title: MGMT enrichment and second gene co-expression in hematopoietic progenitor cells using separate or dual-gene lentiviral vectors.
abstract: 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.
authors.affiliation: 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.
authors.forename: Justin C
authors.initials: JC
authors.lastname: Roth
authors.affiliation: Division of Pediatric Infectious Diseases, Department of Pediatrics, University of Alabama at Birmingham, Birmingham, AL, USA. Electronic address: malberti@mednet.ucla.edu.
authors.forename: Michael O
authors.initials: MO
authors.lastname: Alberti
authors.affiliation: 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.
authors.forename: Mourad
authors.initials: M
authors.lastname: Ismail
authors.affiliation: 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.
authors.forename: Karen T
authors.initials: KT
authors.lastname: Lingas
authors.affiliation: 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.
authors.forename: Jane S
authors.initials: JS
authors.lastname: Reese
authors.affiliation: 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.
authors.forename: Stanton L
authors.initials: SL
authors.lastname: Gerson
mesh_terms: D000818:Animals; D005472:Fluorouracil / Q000494:pharmacology; D018628:Gene Dosage; D015870:Gene Expression; D023061:Gene Order; D018014:Gene Transfer Techniques; D017930:Genes, Reporter; D005822:Genetic Vectors / Q000235:genetics*; D018380:Hematopoietic Stem Cell Transplantation; D006412:Hematopoietic Stem Cells / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D020014:K562 Cells; D016086:Lentivirus / Q000235:genetics*; D051379:Mice; D019853:O(6)-Methylguanine-DNA Methyltransferase / Q000235:genetics*; D014161:Transduction, Genetic
chemical_list: D019853:O(6)-Methylguanine-DNA Methyltransferase; D005472:Fluorouracil
keywords: F2A; Gene therapy; IRES; Lentivirus; MGMT; O(6)-methylguanine-DNA methyltransferase; P140K