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      Efficient Ex Vivo Engineering and Expansion of Highly Purified Human Hematopoietic Stem and Progenitor Cell Populations for Gene Therapy

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          Summary

          Ex vivo gene therapy based on CD34 + hematopoietic stem cells (HSCs) has shown promising results in clinical trials, but genetic engineering to high levels and in large scale remains challenging. We devised a sorting strategy that captures more than 90% of HSC activity in less than 10% of mobilized peripheral blood (mPB) CD34 + cells, and modeled a transplantation protocol based on highly purified, genetically engineered HSCs co-infused with uncultured progenitor cells. Prostaglandin E 2 stimulation allowed near-complete transduction of HSCs with lentiviral vectors during a culture time of less than 38 hr, mitigating the negative impact of standard culture on progenitor cell function. Exploiting the pyrimidoindole derivative UM171, we show that transduced mPB CD34 +CD38 cells with repopulating potential could be expanded ex vivo. Implementing these findings in clinical gene therapy protocols will improve the efficacy, safety, and sustainability of gene therapy and generate new opportunities in the field of gene editing.

          Highlights

          • CD34 +CD38 cells as an HSC-enriched starting population for ex vivo gene therapy

          • Reduced culture time (<38 hr) alleviates negative impact on progenitor cell potency

          • Prostaglandin E 2 increases LV transduction up to 2× enabling shorter protocols

          • UM171 supports ex vivo expansion of mobilized peripheral blood HSCs

          Abstract

          In this article, Gentner and colleagues undertake a comprehensive strategy to advance ex vivo genetic engineering of HSCs for gene therapy. They experimentally define an optimal strategy to purify HSCs, which allows uncoupling long-term from short-term hematopoietic reconstitution, and implement ex vivo conditions that best preserve their biological properties applying novel transduction-enhancing compounds and pyrimidoindole derivatives to support HSC expansion.

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          Most cited references22

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          Aryl hydrocarbon receptor antagonists promote the expansion of human hematopoietic stem cells.

          Although practiced clinically for more than 40 years, the use of hematopoietic stem cell (HSC) transplants remains limited by the ability to expand these cells ex vivo. An unbiased screen with primary human HSCs identified a purine derivative, StemRegenin 1 (SR1), that promotes the ex vivo expansion of CD34+ cells. Culture of HSCs with SR1 led to a 50-fold increase in cells expressing CD34 and a 17-fold increase in cells that retain the ability to engraft immunodeficient mice. Mechanistic studies show that SR1 acts by antagonizing the aryl hydrocarbon receptor (AHR). The identification of SR1 and AHR modulation as a means to induce ex vivo HSC expansion should facilitate the clinical use of HSC therapy.
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            Non-genotoxic conditioning for hematopoietic stem cell transplantation using a hematopoietic-cell-specific internalizing immunotoxin.

            Hematopoietic stem cell transplantation (HSCT) offers curative therapy for patients with hemoglobinopathies, congenital immunodeficiencies, and other conditions, possibly including AIDS. Autologous HSCT using genetically corrected cells would avoid the risk of graft-versus-host disease (GVHD), but the genotoxicity of conditioning remains a substantial barrier to the development of this approach. Here we report an internalizing immunotoxin targeting the hematopoietic-cell-restricted CD45 receptor that effectively conditions immunocompetent mice. A single dose of the immunotoxin, CD45-saporin (SAP), enabled efficient (>90%) engraftment of donor cells and full correction of a sickle-cell anemia model. In contrast to irradiation, CD45-SAP completely avoided neutropenia and anemia, spared bone marrow and thymic niches, enabling rapid recovery of T and B cells, preserved anti-fungal immunity, and had minimal overall toxicity. This non-genotoxic conditioning method may provide an attractive alternative to current conditioning regimens for HSCT in the treatment of non-malignant blood diseases.
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              Prostaglandin E2 enhances human cord blood stem cell xenotransplants and shows long-term safety in preclinical nonhuman primate transplant models.

              Hematopoietic stem cells (HSCs) are used in transplantation therapy to reconstitute the hematopoietic system. Human cord blood (hCB) transplantation has emerged as an attractive alternative treatment option when traditional HSC sources are unavailable; however, the absolute number of hCB HSCs transplanted is significantly lower than bone marrow or mobilized peripheral blood stem cells (MPBSCs). We previously demonstrated that dimethyl-prostaglandin E2 (dmPGE2) increased HSCs in vertebrate models. Here, we describe preclinical analyses of the therapeutic potential of dmPGE2 treatment by using human and nonhuman primate HSCs. dmPGE2 significantly increased total human hematopoietic colony formation in vitro and enhanced engraftment of unfractionated and CD34(+) hCB after xenotransplantation. In nonhuman primate autologous transplantation, dmPGE2-treated CD34(+) MPBSCs showed stable multilineage engraftment over 1 year postinfusion. Together, our analyses indicated that dmPGE2 mediates conserved responses in HSCs from human and nonhuman primates and provided sufficient preclinical information to support proceeding to an FDA-approved phase 1 clinical trial. Copyright © 2011 Elsevier Inc. All rights reserved.
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                Author and article information

                Contributors
                Journal
                Stem Cell Reports
                Stem Cell Reports
                Stem Cell Reports
                Elsevier
                2213-6711
                16 March 2017
                11 April 2017
                16 March 2017
                : 8
                : 4
                : 977-990
                Affiliations
                [1 ]San Raffaele Telethon Institute for Gene Therapy (SR-TIGET), Milan 20132, Italy
                [2 ]Vita-Salute San Raffaele University, Milan 20132, Italy
                [3 ]Pediatric Immunohematology and Bone Marrow Transplantation Unit, IRCSS Ospedale San Raffaele, Milan 20132, Italy
                [4 ]Hematology and Bone Marrow Transplantation Unit, IRCSS Ospedale San Raffaele, Milan 20132, Italy
                Author notes
                []Corresponding author gentner.bernhard@ 123456hsr.it
                Article
                S2213-6711(17)30077-2
                10.1016/j.stemcr.2017.02.010
                5390102
                28330619
                42b70b99-24eb-4a24-9444-0ff41b1ea0e8
                © 2017 The Author(s)

                This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

                History
                : 22 November 2016
                : 13 February 2017
                : 13 February 2017
                Categories
                Article

                hsc gene therapy,purified hscs,hsc expansion,lentiviral vector transduction,prostaglandin e2,um171

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