1090-0233/01/030269 + 09 $35.00/0 © 2001 Harcourt Publishers Ltd INTRODUCTION Gene therapy, at its simplest level, is the introduc- tion of genes into a cell to ameliorate a disease process. One form of gene therapy used to target the toxicity of cytotoxic drugs is gene directed enzyme prodrug therapy (GDEPT). In this type of therapy, the gene encoding an enzyme is intro- duced into target cells with therapeutic intent. This enzyme can convert a non-toxic prodrug to its toxic form. The non-toxic prodrug is then administered systemically, and converted into its active, toxic form by the drug-metabolizing enzyme expressed in the transformed cells only. However, it is impos- sible to introduce the gene into every cell of a target tumour or tissue. This limitation is to some extent overcome by the bystander effect, in which adjacent cells which have not been transformed with the gene are also subject to cytotoxicity due to local effects of toxic metabolites, and in some cases, immune activation. The bystander effect makes this form of gene therapy especially suitable for the treatment of cancer, as transformation of a relatively small proportion of cells within a tumour can result in a dramatic response. The E. coli nitroreductase/CB1954 system has recently emerged as a potent GDEPT system (Bailey & Hart, 1997). E. coli nitroreductase is a bacterial enzyme that can be expressed without toxicity in mammalian cells from a number of species. CB1954 [5-(aziridin-1-yl)-2,4-dinitrobenz- amide] was originally developed as a cytotoxic drug in its own right, but was only active in rat cell lines later found to possess an endogenous enzyme capa- ble of activation of the compound (Cobb et al., Correspondence to: L. Blackwood, Molecular Therapeutics Research Group, Small Animal Clinical Studies, University of Glasgow, Bearsden Road, Glasgow G61 7QH, UK. Tel.: +44 141 330 5700; Fax: +44 141 942 7215; E-mail: L.Blackwood@vet.gla.ac.uk E. coli Nitroreductase/CB1954: In Vitro Studies into a Potential System for Feline Cancer Gene Therapy L. BLACKWOOD,* P. J. O’SHAUGHNESSY,† S. W. J. REID,‡ and D. J. ARGYLE* *Molecular Therapeutics Research Group, Small Animal Clinical Studies, † Department of Veterinary Physiology, ‡ Veterinary Informatics and Epidemiology, Faculty of Veterinary Medicine, University of Glasgow, Bearsden Road, Glasgow G61 1QH, UK SUMMARY Investigations were carried out to identify a suitable prodrug activating system for feline gene therapy with the eventual aim of treating feline thyroid disease and feline neoplasia. The E. coli nitroreductase (NTR)/CB1954 prodrug activating system was evaluated in vitro in feline cells by transient transfection with a nitroreductase expressing construct and subsequent treatment with the prodrug CB1954. The feline cells successfully expressed E. coli nitroreductase, which was able to activate the prodrug CB1954 resulting in cyto- toxicity to both transformed and adjacent cells (a bystander effect) in vitro. In the absence of nitroreductase, CB1954 was non-toxic to feline cells. In addition, the nitroreductase gene was expressed in rat thyroid cells under the control of the cell type specific feline thyroglobulin promoter. This paper demonstrates that the E. coli nitroreductase/CB1954 system may be suitable for in vivo feline gene therapy, and further investiga- tions are warranted. KEYWORDS: Prodrug activation; nitroreductase; feline; gene therapy. The Veterinary Journal 2001, 161, 269–279 doi: 10.1053/tvjl.2000.0557, available online at http://www.idealibrary.com on © 2001 Harcourt Publishers Ltd