Engineered human tmpk/AZT as a novel enzyme/prodrug axis for suicide gene therapy.
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.
Sato
Takeya
T
Division of Stem Cell and Developmental Biology, Ontario Cancer Institute, Toronto, Ontario, Canada.
Neschadim
Anton
A
Konrad
Manfred
M
Fowler
Daniel H
DH
Lavie
Arnon
A
Medin
Jeffrey A
JA
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
D018941:Antigens, CD19; D000963:Antimetabolites; D011355:Prodrugs; D015215:Zidovudine; D009703:Nucleoside-Phosphate Kinase; C020093:dTMP kinase; D053148:Caspase 3