title: Promoter and lineage independent anti-silencing activity of the A2 ubiquitous chromatin opening element for optimized human pluripotent stem cell-based gene therapy.
abstract: Epigenetic silencing of retroviral transgene expression in pluripotent stem cells (PSC) and their differentiated progeny constitutes a major roadblock for PSC-based gene therapy. As ubiquitous chromatin opening elements (UCOEs) have been successfully employed to stabilize transgene expression in murine hematopoietic and pluripotent stem cells as well as their differentiated progeny, we here investigated UCOE activity in their human counterparts to establish a basis for future clinical application of the element. To this end, we demonstrate profound anti-silencing activity of the A2UCOE in several human iPS and ES cell lines including their progeny obtained upon directed cardiac or hematopoietic differentiation. We also provide evidence for A2UCOE activity in murine iPSC-derived hepatocyte-like cells, thus establishing efficacy of the element in cells of different germ layers. Finally, we investigated combinations of the A2UCOE with viral promoter/enhancer elements again demonstrating profound stabilization of transgene expression. In all these settings the effect of the A2UCOE was associated with strongly reduced promoter DNA-methylation. Thus, our data clearly support the concept of the A2UCOE as a generalized strategy to prevent epigenetic silencing in PSC and their differentiated progeny and strongly favors its application to stabilize transgene expression in PSC-based cell and gene therapy approaches.
authors.affiliation: Reprogramming and Gene Therapy Group, REBIRTH Cluster of Excellence, Hannover Medical School, Hannover, Lower Saxony 30625, Germany; Institute of Experimental Hematology, Hannover Medical School, Hannover, Lower Saxony 30625, Germany.
authors.forename: Mania
authors.initials: M
authors.lastname: Ackermann
authors.affiliation: Reprogramming and Gene Therapy Group, REBIRTH Cluster of Excellence, Hannover Medical School, Hannover, Lower Saxony 30625, Germany; Institute of Experimental Hematology, Hannover Medical School, Hannover, Lower Saxony 30625, Germany.
authors.forename: Nico
authors.initials: N
authors.lastname: Lachmann
authors.affiliation: Leibniz Research Laboratories for Biotechnology and Artificial Organs (LEBAO), Department of Cardiothoracic, Transplantation and Vascular Surgery, Hannover Medical School, Hannover, Lower Saxony 30625, Germany.
authors.forename: Susann
authors.initials: S
authors.lastname: Hartung
authors.affiliation: Translational Hepatology and Stem Cell Biology, REBIRTH Cluster of Excellence, Hannover Medical School, Hannover, Lower Saxony 30625, Germany.
authors.forename: Reto
authors.initials: R
authors.lastname: Eggenschwiler
authors.affiliation: Reprogramming and Gene Therapy Group, REBIRTH Cluster of Excellence, Hannover Medical School, Hannover, Lower Saxony 30625, Germany; Institute of Experimental Hematology, Hannover Medical School, Hannover, Lower Saxony 30625, Germany.
authors.forename: Nils
authors.initials: N
authors.lastname: Pfaff
authors.affiliation: Department of Pediatrics, Hannover Medical School, Hannover, Lower Saxony 30625, Germany; Biomedical Research in Endstage and Obstructive Lung Disease Hannover (BREATH), Member of the Center for Lung Research (DZL), Hannover Medical School, Hannover, Lower Saxony 30625, Germany.
authors.forename: Christine
authors.initials: C
authors.lastname: Happle
authors.affiliation: Reprogramming and Gene Therapy Group, REBIRTH Cluster of Excellence, Hannover Medical School, Hannover, Lower Saxony 30625, Germany.
authors.forename: Adele
authors.initials: A
authors.lastname: Mucci
authors.affiliation: Institute of Cell and Molecular Pathology, Hannover Medical School, Hannover, Lower Saxony 30625, Germany.
authors.forename: Gudrun
authors.initials: G
authors.lastname: Göhring
authors.affiliation: Institute of Farm Animal Genetics, Friedrich-Löffler-Institut, Mariensee/Neustadt, Lower Saxony 31535, Germany.
authors.forename: Heiner
authors.initials: H
authors.lastname: Niemann
authors.affiliation: Department of Pediatrics, Hannover Medical School, Hannover, Lower Saxony 30625, Germany; Biomedical Research in Endstage and Obstructive Lung Disease Hannover (BREATH), Member of the Center for Lung Research (DZL), Hannover Medical School, Hannover, Lower Saxony 30625, Germany.
authors.forename: Gesine
authors.initials: G
authors.lastname: Hansen
authors.affiliation: Institute of Experimental Hematology, Hannover Medical School, Hannover, Lower Saxony 30625, Germany; Division of Pediatric Hematology/Oncology, Boston Children's Hospital, Harvard Medical School, Boston, MA 02115, USA.
authors.forename: Axel
authors.initials: A
authors.lastname: Schambach
authors.affiliation: Translational Hepatology and Stem Cell Biology, REBIRTH Cluster of Excellence, Hannover Medical School, Hannover, Lower Saxony 30625, Germany.
authors.forename: Tobias
authors.initials: T
authors.lastname: Cantz
authors.affiliation: Leibniz Research Laboratories for Biotechnology and Artificial Organs (LEBAO), Department of Cardiothoracic, Transplantation and Vascular Surgery, Hannover Medical School, Hannover, Lower Saxony 30625, Germany.
authors.forename: Robert
authors.initials: R
authors.lastname: Zweigerdt
authors.affiliation: Reprogramming and Gene Therapy Group, REBIRTH Cluster of Excellence, Hannover Medical School, Hannover, Lower Saxony 30625, Germany; Institute of Experimental Hematology, Hannover Medical School, Hannover, Lower Saxony 30625, Germany. Electronic address: moritz.thomas@mh-hannover.de.
authors.forename: Thomas
authors.initials: T
authors.lastname: Moritz
mesh_terms: D000818:Animals; D002454:Cell Differentiation; D019070:Cell Lineage; D002843:Chromatin / Q000378:metabolism*; D020868:Gene Silencing; D015316:Genetic Therapy; D057809:HEK293 Cells; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000166:cytology / Q000378:metabolism*; D051379:Mice; D011401:Promoter Regions, Genetic; D020133:Reverse Transcriptase Polymerase Chain Reaction; D019076:Transgenes
chemical_list: D002843:Chromatin
keywords: Epigenetic silencing; Gene therapy; Lentiviral vector; Pluripotent stem cells; UCOE