Use of a Herpes Thymidine Kinase/Neomycin Phosphotransferase Chimeric Gene for Metabolic Suicide Gene Transfer
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© 2000 Nature America, Inc. 0929-1903/00/$15.00/ϩ0 www.nature.com/cgt Use of a herpes thymidine kinase/neomycin phosphotransferase chimeric gene for metabolic suicide gene transfer Fabio Candotti,1 Riad Agbaria,2 Craig A. Mullen,1 Renaud Touraine,1 Jan Balzarini,3 David G. Johns,2 and R. Michael Blaese1 1Clinical Gene Therapy Branch, National Human Genome Research Institute, and 2Laboratory of Medicinal Chemistry, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892; and 3Rega Institute for Medical Research, Katholieke Universiteit Leuven, Leuven, Belgium. Metabolic suicide gene transfer is widely applied for gene therapy of cancer, and retroviral vectors expressing the herpes simplex virus thymidine kinase (HSV-tk) gene are commonly used in clinical trials. Most of these vectors contain positive selectable markers that undoubtedly facilitate the determination of viral titer and the identification of high-titer producer clones. However, the presence of additional transcriptional units may result in reduced expression of the gene of interest. The use of fusion genes expressing bifunctional proteins may help to overcome this problem. We have constructed a retroviral vector carrying the TNFUS69 chimeric gene, which originates 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 resistance to neomycin and selective sensitivity to the antiherpetic drugs ganciclovir and (E)-5-(2-bromovinyl)-2Ј-deoxyuridine. Cells transduced with the fusion gene, however, showed reduced ability to phosphorylate ganciclovir compared with cells expressing the native HSV-tk. Therefore, although the fusion gene may be used as a constituent of retroviral cassettes for positive and negative selection in vitro, its usefulness for suicide gene transfer applications in vivo may depend upon the possibility of using (E)-5-(2-bromovinyl)-2Ј- deoxyuridine in a clinical context. Cancer Gene Therapy (2000) 7, 574–580 .Key words: Retroviral vector; gene therapy; suicide gene; ganciclovir; (E)-5-(2-bromovinyl)-2-deoxyuridine n the last several years, “suicide” gene transfer sys- tives. GCV-TP, an analog of guanosine-TP, is the ulti- Items have been extensively investigated as a therapeu- mate toxic metabolite of the prodrug that inhibits cellu- tic approach of cancer (reviewed in Refs. 1 and 2). These lar DNA polymerase and DNA synthesis, leading to cell systems are based on the transfer of genes encoding for death.9,10 Preclinical models have demonstrated signifi- non-mammalian metabolic enzymes, which confer a cant antitumoral activity against several tumors (re- novel and selective chemosensitivity to the target cells by viewed in Ref. 11), leading to a series of clinical gene providing intracellular conversion of a relatively non- therapy trials that are currently testing the clinical toxic prodrug to a toxic metabolite. Several suicide efficacy of the HSV-tk/GCV system as a cancer treat- gene/prodrug systems are currently under investiga- ment. Preliminary results have demonstrated that retro- 3–8 tion. Among these, the metabolic suicide strategy viral and adenoviral vectors can be used in vivo to confer based on the use of the herpes simplex virus type 1 GCV sensitivity to cancer cells,12,13 but have also indi- thymidine kinase (HSV-tk) in association with ganciclo- cated the need to improve the overall efficacy of this vir (GCV) is probably the most widely studied. In this suicide system. system, the HSV-tk provides specific phosphorylation of In vitro studies of other antiherpetic compounds that GCV to GCV monophosphate; other cellular kinases are potentially more potent than GCV in arresting the complete the subsequent phosphorylation steps to growth of HSV-tk-transfected cells are underway, diphosphate and triphosphate (GCV-TP) GCV deriva- searching for more and more effective tools for the selective elimination of cancer cells. Among the possible alternative drugs, (E)-5-(2-bromovinyl)-2Ј-deoxyuridine Received May 10, 1999; accepted August 29, 1999. (BVdU) was recently demonstrated to be a very efficient Address correspondence and reprint requests to Dr. Fabio Candotti, Clinical Gene Therapy Branch, National Human Genome Research substrate for HSV-tk; in addition, it has a strong cyto- Institute, Building 10, Room 10C103, National Institutes of Health, 10 static activity against HSV-tk-transfected murine mam- Center Drive MSC 1851, Bethesda, MD 20892-1851. E-mail address: mary carcinoma cells in vitro. The mechanism of action [email protected] of BVdU differs from that described for GCV in that the 574 Cancer Gene Therapy, Vol 7, No 4, 2000: pp 574–580 CANDOTTI, AGBARIA, MULLEN, ET AL: HSV-tk/neo FUSION GENE FOR RETROVIRAL VECTORS 575 principal toxic effect induced by BVdU is the inhibition Dulbecco’s modified Eagle’s medium (Biofluids, Rockville, of the cellular thymidylate synthase by the BVdU 5Ј- Md) supplemented with 10% fetal calf sera and 2 mM L- monophosphate generated by the phosphorylation of glutamine (D10); MC 38 and MCA 205 cells were cultured in BVdU by HSV-tk.10,14 RPMI 1640 (Life Technologies, Rockville, Md) also supple- In addition to their important potential therapeutic mented with 10% fetal calf sera and 2 mM L-glutamine (R10). applications, suicide genes could be also considered as a Transfections and transductions safety feature for those gene transfer methods (retrovi- rus- or adeno-associated virus-based vectors) that in- Retroviral producer cell lines were obtained by conventional volve integration of foreign sequences into the host calcium phosphate coprecipitation of 20 g of plasmid DNA genome. The presence of a negative selection system in on PA317 packaging cell lines. Cells producing the BTK and these vectors would be highly beneficial in the unfortu- BTNfus retroviral vectors were obtained by selecting trans- fected cells in the neomycin analog G418 (Geneticin; Life nate case of an insertional mutagenic event. However, Technologies; 1.0 mg/mL of active metabolite). Titers of BTK the routine inclusion of a suicide gene as a regular and BTNfus retroviral supernatants were assessed by transduc- constituent in the vector cassette would most likely affect tion of NIH 3T3 cells followed by puromycin selection (2.5 the expression of other essential vector components such g/mL; Calbiochem, La Jolla, Calif) and were estimated to be as a positive selectable trait (e.g., neomycin resistance 5 ϫ 105 and 2 ϫ 105 colony-forming units/mL, respectively. gene (neo)) as well as the exogenous (therapeutic) gene Viral particle-containing supernatants were used for transduc- of interest. One possible way to overcome this drawback tions of target cells in the presence of 5 g/mL protamine (Sigma). After transduction, cells were subjected to selection is offered by the utilization of fusion genes that can provide expression of multifunctional proteins, avoiding with 2.5 g/mL puromycin. Cells transduced with the LNL6 the problems created by the coexistence of multiple vector were selected in 1 mg/mL G418. transcriptional units. In the present work, we have Assessment of neo gene expression and explored the applicability of the TNFUS69 chimeric biological activity gene,15 which is created by the fusion of HSV-tk and neo genes, in a retroviral-based suicide gene transfer system All experiments were performed using MC 38 cells unmodified by evaluating the extent of the chemosensitivity induced or subjected to transduction with either the BTK or BTNfus in target cells to GCV and BVdU. vectors. To evaluate the amount of neomycin phosphotransferase II (NPT II) produced by the neo and the HSV-tk/neo fusion MATERIALS AND METHODS genes, we used an enzyme-linked immunosorbent assay (ELISA) based on a rabbit polyclonal antibody specific to the Chemicals NPT II protein encoded by the Escherichia coli Tn5 (5 3 GCV was purchased from Syntex Laboratories (Palo Alto, Prime 3 Prime, Boulder, Colo) according to the manufactur- Calif); BVdU was either obtained from the Rega Institute for er’s specifications. In addition, NPT II enzymatic activity was Medical Research (Leuven, Belgium) or purchased from determined by in situ phosphorylation of the antibiotic kana- Sigma (St. Louis, Mo). [methyl-3H]thymidine ([3H]Thd) (spe- mycin after polyacrylamide gel electrophoresis of crude cell 20 cific activity 6.7 Ci/mmol) was obtained from DuPont-New extracts as described previously. Detection and quantifica- England Nuclear Research Products (Boston, Mass); tion of radiolabeled phosphorylated kanamycin were per- [8-3H]GCV ([3H]GCV) (specific activity 22 Ci/mmol) and formed using a PhosphorImager SI (Molecular Dynamics, [2Ј-3H]BVdU ([3H]BVdU) (3 Ci/mmol) were provided by Sunnyvale, Calif). Moravek Biochemicals (Brea, Calif). To evaluate the neo biological effect in vitro, unmodified or transduced MC 38 cells were plated (104 cells/well) in tripli- Plasmids and cell lines cates in 96-well plates, exposed to different concentrations of G418, and incubated at 37°C in 5% CO for 24 hours. Cell The TNFUS69 plasmid containing the fused sequences of 2 15 proliferation was estimated at the end of the treatment period HSV-tk and neo genes has already been described in detail as a function of radioactive thymidine incorporation into and was a gift of Dr. R. Kucherlapati (Department of Molec- cellular DNA after a 6-hour pulse with [3H]Thd (0.5 Ci/well). ular Genetics, Albert Einstein College of Medicine, Bronx, Results are expressed as the percentage of the cell prolifera- NY); a native copy of HSV-tk was derived from the pSPTK1 tion observed in the control (untreated) population. plasmid, which was a gift of Genetic Therapy Inc. (Gaithers- 16 burg, Md). The pBabe-puro retroviral vector was kindly HSV-tk functional activity provided by H. Land (Imperial Cancer Research Fund, Lon- don, UK) and used to subclone the HSV-tk and TNFU69 The kinase activity provided by the native HSV-tk and the genes to obtain the BTK and BTNfus constructs, respectively.