title: Limited lentiviral transgene expression with increasing copy number in an MGMT selection model: lack of copy number selection by drug treatment.
abstract: Retroviral vector integration into the human genome carries increased risk of oncogenesis with increasing integrations. To boost transgene expression for gene therapy, multiple integrations are often sought. We studied the relationship between the number of vector integrations and transgene expression and the effect that drug selection in an MGMT-selection model would have on vector copy number. K562 cells were transduced using a lentiviral vector and a library of clones was generated. Median proviral copy number was 4 and a positive correlation with transgene expression was observed. Transgene expression increased at a linear rate between 1 and 4 vector copies/cell, but was unpredictable at >4 integrations/cell. When lentivirus MGMT(P140K)-transduced K562 cells were treated with O(6)-benzylguanine (BG)/BCNU, there was no selection for increased median copy number in colony-forming units, despite strong selection pressure and an increase in transgene expression and activity. These data show a direct and linear correlation between MGMT(P140K) transgene expression and vector copy number. Strong BG/BCNU selective pressure does not result in preferential survival of high-copy-number clones but does select for strong transgene expression. Thus drug selection would not be expected to increase the risk of oncogenesis due to exaggerated selection in favor of high-copy-number vector integration.
authors.affiliation: Division of Hematology/Oncology, Case Comprehensive Cancer Center and Center for Stem Cell and Regenerative Medicine, Case School of Medicine, Case Western Reserve University and University Hospitals of Cleveland, Cleveland, OH 44106, USA.
authors.forename: Steven P
authors.initials: SP
authors.lastname: Zielske
authors.forename: Karen T
authors.initials: KT
authors.lastname: Lingas
authors.forename: Yan
authors.initials: Y
authors.lastname: Li
authors.forename: Stanton L
authors.initials: SL
authors.lastname: Gerson
mesh_terms: D019883:Alkyl and Aryl Transferases / Q000032:analysis; D002460:Cell Line; D015870:Gene Expression / Q000235:genetics; D015316:Genetic Therapy / Q000009:adverse effects; D005822:Genetic Vectors / Q000235:genetics*; D006147:Guanine / Q000031:analogs & derivatives* / Q000494:pharmacology*; D006801:Humans; D016086:Lentivirus / Q000235:genetics*; D016254:Mutagenesis, Insertional / Q000235:genetics; D019853:O(6)-Methylguanine-DNA Methyltransferase / Q000032:analysis / Q000235:genetics*; D016133:Polymerase Chain Reaction; D011533:Proviruses / Q000235:genetics; D014161:Transduction, Genetic; D019076:Transgenes / Q000235:genetics; D016662:Virus Integration / Q000187:drug effects / Q000235:genetics
chemical_list: C064976:O(6)-benzylguanine; D006147:Guanine; D019853:O(6)-Methylguanine-DNA Methyltransferase; D019883:Alkyl and Aryl Transferases; C050956:DNA alkyltransferase