adult, 03 medical and health sciences, Ophthalmology, Genetics, medicine, Humans, Molecular Biology, Retinal thinning, Pharmacology, Analysis of Variance, business.industry, Genetic Therapy, medicine.disease, eye diseases, Clinical trial, 030104 developmental biology, 030221 ophthalmology & optometry, sense organs, Visual Fields, business, [SDV.MHEP]Life Sciences [q-bio]/Human health and pathology, Follow-Up Studies
Abstract: International audience; The aim of this study was the evaluation of the safety and efficacy of unilateral subretinal injection of the adeno-associated vector (AAV) serotypes 2 and 4 (AAV2/4) RPE65-RPE65 vector in patients with Leber congenital amaurosis (LCA) associated with RPE65 gene deficiency. We evaluated ocular and general tolerance and visual function up to 1 year after vector administration in the most severely affected eye in nine patients with retinal degeneration associated with mutations in the RPE65 gene. Patients received either low (1.22 × 1010 to 2 × 1010 vector genomes [vg]) or high (between 3.27 × 1010 and 4.8 × 1010 vg) vector doses. An ancillary study, in which six of the original nine patients participated, extended the follow-up period to 2-3.5 years. All patients showed good ophthalmological and general tolerance to the rAAV2/4-RPE65-RPE65 vector. We observed a trend toward improved visual acuity in patients with nystagmus, stabilization and improvement of the visual field, and cortical activation along visual pathways during fMRI analysis. OCT analysis after vector administration revealed no retinal thinning, except in cases of macular detachment. Our findings show that the rAAV2/4.RPE65.RPE65 vector was well tolerated in nine patients with RPE65-associated LCA. Efficacy parameters varied between patients during follow-up.
Published: 2018

40. Relevance of Assembly-Activating Protein for Adeno-associated Virus Vector Production and Capsid Protein Stability in Mammalian and Insect Cells

Author: Manuel Gunkel, Kathleen Börner, Stefanie Grosse, Lucie Ménard, Ellen Wiedtke, Eduard Ayuso, Chiara Krämer, Julia Fakhiri, Anne-Kathrin Herrmann, Magalie Penaud-Budloo, Dirk Grimm, Vibor Laketa, Department of Infectious Diseases/Virology [Heidelberg, Germany] (Cluster of Excellence CellNetworks), Universität Heidelberg [Heidelberg], BioQuant Center [Heidelberg, Germany], Laboratoire de Thérapie Génique Translationnelle des Maladies Génétiques (Inserm UMR 1089), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Nantes - UFR de Médecine et des Techniques Médicales (UFR MEDECINE), Université de Nantes (UN)-Université de Nantes (UN), German Center for Infection Research [Heidelberg, Germany] (DZIF), Heidelberg University, CellNetworks Advanced Biological Screening Facility [Heidelberg, Germany], A.-K.H. and D.G. gratefully acknowledge support from Collaborative Research Center SFB1129 (project TP2, Deutsche Forschungsgemeinschaft [DFG]). D.G. and his lab are further thankful for support from Collaborative Research Center TRR179 (project TP18, DFG). S.G., A.-K.H., E.W., and D.G. are grateful for funding from the Cluster of Excellence CellNetworks at Heidelberg University (funded by the DFG [EXC81]). J.F. appreciates a PhD stipend from the Hartmut Hoffmann-Berling International Graduate School (HBIGS) at Heidelberg University. M.P.-B. and E.A. acknowledge support from Institut National de la Santé et la Recherche Médicale (INSERM), CHU Nantes, University of Nantes, and the Association Française contre les Myopathies (AFM). K.B., V.L. and D.G. gratefully acknowledge funding through the German Center for Infection Research (DZIF, TTU HIV). S.G., C.K., and D.G. further acknowledge support through a research collaboration with the company Baxalta Inc./Shire., JAULIN, Nicolas, Université de Nantes - UFR de Médecine et des Techniques Médicales (UFR MEDECINE), and Université de Nantes (UN)-Université de Nantes (UN)-Institut National de la Santé et de la Recherche Médicale (INSERM)
Subjects: 0301 basic medicine, Proteasome Endopeptidase Complex, Insecta, viruses, [SDV]Life Sciences [q-bio], Genetic Vectors, Immunology, adeno-associated virus, Biology, medicine.disease_cause, Microbiology, Virus, law.invention, Gene Delivery, 03 medical and health sciences, Plasmid, law, Virology, Sf9 Cells, medicine, Animals, Humans, Vector (molecular biology), capsid assembly, Adeno-associated virus, Mammals, AAP, Protein Stability, Virus Assembly, parvovirus, Virion, AAV, Dependovirus, assembly-activating protein, [SDV] Life Sciences [q-bio], Open reading frame, 030104 developmental biology, Capsid, Insect Science, Recombinant DNA, Capsid Proteins, Proteasome Inhibitors, Nuclear localization sequence, HeLa Cells
Abstract: The discovery that adeno-associated virus 2 (AAV2) encodes an eighth protein, called assembly-activating protein (AAP), transformed our understanding of wild-type AAV biology. Concurrently, it raised questions about the role of AAP during production of recombinant vectors based on natural or molecularly engineered AAV capsids. Here, we show that AAP is indeed essential for generation of functional recombinant AAV2 vectors in both mammalian and insect cell-based vector production systems. Surprisingly, we observed that AAV2 capsid proteins VP1 to -3 are unstable in the absence of AAP2, likely due to rapid proteasomal degradation. Inhibition of the proteasome led to an increase of intracellular VP1 to -3 but neither triggered assembly of functional capsids nor promoted nuclear localization of the capsid proteins. Together, this underscores the crucial and unique role of AAP in the AAV life cycle, where it rapidly chaperones capsid assembly, thus preventing degradation of free capsid proteins. An expanded analysis comprising nine alternative AAV serotypes (1, 3 to 9, and rh10) showed that vector production always depends on the presence of AAP, with the exceptions of AAV4 and AAV5, which exhibited AAP-independent, albeit low-level, particle assembly. Interestingly, AAPs from all 10 serotypes could cross-complement AAP-depleted helper plasmids during vector production, despite there being distinct intracellular AAP localization patterns. These were most pronounced for AAP4 and AAP5, congruent with their inability to rescue an AAV2/AAP2 knockout. We conclude that AAP is key for assembly of genuine capsids from at least 10 different AAV serotypes, which has implications for vectors derived from wild-type or synthetic AAV capsids. IMPORTANCE Assembly of adeno-associated virus 2 (AAV2) is regulated by the assembly-activating protein (AAP), whose open reading frame overlaps with that of the viral capsid proteins. As the majority of evidence was obtained using virus-like particles composed solely of the major capsid protein VP3, AAP's role in and relevance for assembly of genuine AAV capsids have remained largely unclear. Thus, we established a trans -complementation assay permitting assessment of AAP functionality during production of recombinant vectors based on complete AAV capsids and derived from any serotype. We find that AAP is indeed a critical factor not only for AAV2, but also for generation of vectors derived from nine other AAV serotypes. Moreover, we identify a new role of AAP in maintaining capsid protein stability in mammalian and insect cells. Thereby, our study expands our current understanding of AAV/AAP biology, and it concomitantly provides insights into the importance of AAP for AAV vector production.
Published: 2017

41. AAV-ID: A Rapid and Robust Assay for Batch-to-Batch Consistency Evaluation of AAV Preparations

Author: Eva Andres-Mateos, Frédéric Broucque, Rajani Shelke, Ru Xiao, Magalie Penaud-Budloo, Trisha Barungi, Eduard Ayuso, Anna C. Maurer, Heikki Turunen, Véronique Blouin, Mathieu Mével, Luk H. Vandenberghe, Philippe Moullier, Mohammed Bouzelha, Simon Pacouret, Laboratoire de Thérapie Génique Translationnelle des Maladies Génétiques (Inserm UMR 1089), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Nantes - UFR de Médecine et des Techniques Médicales (UFR MEDECINE), Université de Nantes (UN)-Université de Nantes (UN), Université de Nantes - UFR de Médecine et des Techniques Médicales (UFR MEDECINE), Université de Nantes (UN)-Université de Nantes (UN)-Institut National de la Santé et de la Recherche Médicale (INSERM), and JAULIN, Nicolas
Subjects: 0301 basic medicine, [SDV]Life Sciences [q-bio], viruses, Genetic Vectors, Gene transfer, Computational biology, adeno-associated virus, Biology, medicine.disease_cause, identity assay, AAV vectors, 03 medical and health sciences, Consistency (database systems), Structure-Activity Relationship, Capsid, CMC, Drug Discovery, Genetics, medicine, Humans, quality control, Molecular Biology, Adeno-associated virus, identity, Pharmacology, capsid thermostability, Protein Stability, Protein level, Reproducibility of Results, Dependovirus, Virology, gene therapy, [SDV] Life Sciences [q-bio], manufacturing, 030104 developmental biology, homogeneity, Spectrometry, Fluorescence, Mutation, Molecular Medicine, Thermodynamics, Original Article, Capsid Proteins
Abstract: International audience; Adeno-associated virus (AAV) vectors are promising clinical candidates for therapeutic gene transfer, and a number of AAV-based drugs may emerge on the market over the coming years. To insure the consistency in efficacy and safety of any drug vial that reaches the patient, regulatory agencies require extensive characterization of the final product. Identity is a key characteristic of a therapeutic product, as it ensures its proper labeling and batch-to-batch consistency. Currently, there is no facile, fast, and robust characterization assay enabling to probe the identity of AAV products at the protein level. Here, we investigated whether the thermostability of AAV particles could inform us on the composition of vector preparations. AAV-ID, an assay based on differential scanning fluorimetry (DSF), was evaluated in two AAV research laboratories for specificity, sensitivity, and reproducibility, for six different serotypes (AAV1, 2, 5, 6.2, 8, and 9), using 67 randomly selected AAV preparations. In addition to enabling discrimination of AAV serotypes based on their melting temperatures, the obtained fluorescent fingerprints also provided information on sample homogeneity, particle concentration, and buffer composition. Our data support the use of AAV-ID as a reproducible, fast, and low-cost method to ensure batch-to-batch consistency in manufacturing facilities and academic laboratories.
Published: 2017

42. An AAVrh10-CAG-CYP21-HA vector allows persistent correction of 21-hydroxylase deficiency in a Cyp21−/− mouse model

Author: C Goumeaux, Véronique Blouin, M Perdomini, C Dos Santos, Pierre Bougnères, JAULIN, Nicolas, Laboratoire de Thérapie Génique Translationnelle des Maladies Génétiques (Inserm UMR 1089), Université de Nantes - UFR de Médecine et des Techniques Médicales (UFR MEDECINE), Université de Nantes (UN)-Université de Nantes (UN)-Institut National de la Santé et de la Recherche Médicale (INSERM), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Nantes - UFR de Médecine et des Techniques Médicales (UFR MEDECINE), and Université de Nantes (UN)-Université de Nantes (UN)
Subjects: 0301 basic medicine, Adrenal cortex, Genetic enhancement, [SDV]Life Sciences [q-bio], 21-Hydroxylase, Biology, Phenotype, Molecular biology, 3. Good health, Green fluorescent protein, [SDV] Life Sciences [q-bio], 03 medical and health sciences, 030104 developmental biology, medicine.anatomical_structure, Cell culture, Genetics, biology.protein, medicine, Molecular Medicine, Molecular Biology, Gene, Tropism
Abstract: International audience; The treatment of severe forms of 21-hydroxylase deficiency (21OHD) remains unsatisfactory in many respects. As a monogenic disease caused by loss-of-function mutations, 21OHD is a potential candidate for a gene therapy (GT) approach. The first step of GT is to demonstrate positive effects of the therapeutic vector in the Cyp21-/- mouse model. Thus, we tested the adrenal tropism of an AAVrh10-CAG-GFP vector ('GFP vector') then attempted to correct the phenotypic and biochemical alterations in Cyp21-/- mice using an AAVrh10-CAG-humanCYP21A2-HA vector ('CYP21 vector'). Cyp21-/- mice had decreased body mass, high progesterone (4 ×), impaired stress response, increased adrenal expression of genes involved in steroidogenesis or ACTH signaling. Following injection of the GFP vector, Cyp21-/- mice showed abundant GFP expression in the adrenal cortex. Intravenous injection of the therapeutic CYP21 vector allowed 21OH expression in adrenal tissue, resulting in increased body weight and near normalization of urinary progesterone for more than 15 weeks, improved response to stress and restoration of near-normal expression of (several important genes) in the adrenal cortex. The adrenal tropism of AAVrh10 and the persistent correction of phenotypic and biochemical traits in Cyp21-/- mice pave a first step on the way to GT of 21OHD in humans.
Published: 2017

43. Vitrectomy Before Intravitreal Injection of AAV2/2 Vector Promotes Efficient Transduction of Retinal Ganglion Cells in Dogs and Nonhuman Primates

Author: Steven Nedellec, Baptiste Ameline, Fabienne Rolling, Guylène Le Meur, Nathalie Provost, Jack-Yves Deschamps, Véronique Blouin, Lyse Libeau, Marine Biget, Michel Weber, Alexandra Mendes-Madeira, Marie-Anne Colle, Kizito-Tshitoko Tshilenge, Philippe Moullier, Virginie Pichard, Laboratoire de Thérapie Génique Translationnelle des Maladies Génétiques (Inserm UMR 1089), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Nantes (UN), Service d'ophtalmologie [CHU Nantes], Centre hospitalier universitaire de Nantes (CHU Nantes), 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), ONIRIS, Ecole Nationale Vétérinaire, Agroalimentaire et de l'alimentation Nantes-Atlantique (ONIRIS), Physiopathologie Animale et bioThérapie du muscle et du système nerveux (INRA UMR703 PAnTher), Institut National de la Recherche Agronomique (INRA)-Ecole Nationale Vétérinaire de Nantes-ONIRIS, Department of Molecular Genetics and Microbiology [Gainesville, FL, USA] (Center for NeuroGenetics ), University of Florida [Gainesville], This work was supported by unrestricted grants from the Association Française contre les Myopathies, the INSERM, the Fondation pour la Thérapie Génique en Pays de la Loire, and the Agence Nationale pour la Recherche., Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Nantes - UFR de Médecine et des Techniques Médicales (UFR MEDECINE), Université de Nantes (UN)-Université de Nantes (UN), Physiopathologie Animale et bioThérapie du muscle et du système nerveux (PAnTher), Institut National de la Recherche Agronomique (INRA)-Ecole Nationale Vétérinaire, Agroalimentaire et de l'alimentation Nantes-Atlantique (ONIRIS), University of Florida [Gainesville] (UF), JAULIN, Nicolas, École nationale vétérinaire, agroalimentaire et de l'alimentation Nantes-Atlantique (ONIRIS), and Institut National de la Recherche Agronomique (INRA)-École nationale vétérinaire, agroalimentaire et de l'alimentation Nantes-Atlantique (ONIRIS)
Subjects: Retinal Ganglion Cells, 0301 basic medicine, genetic structures, medicine.medical_treatment, viruses, [SDV]Life Sciences [q-bio], Vitrectomy, retinitis-pigmentosa, Applied Microbiology and Biotechnology, Macaque, ectopic expression, chemistry.chemical_compound, Transduction (genetics), 0302 clinical medicine, Transduction, Genetic, Genetics (clinical), [SDV.MHEP] Life Sciences [q-bio]/Human health and pathology, mouse retina, Gene Transfer Techniques, Anatomy, Dependovirus, 3. Good health, [SDV] Life Sciences [q-bio], medicine.anatomical_structure, Retinal ganglion cell, mediated expression, Intravitreal Injections, Molecular Medicine, lebers congenital amaurosis, Retinal Dystrophies, medicine.medical_specialty, gene-therapy, Genetic Vectors, Green Fluorescent Proteins, Biology, Retinal ganglion, Retina, 03 medical and health sciences, Dogs, Ophthalmology, biology.animal, Genetics, medicine, adenoassociated viral vector, visual function, Animals, Humans, photoreceptor degeneration, Pharmacology, Retinal, Genetic Therapy, eye diseases, 030104 developmental biology, chemistry, Vitreous chamber, 030221 ophthalmology & optometry, Macaca, sense organs, transgene expression, [SDV.MHEP]Life Sciences [q-bio]/Human health and pathology
Abstract: International audience; Recombinant adeno-associated virus (AAV) has emerged as a promising vector for retinal gene delivery to restore visual function in certain forms of inherited retinal dystrophies. Several studies in rodent models have shown that intravitreal injection of the AAV2/2 vector is the optimal route for efficient retinal ganglion cell (RGC) transduction. However, translation of these findings to larger species, including humans, is complicated by anatomical differences in the eye, a key difference being the comparatively smaller volume of the vitreous chamber in rodents. Here, we address the role of the vitreous body as a potential barrier to AAV2/2 diffusion and transduction in the RGCs of dogs and macaques, two of the most relevant preclinical models. We intravitreally administered the AAV2/2 vector carrying the CMV-eGFP reporter cassette in dog and macaque eyes, either directly into the vitreous chamber or after complete vitrectomy, a surgical procedure that removes the vitreous body. Our findings suggest that the vitreous body appears to trap the injected vector, thus impairing the diffusion and transduction of AAV2/2 to inner retinal neurons. We show that vitrectomy before intravitreal vector injection is an effective means of overcoming this physical barrier, improving the transduction of RGCs in dog and macaque retinas. These findings support the use of vitrectomy in clinical trials of intravitreal gene transfer techniques targeting inner retinal neurons.
Published: 2016

44. AAV-mediated Gene Therapy Halts Retinal Degeneration in PDE6β-deficient Dogs

Author: Marine Biget, Guylène Le Meur, Marie-Anne Colle, Philippe Hulin, Philippe Moullier, Virginie Pichard, Fabienne Rolling, Baptiste Ameline, Lyse Libeau, Nathalie Provost, Alexandra Mendes-Madeira, Jack-Yves Deschamps, Michel Weber, Kizito-Tshitoko Tshilenge, Laboratoire de Thérapie Génique Translationnelle des Maladies Génétiques (Inserm UMR 1089), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Nantes - UFR de Médecine et des Techniques Médicales (UFR MEDECINE), Université de Nantes (UN)-Université de Nantes (UN), Physiopathologie Animale et bioThérapie du muscle et du système nerveux (PAnTher), Institut National de la Recherche Agronomique (INRA)-Ecole Nationale Vétérinaire, Agroalimentaire et de l'alimentation Nantes-Atlantique (ONIRIS), Association Francaise contre les Myopathies, INSERM, Fondation d'Entreprises pour la Therapie Genique en Pays de la Loire, JAULIN, Nicolas, Université de Nantes - UFR de Médecine et des Techniques Médicales (UFR MEDECINE), Université de Nantes (UN)-Université de Nantes (UN)-Institut National de la Santé et de la Recherche Médicale (INSERM), and Institut National de la Recherche Agronomique (INRA)-École nationale vétérinaire, agroalimentaire et de l'alimentation Nantes-Atlantique (ONIRIS)
Subjects: 0301 basic medicine, Retinal degeneration, Pathology, genetic structures, Genetic enhancement, [SDV]Life Sciences [q-bio], rod phosphodiesterase, chemistry.chemical_compound, Retinal Rod Photoreceptor Cells, Drug Discovery, Medicine, [SDV.MHEP] Life Sciences [q-bio]/Human health and pathology, Retinal Degeneration, cone photoreceptors, Dependovirus, childhood blindness, Ganglion, [SDV] Life Sciences [q-bio], medicine.anatomical_structure, Molecular Medicine, Original Article, restores vision, photoreceptor cell-death, medicine.medical_specialty, mouse model, Genetic Vectors, congenital amaurosis, visual improvement, Retina, 03 medical and health sciences, Dogs, Genetics, Animals, Humans, Bleb (cell biology), Molecular Biology, Pharmacology, Cyclic Nucleotide Phosphodiesterases, Type 6, business.industry, Retinal, Genetic Therapy, medicine.disease, Disease Models, Animal, 030104 developmental biology, chemistry, Dysplasia, recessive retinitis-pigmentosa, beta-subunit, sense organs, business, [SDV.MHEP]Life Sciences [q-bio]/Human health and pathology
Abstract: International audience; We previously reported that subretinal injection of AAV2/5 RK.cpde6β allowed long-term preservation of photoreceptor function and vision in the rod-cone dysplasia type 1 (rcd1) dog, a large animal model of naturally occurring PDE6β deficiency. The present study builds on these earlier findings to provide a detailed assessment of the long-term effects of gene therapy on the spatiotemporal pattern of retinal degeneration in rcd1 dogs treated at 20 days of age. We analyzed the density distribution of the retinal layers and of particular photoreceptor cells in 3.5-year-old treated and untreated rcd1 dogs. Whereas no rods were observed outside the bleb or in untreated eyes, gene transfer halted rod degeneration in all vector-exposed regions. Moreover, while gene therapy resulted in the preservation of cones, glial cells and both the inner nuclear and ganglion cell layers, no cells remained in vector-unexposed retinas, except in the visual streak. Finally, the retinal structure of treated 3.5-year-old rcd1 dogs was identical to that of unaffected 4-month-old rcd1 dogs, indicating near complete preservation. Our findings indicate that gene therapy arrests the degenerative process even if intervention is initiated after the onset of photoreceptor degeneration, and point to significant potential of this therapeutic approach in future clinical trials.
Published: 2016

45. Practical utilization of recombinant AAV vector reference standards: focus on vector genomes titration by free ITR qPCR

Author: Philippe Moullier, Irene C. Perez, Frédéric Broucque, Christine Le Bec, Eduard Ayuso, Achille Fçranois, Richard O. Snyder, Magalie Penaud-Budloo, Véronique Blouin, Susan M. D'Costa, Center of Excellence for Regenerative Health Biotechnology [Florida, USA], University of Florida [Gainesville] (UF), Laboratoire de Thérapie Génique Translationnelle des Maladies Génétiques (Inserm UMR 1089), Université de Nantes - UFR de Médecine et des Techniques Médicales (UFR MEDECINE), Université de Nantes (UN)-Université de Nantes (UN)-Institut National de la Santé et de la Recherche Médicale (INSERM), Généthon, Department of Molecular Genetics and Microbiology [Gainesville, FL, USA] (Center for NeuroGenetics ), This work was performed under a Cooperative Agreement between CHU-Nantes, INSERM, AFM, and the University of Florida Center of Excellence for Regenerative Health Biotechnology., JAULIN, Nicolas, Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Nantes - UFR de Médecine et des Techniques Médicales (UFR MEDECINE), and Université de Nantes (UN)-Université de Nantes (UN)
Subjects: 0301 basic medicine, Focus (geometry), lcsh:QH426-470, viruses, [SDV]Life Sciences [q-bio], Computational biology, Biology, Genome, Article, law.invention, 03 medical and health sciences, Plasmid, law, Genetics, Vector (molecular biology), lcsh:QH573-671, Molecular Biology, Reference standards, lcsh:Cytology, [SDV] Life Sciences [q-bio], Titer, lcsh:Genetics, 030104 developmental biology, Real-time polymerase chain reaction, Recombinant DNA, Molecular Medicine
Abstract: International audience; Clinical trials using recombinant adeno-associated virus (rAAV) vectors have demonstrated efficacy and a good safety profile. Although the field is advancing quickly, vector analytics and harmonization of dosage units are still a limitation for commercialization. AAV reference standard materials (RSMs) can help ensure product safety by controlling the consistency of assays used to characterize rAAV stocks. The most widely utilized unit of vector dosing is based on the encapsidated vector genome. Quantitative polymerase chain reaction (qPCR) is now the most common method to titer vector genomes (vg); however, significant inter- and intralaboratory variations have been documented using this technique. Here, RSMs and rAAV stocks were titered on the basis of an inverted terminal repeats (ITRs) sequence-specific qPCR and we found an artificial increase in vg titers using a widely utilized approach. The PCR error was introduced by using single-cut linearized plasmid as the standard curve. This bias was eliminated using plasmid standards linearized just outside the ITR region on each end to facilitate the melting of the palindromic ITR sequences during PCR. This new “Free-ITR” qPCR delivers vg titers that are consistent with titers obtained with transgene-specific qPCR and could be used to normalize in-house product-specific AAV vector standards and controls to the rAAV RSMs. The free-ITR method, including well-characterized controls, will help to calibrate doses to compare preclinical and clinical data in the field.
Published: 2016

46. A Fragmented Adeno-Associated Viral Dual Vector Strategy for Treatment of Diseases Caused by Mutations in Large Genes Leads to Expression of Hybrid Transcripts

Author: Robert E MacLaren, Michelle E. McClements, Peter Charbel Issa, Véronique Blouin, Nuffield Department of Clinical Neurosciences [Oxford], University of Oxford [Oxford], The John Radcliffe Hospital [Oxford, UK] (Oxford Eye Hospital), Laboratoire de Thérapie Génique Translationnelle des Maladies Génétiques (Inserm UMR 1089), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Nantes - UFR de Médecine et des Techniques Médicales (UFR MEDECINE), Université de Nantes (UN)-Université de Nantes (UN), MRC DPFS scheme, Fight for Sight (UK), Royal College of Surgeons of Edinburgh, Knoop Fellowship, Marie Curie Foundation., JAULIN, Nicolas, and University of Oxford
Subjects: 0301 basic medicine, education.field_of_study, Messenger RNA, Transgene, [SDV]Life Sciences [q-bio], Population, Computational biology, Biology, Molecular biology, Article, [SDV] Life Sciences [q-bio], 03 medical and health sciences, 030104 developmental biology, Clinical trials, Capsid, Genes, Protein biosynthesis, Coding region, Vector (molecular biology), Vector, education, Gene, Mutations
Abstract: Objective Dual vector AAV systems are being utilised to enable gene therapy for disorders in which the disease gene is too large to ft into a single capsid. Fragmented adeno-associated viral (fAAV) vectors containing single inverted terminal repeat truncated transgenes have been considered as one such gene replacement strategy. Here we aim to add to the current understanding of the molecular mechanisms employed by fAAV dual vector systems. Methods Oversized (>8 kb) transgene constructs containing ABCA4 coding sequence were packaged as truncated fragments Results Transductions with fAAV vector preparations yielded ABCA4 mRNA, but did not generate detectable levels of protein. Sequencing of the transcript population revealed the presence of full length ABCA4 CDS with additional hybrid ABCA4 variants, indicating truncated transgenes without regions of overlap were joining and forming stable hybrid transgenes. In contrast, an ABCA4 overlapping dual vector system (OV) with a defned complementary region generated only full length mRNA transcripts plus detectable ABCA4 protein. Conclusion Despite previous success shown with the fAAV approach, the lack of repeatability and identifcation of stable hybrid transcripts capable of protein production suggests there is more refnement required before considering this approach in a clinical setting.
Published: 2016

47. Manufacturing of recombinant adeno-associated viral vectors: new technologies are welcome

Author: Eduard Ayuso, Laboratoire de Thérapie Génique Translationnelle des Maladies Génétiques (Inserm UMR 1089), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Nantes - UFR de Médecine et des Techniques Médicales (UFR MEDECINE), Université de Nantes (UN)-Université de Nantes (UN), Université de Nantes - UFR de Médecine et des Techniques Médicales (UFR MEDECINE), Université de Nantes (UN)-Université de Nantes (UN)-Institut National de la Santé et de la Recherche Médicale (INSERM), and JAULIN, Nicolas
Subjects: 0301 basic medicine, lcsh:QH426-470, viruses, Genetic enhancement, [SDV]Life Sciences [q-bio], Gene delivery, Viral vector, law.invention, 03 medical and health sciences, law, Genetics, Medicine, lcsh:QH573-671, Molecular Biology, ComputingMilieux_MISCELLANEOUS, business.industry, lcsh:Cytology, Comment, Leber congenital amaurosis, Preclinical data, Virology, 3. Good health, Biotechnology, Clinical trial, Alipogene tiparvovec, [SDV] Life Sciences [q-bio], lcsh:Genetics, 030104 developmental biology, Recombinant DNA, Molecular Medicine, business
Abstract: Recombinant adeno-associated viral vectors (rAAV) are probably the most powerful tools for in vivo gene delivery. Encouraging preclinical data have been followed by successful gene therapy clinical trials including Leber's congenital amaurosis type 2 (refs. 1–3), hemophilia B,4,5 and recently choroideremia.6 These results together with the market authorization of Glybera, an AAV-based product for the treatment of lipoprotein lipase deficiency,7,8 has prompted skeptical investors and biotechnology and pharmaceuticals companies to move into this field.
Published: 2015

48. Use of Adeno-Associated Virus to Enrich Cardiomyocytes Derived from Human Stem Cells

Author: Philippe Moullier, Xuan Guan, Stefan Czerniecki, David L. Mack, Zejing Wang, Virginie François, Véronique Blouin, Martin K. Childers, Laboratoire de Thérapie Génique Translationnelle des Maladies Génétiques (Inserm UMR 1089), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Nantes - UFR de Médecine et des Techniques Médicales (UFR MEDECINE), Université de Nantes (UN)-Université de Nantes (UN), Université de Nantes - UFR de Médecine et des Techniques Médicales (UFR MEDECINE), Université de Nantes (UN)-Université de Nantes (UN)-Institut National de la Santé et de la Recherche Médicale (INSERM), and JAULIN, Nicolas
Subjects: viruses, [SDV]Life Sciences [q-bio], Population, Cell, Genetic Vectors, Induced Pluripotent Stem Cells, Gene Expression, Biology, 030204 cardiovascular system & hematology, medicine.disease_cause, Serogroup, Virus, Cell Line, Immunophenotyping, Transduction (genetics), 03 medical and health sciences, 0302 clinical medicine, Genes, Reporter, Transduction, Genetic, medicine, Humans, Myocytes, Cardiac, education, Induced pluripotent stem cell, Adeno-associated virus, Research Articles, Genetics (clinical), 030304 developmental biology, education.field_of_study, 0303 health sciences, [SDV.MHEP] Life Sciences [q-bio]/Human health and pathology, Stem Cells, Gene Transfer Techniques, Cell Differentiation, Cell sorting, Dependovirus, Molecular biology, Immunohistochemistry, Cell biology, [SDV] Life Sciences [q-bio], Viral Tropism, medicine.anatomical_structure, Stem cell, [SDV.MHEP]Life Sciences [q-bio]/Human health and pathology
Abstract: International audience; Cardiomyocytes derived from human induced pluripotent stem cells (iPSCs) show great promise as autologousdonor cells to treat heart disease. A major technical obstacle to this approach is that availableinduction methods often produce heterogeneous cell population with low percentage of cardiomyocytes.Here we describe a cardiac enrichment approach using nonintegrating adeno-associated virus (AAV). Wefirst examined several AAV serotypes for their ability to selectively transduce iPSC-derived cardiomyocytes.Results showed that AAV1 demonstrated the highest in vitro transduction efficiency among sevenwidely used serotypes. Next, differentiated iPSC derivatives were transduced with drug-selectable AAV1expressing neomycin resistance gene. Selection with G418 enriched the cardiac cell fraction from 27% to57% in 2 weeks. Compared with other enrichment strategies such as integrative genetic selection, mitochondrialabeling, or surface marker cell sorting, this simple AAV method described herein bypassesantibody or dye labeling. These findings provide proof of concept for large-scale cardiomyocyte enrichmentby exploiting AAV’s intrinsic tissue tropism
Published: 2015

49. Advanced Characterization of DNA Molecules in rAAV Vector Preparations by Single-stranded Virus Next-generation Sequencing

Author: Lecomte, Emilie, Tournaire, Benoît, Cogné, Benjamin, Dupont, Jean-Baptiste, Lindenbaum, Pierre, Martin-Fontaine, Mélanie, Broucque, Frédéric, Robin, Cécile, Hebben, Matthias, Merten, Otto-Wilhelm, Blouin, Véronique, François, Achille, Redon, Richard, Moullier, Philippe, Léger, Adrien, Laboratoire de Thérapie Génique Translationnelle des Maladies Génétiques (Inserm UMR 1089), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Nantes - UFR de Médecine et des Techniques Médicales (UFR MEDECINE), Université de Nantes (UN)-Université de Nantes (UN), unité de recherche de l'institut du thorax UMR1087 UMR6291 (ITX), Université de Nantes - UFR de Médecine et des Techniques Médicales (UFR MEDECINE), Université de Nantes (UN)-Université de Nantes (UN)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Généthon, Department of Molecular Genetics and Microbiology [Gainesville, FL, USA] (Center for NeuroGenetics ), University of Florida [Gainesville] (UF), This work was supported by the 'Association Française contre les Myopathies', the 'région Pays-de-la-Loire' (Emergence collective 2012, AGTI-1), and the 'Commissariat général à l’investissement' (ANR-PGT Investissements d’avenir)., JAULIN, Nicolas, Unité de recherche de l'institut du thorax (ITX-lab), and Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université de Nantes - UFR de Médecine et des Techniques Médicales (UFR MEDECINE)
Subjects: [SDV] Life Sciences [q-bio], next generation sequencing, Methods: Original Article, lcsh:Therapeutics. Pharmacology, [SDV]Life Sciences [q-bio], viruses, lcsh:RM1-950, quality control, rAAV, DNA contaminants
Abstract: International audience; Recent successful clinical trials with recombinant adeno-associated viral vectors (rAAVs) have led to a renewed interest in gene therapy. However, despite extensive developments to improve vector-manufacturing processes, undesirable DNA contaminants in rAAV preparations remain a major safety concern. Indeed, the presence of DNA fragments containing antibiotic resistance genes, wild-type AAV, and packaging cell genomes has been found in previous studies using quantitative polymerase chain reaction (qPCR) analyses. However, because qPCR only provides a partial view of the DNA molecules in rAAV preparations, we developed a method based on next-generation sequencing (NGS) to extensively characterize single-stranded DNA virus preparations (SSV-Seq). In order to validate SSV-Seq, we analyzed three rAAV vector preparations produced by transient transfection of mammalian cells. Our data were consistent with qPCR results and showed a quasi-random distribution of contaminants originating from the packaging cells genome. Finally, we found single-nucleotide variants (SNVs) along the vector genome but no evidence of large deletions. Altogether, SSV-Seq could provide a characterization of DNA contaminants and a map of the rAAV genome with unprecedented resolution and exhaustiveness. We expect SSV-Seq to pave the way for a new generation of quality controls, guiding process development toward rAAV preparations of higher potency and with improved safety profiles.
Published: 2015

50. Early Interaction of Adeno-Associated Virus Serotype 8 Vector with the Host Immune System Following Intramuscular Delivery Results in Weak but Detectable Lymphocyte and Dendritic Cell Transduction

Author: GernouxGwladys, LedevinMireille, LarcherThibaut, JaulinNicolas, GuilbaudMickaël, DubreilLaurence, PlanelPierre, MoullierPhilippe, BabaritCandice, AdjaliOumeya, Laboratoire de Thérapie Génique Translationnelle des Maladies Génétiques (Inserm UMR 1089), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Nantes - UFR de Médecine et des Techniques Médicales (UFR MEDECINE), Université de Nantes (UN)-Université de Nantes (UN), Atlantic Gene Therapies [Nantes] (AGT), Institut de Recherche en Santé 2 – Nantes Biotech (IRS2 Nantes Biotech), Physiopathologie Animale et bioThérapie du muscle et du système nerveux (PAnTher), Institut National de la Recherche Agronomique (INRA)-Ecole Nationale Vétérinaire, Agroalimentaire et de l'alimentation Nantes-Atlantique (ONIRIS), Science et génie alimentaire [UNAM, Nantes], Ecole Nationale Vétérinaire de Nantes-PRES Université Nantes Angers Le Mans (UNAM)-Ecole Nationale Vétérinaire, Agroalimentaire et de l'alimentation Nantes-Atlantique (ONIRIS), Department of Molecular Genetics and Microbiology [Gainesville, FL, USA] (Center for NeuroGenetics ), University of Florida [Gainesville] (UF), Financial support was provided by INSERM, the French Ministry of Health, the INRA, the University Hospital of Nantes, the Fondation pour la Thérapie Génique en Pays de Loire, The IHU-CESTI (which is supported by the French National Research Agency [ANR] via the ‘‘Investment into the Future’’ program ANR-10-IBHU-005), and the AFM-Telethon (Association Française contre les Myopathies)., Université de Nantes - UFR de Médecine et des Techniques Médicales (UFR MEDECINE), Université de Nantes (UN)-Université de Nantes (UN)-Institut National de la Santé et de la Recherche Médicale (INSERM), Institut National de la Recherche Agronomique (INRA)-École nationale vétérinaire, agroalimentaire et de l'alimentation Nantes-Atlantique (ONIRIS), Ecole Nationale Vétérinaire de Nantes-PRES Université Nantes Angers Le Mans (UNAM)-École nationale vétérinaire, agroalimentaire et de l'alimentation Nantes-Atlantique (ONIRIS), JAULIN, Nicolas, Université de Nantes (UN)-Institut National de la Santé et de la Recherche Médicale (INSERM), Physiopathologie Animale et bioThérapie du muscle et du système nerveux (INRA UMR703 PAnTher), Institut National de la Recherche Agronomique (INRA)-Ecole Nationale Vétérinaire de Nantes-ONIRIS, University of Florida [Gainesville], and Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Nantes (UN)
Subjects: Male, Transcription, Genetic, Lymphocyte, Transgene, viruses, [SDV]Life Sciences [q-bio], Genetic Vectors, Gene Dosage, Antigen-Presenting Cells, Gene Expression, Genome, Viral, Biology, medicine.disease_cause, Serogroup, Virus, Cell Line, 03 medical and health sciences, Transduction (genetics), Mice, 0302 clinical medicine, Immune system, Immunity, Transduction, Genetic, Genetics, medicine, Animals, Humans, Tissue Distribution, Lymphocytes, Transgenes, Molecular Biology, Adeno-associated virus, 030304 developmental biology, 0303 health sciences, [SDV.MHEP] Life Sciences [q-bio]/Human health and pathology, Gene Transfer Techniques, Dendritic cell, Dendritic Cells, Dependovirus, Virology, [SDV] Life Sciences [q-bio], medicine.anatomical_structure, Liver, 030220 oncology & carcinogenesis, Molecular Medicine, RNA, Viral, [SDV.MHEP]Life Sciences [q-bio]/Human health and pathology
Abstract: International audience; Following in vivo recombinant adeno-associated virus (rAAV)–based gene transfer, adaptive immune responses specific to the vector or the transgene product have emerged as a potential roadblock to successful clinical translation. The occurrence of such responses depends on several parameters, including the route of vector administration as well as the viral serotype and the genome configuration, either self-complementary (sc) or single-stranded (ss). These parameters influence rAAV vector-associated immunity by modulating the crosstalk between the vector and the host immune system, including vector ability to interact or even transduce lymphoid tissues in general and antigen-presenting cells (APCs) in particular. Little is known about immune cell populations that are targeted in vivo by rAAV vectors. Moreover, the transduction of dendritic cells is still controversial and not directly demonstrated. Here, we show that intramuscular administration of an sc rAAV8 vector in the mouse leads to a rapid distribution of viral genomes in the lymphoid tissues that is associated with transgene expression. Transduced cells were detected in follicular areas of the spleen and the draining lymph nodes. In addition to B and T lymphocytes, transduced professional APCs were detected although at very low frequency. In addition, viral genomes and transgene transcripts were also detected in these cell populations after ss rAAV8 vector administration. Although the functional significance of those observations needs further explorations, our results highlight an early and intricate interaction between the rAAV vector upon its in vivo delivery and the host immune system.
Published: 2015

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