Use of a herpes thymidine kinase/neomycin phosphotransferase chimeric genefor metabolic suicide gene transfer

Metabolic suicide gene transfer is widely applied for gene therapy of cance r, and retroviral vectors expressing the herpes simplex virus thymidine kin ase (HSV-tk) gene are commonly used in clinical trials. Most of these vecto rs contain positive selectable markers that undoubtedly facilitate the dete rmination of viral titer and the identification of high-titer producer clon es. However, the presence of additional transcriptional units may result in reduced expression of the gene of interest. The use of fusion genes expres sing bifunctional proteins may help to overcome this problem. We have const ructed a retroviral vector carrying the TNFUS69 chimeric gene, which origin ates From the fusion of the HSV-tk and neomycin phosphotransferase II genes , and evaluated the functional expression of the encoded fusion protein. In vitro, expression of the fusion gene conferred to target cells both resist ance to neomycin and selective sensitivity to the antiherpetic drugs gancic lovir and (E)-5-(2-bromovinyl)-2'-deoxyuridine. Cells transduced with the f usion gene, however, showed reduced ability to phosphorylate ganciclovir co mpared with cells expressing the native HSV-tk. Therefore, although the fus ion gene may be used as a constituent of retroviral cassettes for positive and negative selection in vitro, its usefulness for suicide gene transfer a pplications in vivo may depend upon the possibility of using (E)-5-(2-bromo vinyl)-2'-deoxyuridine in a clinical context.