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1. A case of T-cell acute lymphoblastic leukemia in retroviral gene therapy for ADA-SCID

Author

Cesana, Daniela, Cicalese, Maria Pia, Calabria, Andrea, Merli, Pietro, Caruso, Roberta, Volpin, Monica, Rudilosso, Laura, Migliavacca, Maddalena, Barzaghi, Federica, Fossati, Claudia, Gazzo, Francesco, Pizzi, Simone, Ciolfi, Andrea, Bruselles, Alessandro, Tucci, Francesca, Spinozzi, Giulio, Pais, Giulia, Benedicenti, Fabrizio, Barcella, Matteo, Merelli, Ivan, Gallina, Pierangela, Giannelli, Stefania, Dionisio, Francesca, Scala, Serena, Casiraghi, Miriam, Strocchio, Luisa, Vinti, Luciana, Pacillo, Lucia, Draghi, Eleonora, Cesana, Marcella, Riccardo, Sara, Colantuono, Chiara, Six, Emmanuelle, Cavazzana, Marina, Carlucci, Filippo, Schmidt, Manfred, Cancrini, Caterina, Ciceri, Fabio, Vago, Luca, Cacchiarelli, Davide, Gentner, Bernhard, Naldini, Luigi, Tartaglia, Marco, Montini, Eugenio, Locatelli, Franco, and Aiuti, Alessandro

Published
2024
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2. Hematopoietic reconstitution dynamics of mobilized- and bone marrow-derived human hematopoietic stem cells after gene therapy

Author

Scala, Serena, Ferrua, Francesca, Basso-Ricci, Luca, Dionisio, Francesca, Omrani, Maryam, Quaranta, Pamela, Jofra Hernandez, Raisa, Del Core, Luca, Benedicenti, Fabrizio, Monti, Ilaria, Giannelli, Stefania, Fraschetta, Federico, Darin, Silvia, Albertazzi, Elena, Galimberti, Stefania, Montini, Eugenio, Calabria, Andrea, Cicalese, Maria Pia, and Aiuti, Alessandro

Published
2023
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3. Circulating hematopoietic stem/progenitor cell subsets contribute to human hematopoietic homeostasis

Author

Quaranta, Pamela, Basso-Ricci, Luca, Jofra Hernandez, Raisa, Pacini, Guido, Naldini, Matteo Maria, Barcella, Matteo, Seffin, Luca, Pais, Giulia, Spinozzi, Giulio, Benedicenti, Fabrizio, Pietrasanta, Carlo, Cheong, Jin Gyu, Ronchi, Andrea, Pugni, Lorenza, Dionisio, Francesca, Monti, Ilaria, Giannelli, Stefania, Darin, Silvia, Fraschetta, Federico, Barera, Graziano, Ferrua, Francesca, Calbi, Valeria, Ometti, Marco, Di Micco, Raffaella, Mosca, Fabio, Josefowicz, Steven Zvi, Montini, Eugenio, Calabria, Andrea, Bernardo, Maria Ester, Cicalese, Maria Pia, Gentner, Bernhard, Merelli, Ivan, Aiuti, Alessandro, and Scala, Serena

Published
2024
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7. Retrieval of vector integration sites from cell-free DNA

Author

Cesana, Daniela, Calabria, Andrea, Rudilosso, Laura, Gallina, Pierangela, Benedicenti, Fabrizio, Spinozzi, Giulio, and Schiroli, Giulia

Subjects
Nucleotide sequencing -- Health aspects, Gene therapy -- Innovations, DNA sequencing -- Health aspects, Genetic vectors -- Identification and classification, Biological sciences, Health
Abstract

Gene therapy (GT) has rapidly attracted renewed interest as a treatment for otherwise incurable diseases, with several GT products already on the market and many more entering clinical testing for selected indications. Clonal tracking techniques based on vector integration enable monitoring of the fate of engineered cells in the blood of patients receiving GT and allow assessment of the safety and efficacy of these procedures. However, owing to the limited number of cells that can be tested and the impracticality of studying cells residing in peripheral organs without performing invasive biopsies, this approach provides only a partial snapshot of the clonal repertoire and dynamics of genetically modified cells and reduces the predictive power as a safety readout. In this study, we developed liquid biopsy integration site sequencing, or LiBIS-seq, a polymerase chain reaction technique optimized to quantitatively retrieve vector integration sites from cell-free DNA released into the bloodstream by dying cells residing in several tissues. This approach enabled longitudinal monitoring of in vivo liver-directed GT and clonal tracking in patients receiving hematopoietic stem cell GT, improving our understanding of the clonal composition and turnover of genetically modified cells in solid tissues and, in contrast to conventional analyses based only on circulating blood cells, enabling earlier detection of vector-marked clones that are aberrantly expanding in peripheral tissues. A new approach called liquid biopsy integration site sequencing enables monitoring of genetically modified cells in solid tissues of patients receiving gene therapy., Author(s): Daniela Cesana [sup.1] , Andrea Calabria [sup.1] , Laura Rudilosso [sup.1] , Pierangela Gallina [sup.1] , Fabrizio Benedicenti [sup.1] , Giulio Spinozzi [sup.1] , Giulia Schiroli [sup.1] , Alessandra [...]

Published
2021
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9. Pervasive supply of therapeutic lysosomal enzymes in the CNS of normal and Krabbe‐affected non‐human primates by intracerebral lentiviral gene therapy

Author

Meneghini, Vasco, Lattanzi, Annalisa, Tiradani, Luigi, Bravo, Gabriele, Morena, Francesco, Sanvito, Francesca, Calabria, Andrea, Bringas, John, Fisher-Perkins, Jeanne M, Dufour, Jason P, Baker, Kate C, Doglioni, Claudio, Montini, Eugenio, Bunnell, Bruce A, Bankiewicz, Krystof, Martino, Sabata, Naldini, Luigi, and Gritti, Angela

Subjects
Biochemistry and Cell Biology, Biological Sciences, Neurosciences, Neurodegenerative, Stem Cell Research, Gene Therapy, Genetics, Biotechnology, Stem Cell Research - Nonembryonic - Non-Human, Brain Disorders, Development of treatments and therapeutic interventions, 5.2 Cellular and gene therapies, Neurological, Animals, Cerebroside-Sulfatase, Disease Models, Animal, Galactosylceramidase, Genetic Therapy, Genetic Vectors, Humans, Lentivirus, Leukodystrophy, Globoid Cell, Leukodystrophy, Metachromatic, Macaca mulatta, Mice, Transduction, Genetic, Treatment Outcome, brain, gene therapy, lentiviral vectors, leukodystrophy, non-human primates, non‐human primates, Medical and Health Sciences, Biochemistry and cell biology
Abstract

Metachromatic leukodystrophy (MLD) and globoid cell leukodystrophy (GLD or Krabbe disease) are severe neurodegenerative lysosomal storage diseases (LSD) caused by arylsulfatase A (ARSA) and galactosylceramidase (GALC) deficiency, respectively. Our previous studies established lentiviral gene therapy (GT) as a rapid and effective intervention to provide pervasive supply of therapeutic lysosomal enzymes in CNS tissues of MLD and GLD mice. Here, we investigated whether this strategy is similarly effective in juvenile non-human primates (NHP). To provide proof of principle for tolerability and biological efficacy of the strategy, we established a comprehensive study in normal NHP delivering a clinically relevant lentiviral vector encoding for the human ARSA transgene. Then, we injected a lentiviral vector coding for the human GALC transgene in Krabbe-affected rhesus macaques, evaluating for the first time the therapeutic potential of lentiviral GT in this unique LSD model. We showed favorable safety profile and consistent pattern of LV transduction and enzyme biodistribution in the two models, supporting the robustness of the proposed GT platform. We documented moderate inflammation at the injection sites, mild immune response to vector particles in few treated animals, no indication of immune response against transgenic products, and no molecular evidence of insertional genotoxicity. Efficient gene transfer in neurons, astrocytes, and oligodendrocytes close to the injection sites resulted in robust production and extensive spreading of transgenic enzymes in the whole CNS and in CSF, leading to supraphysiological ARSA activity in normal NHP and close to physiological GALC activity in the Krabbe NHP, in which biological efficacy was associated with preliminary indication of therapeutic benefit. These results support the rationale for the clinical translation of intracerebral lentiviral GT to address CNS pathology in MLD, GLD, and other neurodegenerative LSD.

Published
2016

10. Sleeping Beauty-engineered CAR T cells achieve antileukemic activity without severe toxicities

Author

Biondi, Andrea, Magnani, Chiara F., Gaipa, Gissuppe, Lussana, Federico, Belotti, Daniela, Gritti, Giuseppe, Napolitano, Sara, Matera, Giada, Cabiati, Benedetta, Buracchi, Chiara, Borleri, Gianmaria, Fazio, Grazia, Zaninelli, Silvia, Tettamanti, Sarah, Cesana, Stefania, Colombo, Valentina, Quaroni, Michele, Cazzaniga, Giovanni, Rovelli, Attilio, Biagi, Ettore, Galimberti, Stefania, Benedicenti, Fabrizio, Montini, Eugenio, Ferrari, Silvia, Introna, Martino, Balduzzi, Adriana, Valsecchi, Maria Grazia, Dastoli, Giuseppe, Rambaldi, Alessandro, and Calabria, Andrea

Subjects
Transposons -- Research, Immunological research, T cells -- Health aspects, Biological products -- Usage, Cellular therapy -- Methods, Immunotherapy -- Methods, Acute lymphocytic leukemia -- Care and treatment, Biomedical engineering -- Research, Health care industry
Abstract

BACKGROUND. Chimeric antigen receptor (CAR) T cell immunotherapy has resulted in complete remission (CR) and durable response in highly refractory patients. However, logistical complexity and high costs of manufacturing autologous viral products limit CAR T cell availability. METHODS. We report the early results of a phase I/II trial in B cell acute lymphoblastic leukemia (B-ALL) patients relapsed after allogeneic hematopoietic stem cell transplantation (HSCT) using donor-derived CD19 CAR T cells generated with the Sleeping Beauty (SB) transposon and differentiated into cytokine-induced killer (CIK) cells. RESULTS. The cellular product was produced successfully for all patients from the donor peripheral blood (PB) and consisted mostly of [CD3.sup.+] lymphocytes with 43% CAR expression. Four pediatric and 9 adult patients were infused with a single dose of CAR T cells. Toxicities reported were 2 grade I and 1 grade II cytokine-release syndrome (CRS) cases at the highest dose in the absence of graft-versus-host disease (GVHD), neurotoxicity, or dose-limiting toxicities. Six out of 7 patients receiving the highest doses achieved CR and CR with incomplete blood count recovery (CRi) at day 28. Five out of 6 patients in CR were also minimal residual disease negative (MRD). Robust expansion was achieved in the majority of the patients. CAR T cells were measurable by transgene copy PCR up to 10 months. Integration site analysis showed a positive safety profile and highly polyclonal repertoire in vitro and at early time points after infusion. CONCLUSION. SB-engineered CAR T cells expand and persist in pediatric and adult B-ALL patients relapsed after HSCT. Antileukemic activity was achieved without severe toxicities. TRIAL REGISTRATION. ClinicalTrials.gov NCT03389035. FUNDING. This study was supported by grants from the Fondazione AIRC per la Ricerca sul Cancro (AIRC); Cancer Research UK (CRUK); the Fundacion Cientifica de la Asociacion Espanola Contra el Cancer (FC AECC); Ministero Della Salute; Fondazione Regionale per la Ricerca Biomedica (FRRB)., Introduction Substantial efforts over the past few years have led chimeric antigen receptor (CAR) T cell therapy to success in relapsed and refractory (r/r) B cell malignancies. The early concept [...]

Published
2020
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11. Intrabone hematopoietic stem cell gene therapy for adult and pediatric patients affected by transfusion-dependent ß-thalassemia

Author

Marktel, Sarah, Scaramuzza, Samantha, Cicalese, Maria Pia, Giglio, Fabio, Galimberti, Stefania, Lidonnici, Maria Rosa, Calbi, Valeria, Assanelli, Andrea, Bernardo, Maria Ester, Rossi, Claudia, Calabria, Andrea, Milani, Raffaella, Gattillo, Salvatore, Benedicenti, Fabrizio, Spinozzi, Giulio, Aprile, Annamaria, Bergami, Alessandra, Casiraghi, Miriam, Consiglieri, Giulia, Masera, Nicoletta, D’Angelo, Emanuela, Mirra, Nadia, Origa, Raffaella, Tartaglione, Immacolata, Perrotta, Silverio, Winter, Robert, Coppola, Milena, Viarengo, Gianluca, Santoleri, Luca, Graziadei, Giovanna, Gabaldo, Michela, Valsecchi, Maria Grazia, Montini, Eugenio, Naldini, Luigi, Cappellini, Maria Domenica, Ciceri, Fabio, Aiuti, Alessandro, and Ferrari, Giuliana

Published
2019
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12. S250: UNVEILING THE BIOLOGICAL ROLE OF PERIPHERAL BLOOD HUMAN CIRCULATING HEMATOPOIETIC STEM AND PROGENITOR CELLS

Author

Quaranta, Pamela, primary, Basso-Ricci, Luca, additional, Jofra Hernandez, Raisa, additional, Maria Naldini, Matteo, additional, Barcella, Matteo, additional, Pacini, Guido, additional, Pietrasanta, Carlo, additional, Pugni, Lorenza, additional, Pais, Giulia, additional, Benedicenti, Fabrizio, additional, Giannelli, Stefania, additional, Monti, Ilaria, additional, Darin, Silvia, additional, Barera, Graziano, additional, Tucci, Francesca, additional, Ferrua, Francesca, additional, Calbi, Valeria, additional, Gentner, Bernhard, additional, DI Micco, Raffaella, additional, Montini, Eugenio, additional, Calabria, Andrea, additional, Merelli, Ivan, additional, Mosca, Fabio, additional, Ester Bernardo, Maria, additional, Pia Cicalese, Maria, additional, Aiuti, Alessandro, additional, and Serena, Scala, additional

Published
2023
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13. Hematopoietic reconstitution dynamics of mobilized- and bone marrow-derived human hematopoietic stem cells after gene therapy

Author

Scala, S, Ferrua, F, Basso-Ricci, L, Dionisio, F, Omrani, M, Quaranta, P, Jofra Hernandez, R, Del Core, L, Benedicenti, F, Monti, I, Giannelli, S, Fraschetta, F, Darin, S, Albertazzi, E, Galimberti, S, Montini, E, Calabria, A, Cicalese, M, Aiuti, A, Scala, Serena, Ferrua, Francesca, Basso-Ricci, Luca, Dionisio, Francesca, Omrani, Maryam, Quaranta, Pamela, Jofra Hernandez, Raisa, Del Core, Luca, Benedicenti, Fabrizio, Monti, Ilaria, Giannelli, Stefania, Fraschetta, Federico, Darin, Silvia, Albertazzi, Elena, Galimberti, Stefania, Montini, Eugenio, Calabria, Andrea, Cicalese, Maria Pia, Aiuti, Alessandro, Scala, S, Ferrua, F, Basso-Ricci, L, Dionisio, F, Omrani, M, Quaranta, P, Jofra Hernandez, R, Del Core, L, Benedicenti, F, Monti, I, Giannelli, S, Fraschetta, F, Darin, S, Albertazzi, E, Galimberti, S, Montini, E, Calabria, A, Cicalese, M, Aiuti, A, Scala, Serena, Ferrua, Francesca, Basso-Ricci, Luca, Dionisio, Francesca, Omrani, Maryam, Quaranta, Pamela, Jofra Hernandez, Raisa, Del Core, Luca, Benedicenti, Fabrizio, Monti, Ilaria, Giannelli, Stefania, Fraschetta, Federico, Darin, Silvia, Albertazzi, Elena, Galimberti, Stefania, Montini, Eugenio, Calabria, Andrea, Cicalese, Maria Pia, and Aiuti, Alessandro

Abstract

Mobilized peripheral blood is increasingly used instead of bone marrow as a source of autologous hematopoietic stem/progenitor cells for ex vivo gene therapy. Here, we present an unplanned exploratory analysis evaluating the hematopoietic reconstitution kinetics, engraftment and clonality in 13 pediatric Wiskott-Aldrich syndrome patients treated with autologous lentiviral-vector transduced hematopoietic stem/progenitor cells derived from mobilized peripheral blood (n = 7), bone marrow (n = 5) or the combination of the two sources (n = 1). 8 out of 13 gene therapy patients were enrolled in an open-label, non-randomized, phase 1/2 clinical study (NCT01515462) and the remaining 5 patients were treated under expanded access programs. Although mobilized peripheral blood- and bone marrow- hematopoietic stem/progenitor cells display similar capability of being gene-corrected, maintaining the engineered grafts up to 3 years after gene therapy, mobilized peripheral blood-gene therapy group shows faster neutrophil and platelet recovery, higher number of engrafted clones and increased gene correction in the myeloid lineage which correlate with higher amount of primitive and myeloid progenitors contained in hematopoietic stem/progenitor cells derived from mobilized peripheral blood. In vitro differentiation and transplantation studies in mice confirm that primitive hematopoietic stem/progenitor cells from both sources have comparable engraftment and multilineage differentiation potential. Altogether, our analyses reveal that the differential behavior after gene therapy of hematopoietic stem/progenitor cells derived from either bone marrow or mobilized peripheral blood is mainly due to the distinct cell composition rather than functional differences of the infused cell products, providing new frames of references for clinical interpretation of hematopoietic stem/progenitor cell transplantation outcome.

Published
2023

14. Lentiviral haemopoietic stem-cell gene therapy in early-onset metachromatic leukodystrophy: an ad-hoc analysis of a non-randomised, open-label, phase 1/2 trial

Author

Sessa, Maria, Lorioli, Laura, Fumagalli, Francesca, Acquati, Serena, Redaelli, Daniela, Baldoli, Cristina, Canale, Sabrina, Lopez, Ignazio D, Morena, Francesco, Calabria, Andrea, Fiori, Rossana, Silvani, Paolo, Rancoita, Paola M V, Gabaldo, Michela, Benedicenti, Fabrizio, Antonioli, Gigliola, Assanelli, Andrea, Cicalese, Maria Pia, del Carro, Ubaldo, Sora, Maria Grazia Natali, Martino, Sabata, Quattrini, Angelo, Montini, Eugenio, Di Serio, Clelia, Ciceri, Fabio, Roncarolo, Maria Grazia, Aiuti, Alessandro, Naldini, Luigi, and Biffi, Alessandra

Published
2016
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15. Long-term clinical outcomes of atidarsagene autotemcel (autologous hematopoietic stem cell gene therapy [HSC-GT] for metachromatic leukodystrophy) with up to 11 years follow-up

Author

Fumagalli, Francesca, primary, Calbi, Valeria, additional, De Mattia, Fabiola, additional, Zambon, Alberto A., additional, Gallo, Vera, additional, Recupero, Salvatore, additional, Baldoli, Cristina, additional, Fratini, Elena Sophia, additional, Ciotti, Francesca, additional, Fraschini, Maddalena, additional, Sarzana, Marina, additional, Scarparo, Stefano, additional, Calabria, Andrea, additional, Montini, Eugenio, additional, Locatelli, Sara, additional, Facchini, Marcella, additional, Clerici, Alessandra, additional, Morena, Francesco, additional, Martino, Sabata, additional, Claros, Angelica, additional, Kaviya, Arpeat, additional, Moro, Sean, additional, Brooks, Jean, additional, Richardson, Alan, additional, and Aiuti, Alessandro, additional

Published
2023
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18. A Whole-Genome Sequencing Study Implicates GRAMD1B in Multiple Sclerosis Susceptibility

Author

Esposito, Federica, primary, Osiceanu, Ana Maria, additional, Sorosina, Melissa, additional, Ottoboni, Linda, additional, Bollman, Bryan, additional, Santoro, Silvia, additional, Bettegazzi, Barbara, additional, Zauli, Andrea, additional, Clarelli, Ferdinando, additional, Mascia, Elisabetta, additional, Calabria, Andrea, additional, Zacchetti, Daniele, additional, Capra, Ruggero, additional, Ferrari, Maurizio, additional, Provero, Paolo, additional, Lazarevic, Dejan, additional, Cittaro, Davide, additional, Carrera, Paola, additional, Patsopoulos, Nikolaos, additional, Toniolo, Daniela, additional, Sadovnick, A Dessa, additional, Martino, Gianvito, additional, De Jager, Philip L., additional, Comi, Giancarlo, additional, Stupka, Elia, additional, Vilariño-Güell, Carles, additional, Piccio, Laura, additional, and Martinelli Boneschi, Filippo, additional

Published
2022
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19. Adaptive Routes of Hematopoietic Stem Cell Differentiation to Disease Conditions and Age in Gene Therapy Patients

Author

Calabria, Andrea, primary, Spinozzi, Giulio, additional, Cesana, Daniela, additional, Benedicenti, Fabrizio, additional, Pais, Giulia, additional, Scala, Serena, additional, Lidonnici, Maria Rosa, additional, Scaramuzza, Samantha, additional, Albertini, Alessandra, additional, Esposito, Simona, additional, De Mattia, Fabiola, additional, Canarutto, Daniele, additional, Tucci, Francesca, additional, Omrani, Maryam, additional, Dionisio, Francesca, additional, Giannelli, Stefania, additional, Marktel, Sarah, additional, Calbi, Valeria, additional, Ferrua, Francesca, additional, Gentner, Bernhard, additional, Ciceri, Fabio, additional, Naldini, Luigi, additional, Ferrari, Giuliana, additional, Aiuti, Alessandro, additional, and Montini, Eugenio, additional

Published
2022
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20. Unveiling the Biological Role of Peripheral Blood Human Circulating Hematopoietic Stem and Progenitor Cells

Author

Quaranta, Pamela, primary, Basso-Ricci, Luca, additional, Jofra Hernández, Raisa, additional, Naldini, Matteo Maria, additional, Barcella, Matteo, additional, Pacini, Guido, additional, Pietrasanta, Carlo, additional, Pugni, Lorenza, additional, Pais, Giulia, additional, Benedicenti, Fabrizio, additional, Dionisio, Francesca, additional, Giannelli, Stefania, additional, Monti, Ilaria, additional, Darin, Silvia, additional, Barera, Graziano, additional, Tucci, Francesca, additional, Ferrua, Francesca, additional, Calbi, Valeria, additional, Ometti, Marco, additional, Gentner, Bernhard, additional, Di Micco, Raffaella, additional, Montini, Eugenio, additional, Calabria, Andrea, additional, Merelli, Ivan, additional, Mosca, Fabio, additional, Bernardo, Maria Ester, additional, Cicalese, Maria Pia, additional, Aiuti, Alessandro, additional, and Scala, Serena, additional

Published
2022
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21. Choice of template delivery mitigates the genotoxic risk and adverse impact of editing in human hematopoietic stem cells

Author

Ferrari, Samuele, primary, Jacob, Aurelien, additional, Cesana, Daniela, additional, Laugel, Marianne, additional, Beretta, Stefano, additional, Varesi, Angelica, additional, Unali, Giulia, additional, Conti, Anastasia, additional, Canarutto, Daniele, additional, Albano, Luisa, additional, Calabria, Andrea, additional, Vavassori, Valentina, additional, Cipriani, Carlo, additional, Castiello, Maria Carmina, additional, Esposito, Simona, additional, Brombin, Chiara, additional, Cugnata, Federica, additional, Adjali, Oumeya, additional, Ayuso, Eduard, additional, Merelli, Ivan, additional, Villa, Anna, additional, Di Micco, Raffaella, additional, Kajaste-Rudnitski, Anna, additional, Montini, Eugenio, additional, Penaud-Budloo, Magalie, additional, and Naldini, Luigi, additional

Published
2022
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23. Lentiviral Hematopoietic Stem Cell Gene Therapy Benefits Metachromatic Leukodystrophy

Author

Biffi, Alessandra, Montini, Eugenio, Lorioli, Laura, Cesani, Martina, Fumagalli, Francesca, Plati, Tiziana, Baldoli, Cristina, Martino, Sabata, Calabria, Andrea, Canale, Sabrina, Benedicenti, Fabrizio, Vallanti, Giuliana, Biasco, Luca, Leo, Simone, Kabbara, Nabil, Zanetti, Gianluigi, Rizzo, William B., Mehta, Nalini A. L., Cicalese, Maria Pia, Casiraghi, Miriam, Boelens, Jaap J., Del Carro, Ubaldo, Dow, David J., Schmidt, Manfred, Assanelli, Andrea, Neduva, Victor, Di Serio, Clelia, Stupka, Elia, Gardner, Jason, von Kalle, Christof, Bordignon, Claudio, Ciceri, Fabio, Rovelli, Attilio, Roncarolo, Maria Grazia, Aiuti, Alessandro, Sessa, Maria, and Naldini, Luigi

Published
2013

24. Lentiviral Hematopoietic Stem Cell Gene Therapy in Patients with Wiskott-Aldrich Syndrome

Author

Aiuti, Alessandro, Biasco, Luca, Scaramuzza, Samantha, Ferrua, Francesca, Cicalese, Maria Pia, Baricordi, Cristina, Dionisio, Francesca, Calabria, Andrea, Giannelli, Stefania, Castiello, Maria Carmina, Bosticardo, Marita, Evangelio, Costanza, Assanelli, Andrea, Casiraghi, Miriam, Di Nunzio, Sara, Callegaro, Luciano, Benati, Claudia, Rizzardi, Paolo, Pellin, Danilo, Di Serio, Clelia, Schmidt, Manfred, Von Kalle, Christof, Gardner, Jason, Mehta, Nalini, Neduva, Victor, Dow, David J., Galy, Anne, Miniero, Roberto, Finocchi, Andrea, Metin, Ayse, Banerjee, Pinaki P., Orange, Jordan S., Galimberti, Stefania, Valsecchi, Maria Grazia, Biffi, Alessandra, Montini, Eugenio, Villa, Anna, Ciceri, Fabio, Roncarolo, Maria Grazia, and Naldini, Luigi

Published
2013

25. Normalization of clonal diversity in gene therapy studies using shape constrained splines

Author

Del Core, Luca, Cesana, D., Gallina, P., Serina Secanechia, Y. N., Rudilosso, L., Montini, Eugenio, Wit, Ernst C., Calabria, Andrea, Grzegorczyk, Marco, and Stochastic Studies and Statistics

Abstract

Viral vectors are used to insert genetic material into semirandom genomic positions of hematopoietic stem cells which, after reinfusion into patients, regenerate the entire hematopoietic system. Hematopoietic cells originating from genetically modified stem cells will harbor insertions in specific genomic positions called integration sites, which represent unique genetic marks of clonal identity. Therefore, the analysis of vector integration sites present in the genomic DNA of circulating cells allows to determine the number of clones in the blood ecosystem. Shannon diversity index is adopted to evaluate the heterogeneity of the transduced population of gene corrected cells. However, this measure can be affected by several technical variables such as the DNA amount used and the sequencing depth of the library analyzed and therefore the comparison across samples may be affected by these confounding factors. We developed an advanced spline-regression approach that leverages on confounding effects to provide a normalized entropy index. Our proposed method was first validated and compared with two state of the art approaches in a specifically designed in vitro assay. Subsequently our approach allowed to observe the expected impact of vector genotoxicity on entropy level decay in an in vivo model of hematopoietic stem cell gene therapy based on tumor prone mice.

Published
2022

26. SNPRanker: a tool for identification and scoring of SNPs associated to target genes

Author

Calabria Andrea, Mosca Ettore, Viti Federica, Merelli Ivan, and Milanesi Luciano

Subjects
Biotechnology, TP248.13-248.65
Abstract

The identification of genes and SNPs involved in human diseases remains a challenge. Many public resources, databases and applications, collect biological data and perform annotations, increasing the global biological knowledge. The need of SNPs prioritization is emerging with the development of new high-throughput genotyping technologies, which allow to develop customized disease-oriented chips. Therefore, given a list of genes related to a specific biological process or disease as input, a crucial issue is finding the most relevant SNPs to analyse. The selection of these SNPs may rely on the relevant a-priori knowledge of biomolecular features characterising all the annotated SNPs and genes of the provided list. The bioinformatics approach described here allows to retrieve a ranked list of significant SNPs from a set of input genes, such as candidate genes associated with a specific disease. The system enriches the genes set by including other genes, associated to the original ones by ontological similarity evaluation. The proposed method relies on the integration of data from public resources in a vertical perspective (from genomics to systems biology data), the evaluation of features from biomolecular knowledge, the computation of partial scores for SNPs and finally their ranking, relying on their global score. Our approach has been implemented into a web based tool called SNPRanker, which is accessible through at the URL http://www.itb.cnr.it/snpranker. An interesting application of the presented system is the prioritisation of SNPs related to genes involved in specific pathologies, in order to produce custom arrays.

Published
2010
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28. Efficient and safe correction of hemophilia A by lentiviral vector-transduced BOECs in an implantable device

Author

Olgasi, Cristina, primary, Borsotti, Chiara, additional, Merlin, Simone, additional, Bergmann, Thorsten, additional, Bittorf, Patrick, additional, Adewoye, Adeolu Badi, additional, Wragg, Nicholas, additional, Patterson, Kelcey, additional, Calabria, Andrea, additional, Benedicenti, Fabrizio, additional, Cucci, Alessia, additional, Borchiellini, Alessandra, additional, Pollio, Berardino, additional, Montini, Eugenio, additional, Mazzuca, Delfina M., additional, Zierau, Martin, additional, Stolzing, Alexandra, additional, Toleikis, Philip.M., additional, Braspenning, Joris, additional, and Follenzi, Antonia, additional

Published
2021
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29. Targeted genome editing in human repopulating haematopoietic stem cells

Author

Genovese, Pietro, Schiroli, Giulia, Escobar, Giulia, Tomaso, Tiziano Di, Firrito, Claudia, Calabria, Andrea, Moi, Davide, Mazzieri, Roberta, Bonini, Chiara, Holmes, Michael C., Gregory, Philip D., van der Burg, Mirjam, Gentner, Bernhard, Montini, Eugenio, Lombardo, Angelo, and Naldini, Luigi

Subjects
DNA repair -- Research, Gene therapy -- Research, Hematopoietic stem cells -- Research, Stem cells -- Transplantation, Environmental issues, Science and technology, Zoology and wildlife conservation
Abstract

Targeted genome editing by artificial nucleases has brought the goal of site-specific transgene integration and gene correction within the reach of gene therapy. However, its application to long-term repopulating haematopoietic stem cells (HSCs) has remained elusive. Here we show that poor permissiveness to gene transfer and limited proficiency of the homologydirected DNA repair pathway constrain gene targeting in human HSCs. By tailoring delivery platforms and culture conditions we overcame these barriers and provide stringent evidence of targeted integration in human HSCs by long-term multilineage repopulation of transplanted mice. We demonstrate the therapeutic potential of our strategy by targeting a corrective complementary DNA into the IL2RG gene of HSCs from healthy donors and a subject with X-linked severe combined immunodeficiency (SCID-X1). Gene-edited HSCs sustained normal haematopoiesis and gave rise to functional lymphoid cells that possess a selective growth advantage over those carrying disruptive IL2RG mutations. These results open up new avenues for treating SCID-X1 and other diseases., Haematopoietic stem cell (HSC)-based gene therapy has provided therapeutic benefit in primary immunodeficiencies (1,2), thalassaemia (3) and leukodystrophies (4,5). Whereas more advanced vectors, such as lentiviral vectors (6), have shown [...]

Published
2014
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30. ISAnalytics enables longitudinal and high-throughput clonal tracking studies in hematopoietic stem cell gene therapy applications.

Author

Pais, Giulia, Spinozzi, Giulio, Cesana, Daniela, Benedicenti, Fabrizio, Albertini, Alessandra, Bernardo, Maria Ester, Gentner, Bernhard, Montini, Eugenio, and Calabria, Andrea

Subjects
STEM cell treatment, GENETIC vectors, GENE therapy, MUCOPOLYSACCHARIDOSIS I, CYTOLOGY, GENETIC barcoding
Abstract

Longitudinal clonal tracking studies based on high-throughput sequencing technologies supported safety and long-term efficacy and unraveled hematopoietic reconstitution in many gene therapy applications with unprecedented resolution. However, monitoring patients over a decade-long follow-up entails a constant increase of large data volume with the emergence of critical computational challenges, unfortunately not addressed by currently available tools. Here we present ISAnalytics, a new R package for comprehensive and high-throughput clonal tracking studies using vector integration sites as markers of cellular identity. Once identified the clones externally from ISAnalytics and imported in the package, a wide range of implemented functionalities are available to users for assessing the safety and long-term efficacy of the treatment, here described in a clinical trial use case for Hurler disease, and for supporting hematopoietic stem cell biology in vivo with longitudinal analysis of clones over time, proliferation and differentiation. ISAnalytics is conceived to be metadata-driven, enabling users to focus on biological questions and hypotheses rather than on computational aspects. ISAnalytics can be fully integrated within laboratory workflows and standard procedures. Moreover, ISAnalytics is designed with efficient and scalable data structures, benchmarked with previous methods, and grants reproducibility and full analytical control through interactive web-reports and a module with Shiny interface. The implemented functionalities are flexible for all viral vector-based clonal tracking applications as well as genetic barcoding or cancer immunotherapies. [ABSTRACT FROM AUTHOR]

Published
2023
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31. Hematopoietic Stem- and Progenitor-Cell Gene Therapy for Hurler Syndrome

Author

Gentner, Bernhard, primary, Tucci, Francesca, additional, Galimberti, Stefania, additional, Fumagalli, Francesca, additional, De Pellegrin, Maurizio, additional, Silvani, Paolo, additional, Camesasca, Chiara, additional, Pontesilli, Silvia, additional, Darin, Silvia, additional, Ciotti, Francesca, additional, Sarzana, Marina, additional, Consiglieri, Giulia, additional, Filisetti, Chiara, additional, Forni, Giulia, additional, Passerini, Laura, additional, Tomasoni, Daniela, additional, Cesana, Daniela, additional, Calabria, Andrea, additional, Spinozzi, Giulio, additional, Cicalese, Maria-Pia, additional, Calbi, Valeria, additional, Migliavacca, Maddalena, additional, Barzaghi, Federica, additional, Ferrua, Francesca, additional, Gallo, Vera, additional, Miglietta, Simona, additional, Zonari, Erika, additional, Cheruku, Patali S., additional, Forni, Claudia, additional, Facchini, Marcella, additional, Corti, Ambra, additional, Gabaldo, Michela, additional, Zancan, Stefano, additional, Gasperini, Serena, additional, Rovelli, Attilio, additional, Boelens, Jaap-Jan, additional, Jones, Simon A., additional, Wynn, Robert, additional, Baldoli, Cristina, additional, Montini, Eugenio, additional, Gregori, Silvia, additional, Ciceri, Fabio, additional, Valsecchi, Maria G., additional, la Marca, Giancarlo, additional, Parini, Rossella, additional, Naldini, Luigi, additional, Aiuti, Alessandro, additional, and Bernardo, Maria-Ester, additional

Published
2021
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32. Lentiviral Hematopoietic Stem and Progenitor Cell Gene Therapy for Metachromatic Leukodystrophy (MLD): Clinical Outcomes from 38 Patients

Author

Essing, Mirko, additional, Zambon, Alberto, additional, Gallo, Vera, additional, Baldoli, Cristina, additional, Cugnata, Federica, additional, Rancoita, Paola M. V., additional, De Mattia, Fabiola, additional, Fratini, Elena, additional, Recupero, Salvatore, additional, Ferrua, Francesca, additional, Barzaghi, Federica, additional, Cicalese, Maria Pia, additional, Migliavacca, Maddalena, additional, Tucci, Francesca, additional, Ciotti, Francesca, additional, Fraschini, Maddalena, additional, Sarzana, Marina, additional, Scarparo, Stefano, additional, Silvani, Paolo, additional, Locatelli, Sara, additional, Clerici, Alessandra, additional, Antonioli, Gigliola, additional, Sangalli, Mara, additional, Zancan, Stefano, additional, Calabria, Andrea, additional, Montini, Eugenio, additional, Farinelli, Giada, additional, Morena, Francesco, additional, Segovia, Jesus, additional, Schwab, Laetitia C., additional, Downey, Gerald, additional, Gabaldo, Michela, additional, Martino, Sabata, additional, Di Serio, Clelia, additional, Ciceri, Fabio, additional, Filippi, Massimo, additional, Sessa, Maria, additional, Natali Sora, Maria Grazia, additional, Bernardo, Maria Ester, additional, Naldini, Luigi, additional, Biffi, Alessandra, additional, and Aiuti, Alessandro, additional

Published
2021
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34. Hematopoietic Stem- and Progenitor-Cell Gene Therapy for Hurler Syndrome

Author

Gentner, B, Tucci, F, Galimberti, S, Fumagalli, F, De Pellegrin, M, Silvani, P, Camesasca, C, Pontesilli, S, Darin, S, Ciotti, F, Sarzana, M, Consiglieri, G, Filisetti, C, Forni, G, Passerini, L, Tomasoni, D, Cesana, D, Calabria, A, Spinozzi, G, Cicalese, M, Calbi, V, Migliavacca, M, Barzaghi, F, Ferrua, F, Gallo, V, Miglietta, S, Zonari, E, Cheruku, P, Forni, C, Facchini, M, Corti, A, Gabaldo, M, Zancan, S, Gasperini, S, Rovelli, A, Boelens, J, Jones, S, Wynn, R, Baldoli, C, Montini, E, Gregori, S, Ciceri, F, Valsecchi, M, la Marca, G, Parini, R, Naldini, L, Aiuti, A, Bernardo, M, Gentner, Bernhard, Tucci, Francesca, Galimberti, Stefania, Fumagalli, Francesca, De Pellegrin, Maurizio, Silvani, Paolo, Camesasca, Chiara, Pontesilli, Silvia, Darin, Silvia, Ciotti, Francesca, Sarzana, Marina, Consiglieri, Giulia, Filisetti, Chiara, Forni, Giulia, Passerini, Laura, Tomasoni, Daniela, Cesana, Daniela, Calabria, Andrea, Spinozzi, Giulio, Cicalese, Maria-Pia, Calbi, Valeria, Migliavacca, Maddalena, Barzaghi, Federica, Ferrua, Francesca, Gallo, Vera, Miglietta, Simona, Zonari, Erika, Cheruku, Patali S, Forni, Claudia, Facchini, Marcella, Corti, Ambra, Gabaldo, Michela, Zancan, Stefano, Gasperini, Serena, Rovelli, Attilio, Boelens, Jaap-Jan, Jones, Simon A, Wynn, Robert, Baldoli, Cristina, Montini, Eugenio, Gregori, Silvia, Ciceri, Fabio, Valsecchi, Maria G, la Marca, Giancarlo, Parini, Rossella, Naldini, Luigi, Aiuti, Alessandro, Bernardo, Maria-Ester, Gentner, B, Tucci, F, Galimberti, S, Fumagalli, F, De Pellegrin, M, Silvani, P, Camesasca, C, Pontesilli, S, Darin, S, Ciotti, F, Sarzana, M, Consiglieri, G, Filisetti, C, Forni, G, Passerini, L, Tomasoni, D, Cesana, D, Calabria, A, Spinozzi, G, Cicalese, M, Calbi, V, Migliavacca, M, Barzaghi, F, Ferrua, F, Gallo, V, Miglietta, S, Zonari, E, Cheruku, P, Forni, C, Facchini, M, Corti, A, Gabaldo, M, Zancan, S, Gasperini, S, Rovelli, A, Boelens, J, Jones, S, Wynn, R, Baldoli, C, Montini, E, Gregori, S, Ciceri, F, Valsecchi, M, la Marca, G, Parini, R, Naldini, L, Aiuti, A, Bernardo, M, Gentner, Bernhard, Tucci, Francesca, Galimberti, Stefania, Fumagalli, Francesca, De Pellegrin, Maurizio, Silvani, Paolo, Camesasca, Chiara, Pontesilli, Silvia, Darin, Silvia, Ciotti, Francesca, Sarzana, Marina, Consiglieri, Giulia, Filisetti, Chiara, Forni, Giulia, Passerini, Laura, Tomasoni, Daniela, Cesana, Daniela, Calabria, Andrea, Spinozzi, Giulio, Cicalese, Maria-Pia, Calbi, Valeria, Migliavacca, Maddalena, Barzaghi, Federica, Ferrua, Francesca, Gallo, Vera, Miglietta, Simona, Zonari, Erika, Cheruku, Patali S, Forni, Claudia, Facchini, Marcella, Corti, Ambra, Gabaldo, Michela, Zancan, Stefano, Gasperini, Serena, Rovelli, Attilio, Boelens, Jaap-Jan, Jones, Simon A, Wynn, Robert, Baldoli, Cristina, Montini, Eugenio, Gregori, Silvia, Ciceri, Fabio, Valsecchi, Maria G, la Marca, Giancarlo, Parini, Rossella, Naldini, Luigi, Aiuti, Alessandro, and Bernardo, Maria-Ester

Abstract

Background Allogeneic hematopoietic stem-cell transplantation is the standard of care for Hurler syndrome (mucopolysaccharidosis type I, Hurler variant [MPSIH]). However, this treatment is only partially curative and is associated with complications. Methods We are conducting an ongoing study involving eight children with MPSIH. At enrollment, the children lacked a suitable allogeneic donor and had a Developmental Quotient or Intelligence Quotient score above 70 (i.e., none had moderate or severe cognitive impairment). The children received autologous hematopoietic stem and progenitor cells (HSPCs) transduced ex vivo with an alpha-L-iduronidase (IDUA)-encoding lentiviral vector after myeloablative conditioning. Safety and correction of blood IDUA activity up to supraphysiologic levels were the primary end points. Clearance of lysosomal storage material as well as skeletal and neurophysiological development were assessed as secondary and exploratory end points. The planned duration of the study is 5 years. Results We now report interim results. The children's mean (+/- SD) age at the time of HSPC gene therapy was 1.9 +/- 0.5 years. At a median follow-up of 2.10 years, the procedure had a safety profile similar to that known for autologous hematopoietic stem-cell transplantation. All the patients showed prompt and sustained engraftment of gene-corrected cells and had supraphysiologic blood IDUA activity within a month, which was maintained up to the latest follow-up. Urinary glycosaminoglycan (GAG) excretion decreased steeply, reaching normal levels at 12 months in four of five patients who could be evaluated. Previously undetectable levels of IDUA activity in the cerebrospinal fluid became detectable after gene therapy and were associated with local clearance of GAGs. Patients showed stable cognitive performance, stable motor skills corresponding to continued motor development, improved or stable findings on magnetic resonance imaging of the brain and spine, reduced

Published
2021

36. Targeted next-generation sequencing appoints C16orf57 as Clericuzio-type poikiloderma with neutropenia gene

Author

Volpi, Ludovica, Roversi, Gaia, Colombo, Elisa Adele, Leijsten, Nico, Concolino, Daniela, Calabria, Andrea, Mencarelli, Maria Antonietta, Fimiani, Michele, Macciardi, Fabio, Pfundt, Rolph, Schoenmakers, Eric F.P.M., and Larizza, Lidia

Subjects
Chromosome mapping -- Usage, Gene mutations -- Analysis, Italians -- Genetic aspects, Neutropenia -- Genetic aspects, Neutropenia -- Diagnosis, Neutropenia -- Care and treatment, Biological sciences
Abstract

A study uses targeted next-generation sequencing and autozygosity mapping on an inbred Italian family to identify the disease genes in members affected with the rare autosomal-recessive genodermatosis Clericuzio-type poikiloderma with neutropenia (PN). Findings reveal that two deleterious mutations in the C16orf57 gene are associated with PN in the affected individuals.

Published
2010

37. Sleeping Beauty-engineered CAR T cells achieve anti-leukemic activity without severe toxicities

Author

Magnani, C, Gaipa, G, Lussana, F, Belotti, D, Gritti, G, Napolitano, S, Matera, G, Cabiati, B, Buracchi, C, Borleri, G, Fazio, G, Zaninelli, S, Tettamanti, S, Cesana, S, Colombo, V, Quaroni, M, Cazzaniga, G, Rovelli, A, Biagi, E, Galimberti, S, Calabria, A, Benedicenti, F, Montini, E, Ferrari, S, Introna, M, Balduzzi, A, Valsecchi, M, Dastoli, G, Rambaldi, A, Biondi, A, Magnani, Chiara F, Gaipa, Giuseppe, Lussana, Federico, Belotti, Daniela, Gritti, Giuseppe, Napolitano, Sara, Matera, Giada, Cabiati, Benedetta, Buracchi, Chiara, Borleri, Gianmaria, Fazio, Grazia, Zaninelli, Silvia, Tettamanti, Sarah, Cesana, Stefania, Colombo, Valentina, Quaroni, Michele, Cazzaniga, Giovanni, Rovelli, Attilio, Biagi, Ettore, Galimberti, Stefania, Calabria, Andrea, Benedicenti, Fabrizio, Montini, Eugenio, Ferrari, Silvia, Introna, Martino, Balduzzi, Adriana, Valsecchi, Maria Grazia, Dastoli, Giuseppe, Rambaldi, Alessandro, Biondi, Andrea, Magnani, C, Gaipa, G, Lussana, F, Belotti, D, Gritti, G, Napolitano, S, Matera, G, Cabiati, B, Buracchi, C, Borleri, G, Fazio, G, Zaninelli, S, Tettamanti, S, Cesana, S, Colombo, V, Quaroni, M, Cazzaniga, G, Rovelli, A, Biagi, E, Galimberti, S, Calabria, A, Benedicenti, F, Montini, E, Ferrari, S, Introna, M, Balduzzi, A, Valsecchi, M, Dastoli, G, Rambaldi, A, Biondi, A, Magnani, Chiara F, Gaipa, Giuseppe, Lussana, Federico, Belotti, Daniela, Gritti, Giuseppe, Napolitano, Sara, Matera, Giada, Cabiati, Benedetta, Buracchi, Chiara, Borleri, Gianmaria, Fazio, Grazia, Zaninelli, Silvia, Tettamanti, Sarah, Cesana, Stefania, Colombo, Valentina, Quaroni, Michele, Cazzaniga, Giovanni, Rovelli, Attilio, Biagi, Ettore, Galimberti, Stefania, Calabria, Andrea, Benedicenti, Fabrizio, Montini, Eugenio, Ferrari, Silvia, Introna, Martino, Balduzzi, Adriana, Valsecchi, Maria Grazia, Dastoli, Giuseppe, Rambaldi, Alessandro, and Biondi, Andrea

Abstract

BACKGROUND. Chimeric antigen receptor (CAR) T cell immunotherapy has resulted in complete remission (CR) and durable response in highly refractory patients. However, logistical complexity and high costs of manufacturing autologous viral products limit CAR T cell availability. METHODS. We report the early results of a phase I/II trial in B cell acute lymphoblastic leukemia (B-ALL) patients relapsed after allogeneic hematopoietic stem cell transplantation (HSCT) using donor-derived CD19 CAR T cells generated with the Sleeping Beauty (SB) transposon and differentiated into cytokine-induced killer (CIK) cells. RESULTS. The cellular product was produced successfully for all patients from the donor peripheral blood (PB) and consisted mostly of CD3+ lymphocytes with 43% CAR expression. Four pediatric and 9 adult patients were infused with a single dose of CAR T cells. Toxicities reported were 2 grade I and 1 grade II cytokine-release syndrome (CRS) cases at the highest dose in the absence of graft-versus-host disease (GVHD), neurotoxicity, or dose-limiting toxicities. Six out of 7 patients receiving the highest doses achieved CR and CR with incomplete blood count recovery (CRi) at day 28. Five out of 6 patients in CR were also minimal residual disease negative (MRD–). Robust expansion was achieved in the majority of the patients. CAR T cells were measurable by transgene copy PCR up to 10 months. Integration site analysis showed a positive safety profile and highly polyclonal repertoire in vitro and at early time points after infusion. CONCLUSION. SB-engineered CAR T cells expand and persist in pediatric and adult B-ALL patients relapsed after HSCT. Antileukemic activity was achieved without severe toxicities.

Published
2020

38. Hematopoietic Tumors in a Mouse Model of X-linked Chronic Granulomatous Disease after Lentiviral Vector-Mediated Gene Therapy

Author

Jofra Hernández, Raisa, primary, Calabria, Andrea, additional, Sanvito, Francesca, additional, De Mattia, Fabiola, additional, Farinelli, Giada, additional, Scala, Serena, additional, Visigalli, Ilaria, additional, Carriglio, Nicola, additional, De Simone, Maura, additional, Vezzoli, Michela, additional, Cecere, Francesca, additional, Migliavacca, Maddalena, additional, Basso-Ricci, Luca, additional, Omrani, Maryam, additional, Benedicenti, Fabrizio, additional, Norata, Rossana, additional, Rancoita, Paola Maria Vittoria, additional, Di Serio, Clelia, additional, Albertini, Paola, additional, Cristofori, Patrizia, additional, Naldini, Luigi, additional, Gentner, Bernhard, additional, Montini, Eugenio, additional, Aiuti, Alessandro, additional, and Mortellaro, Alessandra, additional

Published
2021
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40. MYO1E Mutations and Childhood Familial Focal Segmental Glomerulosclerosis

Author

Mele, Caterina, Iatropoulos, Paraskevas, Donadelli, Roberta, Calabria, Andrea, Maranta, Ramona, Cassis, Paola, Buelli, Simona, Tomasoni, Susanna, Piras, Rossella, Krendel, Mira, Bettoni, Serena, Morigi, Marina, Delledonne, Massimo, Pecoraro, Carmine, Abbate, Isabella, Capobianchi, Maria Rosaria, Hildebrandt, Friedhelm, Otto, Edgar, Schaefer, Franz, Macciardi, Fabio, Ozaltin, Fatih, Emre, Sevinc, Ibsirlioglu, Tulin, Benigni, Ariela, Remuzzi, Giuseppe, and Noris, Marina

Published
2011
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41. Sleeping Beauty–engineered CAR T cells achieve antileukemic activity without severe toxicities

Author

Magnani, Chiara F., primary, Gaipa, Giuseppe, additional, Lussana, Federico, additional, Belotti, Daniela, additional, Gritti, Giuseppe, additional, Napolitano, Sara, additional, Matera, Giada, additional, Cabiati, Benedetta, additional, Buracchi, Chiara, additional, Borleri, Gianmaria, additional, Fazio, Grazia, additional, Zaninelli, Silvia, additional, Tettamanti, Sarah, additional, Cesana, Stefania, additional, Colombo, Valentina, additional, Quaroni, Michele, additional, Cazzaniga, Giovanni, additional, Rovelli, Attilio, additional, Biagi, Ettore, additional, Galimberti, Stefania, additional, Calabria, Andrea, additional, Benedicenti, Fabrizio, additional, Montini, Eugenio, additional, Ferrari, Silvia, additional, Introna, Martino, additional, Balduzzi, Adriana, additional, Valsecchi, Maria Grazia, additional, Dastoli, Giuseppe, additional, Rambaldi, Alessandro, additional, and Biondi, Andrea, additional

Published
2020
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43. Assessing the Impact of Cyclosporin A on Lentiviral Transduction and Preservation of Human Hematopoietic Stem Cells in Clinically RelevantEx VivoGene Therapy Settings

Author

Petrillo, Carolina, primary, Calabria, Andrea, additional, Piras, Francesco, additional, Capotondo, Alessia, additional, Spinozzi, Giulio, additional, Cuccovillo, Ivan, additional, Benedicenti, Fabrizio, additional, Naldini, Luigi, additional, Montini, Eugenio, additional, Biffi, Alessandra, additional, Gentner, Bernhard, additional, and Kajaste-Rudnitski, Anna, additional

Published
2019
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44. Phagocytosis-shielded lentiviral vectors improve liver gene therapy in nonhuman primates

Author

Milani, Michela, primary, Annoni, Andrea, additional, Moalli, Federica, additional, Liu, Tongyao, additional, Cesana, Daniela, additional, Calabria, Andrea, additional, Bartolaccini, Sara, additional, Biffi, Mauro, additional, Russo, Fabio, additional, Visigalli, Ilaria, additional, Raimondi, Andrea, additional, Patarroyo-White, Susannah, additional, Drager, Douglas, additional, Cristofori, Patrizia, additional, Ayuso, Eduard, additional, Montini, Eugenio, additional, Peters, Robert, additional, Iannacone, Matteo, additional, Cantore, Alessio, additional, and Naldini, Luigi, additional

Published
2019
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45. Precise Gene Editing Preserves Hematopoietic Stem Cell Function following Transient p53-Mediated DNA Damage Response

Author

Schiroli, Giulia, primary, Conti, Anastasia, additional, Ferrari, Samuele, additional, della Volpe, Lucrezia, additional, Jacob, Aurelien, additional, Albano, Luisa, additional, Beretta, Stefano, additional, Calabria, Andrea, additional, Vavassori, Valentina, additional, Gasparini, Patrizia, additional, Salataj, Eralda, additional, Ndiaye-Lobry, Delphine, additional, Brombin, Chiara, additional, Chaumeil, Julie, additional, Montini, Eugenio, additional, Merelli, Ivan, additional, Genovese, Pietro, additional, Naldini, Luigi, additional, and Di Micco, Raffaella, additional

Published
2019
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46. Multiple Integrated Non-clinical Studies Predict the Safety of Lentivirus-Mediated Gene Therapy for β-Thalassemia

Author

Lidonnici, Maria Rosa, primary, Paleari, Ylenia, additional, Tiboni, Francesca, additional, Mandelli, Giacomo, additional, Rossi, Claudia, additional, Vezzoli, Michela, additional, Aprile, Annamaria, additional, Lederer, Carsten Werner, additional, Ambrosi, Alessandro, additional, Chanut, Franck, additional, Sanvito, Francesca, additional, Calabria, Andrea, additional, Poletti, Valentina, additional, Mavilio, Fulvio, additional, Montini, Eugenio, additional, Naldini, Luigi, additional, Cristofori, Patrizia, additional, and Ferrari, Giuliana, additional

Published
2018
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47. Pervasive supply of therapeutic lysosomal enzymes in the CNS of normal and Krabbe-affected non-human primates by intracerebral lentiviral gene therapy

Author

Lattanzi Annalisa, Tiradani Luigi, Bravo Gabriele, Morena Francesco, Sanvito Francesca, Calabria Andrea, Bringas John, Fisher Perkins Jeanne M., Dufour Jason P., Baker Kate C., Montini Eugenio, Bunnell Bruce A., Bankiewicz Krystof, Martino Sabata, Naldini Luigi, Gritti Angela, DOGLIONI , CLAUDIO, Lattanzi, Annalisa, Tiradani, Luigi, Bravo, Gabriele, Morena, Francesco, Sanvito, Francesca, Calabria, Andrea, Bringas, John, Fisher Perkins Jeanne, M., Dufour Jason, P., Baker Kate, C., Doglioni, Claudio, Montini, Eugenio, Bunnell Bruce, A., Bankiewicz, Krystof, Martino, Sabata, Naldini, Luigi, and Gritti, Angela

Abstract

Metachromatic leukodystrophy (MLD) and globoid cell leukodystrophy (GLD or Krabbe disease) are severe neurodegenerative lysosomal storage diseases (LSD) caused by arylsulfatase A (ARSA) and galactosylceramidase (GALC) deficiency, respectively. Our previous studies established lentiviral gene therapy (GT) as a rapid and effective intervention to provide pervasive supply of therapeutic lysosomal enzymes in CNS tissues of MLD and GLD mice. Here, we investigated whether this strategy is similarly effective in juvenile non-human primates (NHP). To provide proof of principle for tolerability and biological efficacy of the strategy, we established a comprehensive study in normal NHP delivering a clinically relevantlentiviral vector encoding for the human ARSA transgene. Then, we injected a lentiviral vector coding for the human GALC transgene in Krabbe-affected rhesus macaques, evaluating for the first time the therapeutic potential of lentiviral GT in this unique LSD model. We showed favorable safety profile and consistent pattern of LV transduction and enzyme biodistribution in the two models, supporting the robustness of the proposed GT platform. We documented moderate inflammation at the injection sites, mild immune response to vector particles in few treated animals, no indication of immune response against transgenic products, and no molecular evidence of insertional genotoxicity. Efficient gene transfer in neurons, astrocytes, and oligodendrocytes close to the injection sites resulted in robust production and extensive spreading of transgenic enzymes in the whole CNS and in CSF, leading to supraphysiological ARSA activity in normal NHP and close to physiological GALC activity in the Krabbe NHP, in which biological efficacy was associated with preliminary indication of therapeutic benefit. These results support the rationale for the clinical translation of intracerebral lentiviral GT to address CNS pathology in MLD, GLD, and other neurodegenerative LSD.

Published
2016

49. In vivo tracking of T cells in humans unveils decade-long survival and activity of genetically modified T memory stem cells

Author

Biasco, Luca, Scala, Serena, Basso Ricci, Luca, Dionisio, Francesca, Baricordi, Cristina, Calabria, Andrea, Giannelli, Stefania, Cieri, Nicoletta, Barzaghi, Federica, Pajno, Roberta, Al-Mousa, Hamoud, Scarselli, Alessia, Cancrini, Caterina, Bordignon, Claudio, Roncarolo, Maria Grazia, Montini, Eugenio, BONINI, CHIARA, Aiuti, Alessandro, Biasco, Luca, Scala, Serena, Basso Ricci, Luca, Dionisio, Francesca, Baricordi, Cristina, Calabria, Andrea, Giannelli, Stefania, Cieri, Nicoletta, Barzaghi, Federica, Pajno, Roberta, Al-Mousa, Hamoud, Scarselli, Alessia, Cancrini, Caterina, Bordignon, Claudio, Roncarolo, Maria Grazia, Montini, Eugenio, Bonini, Chiara, Aiuti, Alessandro, Biasco, L, Scala, S, Basso, Ricci L, Dionisio, F, Baricordi, C, Calabria, A, Giannelli, S, Cieri, N, Barzaghi, F, Pajno, R, Al-Mousa, H, Scarselli, A, Cancrini, C, Bordignon, C, Roncarolo, M G, Montini, E, Bonini, C, and Aiuti, A

Subjects
Time Factors, T-Lymphocytes, Genetic enhancement, Cell, Longitudinal Studie, Biochemistry, Longitudinal Studies, Child, Medicine (all), Hematology, General Medicine, Phenotype, Tissue Donors, Haematopoiesis, Settore MED/02, medicine.anatomical_structure, Cell Tracking, Tetradecanoylphorbol Acetate, Stem cell, Genetic Engineering, Human, Adult, Time Factor, Cell Survival, T cell, Immunology, Tissue Donor, Biology, Viral vector, Clone Cell, In vivo, medicine, Humans, Severe combined immunodeficiency, Hematopoietic Stem Cell, Cell Biology, Genetic Therapy, Hematopoietic Stem Cells, medicine.disease, Lymphocyte Subsets, In vitro, Clone Cells, T-Lymphocyte, Lymphocyte Subset, Cancer research, Interleukin-2, T memory stem cells, Bone marrow, Immunologic Memory, Ex vivo
Abstract

A deeper understanding of T lymphocytes survival and differentiation potential in humans is paramount for the development of effective gene/cell therapies based on T-cell engineering. We here performed a comprehensive study of T-cells dynamics and plasticity in humans by a unique combination of phenotypic/functional studies and high-throughput integration sites (IS) analyses. We analyzed samples from hematopoietic stem cells (HSC) (n=10) or mature lymphocytes (PBL) gene therapy (GT) (n=4) treated ADA (adenosine deaminase) deficient-SCID patients. For comparative analyses, we also collected data from pediatric (n=19) and adult (n=52) healthy donors (HD), and from bone marrow transplanted patients (BMT) with primary immunodeficiencies (n=10, 4 with ADA-SCID). We observed that vector-positive CD62L+/CD45RA+ putative T naïve cells were detectable 12 years after last infusion of gene-corrected lymphocytes in peripheral blood of PBL-GT patients that lack the support of transduced lymphocytes precursors. We then unveiled that the vast majority of these CD62L+/CD45RA+ cells (80.3%) in PBL-GT patients could be actually classified phenotypically (CD95, IL2Rβ and IL7Rα surface expression) and functionally (IFNγ production and aCD3/aCD28 in vitro differentiation) as active long-lasting T memory stem cells (Tscm). The peculiar Tscm frequency found in PBL-GT patients was most likely due to a combinatorial in vitro and in vivo effect. Indeed, by a series of in vitro assays, we showed that Tscm relative enrichment in CD45RA+CD62L+ compartment have occurred during the in vitro manipulation of T cells before infusion. Additionally, we found higher-then-normal Tscm contribution among CD45RA+/CD62L+ cells even in ADA-SCID patients receiving HSC-GT and BMT, suggesting a role of disease background on in vivo Tscm persistence. Analyzing our cohorts of healthy donors and treated individuals we were able to further correlate Tscm contribution in vivo with age, conditioning regimen, disease background, cell source, and long-term T-cell reconstitution. One unique aspect of our study consisted in the opportunity to track Tscm clonal dynamics in vivo in humans since each gene-corrected cell infused in our GT patients is univocally and permanently tagged by a retroviral integration site.To perform in vivo molecular tracing of individual T-cell clones we sorted T naïve, Tscm, central memory and effector memory subtypes. We then collected from these subpopulations, by LAM-PCR+Illumina-Miseq sequencing, 2.584.137 integration sites (IS) sequences mapped to 1.746 unique chromosomal positions, corresponding to 910 integrations from 5 HSC-GT patients in vivo, 79 integrations from 2 PBL-GT samples of transduced cell products prior to infusion and 754 integrations from 4 PBL-GT patients in vivo. Firstly, to establish a relationship between precursors and terminally differentiated T cells we searched for the presence of identical insertion sites detected in multiple T-cell subtypes, applying stringent analytical filters for cross-contaminations. Strikingly, the level of shared integrations in each subtype was directly correlated to its stage of differentiation with Tscm, isolated from PBL-GT patients, showing the highest proportion of integration sites shared with the other T-cell subsets. Importantly, the results of the same analysis performed on HSC-GT patients were outstandingly coherent with the progressive developmental model of memory T-cell differentiation. We then assessed the survival of individual Tscm clones by performing a longitudinal IS analysis of different T-cell subtypes isolated from 3 PBL-GT patients over a 2 to 5 years timeframe up to 12 years after last infusion. We were able to formally prove the persistence of individual Tscm by re-capturing identical IS tagging specific Tscm clones in two independent timepoints in a 5- years window. Importantly, the same IS were also detected in multiple T-cell subtypes, representing the best indirect evidence that these clones were endowed with long-term precursor activity. We also documented, by IS sequencing reads, the long-term polyclonal composition of each subtype and we did not observe enrichment for IS flanking proto-oncogenes. Overall, this study validates, for the first time in humans, the safe and functional decade-long survival of engineered Tscm, paving the way for their future application in clinical settings. Disclosures No relevant conflicts of interest to declare.

Published
2015

50. VISPA2: a scalable pipeline for high-throughput identification and annotation of vector integration sites

Author

Spinozzi, G, Calabria, A, Brasca, S, Beretta, S, Merelli, I, Milanesi, L, Montini, E, SPINOZZI, GIULIO, CALABRIA, ANDREA, Montini, E., Spinozzi, G, Calabria, A, Brasca, S, Beretta, S, Merelli, I, Milanesi, L, Montini, E, SPINOZZI, GIULIO, CALABRIA, ANDREA, and Montini, E.

Abstract

Background: Bioinformatics tools designed to identify lentiviral or retroviral vector insertion sites in the genome of host cells are used to address the safety and long-term efficacy of hematopoietic stem cell gene therapy applications and to study the clonal dynamics of hematopoietic reconstitution. The increasing number of gene therapy clinical trials combined with the increasing amount of Next Generation Sequencing data, aimed at identifying integration sites, require both highly accurate and efficient computational software able to correctly process "big data" in a reasonable computational time. Results: Here we present VISPA2 (Vector Integration Site Parallel Analysis, version 2), the latest optimized computational pipeline for integration site identification and analysis with the following features: (1) the sequence analysis for the integration site processing is fully compliant with paired-end reads and includes a sequence quality filter before and after the alignment on the target genome; (2) an heuristic algorithm to reduce false positive integration sites at nucleotide level to reduce the impact of Polymerase Chain Reaction or trimming/alignment artifacts; (3) a classification and annotation module for integration sites; (4) a user friendly web interface as researcher front-end to perform integration site analyses without computational skills; (5) the time speedup of all steps through parallelization (Hadoop free). Conclusions: We tested VISPA2 performances using simulated and real datasets of lentiviral vector integration sites, previously obtained from patients enrolled in a hematopoietic stem cell gene therapy clinical trial and compared the results with other preexisting tools for integration site analysis. On the computational side, VISPA2 showed a>6-fold speedup and improved precision and recall metrics (1 and 0.97 respectively) compared to previously developed computational pipelines. These performances indicate that VISPA2 is a fast, reliable a

Published
2017

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