Use of a Herpes Thymidine Kinase/Neomycin Phosphotransferase Chimeric Gene for Metabolic Suicide Gene Transfer

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 (retrovirus- 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 transfected 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. TNFUS69 genes was investigated by high performance liquid The retroviral vector pLNL6 (encoding only for neo)17 and the chromatography (HPLC) analysis of [3H]Thd, [3H]GCV, and PA31718 packaging cell line were a gift of A. D. Miller (Fred [3H]BVdU phosphorylated metabolites produced by retrovi- Hutchinson Cancer Research Center, Seattle, Wash); MC 38 rally transduced cells. Experiments assessing the production of and MCA 205 are murine methylcholanthrene-induced colon [3H]Thd phosphorylated metabolites were performed using Ϫ adenocarcinoma and fibrosarcoma cell lines, respectively, L-M(tk ) cells, unmodified or transduced with the BTK or which originated from a C57BL/6 female mouse;19 both were BTNfus vectors. The generation of [3H]GCV and [3H]BVdU kindly donated by S. A. Rosenberg (Surgery Branch, National phosphorylated metabolites was studied in MC 38 cells, also Ϫ Cancer Institute); L-M cellular TK-negative cells (L-M(tk )) unmodified, or after transduction with BTK or BTNfus. Cells were obtained from the American Type Culture Collection (106/mL) were cultured at 37°C in 5% CO in the presence of Ϫ 2 (Manassas, Va). PA317 and L-M(tk ) cells were maintained in [3H]Thd, [3H]GCV, or [3H]BVdU (2 ␮Ci/mL) for 15 minutes,

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24 hours, and 5 hours, respectively. Cells were then harvested, and pellets were extracted with 66% methanol and heated at 95°C for 2 minutes. After centrifugation, the clarified supernatants were evaporated under vacuum and redissolved in water. Reconstituted samples were subjected to HPLC using a Partisphere 5-SAX column (Whatman, Clifton, NJ) as described previously.21 Eluted, radiolabeled, phosphorylated thymidine, GCV, and BVdU metabolites were monitored with an in-line radioactivity flow detector and quantitated from the areas under the respective elution peaks. The precipitates of the methanol extractions (containing [3H]Thd, [3H]GCV, or Figure 1. Schematic representation of the BTK and BTNfus retro- [3H]BVdU incorporated into methanol insoluble cell material viral vectors. The HSV-tk gene and the TNFUS69 fusion construct15 (e.g., nucleic acids)) were washed three times with 66% cold were inserted into pBabe-puro16 under the transcriptional control of methanol and further assayed for radioactivity.10 the Moloney murine leukemia virus long tandem terminal repeat, using the BamHI-EcoRI sites. In both vectors, the expression of the HSV-tk biological activity drug resistance gene pac (puromycin N-acetyl transferase) is driven ␺ Ϫ by the internal simian virus 40 early region promoter (SV40p). , HAT selection medium survival. L-M(tk ) cells were trans- packaging signal. duced with either the BTK or BTNfus vectors. A total of 500 cells were then plated in triplicate in 6-well plates, maintained in D10 or in Dulbecco’s modified Eagle’s medium supple- Furthermore, the two ATG start codons were placed in mented with 100 ␮M hypoxanthine, 0.4 ␮M aminopterin, and ␮ the context of a “Kozak box” (GCCGCCGCCATGG), 16 M thymidine (HAT media supplement; Sigma), and known as a favorable sequence for translation initiation incubated at 37°C in 5% CO2. After 7–10 days, colonies were 23 fixed with methanol, stained with methylene blue, and counted. in eukaryotic organisms. Desired changes of the nucle- otide sequences were confirmed by sequencing of both Inhibition of DNA synthesis and metabolic activity by GCV and BTK and BTNfus vectors. BVdU. MC 38 cells transduced either with the BTK or BTNfus ϫ 3 4 vectors were plated in 96-well plates (5 10 or 10 cells/well) The BTNfus vector confers neomycin resistance to in triplicates, treated with different concentrations of GCV or transduced cells BVdU, and incubated at 37°C in 5% CO2 for 72 hours. Cell viability at the end of the treatment was assessed as function of We first analyzed the efficacy of the fusion gene to Ϫ their ability to reduce 3-(4,5-dimethylthiazol-2-yl) 2,5-diphe- provide expression of NPT II. Comparison between nyltetrazolium bromide (MTT) according to the MTT colori- LNL6- and BTNfus-transduced MC 38 cells indicated metric assay.22 Briefly, after 69 hours of incubation, 10 ␮Lofa that the fusion gene was generating a lower amount of 10 mg/mL solution of MTT (Sigma) was added to the culture ϳ medium, followed by further incubation for 3 hours. After NPT II protein, as determined by ELISA ( 33% of the removing the supernatants, 100 ␮L of dimethylsulfoxide was control LNL6-transduced cells, data not shown), and a added to each well to dissolve the water-insoluble formazan decreased kanamycin phosphorylation activity (ϳ21% of crystals. Optical density was determined with a microtiter plate the control, Fig 2A). Surprisingly, no difference in the reader using a 560- to 650-nm dual wavelength detector. All sensitivity to the neomycin analog G418 was demon- experiments were repeated at least twice. strated in a [3H]Thd DNA incorporation assay. Cells Inhibition of cell proliferation by GCV and BVdU. MC 38 and transduced with the fusion gene were just as resistant to MCA 205 cells transduced with either the BTK or BTNfus inhibition by G418 as those carrying the original neo vectors were plated to 6-well plates (2.5 ϫ 105 cell/well) and gene, even in the presence of relatively high drug allowed to grow overnight at 37°C in 5% CO2; cells were then concentrations (Fig 2B). Furthermore, we could easily exposed to GCV or BVdU at concentrations from 0.0005 ␮M recover G418-resistant colonies from cells retrovirally to 200 ␮M. After 48 hours of incubation, cells were counted in transduced with the BTNfus vector, thus allowing us to a Coulter counter (Coulter Electronics, Luton, Beds, UK). The conclude that the fusion gene, when inserted into retro- drug concentration required to inhibit cell proliferation by viral vectors, is able to provide efficient positive selection 50% was defined as the IC . 50 despite a somewhat lower NPT II expression. Statistical analysis Thymidine, GCV, and BVdU phosphorylation in Analysis of significant differences among data groups was BTNfus-transduced cells performed using the Student’s t test for unpaired observations. To assess whether the BTNfus retroviral vector was able to confer HSV-tk activity to target cells, we performed RESULTS HPLC analysis of Thd, GCV, and BVdU phosphorylated metabolites generated in transduced cells. The Retroviral vector construction results of this analysis are shown in Table 1. Compared Using the polymerase chain reaction technique, the with the fusion gene, cells transduced with the HSV-tk restriction enzyme sites BamHI (at the 5Ј) and EcoRI (at gene in its native form showed a markedly higher ability the 3Ј) were added to both TNFUS69 and HSV-tk genes to phosphorylate Thd and GCV. Surprisingly however, to facilitate subcloning into the pBabe-puro cassette to cells transduced with the BTNfus vector were slightly obtain BTK and BTNfus retroviral vectors (Fig 1). more efficient than cells expressing native HSV-tk in the

Cancer Gene Therapy, Vol 7, No 4, 2000 CANDOTTI, AGBARIA, MULLEN, ET AL: HSV-tk/neo FUSION GENE FOR RETROVIRAL VECTORS 577

Table 1. HPLC Analysis of Phosphorylated Thymidine, GCV, and BVdU Metabolites in Cells Transduced with BTK or BTNfus Vectors

[3H]Thd (disintegrations per minute/106 cells)

Cell line TP* Bound†

L-M(tkϪ) 0.0 97.5 L-M(tkϪ) BTK 4,666.5 19,075.5 L-M(tkϪ) BTNfus 387.0 3,118.0

[3H]GCV (pmol/ 106 cells) [3H]BVdU (pmol/106 cells)

Cell line TP Bound Monophosphate‡ Bound

MC 38 0.0 9.6 0.8 2.3 MC 38 BTK 527.3 41.3 58.6 16.2 MC 38 BTNfus 13.8 20.1 77.5 11.6 *Cytoplasmic (methanol-soluble) TP metabolites. †Methanol-insoluble extracts (nucleic acid-bound radioactivity). ‡Cytoplasmic monophosphate metabolites.

vector, while in the case of BTNfus-transduced cells, ϳ55% of colonies were HAT-resistant (Fig 3). In addition, MC 38 cells transduced with the BTNfus vector showed metabolic inhibition when treated with GCV or BVdU (Fig 4), thus indicating the effective generation of phosphorylated GCV and BVdU metabolites. Interestingly, in GCV-treated cells, we observed signiﬁcant differences between the effects of the native HSV-tk and the TNFUS69 chimeric gene. The reduced efﬁcacy of the latter in providing metabolic suicide effects in the presence of GCV was less important at higher drug concentrations, but never negligible (Fig 4A). In contrast, BVdU treatment resulted in similar toxicity in both BTK- and BTNfus-transduced cells and

Figure 2. A: In situ kanamycin phosphorylation activity of MC 38 cells unmodified or transduced with the LNL6 or BTNfus retroviral vectors. The total signal intensities of indicated areas are as follows: a1 ϭ 172,999; a2 ϭ 37,690; a3 ϭ 23,202. Band “a3” probably represents the protein aggregate or enzymatically active degrada- tion product of the TK-NEO fusion protein.20 B: In vitro neomycin resistance in MC 38 cells expressing the neo gene (MC 38 LNL6) or the TNFUS69 fusion gene (MC 38 BTNfus). Ability to proliferate in the presence of various concentrations of G418 is expressed as the percentage of incorporated radioactivity ([3H]Thd) in regular culture medium. Observations represent the mean values of triplicate cultures. Error bars indicate SDs (not visible if Ͻ3%). phosphorylation of BVdU. We subsequently evaluated the biological activity of the fusion gene in a series of functional assays indicative of Thd, GCV, and BVdU phosphorylation. Expression of the fusion gene resulted in efficient Thd phosphorylation, as demonstrated by the Ϫ finding that L-M (tk ) cells transduced with the BTNfus Figure 3. Clonogenic potential of BTK- or BTNfus-transduced retroviral vector were only slightly less efficient in form- L-M(tkϪ) cells in HAT selection medium. Values are expressed as ing colonies in HAT selection medium compared with “% of unselected control” given by the following ratio: average BTK-transduced cells. On average, ϳ69% of colonies number of colonies in HAT medium/average number of colonies in were rescued if cells were transduced with the BTK regular culture medium.

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Figure 4. Metabolic inhibition induced by GCV (A) and BVdU (B) in MC 38 cells transduced with BTK or BTNfus retroviral vectors. Results (mean and SD of triplicates samples) are expressed as the percentage of MTT reduction observed in the absence of GCV or in the absence of BVdU. If SDs PϽ,ءءء .are Ͻ3%, they are not visible .P Ͻ .01; F, P Ͻ .05 ء ;P Ͻ .005 ,ءء ;0005.

showed significant superiority of the fusion gene at lower acterization of retroviral producer cells in the absence of BVdU concentrations (Fig 4B). positive selection is cumbersome and extremely time Determination of the IC50 values of GCV and BVdU consuming. However, the occurrence of transcriptional was performed in BTK and BTNfus MC 38 and MC 205 interference between multiple expression units within transduced cells. The results obtained are reported in the same construct has been demonstrated in many Table 2 and are in line with the in vitro sensitivity assays. systems25–27 and justifies the current common efforts to In particular, the reduced sensitivity of BTNfus-trans- develop “simplified,” single-gene vectors. Clearly, the duced cells to GCV (compared with BTK-transduced use of bifunctional fusion genes would represent an counterparts) was confirmed in both cell lines, thus advantageous solution to these problems. We have taken demonstrating that this finding was independent of cell advantage of the TNFUS69 plasmid,15 from which the type. In addition, the IC50 values for BVdU were similar HSV-tk/neo fusion gene was excised and subcloned in a for BTNfus- and BTK-transduced cells, indicating that retroviral cassette under the transcriptional control of the suicide function of the fusion gene in combination the Moloney long tandem terminal repeat, in the context with BVdU could be considered to be comparable with of a Kozak sequence. We show that the resulting retro- that of the native HSV-tk gene. viral vector, BTNfus, can efficiently confer G418-resistance to transduced cells and that, therefore, the fusion gene can be used as a regular positive selectable marker. DISCUSSION Quantitation of the amount of NPT II protein and its ability to phosphorylate kanamycin indicated that there More than 30 cancer gene therapy clinical trials have were lower levels of NPT II in cells transduced with the been proposed that use retroviral vectors expressing the 24 fusion gene compared with cells expressing native neo. suicide gene HSV-tk. Most of these vectors also con- However, the biological activity of the NPT II compo- tain a selectable marker, because production and char- nent of the fusion protein encoded by TNFUS69 was substantial, because BTNfus-transduced cells were as Table 2. Inhibitory Effects of GCV and BVdU on BTK- or resistant to the inhibitory effects of G418 on cell prolif- BTNfus-Transduced Cells eration as LNL6-transduced control cells. The reduced ␮ IC50 ( M)* ability of the anti-NPT II antiserum used in the ELISA to recognize the NPT II “new” structure as part of a Cell line GCV BVdU fusion protein, as well as a potentially decreased affinity MC 38 200 5–10 of the TK-NEO protein for kanamycin, are possible MC 38 BTK 0.05 0.01–0.05 explanations for these apparent discrepancies. Never- MC 38 BTNfus 0.5–1 0.001–0.005 theless, our data clearly demonstrate that the fusion gene can provide useful positive selection. MC 205 100–200 50–100 The reduced ability of BTNfus-transduced cells to MC 205 BTK 0.1–0.25 0.01–0.05 generate phosphorylated thymidine and GCV metabo- MC 205 BTNfus 25 0.05 lites could similarly be ascribed to the modifications

*Drug concentration sufﬁcient to achieve IC50 (see Materials and made to the HSV-tk carboxyl terminus to originate the Methods). TNFUS69 gene15 or to a lower afﬁnity (or catalytic

Cancer Gene Therapy, Vol 7, No 4, 2000 CANDOTTI, AGBARIA, MULLEN, ET AL: HSV-tk/neo FUSION GENE FOR RETROVIRAL VECTORS 579 efficacy) of the TK component of the fusion protein for animal models and/or in human studies. Among these the substrates thymidine and GCV. Verification of this drugs, BVdU was found to be at least 100-fold more supposition would require further biochemical studies potent than GCV in inhibiting the proliferation of involving enzyme purification, which are beyond the HSV-tk-transformed cells.10,31 scope of this work. Although reduced when compared In our studies, in contrast to GCV, BVdU was as with HSV-tk-carrying cells, the phosphorylation of Thd efficiently phosphorylated by the fusion gene-transduced and GCV provided by the fusion gene was biologically cells as it was by HSV-tk-transduced control cells. This active in vitro, allowing mutant cellular TK-negative cells observation, along with the remarkable efficiency of to survive HAT selection and conferring GCV sensitivity BVdU in inhibiting the in vitro proliferation of BTNfus- to BTNfus-transduced cells. transduced cells, suggests that the use of the fusion gene Taken together, we conclude that the BTNfus retro- in association with BVdU may be useful in metabolic viral vector can be used in vitro for both positive and suicide gene transfer. This is particularly relevant in light negative selection of transduced cells. More commonly of the impaired generation of phosphorylated GCV used approaches to achieve combined expression of metabolites observed in BTNfus-transduced cells. The positive and negative selectable markers by retroviral reduced sensitivity to GCV may result in inefficient transduction are based on the inclusion in the retroviral metabolic suicide effects if this drug is used in combina- cassette of two transcriptional units involving the need tion with the fusion gene for in vivo applications. In this for two different promoter elements. Alternative strate- context, BVdU may represent a better choice. gies rely on the use of internal ribosomal entry site It is hoped that further biochemical studies, now made sequences, which allow the synthesis of two different possible by the recent generation of [3H]-labeled BVdU, protein products from the translation of a bicistronic will help to better define the mechanism of action of mRNA transcribed under the control of a single pro- BVdU and its derivatives, as well as to clarify the real moter. In both of these approaches, the level of expres- possibility of its use for applications in vivo. sion of the two marker genes can vary dramatically as a consequence of promoter interference or competition of gene expression. The use of a bifunctional protein may ACKNOWLEDGMENTS overcome these potential problems because of the presence of a single transcriptional unit driven by a single We thank Drs. R. Kucherlapati, H. Land, A. D. Miller, and promoter and expressing a single gene product. S. A. Rosenberg for kindly donating reagents and Dr. Hiroyuki Other fusion genes containing HSV-tk have been Ishii for helpful discussions and advice. described. Among these, the tgLS(ϩ)HyTK retroviral vector encodes a gene originated by the fusion of the hygromycin resistance gene (Hy) and HSV-tk,28 whereas REFERENCES the pETLGB expression cassette29 was generated by fusing the HSV-tk sequence to that of green fluorescent 1. Blaese RM, Mullen CA, Ramsey WJ. Strategies for gene therapy. Pathol Biol. 1993;41:672–676. protein. Both the HyTK and the TK-green fluorescent 2. Blaese RM, Ishii-Morita H, Mullen CA, et al. In situ protein fusion genes were demonstrated to originate delivery of suicide genes for cancer treatment. Eur J bifunctional fusion proteins with potential applications Cancer. 1994;30A:1190–1193. that were similar to the TK-NEO protein encoded by the 3. Moolten FL, Wells JM. Curability of tumors bearing fusion gene inserted in our BTNfus retroviral vector. In herpes thymidine kinase genes transferred by retroviral addition, an attractive application for the same fusion vectors. J Natl Cancer Inst. 1990;82:297–300. gene used in our studies has been described recently by 4. Huber BE, Richards CA, Krenitsky TA. Retroviral-medi- Bonini et al.30 The fusion gene was introduced in a ated gene therapy for the treatment of hepatocellular retroviral vector used to transduced peripheral blood carcinoma: an innovative approach for cancer therapy. lymphocytes of bone marrow donors as part of a clinical Proc Natl Acad Sci USA. 1991;88:8039–8043. 5. Mullen CA, Kilstrup M, Blaese RM. Transfer of the protocol of allogeneic bone marrow transplantation for bacterial gene for cytosine deaminase to mammalian cells leukemia. These engineered lymphocytes were success- confers lethal sensitivity to 5-fluorocytosine: a negative fully used to treat leukemia relapse after bone marrow selection system. Proc Natl Acad Sci USA. 1992;89:33–37. transplantation and were efficiently eliminated by GCV 6. Mroz PJ, Moolten FL. Retrovirally transduced Escherichia administration when they induced a severe graft-versus- coli gpt genes combine selectability with chemosensitivity host reaction. capable of mediating tumor eradication. Hum Gene Ther. Other very recent results of clinical trials using the 1993;4:589–595. HSV-tk/GCV suicide system for the treatment of cancer 7. Sorscher EJ, Peng S, Bebok Z, Allan PW, Bennett LL Jr, have underlined the need to improve the efficiency of the Parker WB. Tumor cell bystander killing in colonic carci- system.12,13 It is clear that both the delivery of the noma utilizing the Escherichia coli DeoD gene to generate toxic purines. Gene Ther. 1994;1:233–238. HSV-tk-carrying viral vector and the choice and/or the 8. Wei MX, Tamiya T, Chase M, et al. Experimental tumor administration schedule of the active prodrug can be therapy in mice using the cyclophosphamide-activating refined. Recent pharmacological studies have led to the cytochrome P450 2B1 gene. Hum Gene Ther. 1994;5:969– identification of several compounds of demonstrated 978. efficacy in the treatment of herpesvirus infection in 9. Mar EC, Chiou JF, Cheng YC, Huang ES. Inhibition of

Cancer Gene Therapy, Vol 7, No 4, 2000 580 CANDOTTI, AGBARIA, MULLEN, ET AL: HSV-tk/neo FUSION GENE FOR RETROVIRAL VECTORS

cellular DNA polymerase ␣ and human cytomegalovirus- method for qualitative and quantitative assay of neomycin induced DNA polymerase by the triphosphates of 9-(2- phosphotransferase in crude cell extracts. Gene. 1984;30: hydroxyethoxymethyl)guanine and 9-(1,3-dihydroxy-2- 211–217. propoxymethyl)guanine. J Virol. 1985;53:776–780. 21. Agbaria R, Mullen CA, Hartman NR, et al. Effects of IMP 10. Balzarini J, Bohman C, De Clercq E. Differential mecha- dehydrogenase inhibitors on the phosphorylation of gan- nism of cytostatic effect of (E)-5-(2-bromovinyl)-2Ј-de- ciclovir in MOLT-4 cells before and after herpes simplex oxyuridine, 9-(1,3-dihydroxy-2-propoxymethyl)guanine, virus thymidine kinase gene transduction. Mol Pharmacol. and other antiherpetic drugs on tumor cells transfected by 1994;45:777–782. the thymidine kinase gene of herpes simplex virus type 1 or 22. Mosmann T. Rapid colorimetric assay for cellular growth type 2. J Biol Chem. 1993;268:6332–6337. and survival: application to proliferation and cytotoxicity 11. Singhal S, Kaiser LR. Cancer chemotherapy using suicide assays. J Immunol Methods. 1983;65:55–63. genes. Surg Oncol Clin N Am. 1998;7:505–536. 23. Kozak M. The scanning model for translation: an update. 12. Ram Z, Culver KW, Oshiro EM, et al. Therapy of malig- J Cell Biol. 1989;108:229–241. nant brain tumors by intratumoral implantation of retro- 24. Human gene marker/therapy protocols. Hum Gene Ther. viral vector-producing cells. Nat Med. 1997;3:1354–1361. 1998;9:2805–2852. 13. Sterman DH, Treat J, Litzky LA, et al. Adenovirus- 25. Wu X, Holschen J, Kennedy SC, Ponder KP. Retroviral mediated herpes simplex virus thymidine kinase/ganciclo- vector sequences may interact with some internal promot- vir gene therapy in patients with localized malignancy: ers and inﬂuence expression. Hum Gene Ther. 1996;7:159– results of a phase I clinical trial in malignant mesotheli- 171. oma. Hum Gene Ther. 1998;9:1083–1092. 26. Yee JK, Moores JC, Jolly DJ, Wolff JA, Respess JG, 14. Balzarini J, Bohman C, Walker RT, De Clercq E. Com- Friedmann T. Gene expression from transcriptionally dis- parative cytostatic activity of different antiherpetic drugs abled retroviral vectors. Proc Natl Acad Sci USA. 1987;84: against herpes simplex virus thymidine kinase gene-trans- 5197–5201. fected tumor cells. Mol Pharmacol. 1994;45:1253–1258. 27. Chen BF, Hsieh CL, Chen DS, Hwang LH. Improved gene 15. Schwartz F, Maeda N, Smithies O, et al. A dominant expression by a U3-based retroviral vector. Biochem Bio- positive and negative selectable gene for use in mamma- phys Res Commun. 1992;184:330–337. lian cells. Proc Natl Acad Sci USA. 1991;88:10416–10420. 28. Lupton SD, Brunton LL, Kalberg VA, Overell RW. Dom- 16. Morgenstern JP, Land H. Advanced mammalian gene inant positive and negative selection using a hygromycin transfer: high titre retroviral vectors with multiple drug phosphotransferase-thymidine kinase fusion gene. Mol selection markers and a complementary helper-free pack- Cell Biol. 1991;11:3374–3378. aging cell line. Nucleic Acids Res. 1990;18:3587–3596. 29. Loimas S, Wahlfors J, Janne J. Herpes simplex virus 17. Bender MA, Palmer TD, Gelinas RE, Miller AD. Evi- thymidine kinase-green ﬂuorescent protein fusion gene: dence that the packaging signal of Moloney murine leuke- new tool for gene transfer studies and gene therapy. mia virus extends into the gag region. J Virol. 1987;61: Biotechniques. 1998;24:614–618. 1639–1646. 30. Bonini C, Ferrari G, Verzeletti S, et al. HSV-TK gene 18. Miller AD, Buttimore C. Redesign of retrovirus packaging transfer into donor lymphocytes for control of allogeneic cell lines to avoid recombination leading to helper virus graft-versus-leukemia. Science. 1997;276:1719–1724. production. Mol Cell Biol. 1986;6:2895–2902. 31. Balzarini J, De Clercq E, Ayusawa D, Seno T. Murine 19. Restifo NP, Esquivel F, Asher AL, et al. Defective pre- mammary FM3A carcinoma cells transformed with the sentation of endogenous antigens by a murine sarcoma: herpes simplex virus type 1 thymidine kinase gene are implications for the failure of an anti-tumor immune highly sensitive to the growth-inhibitory properties of response. J Immunol. 1991;147:1453–1459. (E)-5-(2-bromovinyl)-2Ј-deoxyuridine and related com- 20. Reiss B, Sprengel R, Will H, Schaller H. A new sensitive pounds. FEBS Lett. 1985;185:95–100.

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