Human nerve growth factor receptor and cytosine deaminase fusion genes.
Cytosine deaminase (CD) converts 5-fluorocytosine (5-FC) to the toxic metabolite 5-fluorouracil (5-FU), and has been investigated extensively as a potential tool for selective cellular eradication. In this paper, genetic constructs were designed to express the CD enzyme fused to the transmembrane and extracellular domains of the human nerve growth factor receptor (NGFR), thus allowing for positive identification of transduced cells by flow cytometry and positive selection by magnetic bead technology. Constructs were designed to encode a [Gly(4)Ser](2) flexible linker between the nucleic acid coding sequences for the NGFR and CD genes. Retroviral vectors constructed with wild-type CD and NG/CD fusion genes were used to transduce 3T3 fibroblasts and the human T cell line CEM. The function of CD fusion genes was comparable to that of wild-type genes as determined in cytotoxicity assays. By flow cytometry, the NGFR antigen was detectable after expression of the fusion gene derived from either Escherichia coli (NG/CDe) or Saccharomyces cerevisiae (NG/CDs), but the greatest antigen density was observed in cells transduced with the NG/CDs vector. Similarly, superior 5-FC sensitivity was observed with NG/CDs fusion gene in both murine fibroblasts and human T cells. In addition, CEM cells expressing NG/CDs were more efficiently eliminated in vivo. Engineering of cells utilizing the chimeric NG/CD genes provides a new modality in gene therapy allowing positive and negative selection using a single protein-coding sequence.
Lewis
Victor A
VA
Program in Blood and Marrow Transplantation and Department of Pediatrics, University of Minnesota, Minneapolis, MN 55455, USA.
Basso
Lisa
L
Blake
Noel
N
Salo
Jill
J
Lund
Troy C
TC
McIvor
R Scott
RS
Orchard
Paul J
PJ
D000818:Animals; D017209:Apoptosis; D019677:Artificial Gene Fusion; D002470:Cell Survival; D002478:Cells, Cultured; D043525:Cytosine Deaminase; D004926:Escherichia coli / Q000235:genetics; D005434:Flow Cytometry; D005437:Flucytosine / Q000494:pharmacology; D015316:Genetic Therapy; D005822:Genetic Vectors; D006801:Humans; D018189:Immunomagnetic Separation; D015458:Leukemia, T-Cell / Q000628:therapy; D051379:Mice; D009700:Nucleoside Deaminases / Q000235:genetics*; D010957:Plasmids; D011401:Promoter Regions, Genetic; D020800:Receptor, Nerve Growth Factor / Q000032:analysis / Q000235:genetics* / Q000276:immunology; D012190:Retroviridae / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics; D013539:Simian virus 40 / Q000235:genetics; D014161:Transduction, Genetic; D014407:Tumor Cells, Cultured
D020800:Receptor, Nerve Growth Factor; D005437:Flucytosine; D009700:Nucleoside Deaminases; D043525:Cytosine Deaminase