PMID- 25479595 OWN - NLM STAT- MEDLINE DCOM- 20150916 LR - 20221117 IS - 1872-7492 (Electronic) IS - 0168-1702 (Print) IS - 0168-1702 (Linking) VI - 196 DP - 2015 Jan 22 TI - MGMT enrichment and second gene co-expression in hematopoietic progenitor cells using separate or dual-gene lentiviral vectors. PG - 170-80 LID - S0168-1702(14)00502-4 [pii] LID - 10.1016/j.virusres.2014.11.027 [doi] AB - The DNA repair gene O(6)-methylguanine-DNA methyltransferase (MGMT) allows efficient in vivo enrichment of transduced hematopoietic stem cells (HSC). Thus, linking this selection strategy to therapeutic gene expression offers the potential to reconstitute diseased hematopoietic tissue with gene-corrected cells. However, different dual-gene expression vector strategies are limited by poor expression of one or both transgenes. To evaluate different co-expression strategies in the context of MGMT-mediated HSC enrichment, we compared selection and expression efficacies in cells cotransduced with separate single-gene MGMT and GFP lentivectors to those obtained with dual-gene vectors employing either encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES) or foot and mouth disease virus (FMDV) 2A elements for co-expression strategies. Each strategy was evaluated in vitro and in vivo using equivalent multiplicities of infection (MOI) to transduce 5-fluorouracil (5-FU) or Lin(-)Sca-1(+)c-kit(+) (LSK)-enriched murine bone marrow cells (BMCs). The highest dual-gene expression (MGMT(+)GFP(+)) percentages were obtained with the FMDV-2A dual-gene vector, but half of the resulting gene products existed as fusion proteins. Following selection, dual-gene expression percentages in single-gene vector cotransduced and dual-gene vector transduced populations were similar. Equivalent MGMT expression levels were obtained with each strategy, but GFP expression levels derived from the IRES dual-gene vector were significantly lower. In mice, vector-insertion averages were similar among cells enriched after dual-gene vectors and those cotransduced with single-gene vectors. These data demonstrate the limitations and advantages of each strategy in the context of MGMT-mediated selection, and may provide insights into vector design with respect to a particular therapeutic gene or hematologic defect. CI - Copyright (c) 2014 The Authors. Published by Elsevier B.V. All rights reserved. FAU - Roth, Justin C AU - Roth JC AD - Molecular Virology Training Program, Case Western Reserve University, Cleveland, OH, USA; Division of Hematology and Oncology, Department of Medicine, Case Comprehensive Cancer Center, Case Western Reserve University, Cleveland, OH, USA; Division of Pediatric Infectious Diseases, Department of Pediatrics, University of Alabama at Birmingham, Birmingham, AL, USA. Electronic address: jroth@peds.uab.edu. FAU - Alberti, Michael O AU - Alberti MO AD - Division of Pediatric Infectious Diseases, Department of Pediatrics, University of Alabama at Birmingham, Birmingham, AL, USA. Electronic address: malberti@mednet.ucla.edu. FAU - Ismail, Mourad AU - Ismail M AD - Division of Hematology and Oncology, Department of Medicine, Case Comprehensive Cancer Center, Case Western Reserve University, Cleveland, OH, USA. Electronic address: mourad.ismail@gmail.com. FAU - Lingas, Karen T AU - Lingas KT AD - Division of Hematology and Oncology, Department of Medicine, Case Comprehensive Cancer Center, Case Western Reserve University, Cleveland, OH, USA. Electronic address: karen.lingas@case.edu. FAU - Reese, Jane S AU - Reese JS AD - Division of Hematology and Oncology, Department of Medicine, Case Comprehensive Cancer Center, Case Western Reserve University, Cleveland, OH, USA; Seidman Cancer Center, University Hospitals Case Medical Center, Cleveland, OH, USA. Electronic address: jane.reese@case.edu. FAU - Gerson, Stanton L AU - Gerson SL AD - Division of Hematology and Oncology, Department of Medicine, Case Comprehensive Cancer Center, Case Western Reserve University, Cleveland, OH, USA; Seidman Cancer Center, University Hospitals Case Medical Center, Cleveland, OH, USA; National Center for Regenerative Medicine, Case Comprehensive Cancer Center, Case Western Reserve University, Cleveland, OH, USA; The Center for Stem Cell and Regenerative Medicine, Cleveland, OH, USA. Electronic address: stanton.gerson@case.edu. LA - eng GR - P30CA43703/CA/NCI NIH HHS/United States GR - R01CA073062/CA/NCI NIH HHS/United States GR - T32CA059366/CA/NCI NIH HHS/United States GR - P30 CA043703/CA/NCI NIH HHS/United States GR - T32 CA059366/CA/NCI NIH HHS/United States GR - T32HL07147/HL/NHLBI NIH HHS/United States GR - T32 HL007147/HL/NHLBI NIH HHS/United States GR - R01 CA073062/CA/NCI NIH HHS/United States PT - Journal Article PT - Research Support, N.I.H., Extramural DEP - 20141203 PL - Netherlands TA - Virus Res JT - Virus research JID - 8410979 RN - EC 2.1.1.63 (O(6)-Methylguanine-DNA Methyltransferase) RN - U3P01618RT (Fluorouracil) SB - IM MH - Animals MH - Fluorouracil/pharmacology MH - Gene Dosage MH - *Gene Expression MH - Gene Order MH - Gene Transfer Techniques MH - Genes, Reporter MH - Genetic Vectors/*genetics MH - Hematopoietic Stem Cell Transplantation MH - Hematopoietic Stem Cells/drug effects/*metabolism MH - Humans MH - K562 Cells MH - Lentivirus/*genetics MH - Mice MH - O(6)-Methylguanine-DNA Methyltransferase/*genetics MH - Transduction, Genetic PMC - PMC9667281 MID - NIHMS649307 OTO - NOTNLM OT - F2A OT - Gene therapy OT - IRES OT - Lentivirus OT - MGMT OT - O(6)-methylguanine-DNA methyltransferase OT - P140K EDAT- 2014/12/06 06:00 MHDA- 2015/09/17 06:00 PMCR- 2022/11/16 CRDT- 2014/12/06 06:00 PHST- 2014/08/12 00:00 [received] PHST- 2014/11/20 00:00 [revised] PHST- 2014/11/25 00:00 [accepted] PHST- 2014/12/06 06:00 [entrez] PHST- 2014/12/06 06:00 [pubmed] PHST- 2015/09/17 06:00 [medline] PHST- 2022/11/16 00:00 [pmc-release] AID - S0168-1702(14)00502-4 [pii] AID - 10.1016/j.virusres.2014.11.027 [doi] PST - ppublish SO - Virus Res. 2015 Jan 22;196:170-80. doi: 10.1016/j.virusres.2014.11.027. Epub 2014 Dec 3.