title: Lentiviral MGMT(P140K)-mediated in vivo selection employing a ubiquitous chromatin opening element (A2UCOE) linked to a cellular promoter.
abstract: Notwithstanding recent successes, insertional mutagenesis as well as silencing and variegation of transgene expression still represent considerable obstacles to hematopoietic gene therapy. This also applies to O(6)-methylguanine DNA methyltransferase (MGMT)-mediated myeloprotection, a concept recently proven clinically effective in the context of glioblastoma therapy. To improve on this situation we here evaluate a SIN-lentiviral vector expressing the MGMT(P140K)-cDNA from a combined A2UCOE/PGK-promoter. In a murine in vivo chemoselection model the A2UCOE.PGK.MGMT construct allowed for significant myeloprotection as well as robust and stable selection of transgenic hematopoietic cells. In contrast, only transient enrichment and severe myelotoxicity was observed for a PGK.MGMT control vector. Selection of A2UCOE.PGK.MGMT-transduced myeloid and lymphoid mature and progenitor cells was demonstrated in the peripheral blood, bone marrow, spleen, and thymus. Unlike the PGK and SFFV promoters used as controls, the A2UCOE.PGK promoter allowed for sustained vector copy number-related transgene expression throughout the experiment indicating an increased resistance to silencing, which was further confirmed by CpG methylation studies of the PGK promoter. Thus, our data support a potential role of the A2UCOE.PGK.MGMT-vector in future MGMT-based myeloprotection and chemoselection strategies, and underlines the suitability of the A2UCOE element to stabilize lentiviral transgene expression in hematopoietic gene therapy.
authors.affiliation: Reprogramming and Gene Therapy Group, REBIRTH Cluster of Excellence, Hannover Medical School, 30625 Hannover, Germany; Institute of Experimental Hematology, Hannover Medical School, 30625 Hannover, Germany.
authors.forename: Ruhi
authors.initials: R
authors.lastname: Phaltane
authors.affiliation: Reprogramming and Gene Therapy Group, REBIRTH Cluster of Excellence, Hannover Medical School, 30625 Hannover, Germany; Institute of Experimental Hematology, Hannover Medical School, 30625 Hannover, Germany.
authors.forename: Nico
authors.initials: N
authors.lastname: Lachmann
authors.affiliation: Reprogramming and Gene Therapy Group, REBIRTH Cluster of Excellence, Hannover Medical School, 30625 Hannover, Germany; Institute of Experimental Hematology, Hannover Medical School, 30625 Hannover, Germany.
authors.forename: Sebastian
authors.initials: S
authors.lastname: Brennig
authors.affiliation: Reprogramming and Gene Therapy Group, REBIRTH Cluster of Excellence, Hannover Medical School, 30625 Hannover, Germany; Institute of Experimental Hematology, Hannover Medical School, 30625 Hannover, Germany.
authors.forename: Mania
authors.initials: M
authors.lastname: Ackermann
authors.affiliation: Institute of Experimental Hematology, Hannover Medical School, 30625 Hannover, Germany; LOEWE Research Group Genetic Modifications in Stem Cells, Paul-Ehrlich-Institute, 63225 Langen, Germany.
authors.forename: Ute
authors.initials: U
authors.lastname: Modlich
authors.affiliation: Reprogramming and Gene Therapy Group, REBIRTH Cluster of Excellence, Hannover Medical School, 30625 Hannover, Germany; Institute of Experimental Hematology, Hannover Medical School, 30625 Hannover, Germany. Electronic address: moritz.thomas@mh-hannover.de.
authors.forename: Thomas
authors.initials: T
authors.lastname: Moritz
mesh_terms: D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology; D002843:Chromatin / Q000737:chemistry* / Q000235:genetics; D003001:Cloning, Molecular; D056915:DNA Copy Number Variations; D005260:Female; D015870:Gene Expression; D015316:Genetic Therapy; D005822:Genetic Vectors; D006147:Guanine / Q000031:analogs & derivatives / Q000494:pharmacology; D006412:Hematopoietic Stem Cells / Q000378:metabolism; D016086:Lentivirus / Q000235:genetics*; D051379:Mice; D008810:Mice, Inbred C57BL; D019853:O(6)-Methylguanine-DNA Methyltransferase / Q000737:chemistry / Q000235:genetics*; D010735:Phosphoglycerate Kinase / Q000737:chemistry / Q000235:genetics; D011401:Promoter Regions, Genetic; D017422:Sequence Analysis, DNA; D019076:Transgenes
chemical_list: D000970:Antineoplastic Agents; D002843:Chromatin; C064976:O(6)-benzylguanine; D006147:Guanine; D019853:O(6)-Methylguanine-DNA Methyltransferase; D010735:Phosphoglycerate Kinase
keywords: Gene therapy; In vivo selection; Lentiviral vector; MGMT; UCOE