22 results on '"Sergi LS"'
Search Results
2. New liver cancer genes identified by lentiviral vector-based insertional mutagenesis in mice are associated to differential survival in hepatocellular carcinoma patients
- Author
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Ranzani M, Cesana D, Bartholomae CC, Sanvito F, Pala M, Benedicenti F, Gallina P, Sergi LS, Merella S, Bulfone A, Doglioni C, von Kalle C, Kim YJ, Schmidt M, Tonon G, Naldini L, Montini E, Ranzani, M, Cesana, D, Bartholomae, Cc, Sanvito, F, Pala, M, Benedicenti, F, Gallina, P, Sergi, L, Merella, S, Bulfone, A, Doglioni, C, von Kalle, C, Kim, Yj, Schmidt, M, Tonon, G, Naldini, L, and Montini, E
- Published
- 2012
3. Identification of new human liver cancer genes by a novel lentiviral vector-based insertional mutagenesis approach in three mouse models of hepatocarcinogenesis
- Author
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Ranzani M, Cesana D, Bartholomae C, Sanvito F, Pala M, Benedicenti F, Sergi LS, Bulfone A, Doglioni C, von Kalle C, Schmidt M, Tonon G, Naldini L, Montini E, Ranzani, M, Cesana, D, Bartholomae, C, Sanvito, F, Pala, M, Benedicenti, F, Sergi, L, Bulfone, A, Doglioni, C, von Kalle, C, Schmidt, M, Tonon, G, Naldini, L, and Montini, E
- Published
- 2011
4. In vivo models to assess the risk of insertional mutagenesis in the liver upon vector systemic delivery
- Author
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Ranzani M, Cesana D, Schmidt M, Sanvito F, Benedicenti F, Bartholoma C, Ponzoni M, Cantore A, Sergi LS, Doglioni C, VonKalle C, Naldini L, Montini E, Ranzani, M, Cesana, D, Schmidt, M, Sanvito, F, Benedicenti, F, Bartholoma, C, Ponzoni, M, Cantore, A, Sergi, L, Doglioni, C, Vonkalle, C, Naldini, L, and Montini, E
- Published
- 2009
5. A gene mutatioons reinforces genotype-phenotype correlation in metachromatic leukodystrophy
- Author
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CESANI M, CAPOTONDO A, PLATI T, SERGI LS, FUMAGALLI F, RONCAROLO MG, NALDINI L, COMI , GIANCARLO, SESSA M, BIFFI A., Cesani, M, Capotondo, A, Plati, T, Sergi, L, Fumagalli, F, Roncarolo, Mg, Naldini, L, Comi, Giancarlo, Sessa, M, and Biffi, A.
- Published
- 2009
6. Mouse Models To Assess the Risk of Vector Insertional Mutagenesis upon Systemic Delivery
- Author
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Ranzani M, Cesana D, Schmidt M, Sanvito F, Benedicenti F, Bartholoma C, Ponzoni M, Cantore A, Sergi LS, Doglioni C, VonKalle C, Naldini L, Montini E, Ranzani, M, Cesana, D, Schmidt, M, Sanvito, F, Benedicenti, F, Bartholoma, C, Ponzoni, M, Cantore, A, Sergi, L, Doglioni, C, Vonkalle, C, Naldini, L, and Montini, E
- Published
- 2009
7. The genotoxic potential of retroviral vectors is strongly modulated by vector design and integration site selection
- Author
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Montini, E., Cesana, D., Schmidt, M., Sanvito, F., Bartholomae, C., Ranzani, M., Benedicenti, F., Sergi, Ls, Alessandro Ambrosi, Ponzoni, M., Doglioni, C., Di Serio, C., Kalle, C., Naldini, L., Montini, E, Cesana, D, Schmidt, M, Sanvito, F, Bartholomae, C, Ranzani, M, Benedicenti, F, Sergi, L, Ambrosi, A, Ponzoni, M, Doglioni, C, Di Serio, C, von Kalle, C, and Naldini, L
- Published
- 2008
8. Modeling the genotoxicity of viral vector integration in a tumor prone hematopoietic stem cell transplantation model
- Author
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Montini E, Cesana D, Schmidt M, Sanvito F, Ponzoni M, Sergi LS, Benedicenti F, Bartholomae C, di Serio C, Doglioni C, von Kalle C, Naldini L, Montini, E, Cesana, D, Schmidt, M, Sanvito, F, Ponzoni, M, Sergi, L, Benedicenti, F, Bartholomae, C, di Serio, C, Doglioni, C, von Kalle, C, and Naldini, L
- Published
- 2006
9. Tie2 identifies a hematopoietic monocytes required for tumor lineage of proangiogenic vessel formation and a mesenchymal population of pericyte progenitors
- Author
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De Palma, M, Venneri, MA, Galli, R, Sergi, LS, Politi, LS, Sampaolesi, Maurilio, and Naldini, L
- Subjects
Marrow-Derived Cells ,Stem-Cells ,Endothelial-Cells ,Lentiviral Vectors ,Vascularization ,Precursors ,Angiogenesis ,Growth ,In-Vivo ,Vegf - Abstract
Bone marrow-derived cells contribute to tumor angiogenesis. Here, we demonstrate that monocytes expressing the Tie2 receptor (Tie2-expressing monocytes [TEMs]) (1) are a distinct hematopoietic lineage of proangiogenic cells, (2) are selectively recruited to spontaneous and orthotopic tumors, (3) promote angiogenesis in a paracrine manner, and (4) account for most of the proangiogenic activity of myeloid cells in tumors. Remarkably, TEM knockout completely prevented human glioma neovascularization in the mouse brain and induced substantial tumor regression. Besides TEMs and endothelial cells (ECs), Tie2 expression distinguished a rare population of tumor stroma-derived mesenchymal progenitors representing a primary source of tumor pericytes. Therefore, Tie2 expression characterizes three distinct cell types required for tumor neovascularization: ECs, proangiogenic cells of hematopoietic origin, and pericyte precursors of mesenchymal origin. ispartof: Cancer Cell vol:8 issue:3 pages:211-226 ispartof: location:United States status: published
- Published
- 2005
10. Lentiviral correction of enzymatic activity restrains macrophage inflammation in adenosine deaminase 2 deficiency.
- Author
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Zoccolillo M, Brigida I, Barzaghi F, Scala S, Hernández RJ, Basso-Ricci L, Colantuoni M, Pettinato E, Sergi LS, Milardi G, Capasso P, Lombardo A, Gregori S, Sanvito F, Schena F, Cesaro S, Conti F, Pession A, Benedetti F, Gattorno M, Lee PY, Naldini L, Cicalese MP, Aiuti A, and Mortellaro A
- Subjects
- Animals, Humans, Inflammation, Intercellular Signaling Peptides and Proteins, Macrophages, Mice, Adenosine Deaminase genetics, Vasculitis
- Abstract
Adenosine deaminase 2 deficiency (DADA2) is a rare inherited disorder that is caused by autosomal recessive mutations in the ADA2 gene. Clinical manifestations include early-onset lacunar strokes, vasculitis/vasculopathy, systemic inflammation, immunodeficiency, and hematologic defects. Anti-tumor necrosis factor therapy reduces strokes and systemic inflammation. Allogeneic hematopoietic stem/progenitor cell (HSPC) transplantation can ameliorate most disease manifestations, but patients are at risk for complications. Autologous HSPC gene therapy may be an alternative curative option for patients with DADA2. We designed a lentiviral vector encoding ADA2 (LV-ADA2) to genetically correct HSPCs. Lentiviral transduction allowed efficient delivery of the functional ADA2 enzyme into HSPCs from healthy donors. Supranormal ADA2 expression in human and mouse HSPCs did not affect their multipotency and engraftment potential in vivo. The LV-ADA2 induced stable ADA2 expression and corrected the enzymatic defect in HSPCs derived from DADA2 patients. Patients' HSPCs re-expressing ADA2 retained their potential to differentiate into erythroid and myeloid cells. Delivery of ADA2 enzymatic activity in patients' macrophages led to a complete rescue of the exaggerated inflammatory cytokine production. Our data indicate that HSPCs ectopically expressing ADA2 retain their multipotent differentiation ability, leading to functional correction of macrophage defects. Altogether, these findings support the implementation of HSPC gene therapy for DADA2., (© 2021 by The American Society of Hematology.)
- Published
- 2021
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11. Chromosome Transplantation: A Possible Approach to Treat Human X-linked Disorders.
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Paulis M, Susani L, Castelli A, Suzuki T, Hara T, Straniero L, Duga S, Strina D, Mantero S, Caldana E, Sergi LS, Villa A, and Vezzoni P
- Abstract
Many human genetic diseases are associated with gross mutations such as aneuploidies, deletions, duplications, or inversions. For these "structural" disorders, conventional gene therapy, based on viral vectors and/or on programmable nuclease-mediated homologous recombination, is still unsatisfactory. To correct such disorders, chromosome transplantation (CT), defined as the perfect substitution of an endogenous defective chromosome with an exogenous normal one, could be applied. CT re-establishes a normal diploid cell, leaving no marker of the procedure, as we have recently shown in mouse pluripotent stem cells. To prove the feasibility of the CT approach in human cells, we used human induced pluripotent stem cells (hiPSCs) reprogrammed from Lesch-Nyhan (LN) disease patients, taking advantage of their mutation in the X-linked HPRT gene, making the LN cells selectable and distinguishable from the resistant corrected normal cells. In this study, we demonstrate, for the first time, that CT is feasible in hiPSCs: the normal exogenous X chromosome was first transferred using an improved chromosome transfer system, and the extra sex chromosome was spontaneously lost. These CT cells were functionally corrected and maintained their pluripotency and differentiation capability. By inactivation of the autologous HPRT gene, CT paves the way to the correction of hiPSCs from several X-linked disorders., (© 2020 The Author(s).)
- Published
- 2020
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12. miR-135a Inhibits Cancer Stem Cell-Driven Medulloblastoma Development by Directly Repressing Arhgef6 Expression.
- Author
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Hemmesi K, Squadrito ML, Mestdagh P, Conti V, Cominelli M, Piras IS, Sergi LS, Piccinin S, Maestro R, Poliani PL, Speleman F, De Palma M, and Galli R
- Subjects
- Animals, Carcinogenesis genetics, Cell Aggregation, Cell Transformation, Neoplastic genetics, Down-Regulation, Gene Expression Profiling, Medulloblastoma genetics, Mice, Inbred C57BL, MicroRNAs genetics, Neoplastic Stem Cells pathology, Neural Stem Cells metabolism, Rho Guanine Nucleotide Exchange Factors metabolism, Tumor Suppressor Protein p53 metabolism, Carcinogenesis pathology, Medulloblastoma pathology, MicroRNAs metabolism, Neoplastic Stem Cells metabolism, Rho Guanine Nucleotide Exchange Factors genetics
- Abstract
microRNAs (miRNAs) are short noncoding RNAs, which regulate gene expression post-transcriptionally and play crucial roles in relevant biological and pathological processes. Here, we investigated the putative role of miRNAs in modulating the tumor-initiating potential of mouse medulloblastoma (MB)-derived cancer stem cells (CSCs). We first subjected bona fide highly tumorigenic (HT) CSCs as well as lowly tumorigenic MB CSCs and normal neural stem cells to miRNA profiling, which identified a HT CSC-specific miRNA signature. Next, by cross-checking CSC mRNA/miRNA profiles, we pinpointed miR-135a as a potential tumor suppressor gene, which was strongly downregulated in HT CSCs as well as in the highly malignant experimental tumors derived from them. Remarkably, enforced expression of miR-135a in HT CSCs strongly inhibited tumorigenesis by repressing the miR-135a direct target gene Arhgef6. Considering the upregulation of Arhgef6 in human MBs and its involvement in mediating experimental medulloblastomagenesis, its efficient suppression by miR-135a might make available an effective therapeutic strategy to selectively impair the tumorigenic potential of MB CSCs. Stem Cells 2015;33:1377-1389., (© 2015 AlphaMed Press.)
- Published
- 2015
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13. Liver-directed lentiviral gene therapy in a dog model of hemophilia B.
- Author
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Cantore A, Ranzani M, Bartholomae CC, Volpin M, Valle PD, Sanvito F, Sergi LS, Gallina P, Benedicenti F, Bellinger D, Raymer R, Merricks E, Bellintani F, Martin S, Doglioni C, D'Angelo A, VandenDriessche T, Chuah MK, Schmidt M, Nichols T, Montini E, and Naldini L
- Subjects
- Animals, Blood Coagulation, Disease Models, Animal, Dogs, Female, Genetic Vectors metabolism, Mice, Inbred C57BL, Mutagens toxicity, Time Factors, Transduction, Genetic, Transgenes, Genetic Therapy, Hemophilia B therapy, Lentivirus genetics, Liver pathology
- Abstract
We investigated the efficacy of liver-directed gene therapy using lentiviral vectors in a large animal model of hemophilia B and evaluated the risk of insertional mutagenesis in tumor-prone mouse models. We showed that gene therapy using lentiviral vectors targeting the expression of a canine factor IX transgene in hepatocytes was well tolerated and provided a stable long-term production of coagulation factor IX in dogs with hemophilia B. By exploiting three different mouse models designed to amplify the consequences of insertional mutagenesis, we showed that no genotoxicity was detected with these lentiviral vectors. Our findings suggest that lentiviral vectors may be an attractive candidate for gene therapy targeted to the liver and may be potentially useful for the treatment of hemophilia., (Copyright © 2015, American Association for the Advancement of Science.)
- Published
- 2015
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14. Dual-regulated lentiviral vector for gene therapy of X-linked chronic granulomatosis.
- Author
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Chiriaco M, Farinelli G, Capo V, Zonari E, Scaramuzza S, Di Matteo G, Sergi LS, Migliavacca M, Hernandez RJ, Bombelli F, Giorda E, Kajaste-Rudnitski A, Trono D, Grez M, Rossi P, Finocchi A, Naldini L, Gentner B, and Aiuti A
- Subjects
- Animals, Antigens, CD34 metabolism, Cell Line, Cells, Cultured, Combined Modality Therapy, Disease Models, Animal, Genetic Vectors therapeutic use, Granulomatous Disease, Chronic pathology, Hematopoietic Stem Cells metabolism, Humans, Lentivirus genetics, Mice, Myeloid Cells metabolism, NADPH Oxidase 2, Genetic Therapy methods, Granulomatous Disease, Chronic therapy, Hematopoietic Stem Cell Transplantation methods, Hematopoietic Stem Cells virology, Membrane Glycoproteins metabolism, MicroRNAs genetics, NADPH Oxidases metabolism
- Abstract
Regulated transgene expression may improve the safety and efficacy of hematopoietic stem cell (HSC) gene therapy. Clinical trials for X-linked chronic granulomatous disease (X-CGD) employing gammaretroviral vectors were limited by insertional oncogenesis or lack of persistent engraftment. Our novel strategy, based on regulated lentiviral vectors (LV), targets gp91(phox) expression to the differentiated myeloid compartment while sparing HSC, to reduce the risk of genotoxicity and potential perturbation of reactive oxygen species levels. Targeting was obtained by a myeloid-specific promoter (MSP) and posttranscriptional, microRNA-mediated regulation. We optimized both components in human bone marrow (BM) HSC and their differentiated progeny in vitro and in a xenotransplantation model, and generated therapeutic gp91(phox) expressing LVs for CGD gene therapy. All vectors restored gp91(phox) expression and function in human X-CGD myeloid cell lines, primary monocytes, and differentiated myeloid cells. While unregulated LVs ectopically expressed gp91(phox) in CD34(+) cells, transcriptionally and posttranscriptionally regulated LVs substantially reduced this off-target expression. X-CGD mice transplanted with transduced HSC restored gp91(phox) expression, and MSP-driven vectors maintained regulation during BM development. Combining transcriptional (SP146.gp91-driven) and posttranscriptional (miR-126-restricted) targeting, we achieved high levels of myeloid-specific transgene expression, entirely sparing the CD34(+) HSC compartment. This dual-targeted LV construct represents a promising candidate for further clinical development.
- Published
- 2014
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15. Lentiviral vector-based insertional mutagenesis identifies genes associated with liver cancer.
- Author
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Ranzani M, Cesana D, Bartholomae CC, Sanvito F, Pala M, Benedicenti F, Gallina P, Sergi LS, Merella S, Bulfone A, Doglioni C, von Kalle C, Kim YJ, Schmidt M, Tonon G, Naldini L, and Montini E
- Subjects
- Animals, Cyclin-Dependent Kinase Inhibitor p16 deficiency, Genetic Vectors, Humans, Mice, PTEN Phosphohydrolase deficiency, Prealbumin genetics, Receptor, Interferon alpha-beta deficiency, Carcinoma, Hepatocellular genetics, Lentivirus genetics, Liver Neoplasms genetics, Mutagenesis, Insertional, Oncogenes
- Abstract
Transposons and γ-retroviruses have been efficiently used as insertional mutagens in different tissues to identify molecular culprits of cancer. However, these systems are characterized by recurring integrations that accumulate in tumor cells and that hamper the identification of early cancer-driving events among bystander and progression-related events. We developed an insertional mutagenesis platform based on lentiviral vectors (LVVs) by which we could efficiently induce hepatocellular carcinoma (HCC) in three different mouse models. By virtue of the LVV's replication-deficient nature and broad genome-wide integration pattern, LVV-based insertional mutagenesis allowed identification of four previously unknown liver cancer-associated genes from a limited number of integrations. We validated the oncogenic potential of all the identified genes in vivo, with different levels of penetrance. The newly identified genes are likely to play a role in human cancer because they are upregulated, amplified and/or deleted in human HCCs and can predict clinical outcomes of patients.
- Published
- 2013
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16. Gene signatures associated with mouse postnatal hindbrain neural stem cells and medulloblastoma cancer stem cells identify novel molecular mediators and predict human medulloblastoma molecular classification.
- Author
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Corno D, Pala M, Cominelli M, Cipelletti B, Leto K, Croci L, Barili V, Brandalise F, Melzi R, Di Gregorio A, Sergi LS, Politi LS, Piemonti L, Bulfone A, Rossi P, Rossi F, Consalez GG, Poliani PL, and Galli R
- Subjects
- Animals, Animals, Newborn, Cerebellar Neoplasms classification, Cerebellar Neoplasms pathology, Humans, Medulloblastoma classification, Medulloblastoma pathology, Mice, Neoplastic Stem Cells metabolism, Neural Stem Cells metabolism, Rhombencephalon cytology, Cerebellar Neoplasms genetics, Gene Expression Profiling, Medulloblastoma genetics
- Abstract
Medulloblastoma arises from mutations occurring in stem/progenitor cells located in restricted hindbrain territories. Here we report that the mouse postnatal ventricular zone lining the IV ventricle also harbors bona fide stem cells that, remarkably, share the same molecular profile with cerebellar white matter-derived neural stem cells (NSC). To identify novel molecular mediators involved in medulloblastomagenesis, we compared these distinct postnatal hindbrain-derived NSC populations, which are potentially tumor initiating, with murine compound Ptch/p53 mutant medulloblastoma cancer stem cells (CSC) that faithfully phenocopy the different variants of human medulloblastoma in vivo. Transcriptome analysis of both hindbrain NSCs and medulloblastoma CSCs resulted in the generation of well-defined gene signatures, each reminiscent of a specific human medulloblastoma molecular subclass. Most interestingly, medulloblastoma CSCs upregulated developmentally related genes, such as Ebfs, that were shown to be highly expressed in human medulloblastomas and play a pivotal role in experimental medullo-blastomagenesis. These data indicate that gene expression analysis of medulloblastoma CSCs holds great promise not only for understanding functional differences between distinct CSC populations but also for identifying meaningful signatures that might stratify medulloblastoma patients beyond histopathologic staging.
- Published
- 2012
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17. HIV-1-derived lentiviral vectors directly activate plasmacytoid dendritic cells, which in turn induce the maturation of myeloid dendritic cells.
- Author
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Rossetti M, Gregori S, Hauben E, Brown BD, Sergi LS, Naldini L, and Roncarolo MG
- Subjects
- Cells, Cultured, Humans, Immunity, Innate, Interferon-alpha metabolism, Interleukin-6 metabolism, Monocytes cytology, Toll-Like Receptor 7 immunology, Toll-Like Receptor 9 immunology, Tumor Necrosis Factor-alpha metabolism, Up-Regulation, Dendritic Cells cytology, Dendritic Cells metabolism, Genetic Vectors, HIV-1 genetics, Myeloid Cells metabolism
- Abstract
Lentiviral vectors (LV) can induce type I interferon (IFN I) production from murine plasmacytoid dendritic cells (pDC), but not myeloid (my)DC. Here, we investigated whether this mechanism is conserved in human DC. MyDC and pDC were isolated from peripheral blood and transduced with increasing vector concentrations. Compared with in vitro differentiated monocyte-derived DC, the transduction efficiency of peripheral blood DC was low (ranging from <1% to 45%), with pDC showing the lowest susceptibility to LV transduction. Phenotype and function of myDC were not directly modified by LV transduction; by contrast, pDC produced significant levels of IFN-α and tumor necrosis factor-α. pDC activation was dependent on functional vector particles and was mediated by Toll-like receptor 7/9 triggering. Coculture of myDC with pDC in the presence of LV resulted in myDC activation, with CD86 up-regulation and interleukin-6 secretion. These findings demonstrate that the induction of transgene-specific immunity is triggered by an innate immune response with pDC activation and consequent myDC maturation, a response that closely resembles the one induced by functional viruses. This information is important to design strategies aimed at using LV in humans for gene therapy, where adverse immune responses must be avoided, or for cancer immunotherapy, where inducing immunity is the goal.
- Published
- 2011
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18. In vivo delivery of a microRNA-regulated transgene induces antigen-specific regulatory T cells and promotes immunologic tolerance.
- Author
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Annoni A, Brown BD, Cantore A, Sergi LS, Naldini L, and Roncarolo MG
- Subjects
- Animals, Antigen-Presenting Cells cytology, Antigen-Presenting Cells immunology, Antigen-Presenting Cells metabolism, Forkhead Transcription Factors metabolism, Gene Transfer Techniques, Genetic Vectors administration & dosage, Genetic Vectors genetics, Green Fluorescent Proteins genetics, Green Fluorescent Proteins immunology, Green Fluorescent Proteins metabolism, Hepatocytes cytology, Hepatocytes immunology, Hepatocytes metabolism, Interleukin-2 Receptor alpha Subunit metabolism, Lentivirus genetics, Liver cytology, Liver immunology, Liver metabolism, Mice, Microscopy, Confocal, T-Lymphocytes, Regulatory cytology, T-Lymphocytes, Regulatory metabolism, Transgenes genetics, Antigens immunology, Immune Tolerance immunology, MicroRNAs genetics, T-Lymphocytes, Regulatory immunology, Transgenes immunology
- Abstract
We previously showed that incorporating target sequences for the hematopoietic-specific microRNA miR-142 into an antigen-encoding transgene prevents antigen expression in antigen-presenting cells (APCs). To determine whether this approach induces immunologic tolerance, we treated mice with a miR-142-regulated lentiviral vector encoding green fluorescent protein (GFP), and subsequently vaccinated the mice against GFP. In contrast to control mice, no anti-GFP response was observed, indicating that robust tolerance to the transgene-encoded antigen was achieved. Furthermore, injection of the miR-142-regulated vector induced a population of GFP-specific regulatory T cells. Interestingly, an anti-GFP response was observed when microRNA miR-122a was inserted into the vector and antigen expression was detargeted from hepatocytes as well as APCs. This demonstrates that, in the context of lentiviral vector-mediated gene transfer, detargeting antigen expression from professional APCs, coupled with expression in hepatocytes, can induce antigen-specific immunologic tolerance.
- Published
- 2009
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19. Characterization of new arylsulfatase A gene mutations reinforces genotype-phenotype correlation in metachromatic leukodystrophy.
- Author
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Cesani M, Capotondo A, Plati T, Sergi LS, Fumagalli F, Roncarolo MG, Naldini L, Comi G, Sessa M, and Biffi A
- Subjects
- Animals, Genotype, HeLa Cells, Humans, Leukodystrophy, Metachromatic enzymology, Mice, Mice, Knockout, Phenotype, Polymerase Chain Reaction, Cerebroside-Sulfatase genetics, Leukodystrophy, Metachromatic genetics, Mutation
- Abstract
Metachromatic Leukodystrophy (MLD) is a rare inherited lysosomal storage disorder caused by the deficiency of Arylsulfatase A (ARSA). The disease manifests itself with a broad spectrum of clinical variants, all characterized by progressive neurodegeneration in the central and peripheral nervous systems. The correlation between mutations in the ARSA gene, residual enzymatic activity associated with the mutated alleles and patients' phenotype, which has been extensively drawn for common ARSA mutations, has recently been expanded to rare ones. In this context, functional studies on the rare allelic variances acquire particular relevance for patients' prognostic evaluation. Here we have characterized eight newly identified ARSA mutations, through lentiviral vector-based expression studies on cell lines and ARSA defective murine fibroblasts. In each case, the residual activity associated with the new mutant allele correlates well with the patient's phenotype. Therefore, our results confirm the importance of functional characterization of mutant alleles for a precise genotype-based classification and definition of prognosis in MLD patients, which is particularly relevant for pre-symptomatic diagnosis.
- Published
- 2009
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20. The genotoxic potential of retroviral vectors is strongly modulated by vector design and integration site selection in a mouse model of HSC gene therapy.
- Author
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Montini E, Cesana D, Schmidt M, Sanvito F, Bartholomae CC, Ranzani M, Benedicenti F, Sergi LS, Ambrosi A, Ponzoni M, Doglioni C, Di Serio C, von Kalle C, and Naldini L
- Subjects
- Animals, Base Sequence, Cyclin-Dependent Kinase Inhibitor p16 deficiency, Cyclin-Dependent Kinase Inhibitor p16 genetics, DNA Primers genetics, Genes, p16, Genetic Therapy methods, Hematopoietic Stem Cell Transplantation adverse effects, Lentivirus genetics, Lentivirus pathogenicity, Mice, Mice, Knockout, Mice, Transgenic, Mutation, Neoplasms, Experimental etiology, Neoplasms, Experimental genetics, Terminal Repeat Sequences, Gammaretrovirus genetics, Gammaretrovirus pathogenicity, Genetic Therapy adverse effects, Genetic Vectors adverse effects
- Abstract
gamma-Retroviral vectors (gammaRVs), which are commonly used in gene therapy, can trigger oncogenesis by insertional mutagenesis. Here, we have dissected the contribution of vector design and viral integration site selection (ISS) to oncogenesis using an in vivo genotoxicity assay based on transplantation of vector-transduced tumor-prone mouse hematopoietic stem/progenitor cells. By swapping genetic elements between gammaRV and lentiviral vectors (LVs), we have demonstrated that transcriptionally active long terminal repeats (LTRs) are major determinants of genotoxicity even when reconstituted in LVs and that self-inactivating (SIN) LTRs enhance the safety of gammaRVs. By comparing the genotoxicity of vectors with matched active LTRs, we were able to determine that substantially greater LV integration loads are required to approach the same oncogenic risk as gammaRVs. This difference in facilitating oncogenesis is likely to be explained by the observed preferential targeting of cancer genes by gammaRVs. This integration-site bias was intrinsic to gammaRVs, as it was also observed for SIN gammaRVs that lacked genotoxicity in our model. Our findings strongly support the use of SIN viral vector platforms and show that ISS can substantially modulate genotoxicity.
- Published
- 2009
- Full Text
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21. A microRNA-regulated lentiviral vector mediates stable correction of hemophilia B mice.
- Author
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Brown BD, Cantore A, Annoni A, Sergi LS, Lombardo A, Della Valle P, D'Angelo A, and Naldini L
- Subjects
- Animals, Antibodies, Blood Cells metabolism, Factor IX administration & dosage, Factor IX immunology, Gene Transfer Techniques, Mice, MicroRNAs genetics, Genetic Therapy methods, Genetic Vectors therapeutic use, Hemophilia B therapy, Lentivirus genetics, MicroRNAs pharmacology
- Abstract
A longstanding goal for the treatment of hemophilia B is the development of a gene transfer strategy that can maintain sustained production of clotting factor IX (F.IX) in the absence of an immune response. To this end, we have sought to use lentiviral vectors (LVs) as a means for systemic gene transfer. Unfortunately, initial evaluation of LVs expressing F.IX from hepatocyte-specific promoters failed to achieve sustained F.IX expression in hemophilia B mice due to the induction of an anti-F.IX cellular immune response. Further analysis suggested that this may be a result of off-target transgene expression in hematopoietic-lineage cells of the spleen. In order to overcome this problem, we modified our vector to contain a target sequence for the hematopoietic-specific microRNA, miR-142-3p. This eliminated off-target expression in hematopoietic cells, and enabled sustained gene transfer in hemophilia B mice for more than 280 days after injection. Treated mice had more than 10% normal F.IX activity, no detectable anti-F.IX antibodies, and were unresponsive to F.IX immunization. Importantly, the mice survived tail-clip challenge, thus demonstrating phenotypic correction of their bleeding diathesis. This work, which is among the first applications to exploit the microRNA regulatory pathway, provides the basis for a promising new therapy for the treatment of hemophilia B.
- Published
- 2007
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22. In vivo administration of lentiviral vectors triggers a type I interferon response that restricts hepatocyte gene transfer and promotes vector clearance.
- Author
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Brown BD, Sitia G, Annoni A, Hauben E, Sergi LS, Zingale A, Roncarolo MG, Guidotti LG, and Naldini L
- Subjects
- Animals, Dendritic Cells immunology, Gene Transfer Techniques, Genetic Therapy, In Vitro Techniques, Mice, Mice, Inbred BALB C, Mice, Inbred C57BL, Mice, Knockout, Mice, Nude, Transduction, Genetic, Genetic Vectors immunology, Hepatocytes immunology, Hepatocytes virology, Interferon Type I biosynthesis, Lentivirus genetics, Lentivirus immunology
- Abstract
Liver gene transfer is a highly sought goal for the treatment of inherited and infectious diseases. Lentiviral vectors (LVs) have many desirable properties for hepatocyte-directed gene delivery, including the ability to integrate into nondividing cells. Unfortunately, upon systemic administration, LV transduces hepatocytes relatively inefficiently compared with nonparenchymal cells, and the duration of transgene expression is often limited by immune responses. Here, we investigated the role of innate antiviral responses in these events. We show that administration of LVs to mice triggers a rapid and transient IFNalphabeta response. This effect was dependent on functional vector particles, and in vitro challenge of antigen-presenting cells suggested that plasmacytoid dendritic cells initiated the response. Remarkably, when LVs were administered to animals that lack the capacity to respond to IFNalphabeta, there was a dramatic increase in hepatocyte transduction, and stable transgene expression was achieved. These findings indicate that, even in the setting of acute delivery of replication-defective vectors, IFNs effectively interfere with transduction in a cell-type-specific manner. Moreover, because disabling a single component of the innate/immune network was sufficient to establish persistent xenoantigen expression, our results raise the hope that the immunologic barriers to gene therapy are less insurmountable than expected.
- Published
- 2007
- Full Text
- View/download PDF
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