San Raffaele Telethon Institute for Gene Therapy

Milano, Italy

San Raffaele Telethon Institute for Gene Therapy

Milano, Italy
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Ranzani M.,San Raffaele Telethon Institute for Gene Therapy | Annunziato S.,San Raffaele Telethon Institute for Gene Therapy | Adams D.J.,Wellcome Trust Sanger Institute | Montini E.,San Raffaele Telethon Institute for Gene Therapy
Molecular Cancer Research | Year: 2013

Insertional mutagenesis has been used as a functional forward genetics screen for the identification of novel genes involved in the pathogenesis of human cancers. Different insertional mutagens have been successfully used to reveal new cancer genes. For example, retroviruses are integrating viruses with the capacity to induce the deregulation of genes in the neighborhood of the insertion site. Retroviruses have been used for more than 30 years to identify cancer genes in the hematopoietic system and mammary gland. Similarly, another tool that has revolutionized cancer gene discovery is the cut-and-paste transposons. These DNA elements have been engineered to contain strong promoters and stop cassettes that may function to perturb gene expression upon integration proximal to genes. In addition, complex mouse models characterized by tissue-restricted activity of transposons have been developed to identify oncogenes and tumor suppressor genes that control the development of a wide range of solid tumor types, extending beyond those tissues accessible using retrovirus-based approaches. Most recently, lentiviral vectors have appeared on the scene for use in cancer gene screens. Lentiviral vectors are replication-defective integrating vectors that have the advantage of being able to infect nondividing cells, in a wide range of cell types and tissues. In this review, we describe the various insertional mutagens focusing on their advantages/limitations, and we discuss the new and promising tools that will improve the insertional mutagenesis screens of the future. © 2013 American Association for Cancer Research.

Mazzucchelli R.I.,San Raffaele Telethon Institute for Gene Therapy | Riva A.,University of Milan | Durum S.K.,U.S. National Institutes of Health
Seminars in Immunology | Year: 2012

Most T cell subsets depend on IL-7 for survival. IL-7 binds to IL-7Rα and γc, initiating the signaling cascade. Deletion of IL-7Ra in humans has, for some time, been known to cause severe combined immunodeficiency. More recently, polymorphisms in IL-7R have been shown be a risk factor for a number of diseases that are autoimmune or involve excess immune and inflammatory responses including multiple sclerosis, type 1 diabetes, rheumatoid arthritis, primary biliary cirrhosis, inflammatory bowel disease, atopic dermatitis, inhalation allergy, sarcoidosis and graft-versus host disease. The polymorphism that affects risk to most of these immunopathologies is T244I at the border of the extracellular domain and the transmembrane region. The same region has recently been shown to harbor gain-of-function mutations in acute lymphoblastic leukemia. These studies have suggested new therapies that target the IL-7 pathway. © 2012.

Gregori S.,San Raffaele Telethon Institute for Gene Therapy | Tomasoni D.,San Raffaele Telethon Institute for Gene Therapy | Pacciani V.,San Raffaele Telethon Institute for Gene Therapy | Pacciani V.,University of Rome Tor Vergata | And 7 more authors.
Blood | Year: 2010

Type 1 T regulatory (Tr1) cells suppress immune responses in vivo and in vitro and play a key role in maintaining tolerance to self- and non-self-antigens. Interleukin-10 (IL-10) is the crucial driving factor for Tr1 cell differentiation, but the molecular mechanisms underlying this induction remain unknown. We identified and characterized a subset of IL-10-producing human dendritic cells (DCs), termed DC-10, which are present in vivo and can be induced in vitro in the presence of IL-10. DC-10 are CD14+, CD16 +, CD11c+, CD11b+, HLA-DR+, CD83+, CD1a-, CD1c-, express the Ig-like transcripts (ILTs) ILT2, ILT3, ILT4, and HLA-G antigen, display high levels of CD40 and CD86, and up-regulate CD80 after differentiation in vitro. DC-10 isolated from peripheral blood or generated in vitro are potent inducers of antigen-specific IL-10-producing Tr1 cells. Induction of Tr1 cells by DC-10 is IL-10-dependent and requires the ILT4/HLA-G signaling pathway. Our data indicate that DC-10 represents a novel subset of tolerogenic DCs, which secrete high levels of IL-10, express ILT4 and HLA-G, and have the specific function to induce Tr1 cells. © 2010 by The American Society of Hematology.

De Palma M.,Ecole Polytechnique Federale de Lausanne | De Palma M.,San Raffaele Telethon Institute for Gene Therapy | Nucera S.,San Raffaele Telethon Institute for Gene Therapy | Nucera S.,Vita-Salute San Raffaele University
Cancer Discovery | Year: 2012

Acute mobilization of circulating endothelial progenitors has been implicated in tumor resistance to vascular-disrupting agents. In the current issue of Cancer Discovery, Taylor and colleagues provide novel insight into the kinetics of endothelial progenitor mobilization by vascular-disrupting agents in both mouse tumor models and cancer patients. © 2012 American Association for Cancer Research.

Battaglia M.,San Raffaele Telethon Institute for Gene Therapy | Stabilini A.,San Raffaele Diabetes Research Institute | Tresoldi E.,San Raffaele Telethon Institute for Gene Therapy
Methods in Molecular Biology | Year: 2012

CD4 +CD25 +FOXP3 + T regulatory (Treg) cells are pivotal for the induction and maintenance of peripheral tolerance in both mice and humans. The possibility to use Treg cells for the treatment of T-cell-mediated diseases has recently gained increasing momentum. However, given the limited amount of circulating FOXP3 + Treg cells, efficient methods for their ex vivo expansion are highly desirable. Rapamycin allows for in vitro expansion of murine and human FOXP3 + Treg cells, which maintain their regulatory phenotype and suppressive capacity. Here, we describe in detail the powerful methods for enriching human FOXP3 + Treg cells starting from unfractionated CD4 + T cells or for expanding CD25 +-enriched Treg cells in the presence of rapamycin.

Battaglia M.,San Raffaele Diabetes Research Institute | Roncarolo M.-G.,San Raffaele Telethon Institute for Gene Therapy | Roncarolo M.-G.,Vita-Salute San Raffaele University
Seminars in Immunology | Year: 2011

In type 1 diabetes (T1D), insulin-producing pancreatic β-cells are attacked and destroyed by the immune system. Although man-made insulin is life-saving, it is not a cure and it cannot prevent long-term complications. In addition, most T1D patients would do almost anything to achieve release from the burden of daily glucose monitoring and insulin injection. Despite the formation of very large and promising clinical trials, a means to prevent/cure T1D in humans remains elusive. This has led to an increasing interest in the possibility of using T cells with regulatory properties (Treg cells) as a biological therapy to preserve and restore tolerance to self-antigens. In the present review we will attempt to consolidate learning from the past and to describe what we now believe could in the future become a successful Treg-cell based immune intervention in T1D. © 2011.

Biasco L.,San Raffaele Telethon Institute for Gene Therapy | Baricordi C.,San Raffaele Telethon Institute for Gene Therapy | Aiuti A.,San Raffaele Telethon Institute for Gene Therapy | Aiuti A.,University of Rome Tor Vergata
Molecular Therapy | Year: 2012

γ-Retroviral and lentiviral vectors allow the permanent integration of a therapeutic transgene in target cells and have provided in the last decade a delivery platform for several successful gene therapy (GT) clinical approaches. However, the occurrence of adverse events due to insertional mutagenesis in GT treated patients poses a strong challenge to the scientific community to identify the mechanisms at the basis of vector-driven genotoxicity. Along the last decade, the study of retroviral integration sites became a fundamental tool to monitor vector-host interaction in patients overtime. This review is aimed at critically revising the data derived from insertional profiling, with a particular focus on the evidences collected from GT clinical trials. We discuss the controversies and open issues associated to the interpretation of integration site analysis during patient's follow up, with an update on the latest results derived from the use of high-throughput technologies. Finally, we provide a perspective on the future technical development and on the application of these studies to address broader biological questions, from basic virology to human hematopoiesis. © The American Society of Gene & Cell Therapy.

Montini E.,San Raffaele Telethon Institute for Gene Therapy | Cesana D.,San Raffaele Telethon Institute for Gene Therapy
Methods in Enzymology | Year: 2012

Integrative viral vectors are able to efficiently transduce hematopoietic stem progenitor cells allowing stable transgene expression in the entire hematopoietic system upon transplant in conditioned recipients. For these reasons, integrative vectors based on γ-retroviruses and lentiviruses have been successfully used in gene therapy clinical trials for the treatment of genetic diseases, especially blood disorders. However, in different γ-retroviral-based clinical trials, vector integration into the host cell genome triggered oncogenesis by a mechanism called insertional mutagenesis. Thus, a thorough reassessment of the safety of available gene transfer systems is a crucial outstanding issue for the whole gene therapy field. Sensitive preclinical models of vector genotoxicity are instrumental to achieve a more detailed understanding of the factors that modulate the risks of insertional mutagenesis. Here, we will describe the methodologies used to address the mutagenesis risk of vector integration using a murine in vivo genotoxicity assay based on transduction and transplantation of tumor-prone hematopoietic stem and progenitor cells. © 2012 Elsevier Inc. All rights reserved.

Efficient therapeutic protein delivery is a challenging task in several disease contexts and particularly when the CNS is concerned. Different approaches for brain-directed delivery have been thus far investigated, including direct injection of molecules or of their coding information carried by dedicated vector systems within the brain parenchyma or in the ventricular space, intravenous systemic administration of molecules/vectors modified to target and cross the blood-brainbarrier, and exploitation of allogeneic and/or autologous and genetically modified cells as vehicles for the therapeutic of interest. Among these, we here review one of the most promising approaches based on hematopoietic stem cells, taking advantage of lysosomal storage disorders as representative disease setting. © 2012 Bentham Science Publishers.

Bernardo M.E.,San Raffaele Telethon Institute for Gene Therapy | Aiuti A.,San Raffaele Telethon Institute for Gene Therapy
Human Gene Therapy | Year: 2016

Gene therapy (GT) approaches based on autologous hematopoietic stem cells (HSC) corrected ex vivo have shown therapeutic benefit in a number of inherited disorders. GT bares the advantage of allowing each patient to be her/his own donor while reducing the risks of immune-mediated complications as compared with allogeneic hematopoietic stem-cell transplantation (HSCT). In order to achieve stable engraftment of HSC, patients undergoing transplantation of allogeneic or autologous HSC receive a chemotherapy- and/or radiotherapy-based preparation. With regard to HSC-GT for inherited genetic disorders, the ideal conditioning regimen should aim to contain toxicity by reducing the dosage and/or the number of chemotherapeutic agents administered, in comparison to fully myeloablative preparations employed in conventional allogeneic HSCT. To meet this aim, a profound knowledge of the disease-specific biological background and of the therapeutic transgene levels, as well as of the key principles of transplantation, are required. While low-dose conditioning is sufficient to create a mixed chimerism when gene-corrected cells are endowed with a natural selective advantage, such as in the case of immune deficiencies, myeloablative doses are necessary when high levels of engraftment are required in disease such as lysosomal storage disorders and beta thalassemia. Therefore, the intensity and type of conditioning regimen administered to patients undergoing HSC-GT should be tailored to reach a minimal efficacious therapeutic target level while sparing toxicity. Novel strategies based on monoclonal antibodies selectively depleting blood cells and associated with limited extramedullary toxicity might be successfully employed in the context of HSC-GT in the near future. This review focuses on the role of the conditioning regimen in HSC-GT, and in particular, it highlights the importance of modulating the preparative chemotherapy based on disease biology and transgene expression in order to optimize outcome. © Copyright 2016, Mary Ann Liebert, Inc. 2016.

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