title: Coexpression of the uracil phosphoribosyltransferase gene with a chimeric human nerve growth factor receptor/cytosine deaminase fusion gene, using a single retroviral vector, augments cytotoxicity of transduced human T cells exposed to 5-fluorocytosine.
abstract: Donor T lymphocytes genetically engineered to express a "suicide gene" to facilitate negative selection represent a promising strategy for the management of graft-versus-host disease occurring after allogeneic hematopoietic cell transplantation (HCT). For this purpose, the herpes simplex virus thymidine kinase (HSV-tk) gene, although well studied, has limitations. Cytosine deaminase (CD), an alternative gene for negative selection, converts 5-fluorocytosine (5-FC) to the toxic metabolite 5-fluorouracil (5-FU). Sensitivity of cells to 5-FU can be further increased by expression of uracil phosphoribosyltransferase (UPRT), which catalyzes the conversion of 5-FU to 5-fluorouridine monophosphate. By using a chimeric gene (NG/CD) expressing the truncated human nerve growth factor receptor (NGFR) for positive selection fused to the Saccharomyces cerevisiae CD gene, we investigated strategies to achieve optimal T cell eradication by CD and UPRT expression, utilizing a single retroviral vector. Three vector strategies were compared on the basis of NGFR expression by flow cytometry, western analysis, and enzymatic activity. A construct (NG/CDiU) expressing UPRT and NG/CD, using a bicistronic message, provided the greatest UPRT activity and killing, reducing the lethal dose of 5-FC sufficient to eradicate 90% of cells from 38.7 microg/ml (300 microM) (NG/CD expression alone) to 0.13 microg/ml (1 microM). This approach provides an effective alternative to the HSV-tk system for eradication of donor T lymphocytes after allogeneic HCT.
authors.affiliation: Centre for Children's Cancer and Blood Disorders, Sydney Children's Hospital, Sydney, NSW 2031, Australia.
authors.forename: Tracey A
authors.initials: TA
authors.lastname: O'Brien
authors.forename: Dong T
authors.initials: DT
authors.lastname: Tuong
authors.forename: Lisa M
authors.initials: LM
authors.lastname: Basso
authors.forename: R Scott
authors.initials: RS
authors.lastname: McIvor
authors.forename: Paul J
authors.initials: PJ
authors.lastname: Orchard
mesh_terms: D002460:Cell Line; D049109:Cell Proliferation; D043525:Cytosine Deaminase / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D005437:Flucytosine / Q000633:toxicity*; D041722:Genes, Transgenic, Suicide; D005822:Genetic Vectors; D006801:Humans; D010430:Pentosyltransferases / Q000235:genetics* / Q000378:metabolism / Q000633:toxicity; D011743:Pyrimidines / Q000378:metabolism / Q000633:toxicity; D020800:Receptor, Nerve Growth Factor / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000633:toxicity; D018626:Retroelements / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics; D013601:T-Lymphocytes; D014161:Transduction, Genetic / Q000379:methods
chemical_list: D011743:Pyrimidines; D020800:Receptor, Nerve Growth Factor; D011993:Recombinant Fusion Proteins; D018626:Retroelements; C029269:5-fluoropyrimidine; D005437:Flucytosine; D010430:Pentosyltransferases; C023269:uracil phosphoribosyltransferase; D043525:Cytosine Deaminase