title: Engineered human tmpk/AZT as a novel enzyme/prodrug axis for suicide gene therapy.
abstract: Gene therapy and stem cell transplantation safety could be enhanced by control over the fate of therapeutic cells. Suicide gene therapy uses enzymes that convert prodrugs to cytotoxic entities; however, heterologous moieties with poor kinetics are employed. We describe a novel enzyme/prodrug combination for selectively inducing apoptosis in lentiviral vector-transduced cells. Rationally designed variants of human thymidylate kinase (tmpk) that effectively phosphorylate 3'-azido-3'-deoxythymidine (AZT) were efficiently delivered. Transduced Jurkat cell lines were eliminated by AZT. We demonstrate that this schema targeted both dividing and non-dividing cells, with a novel killing mechanism involving apoptosis induction via disruption of the mitochondrial inner membrane potential and activation of caspase-3. Primary murine and human T cells were also transduced and responded to AZT. Furthermore, low-dose AZT administration to non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice injected with transduced K562 cells suppressed tumor growth. This novel suicide gene therapy approach can thus be integrated as a safety switch into therapeutic vectors.
authors: Division of Stem Cell and Developmental Biology, Ontario Cancer Institute, Toronto, Ontario, Canada.
authors: Takeya
authors: T
authors: Sato
authors: Anton
authors: A
authors: Neschadim
authors: Manfred
authors: M
authors: Konrad
authors: Daniel H
authors: DH
authors: Fowler
authors: Arnon
authors: A
authors: Lavie
authors: Jeffrey A
authors: JA
authors: Medin
mesh_terms: D000818:Animals; D018941:Antigens, CD19 / Q000378:metabolism; D000963:Antimetabolites / Q000378:metabolism / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects / Q000235:genetics; D015153:Blotting, Western; D053148:Caspase 3 / Q000378:metabolism; D045744:Cell Line, Tumor; D002851:Chromatography, High Pressure Liquid; D005260:Female; D005434:Flow Cytometry; D041722:Genes, Transgenic, Suicide; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D019169:Jurkat Cells; D020014:K562 Cells; D008297:Male; D051379:Mice; D016688:Mice, Inbred NOD; D016513:Mice, SCID; D009703:Nucleoside-Phosphate Kinase / Q000235:genetics* / Q000378:metabolism; D011355:Prodrugs / Q000378:metabolism / Q000494:pharmacology* / Q000627:therapeutic use; D013601:T-Lymphocytes / Q000276:immunology; D014162:Transfection; D015215:Zidovudine / Q000378:metabolism / Q000494:pharmacology* / Q000627:therapeutic use
chemical_list: D018941:Antigens, CD19; D000963:Antimetabolites; D011355:Prodrugs; D015215:Zidovudine; D009703:Nucleoside-Phosphate Kinase; C020093:dTMP kinase; D053148:Caspase 3