169 results on '"Radrizzani M"'
Search Results
52. Differences between in vivo and in vitro activation of cancer patient lymphocytes by recombinant interleukin 2: possible role for lymphokine-activated killer cell infusion in the in vivo-induced activation
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CARLO GAMBACORTI PASSERINI, Rivoltini, L., Radrizzani, M., Belli, F., Sciorelli, G., Ravagnani, F., Galazka, A. R., Cascinelli, N., and Parmiani, G.
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Cytotoxicity, Immunologic ,Killer Cells, Natural ,Lymphokines ,Humans ,Interleukin-2 ,Immunotherapy ,Lymphocytes ,Lymphocyte Activation ,Melanoma ,Recombinant Proteins ,Cell Line - Abstract
In this study 15 consecutive melanoma patients were treated with two courses of bolus recombinant interleukin 2 (rIL2) and rIL2 plus in vitro-generated lymphokine-activated killers (LAK), respectively. The immunological monitoring performed after 4 days of rIL2 or rIL2 plus LAK, indicate that the in vivo peripheral blood lymphocyte (PBL), activation (spontaneous proliferation, tumor cytotoxicity, number of DR+ PBL, obtained after the second cycle of rIL2 plus LAK is significantly higher than after the first cycle of rIL2 alone. During the 5-day interval between the two courses, PBL activation returns to baseline levels and no evidence for increased sensitivity of PBL to rIL2 is present. To further confirm this, two additional patients were studied, in whom rIL2 was administered by continuous i.v. infusion. In these two patients the in vitro versus in vivo PBL activation could be directly and simultaneously compared by using in vitro the same concentration of rIL2 reached and maintained in the patients' sera. The PBL activation induced in vivo by a cycle of rIL2 alone was significantly less (about 10 times) than that obtained in vitro with a comparable rIL2 concentration. Thus, the infusion of in vitro highly activated PBL could explain the increased in vivo lymphocyte activation of the second cycle of rIL2 plus LAK over the first cycle of rIL2 alone.
53. Human melanoma-reactive CD4+ and CD8+ CTL clones resist Fas ligand- induced apoptosis and use Fas/Fas ligand-independent mechanisms for tumor killing
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Rivoltini, L., Radrizzani, M., Accornero, P., Squarcina, P., Chiodoni, C., Mazzocchi, A., Castelli, C., Tarsini, P., Viggiano, V., Belli, F., Mario Paolo Colombo, and Parmiani, G.
54. Development of monoclonal oligobodies and chemically synthesized oligobodies
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Radrizzani, M., Mariana Brocardo, Solveyra, C. G., Bianchini, M., Reyes, G. B., Cafferata, E. G., Ortiz, G. V., and Santa-Coloma, T. A.
55. Examination of the natural protein substrates affected by staurosporine in the developing cerebral cortex
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Yakisich, J. S., Radrizzani, M., and Vargas, V. I.
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- 1994
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56. IL-7 receptor expression identifies suicide gene–modified allospecific CD8+ T cells capable of self-renewal and differentiation into antileukemia effectors
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Fabio Ciceri, Bart A. Nijmeijer, Zohara Aghai, Claudio Bordignon, Lothar Hambach, Marina Radrizzani, Attilio Bondanza, Katharina Fleischhauer, Shin Kaneko, Sara Mastaglio, Els Goulmy, Chiara Bonini, Bondanza, Attilio, Hambach, L, Aghai, Z, Nijmeijer, B, Kaneko, S, Mastaglio, S, Radrizzani, M, Fleischhauer, K, Ciceri, Fabio, Bordignon, Claudio, Bonini, MARIA CHIARA, and Goulmy, E.
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Receptor expression ,CD3 ,Genetic Vectors ,Immunology ,Gene Expression ,T-Cell Antigen Receptor Specificity ,Mice, SCID ,CD8-Positive T-Lymphocytes ,Biology ,Immunotherapy, Adoptive ,Biochemistry ,Mice ,Antigen ,Mice, Inbred NOD ,Animals ,Humans ,Transplantation, Homologous ,Cytotoxic T cell ,Interleukin-7 receptor ,Cells, Cultured ,Cell Proliferation ,Leukemia ,Receptors, Interleukin-7 ,Genes, Transgenic, Suicide ,CD28 ,Cell Differentiation ,Genetic Therapy ,Cell Biology ,Hematology ,Suicide gene ,Prognosis ,surgical procedures, operative ,Cancer research ,biology.protein ,Female ,Biomarkers ,CD8 ,T-Lymphocytes, Cytotoxic - Abstract
In allogeneic hematopoietic cell transplantation (HSCT), donor T lymphocytes mediate the graft-versus-leukemia (GVL) effect, but induce graft-versus-host disease (GVHD). Suicide gene therapy—that is, the genetic induction of a conditional suicide phenotype into donor T cells—allows dissociating the GVL effect from GVHD. Genetic modification with retroviral vectors after CD3 activation reduces T-cell alloreactivity. We recently found that alloreactivity is maintained when CD28 costimulation, IL-7, and IL-15 are added. Herein, we used the minor histocompatibility (mH) antigens HA-1 and H-Y as model alloantigens to directly explore the antileukemia efficacy of human T cells modified with the prototypic suicide gene herpes simplex virus thymidine kinase (tk) after activation with different stimuli. Only in the case of CD28 costimulation, IL-7, and IL-15, the repertoire of tk+ T cells contained HA-1– and H-Y–specific CD8+ cytotoxic T cells (CTL) precursors. Thymidine kinase–positive HA-1– and H-Y–specific CTLs were capable of self-renewal and differentiation into potent antileukemia effectors in vitro, and in vivo in a humanized mouse model. Self-renewal and differentiation coincided with IL-7 receptor expression. These results pave the way to the clinical investigation of T cells modified with a suicide gene after CD28 costimulation, IL-7, and IL-15 for a safe and effective GVL effect.
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- 2011
57. Long term follow up of patients after allogeneic stem cell transplantation and transfusion of HSV-TK transduced T-cells
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Fabio Ciceri, Bernd Hertenstein, Wolfgang Kuehnau, Arnold Ganser, Ivonne Fernandez-Munoz, Gernot Beutel, Claudia Benati, Anna Silvani, Patrick Schweier, Eva M. Weissinger, Michael Stadler, Irene Katarina Beckmann, Marina Radrizzani, Susanne Luther, Sylvia Borchers, Joerg Schmidtke, Chiara Bonini, Elena Provasi, Weissinger, Em, Borchers, S, Silvani, A, Provasi, E, Radrizzani, M, Beckmann, Ik, Benati, C, Schmidtke, J, Kuehnau, W, Schweier, P, Luther, S, Fernandez Munoz, I, Beutel, G, Ciceri, Fabio, Bonini, MARIA CHIARA, Ganser, A, Hertenstein, B, and Stadler, M.
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Ganciclovir ,Oncology ,medicine.medical_specialty ,Gene Transfer, Horizontal ,Long term follow up ,Genetic enhancement ,Graft vs Host Disease ,Retrovirus ,allogeneic stem cell transplantation ,hemic and lymphatic diseases ,Internal medicine ,graft vs. host disease ,Medicine ,Pharmacology (medical) ,gene transfer ,horizontal ,Original Research ,Pharmacology ,Haploidentical transplantation ,biology ,business.industry ,lcsh:RM1-950 ,biology.organism_classification ,gene therapy ,proteomics data ,Clinical trial ,Transplantation ,lcsh:Therapeutics. Pharmacology ,surgical procedures, operative ,Immunology ,Stem cell ,business ,medicine.drug - Abstract
Allogeneic stem cell transplantation (allo-HSCT) is one of the curative treatments for hematologic malignancies, but is hampered by severe complications, such as acute or chronic graft-versus-host-disease (aGvHD; cGvHD) and infections. CD34-selection of stem cells reduces the risk of aGvHD, but also leads to increased infectious complications and relapse. Thus, we studied the safety, efficacy, and feasibility of transfer of gene modified donor T-cells shortly after allo-HSCT in two clinical trials between 2002 and 2007 and here we compare the results to unmodified donor leukocyte infusion (DLI). The aim of these trials was to provide patients with the protection of T-cells after T-cell-depleted allo-HSCT in the matched or mismatched donor setting with an option to delete transduced T-cells, if severe aGvHD occurred within the trial period. Donor-T-cells were transduced with the replication-deficient retrovirus SFCMM-3, expressing HSV-TK and the truncated A LNGFR for selection of transduced cells. Transduced cells were transfused either after day +60 (matched donors) or on day +42 (haploidentical donors). Nine patients were included in the first trial (MHH; 2002 until 2007), two were included in TK007 (2005-2009) and six serves as a control group for outcome after haploidentical transplantation without HSV-TK-transduced DLI. Three patients developed acute GvHD, two had grade I of the skin, one had aGvHD on day +131 (post-HSCT; +89 post-HSV-TK DLI) grade II, which was successfully controlled by ganciclovir (GCV). Donor chimerism was stabilized after transfusion of the transduced cells in all patients treated. Functionality of HSV-TK gene expressing T-cells was shown by loss of bcr-able gene expression as well as by control of cytomegalovirus-reactivation. To date, six patients have relapsed and died, two after a second hematopoietic stem cell transplantation without T-cell depletion or administration of unmodified T-cells. Eleven patients (seven post-HSV-TK DLI) are alive and well to date.
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- 2015
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58. Preclinical Safety and Efficacy of Human CD34(+) Cells Transduced With Lentiviral Vector for the Treatment of Wiskott-Aldrich Syndrome
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Maria G. Roncarolo, Robbert G. M. Bredius, Maria Carmina Castiello, Marita Bosticardo, Anna Villa, Monica Salomoni, Marco Ranzani, Mariana Loperfido, Elisa Vicenzi, Anna Ripamonti, Raisa Jofra Hernandez, Luca Biasco, Christof von Kalle, Samantha Scaramuzza, Fabrizio Benedicenti, Alessandra Biffi, Manfred Schmidt, Alessandro Aiuti, Elena Draghici, Luigi Naldini, Eugenio Montini, Andrea Finocchi, Cynthia C. Bartholomae, Marina Radrizzani, Scaramuzza, S1, Biasco, L, Ripamonti, A, Castiello, Mc, Loperfido, M, Draghici, E, Hernandez, Rj, Benedicenti, F, Radrizzani, M, Salomoni, M, Ranzani, M, Bartholomae, Cc, Vicenzi, E, Finocchi, A, Bredius, R, Bosticardo, M, Schmidt, M, von Kalle, C, Montini, E, Biffi, A, Roncarolo, MARIA GRAZIA, Naldini, Luigi, Villa, A, and Aiuti, A.
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Wiskott–Aldrich syndrome ,Genetic enhancement ,Knockout ,Genetic Vectors ,CD34 ,Bone Marrow Cells ,Biology ,Viral vector ,03 medical and health sciences ,Mice ,Transduction ,0302 clinical medicine ,Genetic ,Drug Discovery ,medicine ,Genetics ,Animals ,Progenitor cell ,Antigens ,Molecular Biology ,030304 developmental biology ,Interleukin 3 ,Bone Marrow Transplantation ,Pharmacology ,0303 health sciences ,Antigens, CD34 ,Lentivirus ,Mice, Knockout ,Wiskott-Aldrich Syndrome ,Transduction, Genetic ,medicine.disease ,3. Good health ,Haematopoiesis ,medicine.anatomical_structure ,Settore MED/03 - Genetica Medica ,030220 oncology & carcinogenesis ,Immunology ,Molecular Medicine ,Original Article ,Bone marrow - Abstract
"Gene therapy with ex vivo-transduced hematopoietic stem/progenitor cells may represent a valid therapeutic option for monogenic immunohematological disorders such as Wiskott-Aldrich syndrome (WAS), a primary immunodeficiency associated with thrombocytopenia. We evaluated the preclinical safety and efficacy of human CD34+ cells transduced with lentiviral vectors (LV) encoding WAS protein (WASp). We first set up and validated a transduction protocol for CD34+ cells derived from bone marrow (BM) or mobilized peripheral blood (MPB) using a clinical grade, highly purified LV. Robust transduction of progenitor cells was obtained in normal donors and WAS patients' cells, without evidence of toxicity. To study biodistribution of human cells and exclude vector release in vivo, LV-transduced CD34+ cells were transplanted in immunodeficient mice, showing a normal engraftment and differentiation ability towards transduced lymphoid and myeloid cells in hematopoietic tissues. Vector mobilization to host cells and transmission to germline cells of the LV were excluded by different molecular assays. Analysis of vector integrations showed polyclonal integration patterns in vitro and in human engrafted cells in vivo. In summary, this work establishes the preclinical safety and efficacy of human CD34+ cells gene therapy for the treatment of WAS."
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- 2013
59. Safety of retroviral gene marking with a truncated NGF receptor
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Hans-Jochem Kolb, Fabio Ciceri, Claudio Bordignon, Phil Greenberg, J H F Falkenburg, Mohammed A. Sadat, Marco Bregni, Eva M. Weissinger, M. Lamana, Manuel Grez, Timothy W. Austin, Alexis Grande, Mirjam H.M. Heemskerk, Zulma Magnani, Sabrina Cazzaniga, Alessandro Aiuti, Anton Hagenbeek, Saskia B. Ebeling, Steven M. Kornblau, Claudia Benati, Fulvio Mavilio, Sara Deola, Francesco Lotti, Catia Traversari, Mary C. Dinauer, Anton C.M. Martens, Giuliana Ferrari, Shimon Slavin, M. Weber, J. F. Apperley, Frank C. Marini, Chiara Bonini, Sarah Marktel, Corrado Gallo-Stampino, Salvatore Toma, Marina Radrizzani, Juan A. Bueren, Martino Introna, University of Groningen, Bonini, MARIA CHIARA, Grez, M, Traversari, C, Ciceri, Fabio, Marktel, S, Ferrari, Giuliana, Dinauer, M, Sadat, M, Aiuti, Alessandro, Deola, S, Radrizzani, M, Hagenbeek, A, Apperley, J, Ebeling, S, Martens, A, Kolb, Hj, Weber, M, Lotti, F, Grande, A, Weissinger, E, Bueren, Ja, Lamana, M, Falkenburg, Jh, Heemskerk, Mh, Austin, T, Kornblau, S, Marini, F, Benati, C, Magnani, Z, Cazzaniga, S, Toma, S, GALLO STAMPINO, C, Introna, M, Slavin, S, Greenberg, Pd, Bregni, M, Mavilio, F, Bordignon, Claudio, and Clinical Haematology
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NGF Receptor ,gene therapy ,viral vectors ,NGF receptor ,Bone marrow transplantation ,Genetic enhancement ,General Medicine ,Biology ,Molecular biology ,General Biochemistry, Genetics and Molecular Biology ,Cell biology ,Viral vector ,Transduction (genetics) ,Multicenter study ,Receptor ,Gene - Published
- 2003
60. Genetically modified donor leukocyte transfusion and graft-versus-leukemia effect after allogeneic stem cell transplantation
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Sylvia Borchers, Julia Kontsendorn, Bernd Hertenstein, Michael Stadler, Elena Provasi, Wolfgang Kuehnau, Chiara Bonini, Nils von Neuhoff, Joerg Schmidtke, Eva M. Weissinger, Annika Krons, Arnold Ganser, Marina Radrizzani, Fabio Ciceri, Anna Silvani, Claudia Benati, Elke Dammann, Borchers, S, Provasi, E, Silvani, A, Radrizzani, M, Benati, C, Dammann, E, Krons, A, Kontsendorn, J, Schmidtke, J, Kuehnau, W, von Neuhoff, N, Stadler, M, Ciceri, Fabio, Bonini, C, Ganser, A, Hertenstein, B, and Weissinger, Em
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Adult ,Male ,Proteomics ,Graft-vs-Leukemia Effect ,medicine.medical_treatment ,Receptors, Antigen, T-Cell, alpha-beta ,Genetic Vectors ,CD34 ,Fusion Proteins, bcr-abl ,Graft vs Host Disease ,Graft vs Leukemia Effect ,Hematopoietic stem cell transplantation ,Biology ,Chimerism ,Thymidine Kinase ,Transduction, Genetic ,hemic and lymphatic diseases ,Leukemia, Myelogenous, Chronic, BCR-ABL Positive ,Genetics ,medicine ,Humans ,Transgenes ,Molecular Biology ,Research Articles ,Immunosuppression Therapy ,Remission Induction ,Hematopoietic Stem Cell Transplantation ,Myeloid leukemia ,Immunosuppression ,Middle Aged ,medicine.disease ,Tissue Donors ,Transplantation ,Leukemia, Myeloid, Acute ,Leukocyte Transfusion ,surgical procedures, operative ,Retroviridae ,Immunology ,Molecular Medicine ,Female ,Stem cell ,Chronic myelogenous leukemia - Abstract
Seven patients with acute myeloid leukemia (AML) and two patients with chronic myelogenous leukemia (CML) were transplanted from HLA-identical sibling donors with CD34(+) cell-enriched stem cells (HSCTs) without further immunosuppression. The myeloablative standard transplantation protocol was adapted to include transfusion of gene-modified donor T cells after HSCT. Donor T cells were transduced with the replication-deficient retrovirus SFCMM-3, which expresses herpes simplex thymidine kinase (HSV-Tk) and a truncated version of low-affinity nerve growth factor receptor (Delta LNGFR) for selection and characterization of transduced cells. Transduced T cells were detectable in all patients during follow-up for up to 5 years after transfusion. Proteomic screening for development of acute graft-versus-host disease (aGvHD) was applied to five of the seven patients with AML. No positivity for the aGvHD grade II-specific proteomic pattern was observed. Only one patient developed aGvHD grade I. To date, three of the patients with AML relapsed; one responded to three escalating transfusions of lymphocytes from the original donor and is in complete remission. Two were retransplanted with non-T cell-depleted peripheral blood stem cells from their original donors and died after retransplantation of septic complications or relapse, respectively. In one patient with CML, loss of bcr-abl gene expression was observed after an expansion of transduced cells. Seven of nine patients are alive and in complete remission.
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- 2010
61. Large-scale manufacture and characterization of a lentiviral vector produced for clinical ex vivo gene therapy application
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Anne Galy, Maria Antonietta Zanta-Boussif, Muriel Audit, Otto Wilhelm Merten, Marina Radrizzani, Christine Jenny, Sylvain Fauchille, Patricia Noguiez-Hellin, Céline Dugué, Luigi Naldini, Nicolas Laroudie, Hélène Chautard, Giuliana Vallanti, Sabine Charrier, Merten, Ow, Charrier, S, Laroudie, N, Fauchille, S, Dugue, C, Jenny, C, Audit, M, Zanta Boussif, Ma, Chautard, H, Radrizzani, M, Vallanti, G, Naldini, Luigi, Noguiez Hellin, P, Galy, A., Immunologie moléculaire et biothérapies innovantes (IMBI), Généthon-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université d'Évry-Val-d'Essonne (UEVE)-École pratique des hautes études (EPHE), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL), Laboratoire Environnements Sédimentaires - Géosciences Marines (GM/LES), Institut Français de Recherche pour l'Exploitation de la Mer (IFREMER), Centre de biophysique moléculaire (CBM), Université d'Orléans (UO)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie du CNRS (INC), École Pratique des Hautes Études (EPHE), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Université d'Évry-Val-d'Essonne (UEVE)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Généthon, Laboratoire Environnements Sédimentaires (LES), Géosciences Marines (GM), Institut Français de Recherche pour l'Exploitation de la Mer (IFREMER)-Institut Français de Recherche pour l'Exploitation de la Mer (IFREMER), and Université d'Orléans (UO)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)
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Lentivirus/*genetics/physiology ,Drug Contamination/legislation & ,Wiskott-Aldrich Syndrome/therapy ,[SDV]Life Sciences [q-bio] ,Genetic enhancement ,Cell Culture Techniques ,Industrial Microbiology/*methods ,Transduction (genetics) ,0302 clinical medicine ,Proviruses ,Transduction, Genetic ,Gene Order ,Hematopoietic Stem Cells/metabolism ,Genetic Vectors/*biosynthesis/*genetics/physiology ,Transgenes ,Plasmids/genetics ,0303 health sciences ,biology ,Wiskott-Aldrich Syndrome ,Titer ,Vesicular stomatitis virus ,030220 oncology & carcinogenesis ,Molecular Medicine ,Drug Contamination ,Plasmids ,Quality Control ,Transgenes/genetics ,Genetic Vectors ,Virus ,Viral vector ,Cell Line ,Transduction ,03 medical and health sciences ,Industrial Microbiology ,Genetic ,jurisprudence/prevention & ,Genetics ,Humans ,Molecular Biology ,Proviruses/genetics ,030304 developmental biology ,Genetic Therapy ,Lentivirus ,biology.organism_classification ,Hematopoietic Stem Cells ,Virology ,HEK293 Cells ,Gene Expression Regulation ,Cell culture ,control ,Ex vivo - Abstract
International audience; From the perspective of a pilot clinical gene therapy trial for Wiskott-Aldrich syndrome (WAS), we implemented a process to produce a lentiviral vector under good manufacturing practices (GMP). The process is based on the transient transfection of 293T cells in Cell Factory stacks, scaled up to harvest 50 liters of viral stock per batch, followed by purification of the vesicular stomatitis virus glycoprotein-pseudotyped particles through several membrane-based and chromatographic steps. The process leads to a 200-fold volume concentration and an approximately 3-log reduction in protein and DNA contaminants. An average yield of 13% of infectious particles was obtained in six full-scale preparations. The final product contained low levels of contaminants such as simian virus 40 large T antigen or E1A sequences originating from producer cells. Titers as high as 2 x 10(9) infectious particles per milliliter were obtained, generating up to 6 x 10(11) infectious particles per batch. The purified WAS vector was biologically active, efficiently expressing the genetic insert in WAS protein-deficient B cell lines and transducing CD34(+) cells. The vector introduced 0.3-1 vector copy per cell on average in CD34(+) cells when used at the concentration of 10(8) infectious particles per milliliter, which is comparable to preclinical preparations. There was no evidence of cellular toxicity. These results show the implementation of large-scale GMP production, purification, and control of advanced HIV-1-derived lentiviral technology. Results obtained with the WAS vector provide the initial manufacturing and quality control benchmarking that should be helpful to further development and clinical applications.
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- 2010
62. IL-7 AND IL-15 ALLOW THE GENERATION OF SUICIDE GENE-MODIFIED ALLOREACTIVE SELF-RENEWING CENTRAL MEMORY HUMAN T LYMPHOCYTES
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Catia Traversari, Simona La Seta-Catamancio, Elena Provasi, Vincenzo Russo, Marina Radrizzani, Sara Mastaglio, Claudio Bordignon, Shin Kaneko, Maurilio Ponzoni, Francesca Sanvito, Attilio Bondanza, Luca Aldrighetti, Toshiro Nagasawa, Chiara Bonini, Fabio Ciceri, Anna Mondino, Katharina Fleischhauer, Kaneko, S, Mastaglio, S, Bondanza, Attilio, Ponzoni, Maurilio, Sanvito, F, Aldrighetti, L, Radrizzani, M, La Seta Catamancio, S, Provasi, E, Mondino, A, Nagasawa, T, Fleischhauer, K, Russo, V, Traversari, C, Ciceri, Fabio, Bordignon, Claudio, and Bonini, MARIA CHIARA
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Isoantigens ,T-Lymphocytes ,medicine.medical_treatment ,Genetic enhancement ,Immunology ,Graft vs Host Disease ,Mice, SCID ,Biology ,Lymphocyte Activation ,Biochemistry ,Viral vector ,Mice ,Mice, Inbred NOD ,Neoplasms ,medicine ,Animals ,Humans ,Cells, Cultured ,Interleukin-15 ,Cell Death ,Interleukin-7 ,Genes, Transgenic, Suicide ,CD28 ,Cell Differentiation ,Cell Biology ,Hematology ,Suicide gene ,medicine.disease ,Graft-versus-host disease ,Cytokine ,Interleukin 15 ,Humanized mouse ,Interleukin-2 ,Immunologic Memory ,Stem Cell Transplantation - Abstract
Long-term clinical remissions of leukemia, after allogeneic hematopoietic stem cell transplantation, depend on alloreactive memory T cells able to self-renew and differentiate into antileukemia effectors. This is counterbalanced by detrimental graft-versus-host disease (GVHD). Induction of a selective suicide in donor T cells is a current gene therapy approach to abrogate GVHD. Unfortunately, genetic modification reduces alloreactivity of lymphocytes. This associates with an effector memory (TEM) phenotype of gene-modified lymphocytes and may limit antileukemia effect. We hypothesized that alloreactivity of gene-modified lymphocytes segregates with the central memory (TCM) phenotype. To this, we generated suicide gene–modified TCM lymphocytes with a retroviral vector after CD28 costimulation and culture with IL-2, IL-7, or a combination of IL-7 and IL-15. In vitro, suicide gene–modified TCM cells self-renewed upon alloantigen stimulation and resisted activation-induced cell death. In a humanized mouse model, only suicide gene–modified T cells cultured with IL-7 and IL-15 persisted, differentiated in TEM cells, and were as potent as unmanipulated lymphocytes in causing GVHD. GVHD was halted through the activation of the suicide gene machinery. These results warrant the use of suicide gene–modified TCM cells cultured with IL-7 and IL-15 for the safe exploitation of the alloreactive response against cancer.
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- 2010
63. Quantitative proteomic analysis of lentiviral vectors using 2-DE
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Florence Gonnet, Olivier Danos, Otto Wilhelm Merten, Marina Radrizzani, Luigi Naldini, Anne Galy, Jérôme Denard, Nicolas Laroudie, Fedor Svinartchouk, Stéphanie Rundwasser, Denard, J, Rundwasser, S, Laroudie, N, Gonnet, F, Naldini, Luigi, Radrizzani, M, Galy, A, Merten, Ow, Danos, O, and Svinartchouk, F.
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Proteomics ,Genetic enhancement ,Genetic Vectors ,Lentivirus ,Proteins ,Biology ,Vectors in gene therapy ,biology.organism_classification ,Biochemistry ,Virology ,Virus ,Cell Line ,Viral vector ,law.invention ,Viral Proteins ,law ,Vesicular stomatitis virus ,Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization ,Recombinant DNA ,Humans ,Electrophoresis, Gel, Two-Dimensional ,Vector (molecular biology) ,Molecular Biology - Abstract
Among the integrative gene therapy vectors developed to date, human immunodeficiency virus type 1 (HIV-1)-derived lentiviral vectors (LV) are distinguished by their capacity to infect both dividing and non-dividing cells. Recombinant LV particles contain viral proteins necessary for their packaging, infectious and integrating functions. Like the parental HIV-1 virus they are able to acquire various cellular proteins, but the number and localisation of these proteins are poorly characterised. In the present study we used 2-DE followed by MALDI-TOF to quantify the protein content of several types of vesicular stomatitis virus G-pseudotyped LV including those that were extensively purified in the perspective of clinical gene therapy studies. A proteinase K treatment was used to distinguish between cellular proteins incorporated into virions (I-proteins) and those co-purified with vectors (C-proteins). We found 10 C-proteins and 18 I-proteins associated with LV. Copy numbers for these core I-proteins varied from 5 (AIP-1/ALIX) to 280 (Cyclophilin A) per vector particle. Three novel I-proteins, guanine nucleotide-binding protein 2, L-lactate dehydrogenase B chain and hnRNP core protein A1, were found. This study defines for the first time, the protein stoichiometry of infectious HIV-1-derived LV particles.
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- 2009
64. Suicide gene therapy of graft-versus-host disease induced by central memory human T lymphocytes
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Catia Traversari, Claudio Bordignon, Zulma Magnani, Chiara Bonini, Marina Radrizzani, Salvatore Toma, Fabio Ciceri, Attilio Bondanza, Francesca Sanvito, Katharina Fleischhauer, Veronica Valtolina, Simona La Seta-Catamancio, Maurilio Ponzoni, Mark Bonyhadi, Bondanza, Attilio, Valtolina, V, Magnani, Z, Ponzoni, Maurilio, Fleischhauer, K, Bonyhadi, M, Traversari, C, Sanvito, F, Toma, S, Radrizzani, M, La Seta Catamancio, S, Ciceri, Fabio, Bordignon, Claudio, and Bonini, MARIA CHIARA
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T-Lymphocytes ,Immunology ,Receptors, Antigen, T-Cell ,Graft vs Host Disease ,Graft vs Leukemia Effect ,Mice, SCID ,Biology ,Biochemistry ,Antiviral Agents ,Thymidine Kinase ,Mice ,Viral Proteins ,Antigen ,CD28 Antigens ,Aldesleukin ,Mice, Inbred NOD ,medicine ,Animals ,Humans ,Simplexvirus ,Transplantation, Homologous ,Ganciclovir ,Genes, Transgenic, Suicide ,Hematopoietic Stem Cell Transplantation ,CD28 ,Cell Biology ,Hematology ,T lymphocyte ,Genetic Therapy ,Suicide gene ,medicine.disease ,Transplantation ,surgical procedures, operative ,Graft-versus-host disease ,Retroviridae ,Perforin ,biology.protein ,Female ,Immunologic Memory - Abstract
In allogeneic hematopoietic cell transplantation (allo-HCT), the immune recognition of host antigens by donor T lymphocytes leads to a beneficial graft-versus-leukemia (GvL) effect as well as to life-threatening graft-versus-host disease (GvHD). Genetic modification of T lymphocytes with a retroviral vector (RV) expressing the herpes simplex virus-thymidine kinase (TK) suicide gene confers selective sensitivity to the prodrug ganciclovir (GCV). In patients, the infusion of TK+ lymphocytes and the subsequent administration of GCV resulted in a time-wise modulation of antihost reactivity for a GvL effect, while controlling GvHD. Because activation required for genetic modification with RV may reduce antihost reactivity, we investigated the requirements for maximizing the potency of human TK+ lymphocytes. Whereas T-cell receptor triggering alone led to effector memory (EM) TK+ lymphocytes, the addition of CD28 costimulation through cell-sized beads resulted in the generation of central memory (CM) TK+ lymphocytes. In a quantitative model for GvHD using nonobese diabetic/severely combined immunodeficient mice, CM TK+ lymphocytes were more potent than EM TK+ lymphocytes. GCV administration efficiently controlled GvHD induced by CM TK+ lymphocytes. These results warrant the clinical investigation of CM suicide gene-modified human T lymphocytes for safe and effective allo-HCT. In allogeneic hematopoietic cell transplantation (allo-HCT), the immune recognition of host antigens by donor T lymphocytes leads to a beneficial graft-versus-leukemia (GvL) effect as well as to life-threatening graft-versus-host disease (GvHD). Genetic modification of T lymphocytes with a retroviral vector (RV) expressing the herpes simplex virus-thymidine kinase (TK) suicide gene confers selective sensitivity to the prodrug ganciclovir (GCV). In patients, the infusion of TK+ lymphocytes and the subsequent administration of GCV resulted in a time-wise modulation of antihost reactivity for a GvL effect, while controlling GvHD. Because activation required for genetic modification with RV may reduce antihost reactivity, we investigated the requirements for maximizing the potency of human TK+ lymphocytes. Whereas T-cell receptor triggering alone led to effector memory (EM) TK+ lymphocytes, the addition of CD28 costimulation through cell-sized beads resulted in the generation of central memory (CM) TK+ lymphocytes. In a quantitative model for GvHD using nonobese diabetic/severely combined immunodeficient mice, CM TK+ lymphocytes were more potent than EM TK+ lymphocytes. GCV administration efficiently controlled GvHD induced by CM TK+ lymphocytes. These results warrant the clinical investigation of CM suicide gene-modified human T lymphocytes for safe and effective allo-HCT.
- Published
- 2005
65. Modulation of graft-versus-host disease induced by central memory suicide gene modified human T lymphocytes
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Traversari Catia, Magnani Zulma, La Seta Catamancio Simona, Bondanza Attilio, Bonini Maria Chiara, Toma Salvatore, Valtolina Veronica, Bonyhadi Mark, Bordignon Claudio, Radrizzani Marina, Benati Claudia, Ciceri Fabio, Bondanza, Attilio, Valtolina, V, Magnani, Z, Catamancio, Sl, Benati, C, Toma, S, Traversari, C, Radrizzani, M, Bonyhadi, M, Ciceri, Fabio, Bordignon, Claudio, and Bonini, MARIA CHIARA
- Subjects
biology ,CD3 ,T cell ,education ,Immunology ,CD28 ,chemical and pharmacologic phenomena ,Cell Biology ,Hematology ,Suicide gene ,Biochemistry ,Molecular biology ,medicine.anatomical_structure ,Perforin ,Aldesleukin ,Lymphocyte costimulation ,biology.protein ,medicine ,CD8 - Abstract
Background. Suicide gene therapy is a promising approach for the safe exploitation of the graft-versus-leukemia effect. The insertion of Herpes Simplex Virus thymidine kinase confers an inducible suicidal phenotype upon ganciclovir (GCV) administration, thus enabling the selective elimination of T lymphocytes causing graft-versus-host disease (GvHD). Despite clinical and experimental studies substantiating the efficacy of the strategy, protocols to generate genetically modified cells (GMC) has been shown to reduce alloreactivity. The physiological CD4/CD8 ratio is inverted and GMC are enriched for “effector memory” T cells. Co-stimulation through CD28 has been shown to preserve the functional phenotype GMC. XcyteTM Dynabeads®, 4,5 μm anti-CD3 and anti-CD28 coated paramagnetic beads (bCD3/CD28) sustain T cell proliferation and can be used to obtain GMC. Aim. To in vitro characterize human suicide GMC generated with bCD3/CD28 GMC (XcyteTM Dynabeads®, Xcyte Therapies, Inc.) and to test their ability to engraft and cause GvHD in a xenogeneic mouse model. Results. bCD3/CD28 (bead to T cell ratio 3:1) are a potent stimulus for cell cycle entry for both CD4+ and CD8+ human T cells. This permits retroviral transduction (SFCMM#3 vector, Molmed SpA) and preservation of CD4/CD8 ratio. GMC generated with bCD3/CD28 are enriched for “central memory” T cells (CD45RA+CCR7+ 34±7%, CD28+CD27+ 67±12%, intracytoplasmic IL-2+ 14±5%, IFN-γ+ 10±3% and perforin+ 7±3%) when compared with GMC generated with anti-CD3 (CD3) alone (CD45RA+CCR7+ 17±4%, CD28+CD27+ 21±5%, intracytoplasmic IL-2+ 5±3%, IFN-γ+ 52±11% and perforin+ 22±4%). bCD3/CD28 GMC resist activation induced cell death (AxV+PI+ 12±3% vs 42±13% for CD3 GMC). When injected i.p. in NOD/SCID mice conditioned with irradiation and anti-NK depleting antibodies bCD3/CD28 GMC engraft with a faster kinetics (human chimerism at 2 weeks 14±7%) than observed for for CD3 GMC (5±2%). In this model, mice injected with unmodified human lymphocytes develop signs of xenogeneic (X-) GvHD (ruffled fur, hunched back, weight loss and finally death with massive accumulation of human T cell in lymphoid organs) by week 5. X-GvHD observed in mice injected with CD3 GMC has a significant slower course with a proportion of mice surviving week 8. X-GvHD caused by bCD3/CD28 GMC kill all the animals by week 7 (p Conclusions. GMC generated with bCD3/CD28 display a “central memory” functional phenotype and are significantly more efficient than CD3 GMC in causing lethal X-GvHD. GCV administration is able to abrogate X-GvHD caused by GMC. These results validate a tool for the generation of human suicide GMC with high alloreactive potential to be utilized in clinical protocols of adoptive immunotherapy of tumors.
- Published
- 2004
66. Requirements for retroviral targeting of a suicide gene to alloreactive memory stem T cells for adoptive immunotherapy of leukemia
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Luca Aldrighetti, Bart A. Nijmeijer, Marina Radrizzani, Maurilio Ponzoni, Lothar Hambach, Shin Kaneko, Claudio Bordignon, Chiara Bonini, Salvatore Toma, Sara Mastaglio, Els Goulmy, Attilio Bondanza, Astrid G. S. van Halteren, Fabio Ciceri, Bondanza, Attilio, Kaneko, S, Hambach, L, Mastaglio, S, Nijmeijer, B, Van Halteren, A, Ponzoni, Maurilio, Aldrighetti, L, Toma, S, Radrizzani, M, Ciceri, Fabio, Bordignon, Claudio, Goulmy, E, and Bonini, MARIA CHIARA
- Subjects
business.industry ,ZAP70 ,Immunology ,Adoptive immunotherapy ,CD28 ,chemical and pharmacologic phenomena ,Cell Biology ,Hematology ,Biology ,Suicide gene ,Natural killer T cell ,medicine.disease ,Biochemistry ,Interleukin 21 ,Leukemia ,Cytotoxic T cell ,Molecular Medicine ,Medicine ,IL-2 receptor ,Stem cell ,business ,Molecular Biology ,Interleukin 3 - Abstract
In a phase I/II clinical trial investigating the prophylactic infusion of suicide gene-modified donor T cells in the context of haploidentical hemopoietic cell transplantation (haplo-HCT) for the treatment of high-risk leukemia, we observed a rapid and effective immune reconstitution. After activation with anti-CD3 antibodies, genetic modification of donor T cells was accomplished with a retroviral vector encoding for the Herpes Simplex thymidine kinase (TK). In vitro before infusion and in vivo at immune reconstitution, TK+ cells displayed an effector memory (EM) phenotype (CD45RA–CD62L−, CD28±CD27+, IL-2±IFN–γ+). The graft-versus-leukemia (GvL) effect was substantial in patients transplanted in remission, but failed to cure patients in relapse. Gene targeting with retroviral vectors is limited to memory T cells. Central memory (CM) T cells (CD45RA–CD62L+, CD28+CD27+, IL-2+IFN-γ±) share many characteristics with stem cells, namely the ability to self-renew and to differentiate into a progeny of effector cells. EM TK+ cells have a reduced alloreactivity. Recently, it has been proposed that alloreactivity may be confined to memory T cells with stem cell-features. Since alloantigens are the target not only of graft-versus-host disease (GvHD), but also of the GvL effect, crucial to the success of the strategy is the suicide gene-modification of this subset of memory T cells. We found that addition of CD28 costimulation on cell-sized beads and the use of homeostatic cytokines, such as IL-7 and IL-15, generates central memory (CM) TK+ cells. CM TK+ cells are highly alloreactive, both in vitro and in vivo in a humanized animal model of GvHD based on the grafting of human skin onto NOD/scid mice. Interestingly, CM TK+ cells express the IL7Rα, a marker associated with the stem cell-features of memory T cells. Moreover, IL7Rα expression is maintained after stimulation with alloantigens. Stimulation of CM, but not of EM TK+ cells with autologous dendritic cells pulsed with restricted peptides from the minor histocompatibility alloantigen (mHag) HA-1 or H-Y efficiently induces mHag-specific effector T cells that lyse natural ligand expressing HLA-A2+ targets. TK+ mHag-specific effector T cells also lysed mHag+HLA-A2+ leukemic cells and, when infused in conditioned NOD/scid mice harboring human leukemia, significantly delayed disease progression. Altogether, these data suggest that optimal T-cell receptor triggering and homeostatic cytokines are required for retroviral targeting of a suicide gene to alloreactive memory stem T cells and warrant their use for a safe and powerful GvL effect.
- Published
- 2008
67. Cytotoxic T-lymphocyte clones from different patients display limited T-cell-receptor variable-region gene usage in HLA-A2-restricted recognition of the melanoma antigen Melan-A/MART-1
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Thierry Boon, Cinthia Farina, Catia Traversari, Thomas Wölfel, Marialuisa Sensi, Stefania Salvi, Licia Rivoltini, Roberta Mortarini, Vincent Brichard, Marina Radrizzani, Andrea Anichini, Claudio Bordignon, Giorgio Parmiani, Cristina Maccalli, Gabriella Nicolini, Sensi, M. L., Traversari, C., Radrizzani, M., Salvi, S., Maccalli, C., Mortarini, R., Rivoltini, L., Farina, C., Nicolini, G., Wolfel, T., Brichard, V., Boon, T., Bordignon, Claudio, Anichini, A., and G., Parmiani
- Subjects
Multidisciplinary ,DNA, Complementary ,Base Sequence ,Receptors, Antigen, T-Cell, alpha-beta ,T-cell receptor ,Molecular Sequence Data ,Clone (cell biology) ,Human leukocyte antigen ,Biology ,Molecular biology ,Tumor antigen ,Cell Line ,Clone Cells ,CTL ,Antigen ,Antigens, Neoplasm ,HLA-A2 Antigen ,Tumor Cells, Cultured ,Cytotoxic T cell ,Humans ,Amino Acid Sequence ,Melanoma ,Research Article ,T-Lymphocytes, Cytotoxic - Abstract
To determine whether T-cell-receptor (TCR) usage by T cells recognizing a defined human tumor antigen in the context of the same HLA molecule is conserved, we analyzed the TCR diversity of autologous HLA-A2-restricted cytotoxic T-lymphocyte (CTL) clones derived from five patients with metastatic melanoma and specific for the common melanoma antigen Melan-A/MART-1. These clones were first identified among HLA-A2-restricted anti-melanoma CTL clones by their ability to specifically release tumor necrosis factor in response to HLA-A2.1+ COS-7 cells expressing this tumor antigen. A PCR with variable (V)-region gene subfamily-specific primers was performed on cDNA from each clone followed by DNA sequencing. TCRAV2S1 was the predominant alpha-chain V region, being transcribed in 6 out of 9 Melan-A/MART-1-specific CTL clones obtained from the five patients. beta-chain V-region usage was also restricted, with either TCRBV14 or TCRBV7 expressed by all but one clone. In addition, a conserved TCRAV2S1/TCRBV14 combination was expressed in four CTL clones from three patients. None of these V-region genes was found in a group of four HLA-A2-restricted CTL clones recognizing different antigens (e.g., tyrosinase) on the autologous tumor. TCR joining regions were heterogeneous, although conserved structural features were observed in the complementarity-determining region 3 sequences. These results indicate that a selective repertoire of TCR genes is used in anti-melanoma responses when the response is narrowed to major histocompatibility complex-restricted antigen-specific interactions.
68. Intracoronary delivery of extracellular vesicles from human cardiac progenitor cells reduces infarct size in porcine acute myocardial infarction.
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Emmert MY, Burrello J, Wolint P, Hilbe M, Andriolo G, Balbi C, Provasi E, Turchetto L, Radrizzani M, Nazari-Shafti TZ, Cesarovic N, Neuber S, Falk V, Hoerstrup SP, Hemetsberger R, Gyöngyösi M, Barile L, and Vassalli G
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- Humans, Swine, Animals, Coronary Vessels, Stem Cells, Myocardial Infarction therapy, Extracellular Vesicles
- Published
- 2024
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69. Unbiased assessment of genome integrity and purging of adverse outcomes at the target locus upon editing of CD4 + T-cells for the treatment of Hyper IgM1.
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Canarutto D, Asperti C, Vavassori V, Porcellini S, Rovelli E, Paulis M, Ferrari S, Varesi A, Fiumara M, Jacob A, Sergi Sergi L, Visigalli I, Ferrua F, González-Granado LI, Lougaris V, Finocchi A, Villa A, Radrizzani M, and Naldini L
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- Humans, Genome, T-Lymphocytes, CD4-Positive T-Lymphocytes, CRISPR-Cas Systems, Gene Editing methods
- Abstract
Hyper IgM1 is an X-linked combined immunodeficiency caused by CD40LG mutations, potentially treatable with CD4
+ T-cell gene editing with Cas9 and a "one-size-fits-most" corrective template. Contrary to established gene therapies, there is limited data on the genomic alterations following long-range gene editing, and no consensus on the relevant assays. We developed drop-off digital PCR assays for unbiased detection of large on-target deletions and found them at high frequency upon editing. Large deletions were also common upon editing different loci and cell types and using alternative Cas9 and template delivery methods. In CD40LG edited T cells, on-target deletions were counter-selected in culture and further purged by enrichment for edited cells using a selector coupled to gene correction. We then validated the sensitivity of optical genome mapping for unbiased detection of genome wide rearrangements and uncovered on-target trapping of one or more vector copies, which do not compromise functionality, upon editing using an integrase defective lentiviral donor template. No other recurring events were detected. Edited patient cells showed faithful reconstitution of CD40LG regulated expression and function with a satisfactory safety profile. Large deletions and donor template integrations should be anticipated and accounted for when designing and testing similar gene editing strategies., (© 2023 The Authors. Published under the terms of the CC BY NC ND 4.0 license.)- Published
- 2023
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70. Lipid nanoparticles allow efficient and harmless ex vivo gene editing of human hematopoietic cells.
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Vavassori V, Ferrari S, Beretta S, Asperti C, Albano L, Annoni A, Gaddoni C, Varesi A, Soldi M, Cuomo A, Bonaldi T, Radrizzani M, Merelli I, and Naldini L
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- Humans, Hematopoietic Stem Cells metabolism, RNA metabolism, CRISPR-Cas Systems, Gene Editing methods, Tumor Suppressor Protein p53 genetics, Tumor Suppressor Protein p53 metabolism
- Abstract
Ex vivo gene editing in T cells and hematopoietic stem/progenitor cells (HSPCs) holds promise for treating diseases. Gene editing encompasses the delivery of a programmable editor RNA or ribonucleoprotein, often achieved ex vivo via electroporation, and when aiming for homology-driven correction of a DNA template, often provided by viral vectors together with a nuclease editor. Although HSPCs activate a robust p53-dependent DNA damage response upon nuclease-based editing, the responses triggered in T cells remain poorly characterized. Here, we performed comprehensive multiomics analyses and found that electroporation is the main culprit of cytotoxicity in T cells, causing death and cell cycle delay, perturbing metabolism, and inducing an inflammatory response. Nuclease RNA delivery using lipid nanoparticles (LNPs) nearly abolished cell death and ameliorated cell growth, improving tolerance to the procedure and yielding a higher number of edited cells compared with using electroporation. Transient transcriptomic changes upon LNP treatment were mostly caused by cellular loading with exogenous cholesterol, whose potentially detrimental impact could be overcome by limiting exposure. Notably, LNP-based HSPC editing dampened p53 pathway induction and supported higher clonogenic activity and similar or higher reconstitution by long-term repopulating HSPCs compared with electroporation, reaching comparable editing efficiencies. Overall, LNPs may allow efficient and harmless ex vivo gene editing in hematopoietic cells for the treatment of human diseases., (© 2023 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)
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- 2023
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71. Scalable GMP-compliant gene correction of CD4+ T cells with IDLV template functionally validated in vitro and in vivo .
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Asperti C, Canarutto D, Porcellini S, Sanvito F, Cecere F, Vavassori V, Ferrari S, Rovelli E, Albano L, Jacob A, Sergi Sergi L, Montaldo E, Ferrua F, González-Granado LI, Lougaris V, Badolato R, Finocchi A, Villa A, Radrizzani M, and Naldini L
- Abstract
Hyper-IgM1 is a rare X-linked combined immunodeficiency caused by mutations in the CD40 ligand ( CD40LG ) gene with a median survival of 25 years, potentially treatable with in situ CD4+ T cell gene editing with Cas9 and a one-size-fits-most corrective donor template. Here, starting from our research-grade editing protocol, we pursued the development of a good manufacturing practice (GMP)-compliant, scalable process that allows for correction, selection and expansion of edited cells, using an integrase defective lentiviral vector as donor template. After systematic optimization of reagents and conditions we proved maintenance of stem and central memory phenotypes and expression and function of CD40LG in edited healthy donor and patient cells recapitulating the physiological CD40LG regulation. We then documented the preserved fitness of edited cells by xenotransplantation into immunodeficient mice. Finally, we transitioned to large-scale manufacturing, and developed a panel of quality control assays. Overall, our GMP-compliant process takes long-range gene editing one step closer to clinical application with a reassuring safety profile., Competing Interests: L.N., C.A., M.R., V.V., A.V., S.F., S.P., D.C., and A.J. are inventors of patent applications owned by Ospedale San Raffaele S.r.l. and Fondazione Telethon ETS, including one patent application on CD40LG gene editing. L.N. is founder, quota holder, and consultant of GeneSpire S.r.l., (© 2023 The Authors.)
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- 2023
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72. Two-Step Preparation of Protein-Decorated Biohybrid Quantum Dot Nanoparticles for Cellular Uptake.
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Traverso AN, Fragale DJ, Viale DL, Garate O, Torres P, Valverde G, Berra A, Torbidoni AV, Yakisich JS, Grasselli M, and Radrizzani M
- Abstract
Decoration of nanoparticles with specific molecules such as antibodies, peptides, and proteins that preserve their biological properties is essential for the recognition and internalization of their specific target cells. Inefficient preparation of such decorated nanoparticles leads to nonspecific interactions diverting them from their desired target. We report a simple two-step procedure for the preparation of biohybrid nanoparticles containing a core of hydrophobic quantum dots coated with a multilayer of human serum albumin. These nanoparticles were prepared by ultra-sonication, crosslinked using glutaraldehyde, and decorated with proteins such as human serum albumin or human transferrin in their native conformations. These nanoparticles were homogeneous in size (20-30 nm), retained the fluorescent properties of quantum dots, and did not show a "corona effect" in the presence of serum. The uptake of transferrin-decorated quantum dot nanoparticles was observed in A549 lung cancer and SH-SY5Y neuroblastoma cells but not in non-cancerous 16HB14o- or retinoic acid dopaminergic neurons differentiated SH-SY5Y cells. Furthermore, digitoxin-loaded transferrin-decorated nanoparticles decreased the number of A549 cells without effect on 16HB14o-. Finally, we analyzed the in vivo uptake of these biohybrids by murine retinal cells, demonstrating their capacity to selectively target and deliver into specific cell types with excellent traceability.
- Published
- 2023
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73. Methodologies for Scalable Production of High-Quality Purified Small Extracellular Vesicles from Conditioned Medium.
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Andriolo G, Provasi E, Brambilla A, Panella S, Soncin S, Cicero VL, Radrizzani M, Turchetto L, and Barile L
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- Culture Media, Conditioned analysis, Filtration, Ultracentrifugation, Extracellular Vesicles chemistry
- Abstract
The development of an extracellular vesicles (EV)-based therapeutic product requires the implementation of reproducible and scalable, purification protocols for clinical-grade EV. Commonly used isolation methods including ultracentrifugation, density gradient centrifugation, size exclusion chromatography, and polymer-based precipitation, faced limitations such as yield efficiency, EV purity, and sample volume. We developed a GMP-compatible method for the scalable production, concentration, and isolation of EV through a strategy involving, tangential flow filtration (TFF). We applied this purification method for the isolation of EV from conditioned medium (CM) of cardiac stromal cells, namely cardiac progenitor cells (CPC) which has been shown to possess potential therapeutical application in heart failure. Conditioned medium collection and EV isolation using TFF demonstrated consistent particle recovery (~10
13 particle/mL) enrichment of small/medium-EV subfraction (range size 120-140 nm). EV preparations achieved a 97% reduction of major protein-complex contaminant and showed unaltered biological activity. The protocol describes methods to assess EV identity and purity as well as procedures to perform downstream applications including functional potency assay and quality control tests. The large-scale manufacturing of GMP-grade EV represents a versatile protocol that can be easily applied to different cell sources for wide range of therapeutic areas., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)- Published
- 2023
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74. One Step Histological Detection and Staining of the PTEN Tumor Suppressor Protein by a Single Strand DNA.
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Longinotti G, Ybarra G, Vighi S, Perandones C, Montserrat J, Yakisich JS, Grasselli M, and Radrizzani M
- Abstract
Antibodies are the most used technological tool in histochemistry. However, even with monoclonal antibodies, their standardization is difficult due to variation of biological systems as well as to variability due to the affinity and amplification of the signal arising from secondary peroxidase detection systems. In this article we combined two synthetic molecules to facilitate the standardization of a detection protocol of protein markers in histological sections. The first molecule was an aptamer, a 50-base single-stranded DNA fragment, which recognizes a PTEN tumor suppressor. The second molecule used was also another single stranded 18-base aptamer DNA fragment, which forms a quadruplex structure guanine box. This G-quadruplex recognizes and attaches a molecule of hemin, increasing the catalytic capacity for the hydrogen peroxide. Our results show how the correct structural design of DNA combining an aptamer together with the peroxidase-like DNAzyme allows to detect proteins in histological sections. This tool offers the standardization of the detection of prognostic markers in cancer, in quality and quantity, due to its synthetic nature and its 1:1 antigen:enzyme ratio. This is the first time that reproducible results have been presented in histological sections staining a cancer marker using a single-stranded DNA molecule with dual function.
- Published
- 2021
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75. GMP-Grade Methods for Cardiac Progenitor Cells: Cell Bank Production and Quality Control.
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Andriolo G, Provasi E, Brambilla A, Lo Cicero V, Soncin S, Barile L, Turchetto L, and Radrizzani M
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- Biological Specimen Banks standards, Biomedical Technology methods, Cells, Cultured, Humans, Practice Guidelines as Topic, Primary Cell Culture standards, Tissue Preservation standards, Biomedical Technology standards, Myoblasts cytology, Myocytes, Cardiac cytology, Primary Cell Culture methods
- Abstract
Cardiac explant-derived cells (cEDC), also referred as cardiac progenitors cells (CPC) (Barile et al., Cardiovasc Res 103(4):530-541, 2014; Barile et al., Cardiovasc Res 114(7):992-1005, 2018), represent promising candidates for the development of cell-based therapies, a novel and interesting treatment for cardioprotective strategy in heart failure (Kreke et al., Expert Rev Cardiovasc Ther 10(9):1185-1194, 2012). CPC have been tested in a preclinical setting for direct cell transplantation and tissue engineering or as a source for production of extracellular vesicles (EV) (Oh et al., J Cardiol 68(5):361-367, 2016; Barile et al., Eur Heart J 38(18):1372-1379, 2017; Rosen et al., J Am Coll Cardiol 64(9):922-937, 2014). CPC cultured as cardiospheres derived cells went through favorable Phase 1 and 2 studies demonstrating safety and possible efficacy (Makkar et al., Lancet 379(9819):895-904, 2012; Ishigami et al., Circ Res 120(7):1162-1173, 2017; Ishigami et al., Circ Res 116 (4):653-664, 2015; Tarui et al., J Thorac Cardiovasc Surg 150(5):1198-1207, 1208 e1191-1192, 2015). In this context and in view of clinical applications, cells have to be prepared and released according to Good Manufacturing Practices (GMP) (EudraLex-volume 4-good manufacturing practice (GMP) guidelines-Part I-basic requirements for medicinal products. http://ec.europa.eu/health/documents/eudralex/vol-4 ; EudraLex-volume 4-good manufacturing practice (GMP) guidelines-Part IV-guidelines on good manufacturing practices specific to advanced therapy medicinal products. http://ec.europa.eu/health/documents/eudralex/vol-4 ). This chapter describes GMP-grade methods for production and testing of a CPC Master Cell Bank (MCB), consisting of frozen aliquots of cells that may be used either as a therapeutic product or as source for the manufacturing of Exo for clinical trials.The MCB production method has been designed to isolate and expand CPC from human cardiac tissue in xeno-free conditions (Andriolo et al., Front Physiol 9:1169, 2018). The quality control (QC) methods have been implemented to assess the safety (sterility, endotoxin, mycoplasma, cell senescence, tumorigenicity) and identity/potency/purity (cell count and viability, RT-PCR, immunophenotype) of the cells (Andriolo et al., Front Physiol 9:1169, 2018).
- Published
- 2021
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76. Exosomes From Human Cardiac Progenitor Cells for Therapeutic Applications: Development of a GMP-Grade Manufacturing Method.
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Andriolo G, Provasi E, Lo Cicero V, Brambilla A, Soncin S, Torre T, Milano G, Biemmi V, Vassalli G, Turchetto L, Barile L, and Radrizzani M
- Abstract
Exosomes, nanosized membrane vesicles secreted by cardiac progenitor cells (Exo-CPC), inhibit cardiomyocyte apoptosis under stress conditions, promote angiogenesis in vitro , and prevent the early decline in cardiac function after myocardial infarction in vivo in preclinical rat models. The recognition of exosome-mediated effects has moved attempts at developing cell-free approaches for cardiac repair. Such approaches offer major advantages including the fact that exosomes can be stored as ready-to-use agents and delivered to patients with acute coronary syndromes. The aim of the present work was the development of a good manufacturing practice (GMP)-grade method for the large-scale preparation of Exo-CPC as a medicinal product, for a future clinical translation. A GMP-compliant manufacturing method was set up, based on large-scale cell culture in xeno-free conditions, collection of up to 8 l of exosome-containing conditioned medium and isolation of Exo-CPC through tangential flow filtration. Quality control tests were developed and carried out to evaluate safety, identity, and potency of both cardiac progenitor cells (CPC) as cell source and Exo-CPC as final product (GMP-Exo-CPC). CPC, cultured in xeno-free conditions, showed a lower doubling-time than observed in research-grade condition, while producing exosomes with similar features. Cells showed the typical phenotype of mesenchymal progenitor cells (CD73/CD90/CD105 positive, CD14/CD20/CD34/CD45/HLA-DR negative), and expressed mesodermal (TBX5/TBX18) and cardiac-specific (GATA4/MESP1) transcription factors. Purified GMP-Exo-CPC showed the typical nanoparticle tracking analysis profile and expressed main exosome markers (CD9/CD63/CD81/TSG101). The GMP manufacturing method guaranteed high exosome yield (>10
13 particles) and consistent removal (≥97%) of contaminating proteins. The resulting GMP-Exo-CPC were tested for safety, purity, identity, and potency in vitro , showing functional anti-apoptotic and pro-angiogenic activity. The therapeutic efficacy was validated in vivo in rats, where GMP-Exo-CPC ameliorated heart function after myocardial infarction. Our standardized production method and testing strategy for large-scale manufacturing of GMP-Exo-CPC open new perspectives for reliable human therapeutic applications for acute myocardial infarction syndrome and can be easily applied to other cell sources for different therapeutic areas.- Published
- 2018
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77. In Vitro and In Vivo Development of Horse Cloned Embryos Generated with iPSCs, Mesenchymal Stromal Cells and Fetal or Adult Fibroblasts as Nuclear Donors.
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Olivera R, Moro LN, Jordan R, Luzzani C, Miriuka S, Radrizzani M, Donadeu FX, and Vichera G
- Subjects
- Animals, Blastocyst cytology, Cell Differentiation, Cell Lineage, Cells, Cultured, Embryo Transfer, Female, Horses, Humans, Induced Pluripotent Stem Cells cytology, Kruppel-Like Factor 4, Mesenchymal Stem Cells cytology, Microinjections, Nuclear Transfer Techniques, Oocytes cytology, Plasmids genetics, Plasmids metabolism, Pregnancy, Transcription Factors genetics, Transcription Factors metabolism, Umbilical Cord cytology, Cell Nucleus physiology, Fetus cytology, Fibroblasts cytology, Induced Pluripotent Stem Cells metabolism, Mesenchymal Stem Cells metabolism
- Abstract
The demand for equine cloning as a tool to preserve high genetic value is growing worldwide; however, nuclear transfer efficiency is still very low. To address this issue, we first evaluated the effects of time from cell fusion to activation (<1h, n = 1261; 1-2h, n = 1773; 2-3h, n = 1647) on in vitro and in vivo development of equine embryos generated by cloning. Then, we evaluated the effects of using different nuclear donor cell types in two successive experiments: I) induced pluripotent stem cells (iPSCs) vs. adult fibroblasts (AF) fused to ooplasts injected with the pluripotency-inducing genes OCT4, SOX2, MYC and KLF4, vs. AF alone as controls; II) umbilical cord-derived mesenchymal stromal cells (UC-MSCs) vs. fetal fibroblasts derived from an unborn cloned foetus (FF) vs. AF from the original individual. In the first experiment, both blastocyst production and pregnancy rates were higher in the 2-3h group (11.5% and 9.5%, respectively), respect to <1h (5.2% and 2%, respectively) and 1-2h (5.6% and 4.7%, respectively) groups (P<0.05). However, percentages of born foals/pregnancies were similar when intervals of 2-3h (35.2%) or 1-2h (35.7%) were used. In contrast to AF, the iPSCs did not generate any blastocyst-stage embryos. Moreover, injection of oocytes with the pluripotency-inducing genes did not improve blastocyst production nor pregnancy rates respect to AF controls. Finally, higher blastocyst production was obtained using UC-MSC (15.6%) than using FF (8.9%) or AF (9.3%), (P<0.05). Despite pregnancy rates were similar for these 3 groups (17.6%, 18.2% and 22%, respectively), viable foals (two) were obtained only by using FF. In summary, optimum blastocyst production rates can be obtained using a 2-3h interval between cell fusion and activation as well as using UC-MSCs as nuclear donors. Moreover, FF line can improve the efficiency of an inefficient AF line. Overall, 24 healthy foals were obtained from a total of 29 born foals., Competing Interests: The authors have declared that no competing interests exist.
- Published
- 2016
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78. Effect of Bone Marrow-Derived Mononuclear Cell Treatment, Early or Late After Acute Myocardial Infarction: Twelve Months CMR and Long-Term Clinical Results.
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Sürder D, Manka R, Moccetti T, Lo Cicero V, Emmert MY, Klersy C, Soncin S, Turchetto L, Radrizzani M, Zuber M, Windecker S, Moschovitis A, Bühler I, Kozerke S, Erne P, Lüscher TF, and Corti R
- Subjects
- Bone Marrow Transplantation trends, Female, Follow-Up Studies, Humans, Magnetic Resonance Imaging, Cine trends, Male, Myocardial Infarction epidemiology, Switzerland epidemiology, Time Factors, Treatment Outcome, Bone Marrow Transplantation methods, Leukocytes, Mononuclear transplantation, Magnetic Resonance Imaging, Cine methods, Myocardial Infarction diagnostic imaging, Myocardial Infarction therapy
- Abstract
Rationale: Intracoronary delivery of autologous bone marrow-derived mononuclear cells (BM-MNC) may improve remodeling of the left ventricle (LV) after acute myocardial infarction (AMI)., Objective: To demonstrate long-term efficacy of BM-MNC treatment after AMI., Methods and Results: In a multicenter study, we randomized 200 patients with large AMI in a 1:1:1 pattern into an open-labeled control and 2 BM-MNC treatment groups. In the BM-MNC groups, cells were either administered 5 to 7 days (early) or 3 to 4 weeks (late) after AMI. Cardiac magnetic resonance imaging was performed at baseline and after 12 months. The current analysis investigates the change from baseline to 12 months in global LV ejection fraction, LV volumes, scar size, and N-terminal pro-brain natriuretic peptide values comparing the 2 treatment groups with control in a linear regression model. Besides the complete case analysis, multiple imputation analysis was performed to address for missing data. Furthermore, the long-term clinical event rate was computed. The absolute change in LV ejection fraction from baseline to 12 months was -1.9±9.8% for control (mean±SD), -0.9±10.5% for the early treatment group, and -0.7±10.1% for the late treatment group. The difference between the groups was not significant, both for complete case analysis and multiple imputation analysis. A combined clinical end point occurred equally in all the groups. Overall, 1-year mortality was low (2.25%)., Conclusions: Among patients with AMI and LV dysfunction, treatment with BM-MNC either 5 to 7 days or 3 to 4 weeks after AMI did not improve LV function at 12 months, compared with control. The results are limited by an important drop out rate., Clinical Trial Registration Information: URL: http://www.clinicaltrials.gov. Unique identifier: NCT00355186., (© 2016 American Heart Association, Inc.)
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- 2016
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79. Improvement of bovine semen quality by removal of membrane-damaged sperm cells with DNA aptamers and magnetic nanoparticles.
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Farini VL, Camaño CV, Ybarra G, Viale DL, Vichera G, Yakisich JS, and Radrizzani M
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- Animals, Cattle, Male, Spermatozoa physiology, Aptamers, Nucleotide chemistry, Cell Separation methods, Magnetite Nanoparticles chemistry, SELEX Aptamer Technique methods, Semen Analysis methods, Spermatozoa cytology
- Abstract
In cattle, cryopreservation of semen and sex-sorting kill up to 50% of spermatozoa and decrease the success of assisted insemination (AI). Therefore, significant efforts are being carried out to improve the quality of semen prior to AI. In this work we used the Cell-SELEX technique to select single strand DNA aptamers able to recognize with high affinity and specificity damaged sperm cells generated by heat-treatment. We first isolated aptamers with a conserved two motifs of 6 nucleotides of length that bind to the membrane of heat-treated spermatozoa. Then, we used synthetic biotin-labeled aptamers containing the conserved motif to recognize membrane-damaged cells and separate them from viable cells by the use of avidin-coated superparamagnetic iron oxide nanoparticles (SPION). This procedure improved the quality of semen by significantly increasing the percentage of healthy sperm cells without affecting the rate of blastocyst cleavage. This technique was successfully applied to both unsorted and sex-sorted sperm suspension., (Copyright © 2016. Published by Elsevier B.V.)
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- 2016
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80. Dystonia in a Patient with Autosomal-Dominant Progressive External Ophthalmoplegia Type 1 Caused by Mutation in the POLG Gene.
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Rossi M, Medina Escobar A, Radrizzani M, Tenembaum S, Perandones C, and Merello M
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- 2016
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81. Quality Control Assays for Clinical-Grade Human Mesenchymal Stromal Cells: Methods for ATMP Release.
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Radrizzani M, Soncin S, Lo Cicero V, Andriolo G, Bolis S, and Turchetto L
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- Cell Count, Cell Culture Techniques, Cell Proliferation, Cell Survival, Cells, Cultured, Clinical Trials as Topic, Cryopreservation, Endotoxins analysis, Guideline Adherence, Guidelines as Topic, Humans, Immunophenotyping, Mesenchymal Stem Cells microbiology, Microbiological Techniques methods, Manufactured Materials standards, Mesenchymal Stem Cells cytology, Quality Control
- Abstract
Mesenchymal stromal/stem cells (MSC) are promising candidates for the development of cell-based therapies for various diseases and are currently being evaluated in a number of clinical trials (Sharma et al., Transfusion 54:1418-1437, 2014; Ikebe and Suzuki, Biomed Res Int 2014:951512, 2014). MSC for therapeutic applications are classified as advanced therapy medicinal products (ATMP) (Regulation (EC) No 1394/2007 of the European Parliament and of the Council of 13 November 2007 on advanced therapy medicinal products and amending Directive 2001/83/EC and Regulation (EC) No 726/2004) and must be prepared according to good manufacturing practices ( http://ec.europa.eu/health/documents/eudralex/vol-4 ). They may be derived from different starting materials (mainly bone marrow (BM), adipose tissue, or cord blood) and applied as fresh or cryopreserved products, in the autologous as well as an allogeneic context (Sharma et al., Transfusion 54:1418-1437, 2014; Ikebe and Suzuki, Biomed Res Int 2014:951512, 2014; Sensebé and Bourin, Transplantation 87(9 Suppl):S49-S53, 2009). In any case, they require an approved and well-defined panel of assays in order to be released for clinical use.This chapter describes analytical methods implemented and performed in our cell factory as part of the release strategy for an ATMP consisting of frozen autologous BM-derived MSC. Such methods are designed to assess the safety (sterility, endotoxin, and mycoplasma assays) and identity/potency (cell count and viability, immunophenotype and clonogenic assay) of the final product. Some assays are also applied to the biological starting material (sterility) or carried out as in-process controls (sterility, cell count and viability, immunophenotype, clonogenic assay).The validation strategy for each analytical method is described in the accompanying Chapter 20 .
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- 2016
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82. Quality Control Assays for Clinical-Grade Human Mesenchymal Stromal Cells: Validation Strategy.
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Radrizzani M, Soncin S, Bolis S, Lo Cicero V, Andriolo G, and Turchetto L
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- Bacteria isolation & purification, Cell Count, Cell Culture Techniques methods, Cell Survival, Cells, Cultured, Humans, Immunophenotyping, Mesenchymal Stem Cells microbiology, Microbiological Techniques, Mycoplasma isolation & purification, Endotoxins analysis, Mesenchymal Stem Cells cytology, Quality Control
- Abstract
The present chapter focuses on the validation of the following analytical methods for the control of mesenchymal stromal cells (MSC) for cell therapy clinical trials: Microbiological control for cellular product Endotoxin assay Mycoplasma assay Cell count and viability Immunophenotype Clonogenic potential (CFU-F assay) In our lab, these methods are in use for product release, process control or control of the biological starting materials. They are described in detail in the accompanying Chapter 19.For each method, validation goals and strategy are presented, and a detailed experimental scheme is proposed.
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- 2016
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83. Aptamers: current challenges and future prospects.
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Rozenblum GT, Lopez VG, Vitullo AD, and Radrizzani M
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- Animals, Antibodies administration & dosage, Antibodies metabolism, Aptamers, Nucleotide metabolism, Gene Library, Humans, Aptamers, Nucleotide administration & dosage, Combinatorial Chemistry Techniques methods, SELEX Aptamer Technique methods
- Abstract
Introduction: Aptamers are oligonucleotide molecules raised in vitro from large combinatorial libraries of nucleic acids and developed to bind to targets with high affinity and specificity. Whereas novel target molecules are proposed for therapeutic intervention and diagnostic, aptamer technology has a great potential to become a source of lead compounds., Areas Covered: In this review, the authors address the current status of the technology and highlight the recent progress in aptamer-based technologies. They also discuss the current major technical limitations of aptamer technology and propose original solutions based on existing technologies that could result in a solid aptamer-discovery platform., Expert Opinion: Whereas aptamers have shown to bind to targets with similar affinities and specificities to those of antibodies, aptamers have several advantages that could outweigh antibody technology and open new opportunities for better medical and diagnostic solutions. However, the current status of the aptamer technology suffers from several technical limitations that slowdown the progression of novel aptamers into the clinic and makes the business around aptamers challenging.
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- 2016
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84. Letter to the Editor: Hypothesis: Somatic Mosaicism and Parkinson Disease.
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Perandones C, Pellene LA, Giugni JC, Calvo DS, Raina GB, Cuevas SM, Mata IF, Zabetian CP, Caputo M, Corach D, Micheli FE, and Radrizzani M
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- 2015
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85. Long term follow up of patients after allogeneic stem cell transplantation and transfusion of HSV-TK transduced T-cells.
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Weissinger EM, Borchers S, Silvani A, Provasi E, Radrizzani M, Beckmann IK, Benati C, Schmidtke J, Kuehnau W, Schweier P, Luther S, Fernandez-Munoz I, Beutel G, Ciceri F, Bonini C, Ganser A, Hertenstein B, and Stadler M
- Abstract
Allogeneic stem cell transplantation (allo-HSCT) is one of the curative treatments for hematologic malignancies, but is hampered by severe complications, such as acute or chronic graft-versus-host-disease (aGvHD; cGvHD) and infections. CD34-selection of stem cells reduces the risk of aGvHD, but also leads to increased infectious complications and relapse. Thus, we studied the safety, efficacy, and feasibility of transfer of gene modified donor T-cells shortly after allo-HSCT in two clinical trials between 2002 and 2007 and here we compare the results to unmodified donor leukocyte infusion (DLI). The aim of these trials was to provide patients with the protection of T-cells after T-cell-depleted allo-HSCT in the matched or mismatched donor setting with an option to delete transduced T-cells, if severe aGvHD occurred within the trial period. Donor-T-cells were transduced with the replication-deficient retrovirus SFCMM-3, expressing HSV-TK and the truncated ΔLNGFR for selection of transduced cells. Transduced cells were transfused either after day +60 (matched donors) or on day +42 (haploidentical donors). Nine patients were included in the first trial (MHH; 2002 until 2007), two were included in TK007 (2005-2009) and six serves as a control group for outcome after haploidentical transplantation without HSV-TK-transduced DLI. Three patients developed acute GvHD, two had grade I of the skin, one had aGvHD on day +131 (post-HSCT; +89 post-HSV-TK DLI) grade II, which was successfully controlled by ganciclovir (GCV). Donor chimerism was stabilized after transfusion of the transduced cells in all patients treated. Functionality of HSV-TK gene expressing T-cells was shown by loss of bcr-able gene expression as well as by control of cytomegalovirus-reactivation. To date, six patients have relapsed and died, two after a second hematopoietic stem cell transplantation without T-cell depletion or administration of unmodified T-cells. Eleven patients (seven post-HSV-TK DLI) are alive and well to date.
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- 2015
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86. Bone marrow-derived cells for cardiovascular cell therapy: an optimized GMP method based on low-density gradient improves cell purity and function.
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Radrizzani M, Lo Cicero V, Soncin S, Bolis S, Sürder D, Torre T, Siclari F, Moccetti T, Vassalli G, and Turchetto L
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- Cell Count, Centrifugation, Density Gradient, Humans, Immunophenotyping, Reproducibility of Results, Bone Marrow Cells cytology, Cardiovascular Diseases therapy, Cell Separation methods, Cell Separation standards, Cell- and Tissue-Based Therapy
- Abstract
Background: Cardiovascular cell therapy represents a promising field, with several approaches currently being tested. The advanced therapy medicinal product (ATMP) for the ongoing METHOD clinical study ("Bone marrow derived cell therapy in the stable phase of chronic ischemic heart disease") consists of fresh mononuclear cells (MNC) isolated from autologous bone marrow (BM) through density gradient centrifugation on standard Ficoll-Paque. Cells are tested for safety (sterility, endotoxin), identity/potency (cell count, CD45/CD34/CD133, viability) and purity (contaminant granulocytes and platelets)., Methods: BM-MNC were isolated by density gradient centrifugation on Ficoll-Paque. The following process parameters were optimized throughout the study: gradient medium density; gradient centrifugation speed and duration; washing conditions., Results: A new manufacturing method was set up, based on gradient centrifugation on low density Ficoll-Paque, followed by 2 washing steps, of which the second one at low speed. It led to significantly higher removal of contaminant granulocytes and platelets, improving product purity; the frequencies of CD34+ cells, CD133+ cells and functional hematopoietic and mesenchymal precursors were significantly increased., Conclusions: The methodological optimization described here resulted in a significant improvement of ATMP quality, a crucial issue to clinical applications in cardiovascular cell therapy.
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- 2014
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87. Combined delivery of bone marrow-derived mononuclear cells in chronic ischemic heart disease: rationale and study design.
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Sürder D, Radrizzani M, Turchetto L, Cicero VL, Soncin S, Muzzarelli S, Auricchio A, and Moccetti T
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- Chronic Disease, Clinical Protocols, Feasibility Studies, Humans, Injections, Magnetic Resonance Imaging, Myocardial Contraction, Myocardial Ischemia diagnosis, Myocardial Ischemia physiopathology, Myocardium pathology, Recovery of Function, Stroke Volume, Switzerland, Time Factors, Treatment Outcome, Ventricular Function, Left, Bone Marrow Transplantation methods, Leukocytes, Mononuclear transplantation, Myocardial Ischemia surgery, Research Design
- Abstract
Background: Treatment with bone marrow-derived mononuclear cells (BM-MNC) may improve left ventricular (LV) function in patients with chronic ischemic heart disease (IHD). Delivery method of the cell product may be crucial for efficacy., Hypothesis: We aimed to demonstrate that the combination of intramyocardial and intracoronary injection of BM-MNC is safe and improves LV function in patients with chronic IHD., Methods: After a safety/feasibility phase of 10 patients, 54 patients will be randomly assigned in a 1:1:1 pattern to 1 control and 2 BM-MNC treatment groups. The control group will be treated with state-of-the-art medical management. The treatment groups will receive either exclusively intramyocardial injection or a combination of intramyocardial and intracoronary injection of autologous BM-MNC. Left ventricular function as well as scar size, transmural extension, and regional wall-motion score will be assessed by cardiac magnetic resonance imaging studies at baseline and after 6 months. The primary endpoint is the change in global LV ejection fraction by cardiac magnetic resonance from 6 months to baseline., Results: The results, it is hoped, will have important clinical impact and provide essential information to improve the design of future regenerative-medicine protocols in cardiology., Conclusions: As cell delivery may play an important role in chronic IHD, we aim to demonstrate feasibility and efficacy of a combined cell-delivery approach in patients with decreased LV function., (© 2013 Wiley Periodicals, Inc.)
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- 2013
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88. Intracoronary injection of bone marrow-derived mononuclear cells early or late after acute myocardial infarction: effects on global left ventricular function.
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Sürder D, Manka R, Lo Cicero V, Moccetti T, Rufibach K, Soncin S, Turchetto L, Radrizzani M, Astori G, Schwitter J, Erne P, Zuber M, Auf der Maur C, Jamshidi P, Gaemperli O, Windecker S, Moschovitis A, Wahl A, Bühler I, Wyss C, Kozerke S, Landmesser U, Lüscher TF, and Corti R
- Subjects
- Adult, Aged, Female, Follow-Up Studies, Humans, Injections, Leukocytes, Mononuclear physiology, Male, Middle Aged, Myocardial Infarction pathology, Myocardial Infarction physiopathology, Time Factors, Treatment Outcome, Bone Marrow Cells physiology, Bone Marrow Transplantation methods, Leukocytes, Mononuclear transplantation, Myocardial Infarction surgery, Ventricular Function, Left physiology
- Abstract
Background: Intracoronary administration of autologous bone marrow-derived mononuclear cells (BM-MNC) may improve remodeling of the left ventricle (LV) after acute myocardial infarction. The optimal time point of administration of BM-MNC is still uncertain and has rarely been addressed prospectively in randomized clinical trials., Methods and Results: In a multicenter study, we randomized 200 patients with large, successfully reperfused ST-segment elevation myocardial infarction in a 1:1:1 pattern into an open-labeled control and 2 BM-MNC treatment groups. In the BM-MNC groups, cells were administered either early (i.e., 5 to 7 days) or late (i.e., 3 to 4 weeks) after acute myocardial infarction. Cardiac magnetic resonance imaging was performed at baseline and after 4 months. The primary end point was the change from baseline to 4 months in global LV ejection fraction between the 2 treatment groups and the control group. The absolute change in LV ejection fraction from baseline to 4 months was -0.4±8.8% (mean±SD; P=0.74 versus baseline) in the control group, 1.8±8.4% (P=0.12 versus baseline) in the early group, and 0.8±7.6% (P=0.45 versus baseline) in the late group. The treatment effect of BM-MNC as estimated by ANCOVA was 1.25 (95% confidence interval, -1.83 to 4.32; P=0.42) for the early therapy group and 0.55 (95% confidence interval, -2.61 to 3.71; P=0.73) for the late therapy group., Conclusions: Among patients with ST-segment elevation myocardial infarction and LV dysfunction after successful reperfusion, intracoronary infusion of BM-MNC at either 5 to 7 days or 3 to 4 weeks after acute myocardial infarction did not improve LV function at 4-month follow-up., Clinical Trial Registration: URL: http://www.clinicaltrials.gov. Unique identifier: NCT00355186.
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- 2013
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89. Downregulation of Hsp27 (HSPB1) in MCF-7 human breast cancer cells induces upregulation of PTEN.
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Cayado-Gutiérrez N, Moncalero VL, Rosales EM, Berón W, Salvatierra EE, Alvarez-Olmedo D, Radrizzani M, and Ciocca DR
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- Breast Neoplasms metabolism, Breast Neoplasms pathology, Down-Regulation, Female, Fluorescence Resonance Energy Transfer, HSP27 Heat-Shock Proteins antagonists & inhibitors, HSP27 Heat-Shock Proteins genetics, HSP70 Heat-Shock Proteins metabolism, Humans, Immunohistochemistry, Immunoprecipitation, MCF-7 Cells, Protein Binding, Proto-Oncogene Proteins c-akt metabolism, RNA Interference, RNA, Small Interfering metabolism, Up-Regulation, beta Catenin metabolism, HSP27 Heat-Shock Proteins metabolism, PTEN Phosphohydrolase metabolism
- Abstract
Hsp27 (HSPB1) is usually overexpressed in breast cancers affecting the disease outcome and the sensitivity of tumors to chemotherapy and radiotherapy. Hsp27 interacts with other proteins such as β-catenin, histone deacetylase HDAC6, transcription factor STAT2 and procaspase-3. Phosphatase and tensin homologue (PTEN) is a tumor suppressor gene that is deleted in many human tumors. The PI3K/Akt signaling pathway is negatively regulated by PTEN. Hsp27 is described as a key component of the Akt signaling cascade: Akt, BAD, Forkhead transcription factors, Hsp27, mitogen-activated protein kinase kinase-3 and -6. Here, we have examined whether the downregulation of Hsp27 by siHsp27 affects the PTEN levels in the MCF-7 human breast cancer cell line. PTEN was detected with two different antibodies using western blots and immunocytochemistry. p-Akt was also evaluated by western blot. In addition, Hsp27 and PTEN were immunoprecipitated to know whether these proteins interact. Intracellular colocalization studies were carried out by confocal microscopy. A significant reduction in the Hsp27 levels was noted in the siHsp27 transfected cells. These Hsp27 downregulated cells showed a significant increased expression of PTEN. The MW 76 and 55 kDa PTEN forms were upregulated as revealed by two different antibodies. The phosphatase activity of PTEN seems to be active because p-Akt levels were reduced. Hsp27 immunoprecipitation was bringing PTEN and vice versa, these two proteins seem to interact at cytoplasmic level by FRET. Downregulation of Hsp27 stabilized PTEN protein levels. Chaperone-assisted E3 ligase C terminus of Hsc70-interacting protein (CHIP) levels were not significantly influenced by Hsp27 downregulation. In conclusion, we report a novel function of Hsp27 modulating the PTEN levels in human breast cancer cells suggesting an interaction between these two molecules.
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- 2013
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90. Preclinical safety and efficacy of human CD34(+) cells transduced with lentiviral vector for the treatment of Wiskott-Aldrich syndrome.
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Scaramuzza S, Biasco L, Ripamonti A, Castiello MC, Loperfido M, Draghici E, Hernandez RJ, Benedicenti F, Radrizzani M, Salomoni M, Ranzani M, Bartholomae CC, Vicenzi E, Finocchi A, Bredius R, Bosticardo M, Schmidt M, von Kalle C, Montini E, Biffi A, Roncarolo MG, Naldini L, Villa A, and Aiuti A
- Subjects
- Animals, Bone Marrow Cells immunology, Mice, Mice, Knockout, Antigens, CD34 immunology, Bone Marrow Cells cytology, Bone Marrow Transplantation, Genetic Vectors, Lentivirus genetics, Transduction, Genetic, Wiskott-Aldrich Syndrome therapy
- Abstract
Gene therapy with ex vivo-transduced hematopoietic stem/progenitor cells may represent a valid therapeutic option for monogenic immunohematological disorders such as Wiskott-Aldrich syndrome (WAS), a primary immunodeficiency associated with thrombocytopenia. We evaluated the preclinical safety and efficacy of human CD34(+) cells transduced with lentiviral vectors (LV) encoding WAS protein (WASp). We first set up and validated a transduction protocol for CD34(+) cells derived from bone marrow (BM) or mobilized peripheral blood (MPB) using a clinical grade, highly purified LV. Robust transduction of progenitor cells was obtained in normal donors and WAS patients' cells, without evidence of toxicity. To study biodistribution of human cells and exclude vector release in vivo, LV-transduced CD34(+) cells were transplanted in immunodeficient mice, showing a normal engraftment and differentiation ability towards transduced lymphoid and myeloid cells in hematopoietic tissues. Vector mobilization to host cells and transmission to germline cells of the LV were excluded by different molecular assays. Analysis of vector integrations showed polyclonal integration patterns in vitro and in human engrafted cells in vivo. In summary, this work establishes the preclinical safety and efficacy of human CD34(+) cells gene therapy for the treatment of WAS.
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- 2013
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91. Novel methods to induce exogenous gene expression in SCNT, parthenogenic and IVF preimplantation bovine embryos.
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Pereyra-Bonnet F, Bevacqua R, La Rosa I, Sipowicz P, Radrizzani M, Fernandez-Martin R, and Salamone D
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- Animals, Cattle, Culture Media metabolism, Cumulus Cells cytology, Cumulus Cells metabolism, Cytoplasm genetics, Cytoplasm metabolism, DNA genetics, DNA metabolism, Embryo, Mammalian cytology, Embryo, Mammalian metabolism, Gene Expression, Green Fluorescent Proteins genetics, Green Fluorescent Proteins metabolism, In Situ Hybridization, Fluorescence, Ionomycin pharmacology, Microinjections, Microscopy, Confocal, Nuclear Transfer Techniques, Oocytes cytology, Oocytes drug effects, Oocytes metabolism, Plasmids genetics, Plasmids metabolism, Time Factors, Embryo Culture Techniques methods, Fertilization in Vitro methods, Gene Expression Profiling methods, Gene Transfer Techniques, Parthenogenesis
- Abstract
The import of exogenous DNA (eDNA) from the cytoplasm to the nucleus represents a key intracellular obstacle for efficient gene delivery in mammalian cells. In this study, cumulus cells or oolemma vesicles previously incubated with eDNA, and naked eDNA were injected into the cytoplasm of MII oocytes to evaluate their efficiency for eDNA expressing bovine embryo production. Our study evaluated the potential of short time co-incubation (5 min) of eDNA with; (1) cumulus cells, to be used as donor cells for SCNT and (2) oolemma vesicles (vesicles) to produce parthenogenic transgene expressing embryos. In addition, we included a group consisting of the injection of eDNA alone (plasmid) followed by parthenogenic activation. Two different pCX-EGFP plasmid concentrations (50 and 500 ng/μl) were employed. The results showed that embryos produced by SCNT and by vesicle injection assisted by chemical activation were able to express the eDNA in higher rates than embryos injected with plasmid alone. The lower plasmid concentration allowed the highest development rates in all groups. Using confocal microscopy, we analyzed the interaction of FITC- labeled eDNA with cumulus cells and vesicles as well as oocytes injected with labeled plasmid alone. Our images demonstrated that eDNA interacted with cumulus cells and vesicles, resulting an increase in its expression efficiency. In contrast, oocytes injected with DNA alone did not show signs of transgene accumulation, and their eDNA expression rates were lower. In a further experiment, we evaluated if transgene-expressing embryos could be produced by means of vesicle injection followed by IVF. The lower plasmid concentration (50 ng/μl) injected after IVF, produced the best results. Preliminary FISH analysis indicated detectable integration events in 1/5 of SCNT blastocysts treated. Our studies demonstrate for the first time that short term transgene co-incubation with somatic cells can produce transgene-expressing mammalian SCNT embryos and also that parthenogenic, eDNA- expressing embryos can be obtained by injection of vesicles or eDNA alone. Moreover, eDNA-expressing embryos can be also obtained by cytoplasmic injection vesicles in IVF zygotes, simplifying the traditional IVF pronuclear injection technique.
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- 2011
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92. Genetically modified donor leukocyte transfusion and graft-versus-leukemia effect after allogeneic stem cell transplantation.
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Borchers S, Provasi E, Silvani A, Radrizzani M, Benati C, Dammann E, Krons A, Kontsendorn J, Schmidtke J, Kuehnau W, von Neuhoff N, Stadler M, Ciceri F, Bonini C, Ganser A, Hertenstein B, and Weissinger EM
- Subjects
- Adult, Chimerism, Female, Fusion Proteins, bcr-abl genetics, Genetic Vectors, Graft vs Host Disease mortality, Humans, Immunosuppression Therapy, Male, Middle Aged, Proteomics methods, Receptors, Antigen, T-Cell, alpha-beta metabolism, Remission Induction, Retroviridae genetics, Thymidine Kinase genetics, Tissue Donors, Transduction, Genetic, Transgenes, Graft vs Host Disease prevention & control, Graft vs Leukemia Effect, Hematopoietic Stem Cell Transplantation methods, Leukemia, Myelogenous, Chronic, BCR-ABL Positive therapy, Leukemia, Myeloid, Acute therapy, Leukocyte Transfusion
- Abstract
Seven patients with acute myeloid leukemia (AML) and two patients with chronic myelogenous leukemia (CML) were transplanted from HLA-identical sibling donors with CD34(+) cell-enriched stem cells (HSCTs) without further immunosuppression. The myeloablative standard transplantation protocol was adapted to include transfusion of gene-modified donor T cells after HSCT. Donor T cells were transduced with the replication-deficient retrovirus SFCMM-3, which expresses herpes simplex thymidine kinase (HSV-Tk) and a truncated version of low-affinity nerve growth factor receptor (ΔLNGFR) for selection and characterization of transduced cells. Transduced T cells were detectable in all patients during follow-up for up to 5 years after transfusion. Proteomic screening for development of acute graft-versus-host disease (aGvHD) was applied to five of the seven patients with AML. No positivity for the aGvHD grade II-specific proteomic pattern was observed. Only one patient developed aGvHD grade I. To date, three of the patients with AML relapsed; one responded to three escalating transfusions of lymphocytes from the original donor and is in complete remission. Two were retransplanted with non-T cell-depleted peripheral blood stem cells from their original donors and died after retransplantation of septic complications or relapse, respectively. In one patient with CML, loss of bcr-abl gene expression was observed after an expansion of transduced cells. Seven of nine patients are alive and in complete remission.
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- 2011
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93. IL-7 receptor expression identifies suicide gene-modified allospecific CD8+ T cells capable of self-renewal and differentiation into antileukemia effectors.
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Bondanza A, Hambach L, Aghai Z, Nijmeijer B, Kaneko S, Mastaglio S, Radrizzani M, Fleischhauer K, Ciceri F, Bordignon C, Bonini C, and Goulmy E
- Subjects
- Animals, Biomarkers metabolism, CD8-Positive T-Lymphocytes metabolism, Cells, Cultured, Female, Gene Expression physiology, Genetic Therapy methods, Genetic Vectors immunology, Humans, Immunotherapy, Adoptive methods, Leukemia genetics, Leukemia immunology, Leukemia therapy, Mice, Mice, Inbred NOD, Mice, SCID, Prognosis, Receptors, Interleukin-7 genetics, Receptors, Interleukin-7 metabolism, T-Cell Antigen Receptor Specificity genetics, T-Lymphocytes, Cytotoxic immunology, T-Lymphocytes, Cytotoxic metabolism, T-Lymphocytes, Cytotoxic physiology, Transplantation, Homologous, CD8-Positive T-Lymphocytes immunology, Cell Differentiation genetics, Cell Differentiation immunology, Cell Proliferation, Genes, Transgenic, Suicide immunology, Leukemia diagnosis, Receptors, Interleukin-7 physiology
- Abstract
In allogeneic hematopoietic cell transplantation (HSCT), donor T lymphocytes mediate the graft-versus-leukemia (GVL) effect, but induce graft-versus-host disease (GVHD). Suicide gene therapy-that is, the genetic induction of a conditional suicide phenotype into donor T cells-allows dissociating the GVL effect from GVHD. Genetic modification with retroviral vectors after CD3 activation reduces T-cell alloreactivity. We recently found that alloreactivity is maintained when CD28 costimulation, IL-7, and IL-15 are added. Herein, we used the minor histocompatibility (mH) antigens HA-1 and H-Y as model alloantigens to directly explore the antileukemia efficacy of human T cells modified with the prototypic suicide gene herpes simplex virus thymidine kinase (tk) after activation with different stimuli. Only in the case of CD28 costimulation, IL-7, and IL-15, the repertoire of tk(+) T cells contained HA-1- and H-Y-specific CD8(+) cytotoxic T cells (CTL) precursors. Thymidine kinase-positive HA-1- and H-Y-specific CTLs were capable of self-renewal and differentiation into potent antileukemia effectors in vitro, and in vivo in a humanized mouse model. Self-renewal and differentiation coincided with IL-7 receptor expression. These results pave the way to the clinical investigation of T cells modified with a suicide gene after CD28 costimulation, IL-7, and IL-15 for a safe and effective GVL effect.
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- 2011
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94. Different conformations of phosphatase and tensin homolog, deleted on chromosome 10 (PTEN) protein within the nucleus and cytoplasm of neurons.
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Moncalero VL, Costanzo RV, Perandones C, and Radrizzani M
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- Amino Acid Motifs, Animals, Cell Membrane metabolism, Combinatorial Chemistry Techniques, Epitopes chemistry, Humans, Mice, Models, Biological, Oligonucleotides genetics, Peptides chemistry, Protein Conformation, Cell Nucleus metabolism, Cytoplasm metabolism, Neurons metabolism, PTEN Phosphohydrolase biosynthesis
- Abstract
PTEN is a critical gene involved in the regulation of many cellular processes. The product of this gene has dual phosphatase activity and is able to dephosphorylate the 5' end of the phosphatidylinositol (3,4,5)-trisphosphate. Within the cellular nucleus, this protein has been associated with regulation of the expression of many genes, although the mechanism of this regulation remains unclear. In this paper, two specific oligonucleotide aptamers were developed and selected, using the SELEX procedure, according to their ability to detect the PTEN protein in different subcellular compartments of neurons. While one aptamer was able to detect PTEN in the nucleus, the other recognized PTEN in the cytoplasm. The recognition pattern of PTEN by both aptamers was confirmed using antibodies in western blots of the proteins purified from mouse cerebellar homogenates and subcellular fractions. Additionally, we demonstrated that the two aptamers recognized different epitopes of the target peptide. The results presented here could not be fully explained by the canonical phosphatase structure of PTEN, suggesting the existence of different conformations of phosphatase in the nucleus and the cytoplasm.
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- 2011
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95. Large-scale manufacture and characterization of a lentiviral vector produced for clinical ex vivo gene therapy application.
- Author
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Merten OW, Charrier S, Laroudie N, Fauchille S, Dugué C, Jenny C, Audit M, Zanta-Boussif MA, Chautard H, Radrizzani M, Vallanti G, Naldini L, Noguiez-Hellin P, and Galy A
- Subjects
- Cell Culture Techniques, Cell Line, Drug Contamination legislation & jurisprudence, Drug Contamination prevention & control, Gene Expression Regulation, Gene Order, Genetic Vectors physiology, HEK293 Cells, Hematopoietic Stem Cells metabolism, Humans, Lentivirus physiology, Plasmids genetics, Proviruses genetics, Quality Control, Transduction, Genetic, Transgenes genetics, Wiskott-Aldrich Syndrome therapy, Genetic Therapy, Genetic Vectors biosynthesis, Genetic Vectors genetics, Industrial Microbiology methods, Lentivirus genetics
- Abstract
From the perspective of a pilot clinical gene therapy trial for Wiskott-Aldrich syndrome (WAS), we implemented a process to produce a lentiviral vector under good manufacturing practices (GMP). The process is based on the transient transfection of 293T cells in Cell Factory stacks, scaled up to harvest 50 liters of viral stock per batch, followed by purification of the vesicular stomatitis virus glycoprotein-pseudotyped particles through several membrane-based and chromatographic steps. The process leads to a 200-fold volume concentration and an approximately 3-log reduction in protein and DNA contaminants. An average yield of 13% of infectious particles was obtained in six full-scale preparations. The final product contained low levels of contaminants such as simian virus 40 large T antigen or E1A sequences originating from producer cells. Titers as high as 2 × 10(9) infectious particles per milliliter were obtained, generating up to 6 × 10(11) infectious particles per batch. The purified WAS vector was biologically active, efficiently expressing the genetic insert in WAS protein-deficient B cell lines and transducing CD34(+) cells. The vector introduced 0.3-1 vector copy per cell on average in CD34(+) cells when used at the concentration of 10(8) infectious particles per milliliter, which is comparable to preclinical preparations. There was no evidence of cellular toxicity. These results show the implementation of large-scale GMP production, purification, and control of advanced HIV-1-derived lentiviral technology. Results obtained with the WAS vector provide the initial manufacturing and quality control benchmarking that should be helpful to further development and clinical applications.
- Published
- 2011
- Full Text
- View/download PDF
96. Alpha-synuclein immunoreactivity in minor salivary gland biopsies of Parkinson's disease patients.
- Author
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Cersósimo MG, Perandones C, Micheli FE, Raina GB, Beron AM, Nasswetter G, Radrizzani M, and Benarroch EE
- Subjects
- Aged, Case-Control Studies, Female, Humans, Male, Middle Aged, Intermediate Filament Proteins metabolism, Parkinson Disease pathology, Salivary Glands metabolism
- Published
- 2011
- Full Text
- View/download PDF
97. Sperm genome cloning used in biparental bovine embryo reconstruction.
- Author
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Vichera G, Olivera R, Sipowicz P, Radrizzani M, and Salamone D
- Subjects
- Animals, Blastomeres cytology, Blastomeres metabolism, Cloning, Molecular, Female, Fertilization in Vitro veterinary, Green Fluorescent Proteins metabolism, In Situ Hybridization, Fluorescence, Male, Nuclear Transfer Techniques veterinary, Polymerase Chain Reaction, Pregnancy, Sperm Injections, Intracytoplasmic veterinary, Breeding methods, Cattle, Embryo Culture Techniques veterinary, Embryo, Mammalian physiology, Genome genetics, Haploidy, Spermatozoa chemistry
- Abstract
The generation of androgenetic haploid embryos enables several haploid blastomeres to be obtained as identical copies of a single spermatozoon genome. In the present study, we compared the developmental ability of bovine androgenetic haploid embryos constructed by different methods, namely IVF and intracytoplasmic sperm injection (ICSI) before and after oocyte enucleation. Once obtained, the blastomeres of these androgenetic haploid embryos were used as male genome donors to reconstruct biparental embryos by fusion with matured oocytes. To verify the cytoplasmic contribution of androgenetic haploid blastomeres, we used spermatozoa incubated previously with exogenous DNA that coded for a green fluorescent protein gene (pCX-EGFP) and the enhanced green fluorescent protein (EGFP)-positive androgenetic haploid blastomeres generated were fused with mature oocytes. Of the reconstructed embryos reaching the cleavage and blastocyst stages, 85.1% and 9.0%, respectively, expressed EGFP (P>0.05). EGFP expression was observed in 100% of reconstructed embryos, with 91.2% exhibiting homogenic expression. To confirm sperm genome incorporation, androgenetic haploid blastomeres generated by ICSI prior to enucleation and using Y chromosome sexed spermatozoa were used for biparental embryo reconstruction. Incorporation of the Y chromosome was confirmed by polymerase chain reaction and fluorescence in situ hybridisation analysis. In conclusion, the results of the present study prove that it is possible to use sperm genome replicates to reconstruct biparental bovine embryos and that it is a highly efficient technique to generate homogeneous transgene-expressing embryos., (© CSIRO 2011 Open Access)
- Published
- 2011
- Full Text
- View/download PDF
98. Quantitative proteomic analysis of lentiviral vectors using 2-DE.
- Author
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Denard J, Rundwasser S, Laroudie N, Gonnet F, Naldini L, Radrizzani M, Galy A, Merten OW, Danos O, and Svinartchouk F
- Subjects
- Cell Line, Humans, Proteins metabolism, Proteomics methods, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization, Viral Proteins metabolism, Electrophoresis, Gel, Two-Dimensional methods, Genetic Vectors metabolism, Lentivirus metabolism
- Abstract
Among the integrative gene therapy vectors developed to date, human immunodeficiency virus type 1 (HIV-1)-derived lentiviral vectors (LV) are distinguished by their capacity to infect both dividing and non-dividing cells. Recombinant LV particles contain viral proteins necessary for their packaging, infectious and integrating functions. Like the parental HIV-1 virus they are able to acquire various cellular proteins, but the number and localisation of these proteins are poorly characterised. In the present study we used 2-DE followed by MALDI-TOF to quantify the protein content of several types of vesicular stomatitis virus G-pseudotyped LV including those that were extensively purified in the perspective of clinical gene therapy studies. A proteinase K treatment was used to distinguish between cellular proteins incorporated into virions (I-proteins) and those co-purified with vectors (C-proteins). We found 10 C-proteins and 18 I-proteins associated with LV. Copy numbers for these core I-proteins varied from 5 (AIP-1/ALIX) to 280 (Cyclophilin A) per vector particle. Three novel I-proteins, guanine nucleotide-binding protein 2, L-lactate dehydrogenase B chain and hnRNP core protein A1, were found. This study defines for the first time, the protein stoichiometry of infectious HIV-1-derived LV particles.
- Published
- 2009
- Full Text
- View/download PDF
99. IL-7 and IL-15 allow the generation of suicide gene-modified alloreactive self-renewing central memory human T lymphocytes.
- Author
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Kaneko S, Mastaglio S, Bondanza A, Ponzoni M, Sanvito F, Aldrighetti L, Radrizzani M, La Seta-Catamancio S, Provasi E, Mondino A, Nagasawa T, Fleischhauer K, Russo V, Traversari C, Ciceri F, Bordignon C, and Bonini C
- Subjects
- Animals, Cell Death genetics, Cell Death immunology, Cell Differentiation genetics, Cell Differentiation immunology, Cells, Cultured, Genes, Transgenic, Suicide genetics, Graft vs Host Disease genetics, Graft vs Host Disease therapy, Humans, Interleukin-15 immunology, Interleukin-2 genetics, Interleukin-2 immunology, Interleukin-7 immunology, Isoantigens genetics, Isoantigens immunology, Lymphocyte Activation, Mice, Mice, Inbred NOD, Mice, SCID, Neoplasms genetics, Neoplasms immunology, Neoplasms therapy, Genes, Transgenic, Suicide immunology, Graft vs Host Disease immunology, Immunologic Memory genetics, Interleukin-15 pharmacology, Interleukin-7 pharmacology, Stem Cell Transplantation, T-Lymphocytes immunology
- Abstract
Long-term clinical remissions of leukemia, after allogeneic hematopoietic stem cell transplantation, depend on alloreactive memory T cells able to self-renew and differentiate into antileukemia effectors. This is counterbalanced by detrimental graft-versus-host disease (GVHD). Induction of a selective suicide in donor T cells is a current gene therapy approach to abrogate GVHD. Unfortunately, genetic modification reduces alloreactivity of lymphocytes. This associates with an effector memory (T(EM)) phenotype of gene-modified lymphocytes and may limit antileukemia effect. We hypothesized that alloreactivity of gene-modified lymphocytes segregates with the central memory (T(CM)) phenotype. To this, we generated suicide gene-modified T(CM) lymphocytes with a retroviral vector after CD28 costimulation and culture with IL-2, IL-7, or a combination of IL-7 and IL-15. In vitro, suicide gene-modified T(CM) cells self-renewed upon alloantigen stimulation and resisted activation-induced cell death. In a humanized mouse model, only suicide gene-modified T cells cultured with IL-7 and IL-15 persisted, differentiated in T(EM) cells, and were as potent as unmanipulated lymphocytes in causing GVHD. GVHD was halted through the activation of the suicide gene machinery. These results warrant the use of suicide gene-modified T(CM) cells cultured with IL-7 and IL-15 for the safe exploitation of the alloreactive response against cancer.
- Published
- 2009
- Full Text
- View/download PDF
100. Relative contribution of V-H+ATPase and NA+/H+ exchanger to bicarbonate reabsorption in proximal convoluted tubules of old rats.
- Author
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Fiori M, Radrizzani M, Díaz-Sylvester P, Müller A, Corti T, Monserrat A, and Amorena C
- Subjects
- Amiloride analogs & derivatives, Amiloride pharmacology, Animals, Blotting, Western, Hydrogen-Ion Concentration, Kidney Tubules, Proximal drug effects, Microvilli physiology, Rats, Sodium Channel Blockers pharmacology, Aging physiology, Bicarbonates metabolism, Kidney Tubules, Proximal metabolism, Sodium-Hydrogen Exchangers physiology, Vacuolar Proton-Translocating ATPases physiology
- Abstract
With aging, the kidney develops a progressive deterioration of several structures and functions. Proximal tubular acidification is impaired in old rats with a decrease in the activity of brush border Na+/H+ exchange and a fall of H-ion flux measured with micropuncture experiments. In the present work we evaluate the contribution of 5-N-ethyl-n-isopropyl amiloride- (EIPA) and bafilomycin-sensitive bicarbonate flux (JHCO3-) in proximal convoluted tubules of young and aged rats. We performed micropuncture experiments inhibiting the Na+/H+ exchanger with EIPA (10(-4) M) and the V-H+ATPase with bafilomycin (10(-6) M). We used antibodies against the NHE3 isoform of the Na+/H+ exchanger and the subunit E of the V-H+ATPase for detecting by Western blot the abundance of these proteins in brush border membrane vesicles from proximal convoluted tubules of young and old rats. The abundance of NHE3 and the V-H+ATPase was similar in 18-month-old and 3-month-old rats. The bicarbonate flux in old rats was 30% lower than in young rats. EIPA reduced by 60% and bafilomycin by 30% in young rats; in contrast, EIPA reduced by approximately 40% and bafilomycin by approximately 50% in old rats. The inhibited by bafilomycin was the same in young and old rats: 0.62 nmol.cm-2.s-1 and 0.71 nmol.cm-2.s-1, respectively. However, the EIPA-sensitive fraction was larger in young than in old rats: 1.26 nmol.cm-2.s-1 vs. 0.85 nmol.cm-2.s-1, respectively. These results suggest that the component more affected in bicarbonate reabsorption of proximal convoluted tubules from aged rats is the Na+-H+ exchanger, probably a NHE isoform different from NHE3.
- Published
- 2006
- Full Text
- View/download PDF
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