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1. Gene therapy with secreted acid alpha-glucosidase rescues Pompe disease in a novel mouse model with early-onset spinal cord and respiratory defects

Author

Pasqualina Colella, Pauline Sellier, Manuel J. Gomez, Maria G. Biferi, Guillaume Tanniou, Nicolas Guerchet, Mathilde Cohen-Tannoudji, Maryse Moya-Nilges, Laetitia van Wittenberghe, Natalie Daniele, Bernard Gjata, Jacomina Krijnse-Locker, Fanny Collaud, Marcelo Simon-Sola, Severine Charles, Umut Cagin, and Federico Mingozzi

Subjects
Pompe disease, Mouse model, Respiratory function, Spinal cord, Muscle, AAV, Medicine, Medicine (General), R5-920
Abstract

Background: Pompe disease (PD) is a neuromuscular disorder caused by deficiency of acidalpha-glucosidase (GAA), leading to motor and respiratory dysfunctions. Available Gaa knock-out (KO) mouse models do not accurately mimic PD, particularly its highly impaired respiratory phenotype. Methods: Here we developed a new mouse model of PD crossing Gaa KOB6;129 with DBA2/J mice. We subsequently treated Gaa KODBA2/J mice with adeno-associated virus (AAV) vectors expressing a secretable form of GAA (secGAA). Findings: Male Gaa KODBA2/J mice present most of the key features of the human disease, including early lethality, severe respiratory impairment, cardiac hypertrophy and muscle weakness. Transcriptome analyses of Gaa KODBA2/J, compared to the parental Gaa KOB6;129 mice, revealed a profoundly impaired gene signature in the spinal cord and a similarly deregulated gene expression in skeletal muscle. Muscle and spinal cord transcriptome changes, biochemical defects, respiratory and muscle function in the Gaa KODBA2/J model were significantly improved upon gene therapy with AAV vectors expressing secGAA. Interpretation: These data show that the genetic background impacts on the severity of respiratory function and neuroglial spinal cord defects in the Gaa KO mouse model of PD. Our findings have implications for PD prognosis and treatment, show novel molecular pathophysiology mechanisms of the disease and provide a unique model to study PD respiratory defects, which majorly affect patients. Funding: This work was supported by Genethon, the French Muscular Dystrophy Association (AFM), the European Commission (grant nos. 667751, 617432, and 797144), and Spark Therapeutics.

Published
2020
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2. Restoring neuronal chloride homeostasis with anti-NKCC1 gene therapy rescues cognitive deficits in a mouse model of Down syndrome

Author

Federica Piccardi, Federico Mingozzi, Giovanni Morelli, Giuseppe Ronzitti, Micol Alberti, Ilaria Colombi, Martina Parrini, Anna Rocchi, Marina Nanni, Laura Cancedda, Andrea Contestabile, Shovan Naskar, and Severine Charles

Subjects
Male, Down syndrome, Genetic enhancement, Biology, Chloride, Mice, 03 medical and health sciences, 0302 clinical medicine, Chlorides, RNA interference, Drug Discovery, Genetics, medicine, Animals, Homeostasis, Solute Carrier Family 12, Member 2, Molecular Biology, 030304 developmental biology, Neurons, Pharmacology, 0303 health sciences, Gene knockdown, Transporter, Genetic Therapy, medicine.disease, 3. Good health, Cell biology, Disease Models, Animal, Gene Knockdown Techniques, Molecular Medicine, RNA Interference, Down Syndrome, 030217 neurology & neurosurgery, Intracellular, medicine.drug
Abstract

A common feature of diverse brain disorders is the alteration of GABA-mediated inhibition because of aberrant, intracellular chloride homeostasis induced by changes in the expression and/or function of chloride transporters. Notably, pharmacological inhibition of the chloride importer NKCC1 is able to rescue brain-related core deficits in animal models of these pathologies and in some human clinical studies. Here, we show that reducing NKCC1 expression by RNA interference in the Ts65Dn mouse model of Down syndrome (DS) restores intracellular chloride concentration, efficacy of gamma-aminobutyric acid (GABA)-mediated inhibition, and neuronal network dynamics in vitro and ex vivo. Importantly, adeno-associated virus (AAV)-mediated, neuron-specific NKCC1 knockdown in vivo rescues cognitive deficits in diverse behavioral tasks in Ts65Dn animals. Our results highlight a mechanistic link between NKCC1 expression and behavioral abnormalities in DS mice and establish a molecular target for new therapeutic approaches, including gene therapy, to treat brain disorders characterized by neuronal chloride imbalance.

Published
2021
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3. Development of a dual hybrid AAV vector for endothelial-targeted expression of von Willebrand factor

Author

Federico Mingozzi, Caterina Casari, Aboud Sakkal, Peter J. Lenting, Cécile V. Denis, Charlotte Kawecki, Severine Charles, Elena Barbon, Giuseppe Ronzitti, Solenne Marmier, Olivier D. Christophe, and Fanny Collaud

Subjects
0301 basic medicine, congenital, hereditary, and neonatal diseases and abnormalities, Transgene, Genetic enhancement, Context (language use), 030204 cardiovascular system & hematology, Biology, Gene delivery, medicine.disease, Cell biology, 03 medical and health sciences, Transduction (genetics), 030104 developmental biology, 0302 clinical medicine, Von Willebrand factor, hemic and lymphatic diseases, Genetics, biology.protein, Von Willebrand disease, medicine, Molecular Medicine, Molecular Biology, Gene
Abstract

Von Willebrand disease (VWD), the most common inherited bleeding disorder in humans, is caused by quantitative or qualitative defects in von Willebrand factor (VWF). VWD represents a potential target for gene therapy applications, as a single treatment could potentially result in a long-term correction of the disease. In recent years, several liver-directed gene therapy approaches have been exploited for VWD, but their efficacy was generally limited by the large size of the VWF transgene and the reduced hemostatic activity of the protein produced from hepatocytes. In this context, we aimed at developing a gene therapy strategy for gene delivery into endothelial cells, the natural site of biosynthesis of VWF. We optimized an endothelial-specific dual hybrid AAV vector, in which the large VWF cDNA was put under the control of an endothelial promoter and correctly reconstituted upon cell transduction by a combination of trans-splicing and homologous recombination mechanisms. In addition, we modified the AAV vector capsid by introducing an endothelial-targeting peptide to improve the efficiency for endothelial-directed gene transfer. This vector platform allowed the reconstitution of full-length VWF transgene both in vitro in human umbilical vein endothelial cells and in vivo in VWD mice, resulting in long-term expression of VWF.

Published
2021
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4. Hepatic expression of GAA results in enhanced enzyme bioavailability in mice and non-human primates

Author

Catalina Abad, N. Danièle, Jayme M.L. Nordin, Giuseppe Ronzitti, Bernard Gjata, Helena Costa-Verdera, Simon Barral, Sean M. Armour, Marcelo Simon-Sola, Fanny Collaud, Marco Crosariol, Julien Fabregue, David A. Gross, Mathew Li, Jérémie Cosette, Laetitia van Wittenberghe, Pasqualina Colella, Maryse Moya-Nilges, G. Michael Preston, Christopher Riling, Xavier M. Anguela, Federico Mingozzi, Severine Charles, Pauline Sellier, Tom Antrilli, William J. Quinn, Umut Cagin, Jacomina Krijnse-Locker, Olivier Boyer, Généthon, Approches génétiques intégrées et nouvelles thérapies pour les maladies rares (INTEGRARE), Université d'Évry-Val-d'Essonne (UEVE)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris-Saclay-Généthon, Centre de recherche en Myologie – U974 SU-INSERM, Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU), Spark Therapeutics [Philadelphia, PA, USA], Institut Pasteur [Paris] (IP), Plateforme BioImagerie Ultrastructurale – Ultrastructural BioImaging Platform (UTechS UBI), Institute for Research and Innovation in Biomedicine (IRIB), Université de Rouen Normandie (UNIROUEN), Normandie Université (NU)-Normandie Université (NU)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), This work was supported by Genethon and the French Muscular Dystrophy Association,the European Union’s research and innovation program under grant agreement no.667751 (to F.M.), the European Research Council Consolidator Grant under grantagreement no. 617432 (to F.M.), Marie Skłodowska-Curie Actions Individual Fellowship(MSCA-IF) grant agreement no. 797144 (to U.C.), a grant from the DIM ThérapieGénique (to D.A.G.) and by Spark Therapeutics under a sponsored research agreementto Genethon., ANR-16-CE92-0008,membrane dynamics,Rupture et réparation membranaire : stratégies d'assemblage virale(2016), European Project: 667751,H2020,H2020-PHC-2015-two-stage,MYOCURE(2016), European Project: 617432,EC:FP7:ERC,ERC-2013-CoG,MOMAAV(2014), Gestionnaire, Hal Sorbonne Université, Development of an innovative gene therapy platform to cure rare hereditary muscle disorders - MYOCURE - - H20202016-01-01 - 2019-12-31 - 667751 - VALID, Molecular signatures and Modulation of immunity to Adeno-Associated Virus vectors - MOMAAV - - EC:FP7:ERC2014-07-01 - 2019-06-30 - 617432 - VALID, Thérapie des maladies du muscle strié, Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Sorbonne Université (SU), and Institut Pasteur [Paris]

Subjects
Male, congenital, hereditary, and neonatal diseases and abnormalities, Genetic enhancement, Science, Metabolic disorders, General Physics and Astronomy, Pharmacology, General Biochemistry, Genetics and Molecular Biology, Virus, Article, law.invention, 03 medical and health sciences, Mice, 0302 clinical medicine, Gene therapy, Pharmacokinetics, law, Autophagy, Distribution (pharmacology), Medicine, Animals, Enzyme Replacement Therapy, 030304 developmental biology, chemistry.chemical_classification, 0303 health sciences, Multidisciplinary, [SDV.MHEP] Life Sciences [q-bio]/Human health and pathology, business.industry, Glycogen Storage Disease Type II, nutritional and metabolic diseases, alpha-Glucosidases, General Chemistry, Enzyme replacement therapy, Phenotype, 3. Good health, Enzyme, chemistry, Liver, Recombinant DNA, Female, business, 030217 neurology & neurosurgery, [SDV.MHEP]Life Sciences [q-bio]/Human health and pathology
Abstract

Pompe disease (PD) is a severe neuromuscular disorder caused by deficiency of the lysosomal enzyme acid alpha-glucosidase (GAA). PD is currently treated with enzyme replacement therapy (ERT) with intravenous infusions of recombinant human GAA (rhGAA). Although the introduction of ERT represents a breakthrough in the management of PD, the approach suffers from several shortcomings. Here, we developed a mouse model of PD to compare the efficacy of hepatic gene transfer with adeno-associated virus (AAV) vectors expressing secretable GAA with long-term ERT. Liver expression of GAA results in enhanced pharmacokinetics and uptake of the enzyme in peripheral tissues compared to ERT. Combination of gene transfer with pharmacological chaperones boosts GAA bioavailability, resulting in improved rescue of the PD phenotype. Scale-up of hepatic gene transfer to non-human primates also successfully results in enzyme secretion in blood and uptake in key target tissues, supporting the ongoing clinical translation of the approach., Pompe disease is currently treated with enzyme replacement therapy (ERT) with recombinant human acid alpha-glucosidase (GAA). Here, the authors show hepatic-directed gene therapy with AAV vectors enhances GAA bioavailability compared with ERT, resulting in improved rescue of the disease phenotype in mice and broad enzyme distribution in mice and non-human primates.

Published
2021
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5. A novel therapeutic strategy for skeletal disorders: Proof of concept of gene therapy for X-linked hypophosphatemia

Author

Stéphane Hilliquin, N. Danièle, B. Baroukh, Severine Charles, Louisa Jauze, Fabienne Rajas, Lotfi Slimani, Agnès Linglart, Jérémy Sadoine, Claire Bardet, Giuseppe Ronzitti, Catherine Chaussain, Volha V. Zhukouskaya, Laetitia van Wittenberghe, Federico Mingozzi, Christian Leborgne, Généthon, Approches génétiques intégrées et nouvelles thérapies pour les maladies rares (INTEGRARE), Université d'Évry-Val-d'Essonne (UEVE)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris-Saclay-Généthon, Pathologies, imagerie et biothérapies oro-faciales = Orofacial pathologies, imaging and biotherapies (URP 2496), Université Paris Cité (UPCité), Signalisation Hormonale, Physiopathologie Endocrinienne et Métabolique, Université Paris-Sud - Paris 11 (UP11)-Institut National de la Santé et de la Recherche Médicale (INSERM)-AP-HP Hôpital Bicêtre (Le Kremlin-Bicêtre), Hôpital Bicêtre, Nutrition, diabète et cerveau (NUDICE), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National de la Santé et de la Recherche Médicale (INSERM), Hôpital Cochin [AP-HP], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP), Service d'Odontologie [Bretonneau], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Hôpital Bretonneau, ANR-18-CE14-0018,HYPOSKEL,Hypophosphatémie liée à l'X : des mécanismes pathologiques de la minéralisation aux traitements des manifestations squelettiques(2018), Pathologies, Imagerie et Biothérapies oro-faciales (URP 2496), and Di Carlo, Marie-Ange

Subjects
Multidisciplinary, business.industry, viruses, Genetic enhancement, SciAdv r-articles, Diseases and Disorders, Gene transfer, Bioinformatics, X-linked hypophosphatemia, medicine.disease, Virus, stomatognathic diseases, Text mining, Refractory, Proof of concept, Medicine, [SDV.NEU]Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC], [SDV.NEU] Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC], Biomedicine and Life Sciences, Health and Medicine, business, General Economics, Econometrics and Finance, Research Article, Therapeutic strategy
Abstract

Description, A new therapeutic approach for the treatment of a skeletal disorder with AAVs bypasses bone resistance to gene transfer., Adeno-associated virus (AAV) vectors are a well-established gene transfer approach for rare genetic diseases. Nonetheless, some tissues, such as bone, remain refractory to AAV. X-linked hypophosphatemia (XLH) is a rare skeletal disorder associated with increased levels of fibroblast growth factor 23 (FGF23), resulting in skeletal deformities and short stature. The conventional treatment for XLH, lifelong phosphate and active vitamin D analogs supplementation, partially improves quality of life and is associated with severe long-term side effects. Recently, a monoclonal antibody against FGF23 has been approved for XLH but remains a high-cost lifelong therapy. We developed a liver-targeting AAV vector to inhibit FGF23 signaling. We showed that hepatic expression of the C-terminal tail of FGF23 corrected skeletal manifestations and osteomalacia in a XLH mouse model. Our data provide proof of concept for AAV gene transfer to treat XLH, a prototypical bone disease, further expanding the use of this modality to treat skeletal disorders.

Published
2021
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6. AAV liver gene therapy-mediated inhibition of FGF23 signaling as a therapeutic strategy for X-linked hypophosphatemia

Author

Brigitte Baroukh, Stéphane Hilliquin, Wittenberghe Laetitia van, Christian Leborgne, Giuseppe Ronzitti, Lotfi Slimani, Fabienne Rajas, Volha V. Zhukouskaya, Claire Bardet, Louisa Jauze, N. Danièle, Catherine Chaussain, Federico Mingozzi, Severine Charles, Jérémy Sadoine, Agnès Linglart, Di Carlo, Marie-Ange, Généthon, Approches génétiques intégrées et nouvelles thérapies pour les maladies rares (INTEGRARE), Université d'Évry-Val-d'Essonne (UEVE)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris-Saclay-Généthon, Nutrition, diabète et cerveau (NUDICE), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National de la Santé et de la Recherche Médicale (INSERM), Hôpital Cochin [AP-HP], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP), Pathologies, Imagerie et Biothérapies oro-faciales (URP 2496), Université Paris Cité (UPCité), Université Paris-Saclay, AP-HP Hôpital Bicêtre (Le Kremlin-Bicêtre), Service d'Odontologie [Bretonneau], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Hôpital Bretonneau, Nutrition, diabète et cerveau, Université de Paris (UP), and Pathologies, imagerie et biothérapies oro-faciales = Orofacial pathologies, imaging and biotherapies (URP 2496)

Subjects
business.industry, Genetic enhancement, Cancer research, Medicine, [SDV.NEU]Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC], [SDV.NEU] Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC], business, X-linked hypophosphatemia, medicine.disease, ComputingMilieux_MISCELLANEOUS, 3. Good health, Therapeutic strategy
Abstract

International audience

Published
2021
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7. Gene therapy with secreted acid alpha-glucosidase rescues Pompe disease in a novel mouse model with early-onset spinal cord and respiratory defects

Author

M. Biferi, Bernard Gjata, Nicolas Guerchet, Laetitia van Wittenberghe, Fanny Collaud, N. Danièle, Manuel Gómez, Severine Charles, Pauline Sellier, M. Cohen-Tannoudji, Guillaume Tanniou, Federico Mingozzi, Jacomina Krijnse-Locker, Umut Cagin, Maryse Moya-Nilges, Pasqualina Colella, Marcelo Simon-Sola, Approches génétiques intégrées et nouvelles thérapies pour les maladies rares (INTEGRARE), Université d'Évry-Val-d'Essonne (UEVE)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris-Saclay-Généthon, Centre de Recherche en Myologie, Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU), Thérapie des maladies du muscle strié, Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Sorbonne Université (SU), Institut Pasteur [Paris], Funding: This work was supported by Genethon, the French Muscular Dystrophy Association (AFM), the European Commission (grant nos. 667751, 617432, and 797144), and Spark Therapeutics., European Project: 667751,H2020,H2020-PHC-2015-two-stage,MYOCURE(2016), European Project: 617432,EC:FP7:ERC,ERC-2013-CoG,MOMAAV(2014), Centre de recherche en Myologie – U974 SU-INSERM, Institut Pasteur [Paris] (IP), ANR-16-CE92-0008,membrane dynamics,Rupture et réparation membranaire : stratégies d'assemblage virale(2016), French Muscular Dystrophy Association, European Union, European Research Council, and Unión Europea

Subjects
Male, 0301 basic medicine, Genetic enhancement, [SDV]Life Sciences [q-bio], Gene Expression, lcsh:Medicine, Mice, 0302 clinical medicine, Transduction, Genetic, Medicine, Respiratory function, Muscular dystrophy, Respiratory system, Mice, Knockout, Motor Neurons, lcsh:R5-920, Spinal cord, Glycogen Storage Disease Type II, Homozygote, Gene Transfer Techniques, Pompe disease, AAV, General Medicine, Dependovirus, Prognosis, Immunohistochemistry, 3. Good health, Phenotype, Treatment Outcome, medicine.anatomical_structure, 030220 oncology & carcinogenesis, Acid alpha-glucosidase, Muscle, medicine.symptom, lcsh:Medicine (General), Glycogen, Research Paper, congenital, hereditary, and neonatal diseases and abnormalities, Genetic Vectors, General Biochemistry, Genetics and Molecular Biology, Mouse model, 03 medical and health sciences, Animals, Muscle Strength, Muscle, Skeletal, Alleles, business.industry, lcsh:R, Muscle weakness, Skeletal muscle, nutritional and metabolic diseases, alpha-Glucosidases, Genetic Therapy, medicine.disease, Disease Models, Animal, 030104 developmental biology, Immunology, business
Abstract

Pompe disease (PD) is a neuromuscular disorder caused by deficiency of acidalpha-glucosidase (GAA), leading to motor and respiratory dysfunctions. Available Gaa knock-out (KO) mouse models do not accurately mimic PD, particularly its highly impaired respiratory phenotype. Here we developed a new mouse model of PD crossing Gaa KOB6;129 with DBA2/J mice. We subsequently treated Gaa KODBA2/J mice with adeno-associated virus (AAV) vectors expressing a secretable form of GAA (secGAA). Male Gaa KODBA2/J mice present most of the key features of the human disease, including early lethality, severe respiratory impairment, cardiac hypertrophy and muscle weakness. Transcriptome analyses of Gaa KODBA2/J, compared to the parental Gaa KOB6;129 mice, revealed a profoundly impaired gene signature in the spinal cord and a similarly deregulated gene expression in skeletal muscle. Muscle and spinal cord transcriptome changes, biochemical defects, respiratory and muscle function in the Gaa KODBA2/J model were significantly improved upon gene therapy with AAV vectors expressing secGAA. These data show that the genetic background impacts on the severity of respiratory function and neuroglial spinal cord defects in the Gaa KO mouse model of PD. Our findings have implications for PD prognosis and treatment, show novel molecular pathophysiology mechanisms of the disease and provide a unique model to study PD respiratory defects, which majorly affect patients. This work was supported by Genethon, the French Muscular Dystrophy Association (AFM), the European Commission (grant nos. 667751, 617432, and 797144), and Spark Therapeutics. This work was supported by Genethon and the French Muscular Dystrophy Association (AFM, to F.M.). It was also supported by the European Union’s research and innovation program under grant agreement no. 667751 (to F.M.), the European Research Council Consolidator Grant under grant agreement no. 617432 (to F.M.), Marie Skodowska-Curie Actions Individual Fellowship (MSCA-IF) grant agreement no. 797144 (to U.C.), and by Spark Therapeutics under a sponsored research agreement. Sí

Published
2020
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8. Rescue of Advanced Pompe Disease in Mice with Hepatic Expression of Secretable Acid α-Glucosidase

Author

Severine Charles, Catalina Abad, Nathalie Daniele, Laetitia van Wittenberghe, Jacomina Krijnse-Locker, Maryse Moya-Nilges, Umut Cagin, Marcelo Simon Sola, Fanny Collaud, Bernard Gjata, Francesco Puzzo, Pasqualina Colella, Nicolas Guerchet, Manuel Gómez, Giuseppe Ronzitti, Olivier Boyer, Pauline Sellier, Federico Mingozzi, Genethon (Francia), French Muscular Dystrophy Association, Spark Therapeutics (Estados Unidos), Unión Europea, European Research Council, Ministerio de Ciencia e Innovación (España), Approches génétiques intégrées et nouvelles thérapies pour les maladies rares (INTEGRARE), Université d'Évry-Val-d'Essonne (UEVE)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris-Saclay-Généthon, Sorbonne Université (SU), Centro Nacional de Investigaciones Cardiovasculares Carlos III [Madrid, Spain] (CNIC), Instituto de Salud Carlos III [Madrid] (ISC), Institute for Research and Innovation in Biomedicine (IRIB), Université de Rouen Normandie (UNIROUEN), Normandie Université (NU)-Normandie Université (NU)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), CCSD, Accord Elsevier, Institut Pasteur [Paris] (IP), Genethon, French Muscular Dystro-phy Association, Spark Therapeutics, European Union, and ANR-16-CE92-0008,membrane dynamics,Rupture et réparation membranaire : stratégies d'assemblage virale(2016)

Subjects
Male, liver gene transfer, [SDV]Life Sciences [q-bio], Genetic enhancement, Transcriptome, secretable GAA, chemistry.chemical_compound, transcriptomics, Mice, 0302 clinical medicine, Drug Discovery, chemistry.chemical_classification, Mice, Knockout, 0303 health sciences, Secretory Pathway, Glycogen, Glycogen Storage Disease Type II, Pompe disease, AAV, Dependovirus, Phenotype, gene therapy, Pompe mouse, 3. Good health, [SDV] Life Sciences [q-bio], medicine.anatomical_structure, Treatment Outcome, Liver, 030220 oncology & carcinogenesis, advanced disease, Molecular Medicine, Original Article, Signal Transduction, congenital, hereditary, and neonatal diseases and abnormalities, Genetic Vectors, Biology, Transfection, Virus, 03 medical and health sciences, Genetics, medicine, Animals, Muscle, Skeletal, Molecular Biology, Gene, 030304 developmental biology, Pharmacology, Skeletal muscle, nutritional and metabolic diseases, alpha-Glucosidases, Genetic Therapy, Molecular biology, Disease Models, Animal, Enzyme, chemistry, Lysosomes
Abstract

Pompe disease is a neuromuscular disorder caused by disease-associated variants in the gene encoding for the lysosomal enzyme acid α-glucosidase (GAA), which converts lysosomal glycogen to glucose. We previously reported full rescue of Pompe disease in symptomatic 4-month-old Gaa knockout (Gaa−/−) mice by adeno-associated virus (AAV) vector-mediated liver gene transfer of an engineered secretable form of GAA (secGAA). Here, we showed that hepatic expression of secGAA rescues the phenotype of 4-month-old Gaa−/− mice at vector doses at which the native form of GAA has little to no therapeutic effect. Based on these results, we then treated severely affected 9-month-old Gaa−/− mice with an AAV vector expressing secGAA and followed the animals for 9 months thereafter. AAV-treated Gaa−/− mice showed complete reversal of the Pompe phenotype, with rescue of glycogen accumulation in most tissues, including the central nervous system, and normalization of muscle strength. Transcriptomic profiling of skeletal muscle showed rescue of most altered pathways, including those involved in mitochondrial defects, a finding supported by structural and biochemical analyses, which also showed restoration of lysosomal function. Together, these results provide insight into the reversibility of advanced Pompe disease in the Gaa−/− mouse model via liver gene transfer of secGAA., Graphical Abstract, AAV vector-mediated hepatic expression of secretable GAA drives clearance of glycogen from multiple tissues, resulting in rescue of muscle function in Pompe mice with advanced pathology. Reversal of the Pompe phenotype in mice includes normalization of lysosomal parameters and mitochondrial homeostasis, which is also reflected in the muscle transcriptomics.

Published
2020
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9. Prevalence and long-term monitoring of humoral immunity against adeno-associated virus in Duchenne Muscular Dystrophy patients

Author

Olivier Boyer, Fabienne Jouen, Virginie Latournerie, Carole Masurier, Marcelo Simon Sola, Federico Mingozzi, Diana Desgue, Severine Charles, Philippe Veron, Aliénor Quéré, Elisa Masat, Christian Leborgne, Elodie Pignot, Sylvie Boutin, Fanny Collaud, Généthon, Approches génétiques intégrées et nouvelles thérapies pour les maladies rares (INTEGRARE), Université d'Évry-Val-d'Essonne (UEVE)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Généthon-École pratique des hautes études (EPHE), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL), 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), Physiopathologie et biothérapies des maladies inflammatoires et autoimmunes, Université de Rouen Normandie (UNIROUEN), Normandie Université (NU)-Normandie Université (NU)-Institut National de la Santé et de la Recherche Médicale (INSERM), Physiopathologie, Autoimmunité, maladies Neuromusculaires et THErapies Régénératrices (PANTHER), Généthon, Evry, Centre de recherche et d'applications sur les thérapies géniques (CRATG), Université d'Évry-Val-d'Essonne (UEVE)-GENETHON 3-Centre National de la Recherche Scientifique (CNRS), École pratique des hautes études (EPHE)-Université d'Évry-Val-d'Essonne (UEVE)-GENETHON 3-Institut National de la Santé et de la Recherche Médicale (INSERM), 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 – U974 SU-INSERM, Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU), Association française contre les myopathies (AFM-Téléthon)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS), Immunologie moléculaire et biothérapies innovantes, École pratique des hautes études (EPHE)-Université d'Évry-Val-d'Essonne (UEVE)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Genethon, Laboratoire des Sciences de l'Information et des Systèmes (LSIS), and Aix Marseille Université (AMU)-Université de Toulon (UTLN)-Arts et Métiers Paristech ENSAM Aix-en-Provence-Centre National de la Recherche Scientifique (CNRS)

Subjects
0301 basic medicine, Adult, AAV humoral immunity, Adolescent, Duchenne muscular dystrophy, Genetic enhancement, [SDV]Life Sciences [q-bio], viruses, Immunology, Genetic Vectors, Duchenne Muscular Dystrophy, Biology, medicine.disease_cause, Antibodies, Viral, Virus, Cohort Studies, 03 medical and health sciences, Young Adult, 0302 clinical medicine, Gene therapy, Seroepidemiologic Studies, medicine, Humans, Longitudinal Studies, Seroconversion, Adeno-associated virus, ComputingMilieux_MISCELLANEOUS, Immunosuppression Therapy, Antibody titer, Age Factors, Dependovirus, medicine.disease, Antibodies, Neutralizing, 3. Good health, Immunity, Humoral, Muscular Dystrophy, Duchenne, 030104 developmental biology, [SDV.IMM.IA]Life Sciences [q-bio]/Immunology/Adaptive immunology, Immunoglobulin G, Humoral immunity, biology.protein, [SDV.IMM]Life Sciences [q-bio]/Immunology, Antibody, 030215 immunology
Abstract

International audience; Adeno-associated virus (AAV) vectors are promising candidates for gene therapy and have been explored as gene delivery vehicles in the treatment of Duchenne Muscular Dystrophy (DMD). Recent studies showed compelling evidence of therapeutic efficacy in large animal models following the intravenous delivery of AAV vectors expressing truncated forms of dystrophin. However, to translate these results to humans, careful assessment of the prevalence of anti-AAV neutralizing antibodies (NAbs) is needed, as presence of preexisting NABs to AAV in serum have been associated with a drastic diminution of vector transduction. Here we measured binding and neutralizing antibodies against AAV serotype 1, 2, and 8 in serum from children and young adults with DMD (n = 130). Results were compared with to age-matched healthy donors (HD, n = 113). Overall, approximately 54% of all subjects included in the study presented IgG to AAV2, 49% to AAV1, and 41% to AAV8. A mean of around 80% of IgG positive sera showed neutralizing activity with no statistical difference between DMD and HD. NAb titers for AAV2 were higher than AAV1, and AAV8 in both populations studied. Older DMD patients (13-24 years old) presented significantly lower anti-AAV8 IgG4 subclass. Anti-AAV antibodies were found to be decreased in DMD patients subjected to a 6-month course of corticosteroids and in subjects receiving a variety of immunosuppressive drugs including B cell targeting drugs. Longitudinal follow up of humoral responses to AAV over up to 6 years showed no change in antibody titers, suggesting that in this patient population, seroconversion is a rare event in humans.

Published
2018
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10. Rescue of GSDIII Phenotype with Gene Transfer Requires Liver- and Muscle-Targeted GDE Expression

Author

Federico Mingozzi, Patrice Vidal, Helena Costa Verdera, Bernard Gjata, Francesco Puzzo, Giuseppe Ronzitti, Laetitia van Wittenberghe, Giacomo P. Comi, Gilles Mithieux, Fanny Collaud, Sabrina Lucchiari, Pascal Laforêt, Edoardo Malfatti, Louisa Jauze, Monika Gjorgjieva, Fabienne Rajas, Marcelo Simon Sola, Solenne Marmier, Alban Vignaud, Serena Pagliarani, Severine Charles, Isabelle Richard, Pasqualina Colella, Université Pierre et Marie Curie - Paris 6 (UPMC), 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, Institut National de la Santé et de la Recherche Médicale (INSERM), Department of Physiopatology and Transplantation, University of Milan (DEPT), Università degli Studi di Milano = University of Milan (UNIMI), IRCCS Foundation Ca' Granda Ospedale Maggiore Policlinico, Généthon, Université Pierre et Marie Curie - Paris 6 - UFR de Médecine Pierre et Marie Curie (UPMC), Nutrition et cerveau (U1213, U855), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National de la Santé et de la Recherche Médicale (INSERM), Department of Pathophysiology and Transplantation, Institut de Myologie, Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Association française contre les myopathies (AFM-Téléthon)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS), U855, Nutrition et cerveau, Centre de recherche en Myologie – U974 SU-INSERM, Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU), ANR-17-CE18-0014,TRACeGSDIII,Optimisation translationnelle d'un vecteur AAV pour le traitement de GSDIII(2017), École pratique des hautes études (EPHE), Richard, Isabelle, Centre de recherche en myologie, Association française contre les myopathies (AFM-Téléthon)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS), Institut national de la santé et de la recherche médicale, Université d’Evry Val d’Essonne, Genethon (INSERM), University of Milan, Université de Lyon, Généthon, Evry, Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Assistance publique - Hôpitaux de Paris (AP-HP) (APHP)-Association française contre les myopathies (AFM-Téléthon)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS), Nutrition, diabète et cerveau, École pratique des hautes études (EPHE)-Université d'Évry-Val-d'Essonne (UEVE)-GENETHON 3-Institut National de la Santé et de la Recherche Médicale (INSERM), GENETHON, Genethon, Université d'Évry-Val-d'Essonne (UEVE)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Généthon-École pratique des hautes études (EPHE), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL), and Centre National de la Recherche Scientifique (CNRS)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Association française contre les myopathies (AFM-Téléthon)-Sorbonne Université (SU)

Subjects
Blood Glucose, Male, 0301 basic medicine, [SDV.BIO]Life Sciences [q-bio]/Biotechnology, Genetic enhancement, MESH: Glycogen, Gene Expression, MESH: Dependovirus, Glycogen storage disease type III, [SDV.BBM.BM] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, MESH: Mice, Knockout, MESH: Hepatocytes, Mice, chemistry.chemical_compound, dual AAV vectors, MESH: Genetic Vectors, Drug Discovery, MESH: Animals, Vector (molecular biology), MESH: Glycogen Storage Disease Type III, MESH: Organ Specificity, Mice, Knockout, MESH: Muscle, Skeletal, Glycogen, Gene Transfer Techniques, Dependovirus, Phenotype, Cori disease, gene therapy, 3. Good health, Cell biology, Liver, Organ Specificity, Acid alpha-glucosidase, Molecular Medicine, [SDV.NEU]Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC], Original Article, medicine.symptom, MESH: Enzyme Activation, MESH: Gene Expression, Transgene, Genetic Vectors, adeno-associated vector, MESH: Gene Transfer Techniques, Biology, [SDV.GEN.GH] Life Sciences [q-bio]/Genetics/Human genetics, MESH: Phenotype, 03 medical and health sciences, glycogenosis, Genetics, medicine, Animals, MESH: Glycogen Debranching Enzyme System, Muscle, Skeletal, Molecular Biology, MESH: Mice, glycogen storage disease type III, Pharmacology, MESH: Genetic Therapy, Muscle weakness, Glycogen Debranching Enzyme System, [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, Genetic Therapy, neuromuscular disease, medicine.disease, MESH: Male, [SDV.BIO] Life Sciences [q-bio]/Biotechnology, Enzyme Activation, Disease Models, Animal, 030104 developmental biology, chemistry, [SDV.GEN.GH]Life Sciences [q-bio]/Genetics/Human genetics, acid-alpha-glucosidase, Hepatocytes, MESH: Biomarkers, MESH: Blood Glucose, MESH: Disease Models, Animal, Biomarkers, MESH: Liver
Abstract

International audience; Glycogen storage disease type III (GSDIII) is an autosomal recessive disorder caused by a deficiency of glycogen-debranching enzyme (GDE), which results in profound liver metabolism impairment and muscle weakness. To date, no cure is available for GSDIII and current treatments are mostly based on diet. Here we describe the development of a mouse model of GSDIII, which faithfully recapitulates the main features of the human condition. We used this model to develop and test novel therapies based on adeno-associated virus (AAV) vector-mediated gene transfer. First, we showed that overexpression of the lysosomal enzyme alpha-acid glucosidase (GAA) with an AAV vector led to a decrease in liver glycogen content but failed to reverse the disease phenotype. Using dual overlapping AAV vectors expressing the GDE transgene in muscle, we showed functional rescue with no impact on glucose metabolism. Liver expression of GDE, conversely, had a direct impact on blood glucose levels. These results provide proof of concept of correction of GSDIII with AAV vectors, and they indicate that restoration of the enzyme deficiency in muscle and liver is necessary to address both the metabolic and neuromuscular manifestations of the disease.

Published
2018
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11. Antigen-selective modulation of AAV immunogenicity with tolerogenic rapamycin nanoparticles enables successful vector re-administration

Author

Florence Boisgerault, Federico Mingozzi, Romain Hardet, Helena Costa Verdera, Giuseppe Ronzitti, Alicia M. Michaud, Fanny Collaud, Amine Meliani, Giorgia Manni, Severine Charles, Alban Vignaud, Christian Leborgne, Petr Ilyinskii, Takashi Kei Kishimoto, Marcelo Simon Sola, Francesca Fallarino, Olivier D. Christophe, Solenne Marmier, Christopher J. Roy, Laetitia van Wittenberghe, Approches génétiques intégrées et nouvelles thérapies pour les maladies rares (INTEGRARE), Université d'Évry-Val-d'Essonne (UEVE)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Généthon-École pratique des hautes études (EPHE), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL), 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), Centre de Recherche en Myologie, Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU), Généthon, Coeur, Muscle et Vaisseaux, Université Pierre et Marie Curie - Paris 6 (UPMC)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Institut National de la Santé et de la Recherche Médicale (INSERM), Institut des cellules souches pour le traitement et l'étude des maladies monogéniques (I-STEM), Université d'Évry-Val-d'Essonne (UEVE)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Généthon, Hémostase et biologie vasculaire, Université Paris-Sud - Paris 11 (UP11)-IFR93-Institut National de la Santé et de la Recherche Médicale (INSERM), Exp. Medicine and Bioch. Sciences, École pratique des hautes études (EPHE)-Université d'Évry-Val-d'Essonne (UEVE)-GENETHON 3-Institut National de la Santé et de la Recherche Médicale (INSERM), Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Association française contre les myopathies (AFM-Téléthon)-Université Pierre et Marie Curie - Paris 6 (UPMC), Association française contre les myopathies (AFM-Téléthon)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS), GENETHON, Genethon, Généthon, Evry, Université Pierre et Marie Curie - Paris 6 (UPMC)-Assistance publique - Hôpitaux de Paris (AP-HP) (APHP)-Institut National de la Santé et de la Recherche Médicale (INSERM), Université d'Évry-Val-d'Essonne (UEVE)-Institut National de la Santé et de la Recherche Médicale (INSERM), École Pratique des Hautes Études (EPHE), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Université d'Évry-Val-d'Essonne (UEVE)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Généthon, and Centre de recherche en Myologie – U974 SU-INSERM

Subjects
Male, 0301 basic medicine, Genetics and Molecular Biology (all), Adoptive cell transfer, T-Lymphocytes, Genetic enhancement, viruses, Drug Evaluation, Preclinical, General Physics and Astronomy, MESH: Dependovirus, Biochemistry, MESH: Genetic Vectors, MESH: Animals, IL-2 receptor, Vector (molecular biology), MESH: Immunity, Humoral, lcsh:Science, Immunity, Cellular, Multidisciplinary, Chemistry, Immunogenicity, Chemistry (all), Dependovirus, 3. Good health, Cell biology, medicine.anatomical_structure, MESH: Drug Evaluation, Preclinical, [SDV.IMM]Life Sciences [q-bio]/Immunology, MESH: Immunosuppressive Agents, Immunosuppressive Agents, T cell, Science, Genetic Vectors, chemical and pharmacologic phenomena, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, complex mixtures, Article, General Biochemistry, Genetics and Molecular Biology, MESH: Immunity, Cellular, 03 medical and health sciences, Physics and Astronomy (all), MESH: Mice, Inbred C57BL, medicine, Animals, Sirolimus, MESH: Genetic Therapy, Genetic Therapy, General Chemistry, MESH: Male, Immunity, Humoral, Mice, Inbred C57BL, Macaca fascicularis, 030104 developmental biology, MESH: T-Lymphocytes, MESH: Macaca fascicularis, Nanoparticles, lcsh:Q, MESH: Sirolimus, Biochemistry, Genetics and Molecular Biology (all), Memory T cell, CD8, MESH: Nanoparticles
Abstract

Gene therapy mediated by recombinant adeno-associated virus (AAV) vectors is a promising treatment for systemic monogenic diseases. However, vector immunogenicity represents a major limitation to gene transfer with AAV vectors, particularly for vector re-administration. Here, we demonstrate that synthetic vaccine particles encapsulating rapamycin (SVP[Rapa]), co-administered with AAV vectors, prevents the induction of anti-capsid humoral and cell-mediated responses. This allows successful vector re-administration in mice and nonhuman primates. SVP[Rapa] dosed with AAV vectors reduces B and T cell activation in an antigen-selective manner, inhibits CD8+ T cell infiltration in the liver, and efficiently blocks memory T cell responses. SVP[Rapa] immunomodulatory effects can be transferred from treated to naive mice by adoptive transfer of splenocytes, and is inhibited by depletion of CD25+ T cells, suggesting a role for regulatory T cells. Co-administration of SVP[Rapa] with AAV vector represents a powerful strategy to modulate vector immunogenicity and enable effective vector re-administration., Immunogenicity of AAV vectors renders repeated AAV dosing ineffective. Here the authors show that coadministration of nanoparticle-encapsulated rapamycin overcomes AAV immunogenicity through Treg induction, enabling efficient AAV redosing in mice and nonhuman primates.

Published
2018
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12. A translationally optimized AAV-UGT1A1 vector drives safe and long-lasting correction of Crigler-Najjar syndrome

Author

Laetitia van Wittenberghe, Marcelo Simon Sola, Bernard Gjata, Giulia Bortolussi, Federico Mingozzi, Piter J. Bosma, Giuseppe Ronzitti, Alban Vignaud, Severine Charles, Andrés F. Muro, Patrice Vidal, Fanny Collaud, Remco van Dijk, Christian Leborgne, Philippe Veron, Samia Martin, Généthon, International Centre for Genetic Engineering and Biotechnology (ICGEB) (Trieste), Tytgat Institute for Liver and Intestinal Research, Academic Medical Center, University of Amsterdam [Amsterdam] (UvA), Université Pierre et Marie Curie - Paris 6 (UPMC), Approches génétiques intégrées et nouvelles thérapies pour les maladies rares (INTEGRARE), Université d'Évry-Val-d'Essonne (UEVE)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Généthon-École pratique des hautes études (EPHE), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL), 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, HAL-UPMC, Gestionnaire, École pratique des hautes études (EPHE), Other departments, Tytgat Institute for Liver and Intestinal Research, and Gastroenterology and Hepatology

Subjects
0301 basic medicine, lcsh:QH426-470, Crigler–Najjar syndrome, Transgene, Biology, digestive system, Article, Viral vector, 03 medical and health sciences, In vivo, Gene expression, Genetics, medicine, Vector (molecular biology), lcsh:QH573-671, Molecular Biology, [SDV.MHEP] Life Sciences [q-bio]/Human health and pathology, lcsh:Cytology, Intron, medicine.disease, Molecular biology, 3. Good health, Cell biology, lcsh:Genetics, 030104 developmental biology, Molecular Medicine, Expression cassette, [SDV.MHEP]Life Sciences [q-bio]/Human health and pathology
Abstract

International audience; Crigler-Najjar syndrome is a severe metabolic disease of the liver due to a reduced activity of the UDP Glucuronosyltransferase 1A1 (UGT1A1) enzyme. In an effort to translate to the clinic an adeno-associated virus vector mediated liver gene transfer approach to treat Crigler-Najjar syndrome, we developed and optimized a vector expressing the UGT1A1 transgene. For this purpose, we designed and tested in vitro and in vivo multiple codon-optimized UGT1A1 transgene cDNAs. We also optimized noncoding sequences in the transgene expression cassette. Our results indicate that transgene codon-optimization is a strategy that can improve efficacy of gene transfer but needs to be carefully tested in vitro and in vivo. Additionally, while inclusion of introns can enhance gene expression, optimization of these introns, and in particular removal of cryptic ATGs and splice sites, is an important maneuver to enhance safety and efficacy of gene transfer. Finally, using a translationally optimized adeno-associated virus vector expressing the UGT1A1 transgene, we demonstrated rescue of the phenotype of Crigler-Najjar syndrome in two animal models of the disease, Gunn rats and Ugt1a1-/- mice. We also showed long-term (>1 year) correction of the disease in Gunn rats. These results support further translation of the approach to humans.

Published
2016
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13. Adeno associated vector-based gene therapy strategy for type 3 glycogen storage disease

Author

Edoardo Malfatti, Severine Charles, Patrice Vidal, Federico Mingozzi, Giuseppe Ronzitti, P. Collela, Francesco Puzzo, Monika Gjorgjieva, M. Simon Sola, H. Costa Verdera, Giacomo P. Comi, Fanny Collaud, Fabienne Rajas, P. Laforêt, Bernard Gjata, Alban Vignaud, and L. Van Wittenberghe

Subjects
Neurology, Genetic enhancement, Pediatrics, Perinatology and Child Health, medicine, Glycogen storage disease, Neurology (clinical), Vector (molecular biology), Biology, medicine.disease, Virology, Genetics (clinical)
Published
2017
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14. Whole-body rescue of Pompe disease with AAV liver delivery of engineered secretable GAA transgenes

Author

Nicole K. Paulk, Marcelo Simon-Sola, Dwight D. Koeberl, Mark A. Kay, Fanny Collaud, M. Biferi, Florence Boisgerault, Francesco Puzzo, Patrice Vidal, Deeksha Bali, Christian Leborgne, Federico Mingozzi, Pascal Laforêt, Pauline Sellier, Martine Barkats, Severine Charles, Romain Hardet, L. Van Wittenberghe, Pasqualina Colella, and Giuseppe Ronzitti

Subjects
0301 basic medicine, business.industry, Transgene, Disease, Virology, 03 medical and health sciences, 030104 developmental biology, Neurology, Pediatrics, Perinatology and Child Health, Medicine, Neurology (clinical), Whole body, business, Genetics (clinical)
Published
2017
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15. 704. Long-Term Correction of Crigler-Najjar Syndrome and Scale-Up Production of an Optimized AAV8 Vector Expressing the UGT1A1 Transgene

Author

Piter J. Bosma, Severine Charles, Florence Lacoste, Giulia Bortolussi, Samia Martin, Federico Mingozzi, Christine Le Bec, Remco van Dijk, Matthias Hebben, Alban Vignaud, Andrés F. Muro, Giuseppe Ronzitti, Fulvio Mavilio, and Fanny Collaud

Subjects
Pharmacology, Crigler–Najjar syndrome, Transgene, medicine.medical_treatment, Transfection, Liver transplantation, Biology, medicine.disease, Isotype, Transduction (genetics), In vivo, Drug Discovery, Immunology, Genetics, medicine, Molecular Medicine, Potency, Molecular Biology
Abstract

Crigler-Najjar syndrome (CN) is an autosomal recessive rare disorder caused by mutations in the UDP-glucuronosyltransferase 1 isotype A1 (UGT1A1) gene. In severe CN, lack or reduced activity of UGT1A1 results in high levels of serum unconjugated bilirubin (UCB), which can lead to brain damage and death. Treatment of CN consists of phototherapy for 10-12 hours per day to convert UCB into soluble photoisomers without the need of conjugation, which presents several limitations and has a major impact on life quality of the patients. Liver transplantation is the only curative option for CN. The limited therapeutic options available prompted us to develop a new therapy for CN based on the transfer of a corrected copy of the UGT1A1 gene to hepatocytes.We developed an AAV8 vector optimized for the liver expression of the hUGT1A1 transgene (AAV8-hUGT1A1). Safety and efficacy of correction of total serum bilirubin (TB) levels with AAV-hUGT1A1 were demonstrated in mouse and rat models of CN at vector doses as low as 5×10 11 vector genomes (vg)/kg, a result confirmed also by the detection of conjugated bilirubin in bile of treated rats. In juvenile CN rats, long-term correction of total bilirubin levels were observed for more than 8 months following AAV8-hUGT1A1 gene transfer. In neonate Ugt1a1–/– mice, intraperitoneal delivery of the vector at doses as low as 1×10 9 vg/mouse resulted in correction of TB at 4 weeks. However, vector delivery in P2 and P4 animals resulted in lower efficiency of correction when compared with P11-transduced mice, a finding likely due to the more advanced development of the liver at later time points.Based on these data, dose finding studies were performed in CN rats, which showed a profile of liver transduction with AAV8 vectors similar to humans, i.e. lower efficiency of transduction than mice. No differences in efficacy of correction of TB were observed in male vs. female rats.A scalable process for the production of AAV8-hUGT1A1 production was established based on a protocol of triple transfection of suspension HEK293 cells, leading to high yield vector preparations with excellent purity profile. In vitro transduction assays in human hepatocytes based on Western blot for hUGT1A1 protein and in vivo studies in CN rats demonstrated that vectors produced with the suspension process have potency characteristics undistinguishable from research grade vectors produced by triple transfection of adherent cells and purified by cesium chloride gradient centrifugation.In conclusion, our data demonstrate the safety and efficacy of gene transfer for Crigler-Najjar syndrome in two relevant animal models of the disease and provide tools and a strong rationale for the translation of these results in human subjects.

Published
2015
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16. AAV Gene Transfer with Tandem Promoter Design Prevents Anti-transgene Immunity and Provides Persistent Efficacy in Neonate Pompe Mice

Author

Isabella Ragone, Nathalie Daniele, Bernard Gjata, Marcelo Simon Sola, Francesco Puzzo, Severine Charles, Solenne Marmier, Laetitia van Wittenberghe, Pasqualina Colella, Federico Mingozzi, Pauline Sellier, Nicolas Guerchet, Christian Leborgne, Fanny Collaud, Helena Costa Verdera, 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 – U974 SU-INSERM, Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU), École pratique des hautes études (EPHE), and Centre de Recherche en Myologie

Subjects
0301 basic medicine, [SDV.BIO]Life Sciences [q-bio]/Biotechnology, lcsh:QH426-470, Genetic enhancement, Transgene, Disease, Biology, Genome, Article, 03 medical and health sciences, transgene immunity, 0302 clinical medicine, Immune system, Immunity, Genetics, medicine, lcsh:QH573-671, Molecular Biology, lcsh:Cytology, Promoter, AAV, gene therapy, 3. Good health, lcsh:Genetics, 030104 developmental biology, medicine.anatomical_structure, 030220 oncology & carcinogenesis, Hepatocyte, Cancer research, Molecular Medicine, [SDV.IMM]Life Sciences [q-bio]/Immunology, tandem promoters, transgene persistence
Abstract

Hepatocyte-restricted, AAV-mediated gene transfer is being used to provide sustained, tolerogenic transgene expression in gene therapy. However, given the episomal status of the AAV genome, this approach cannot be applied to pediatric disorders when hepatocyte proliferation may result in significant loss of therapeutic efficacy over time. In addition, many multi-systemic diseases require widespread expression of the therapeutic transgene that, when provided with ubiquitous or tissue-specific non-hepatic promoters, often results in anti-transgene immunity. Here we have developed tandem promoter monocistronic expression cassettes that, packaged in a single AAV, provide combined hepatic and extra-hepatic tissue-specific transgene expression and prevent anti-transgene immunity. We validated our approach in infantile Pompe disease, a prototype disease caused by lack of the ubiquitous enzyme acid-alpha-glucosidase (GAA), presenting multi-systemic manifestations and detrimental anti-GAA immunity. We showed that the use of efficient tandem promoters prevents immune responses to GAA following systemic AAV gene transfer in immunocompetent Gaa−/− mice. Then we demonstrated that neonatal gene therapy with either AAV8 or AAV9 in Gaa−/− mice resulted in persistent therapeutic efficacy when using a tandem liver-muscle promoter (LiMP) that provided high and persistent transgene expression in non-dividing extra-hepatic tissues. In conclusion, the tandem promoter design overcomes important limitations of AAV-mediated gene transfer and can be beneficial when treating pediatric conditions requiring persistent multi-systemic transgene expression and prevention of anti-transgene immunity., Graphical Abstract

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