Electroporation-Mediated and EBV LMP1-Regulated Gene Therapy in A

Electroporation-mediated and EBV LMP1-regulated gene therapy in a syngenic mouse tumor model Yu-hua Hsieh,1 Chang-jer Wu,2 Kai-ping Chow,3 Chia-lung Tsai,1 and Yu-sun Chang3 1Institute of Microbiology and Immunology, National Yang-Ming University, Shih-pai, Taipei 112, Republic of China; 2Institute of Preventive Medicine, National Defense Medical Center, Taipei 114, Republic of China; and 3Graduate Institute of Basic Medical Sciences, Chang-Gung University, Kwei-shan, Taoyuan 333, Taiwan, Republic of China.

Latent membrane protein 1 (LMP1) is an Epstein–Barr virus (EBV)-encoded oncogene expressed in EBV-associated nasopharyngeal carcinoma (NPC). Previous studies indicate that a strategy combining LMP1-mediated NF-kB activation and the HSV thymidine kinase/Ganciclovir (HSVtk/GCV) prodrug system leads to regression of tumor growth in nude mice. To improve the efficacy of this strategy in immunocompetent hosts, we developed a therapeutic cassette, p6kB-EDL1E-tk, containing six copies of the NF-kB binding motif and an epithelial-specific EBV promoter, ED-L1E. The cassette was tested in a murine CT-26 carcinoma model in syngenic Balb/c mice. Coinjection of an LMP1-expressing vector and p6kB-EDL1E-tk by in vivo electroporation in mouse muscle revealed at least two-fold higher TK enzymatic activity than that of previously tested pLTR-tk. Furthermore, growth was attenuated in a group of mice containing LMP1-positive tumors that were intratumorally injected with the p6kB-EDL1E-tk cassette and GCV via in vivo electroporation, but not in mice treated with p6kB-EDL1E-tk or GCV alone. Similarly, no retardation of tumor growth was observed in mice containing LMP1-negative CT-26 tumors injected with both the p6kB-EDL1E-tk cassette and GCV. We propose that intratumoral injection of therapeutic agents, such as DNA of transcription-regulated cassette and GCV, via in vivo electroporation may be used as an alternative treatment for EBV LMP1-expressing cancers. Cancer Gene Therapy (2003) 10, 626–636. doi:10.1038/sj.cgt.7700609 Keywords: EBV; LMP1; HSVtk/GCV; therapeutic gene cassette; in vivo electroporation

uman nasopharyngeal carcinoma (NPC) arises in the (LMP1), an integral membrane protein that has a Hepithelium of the posterior nasopharynx. Histologi- profound effect on the morphology of rodent cells.3 cally, NPC is classified by the World Health Organization LMP1 is additionally essential for the in vitro transforma- (WHO) into three types. Type I NPC is a keratinizing tion of human primary B lymphocytes and downregulates squamous-cell carcinoma similar to those that arise from cytokeratin expression in human epithelial cells.3,4 Trans- other sites of the head and neck. Type II is a genic mice containing the LMP1 gene develop lympho- nonkeratinizing epidermoid carcinoma, whereas Type mas, further supporting a role of LMP1 as a transforming III includes undifferentiated carcinomas.1 Clinically, protein.5,6 Types II and III are the two most prominent diseases in LMP1 is a 62 kDa protein comprising a short the endemic areas, including Taiwan, the Mediterranean cytoplasmic N-terminus of B25 amino acids, a long basin, Alaska, and the southern parts of China.2 NPC is cytoplasmic C-terminus of B200 amino acids, and six primarily treated with radiotherapy. However, metastasis membrane spanning segments.7 The C-terminal cytoplas- occurs in 7% of patients at initial diagnosis and 20% or mic domain of LMP1 contains at least two major more develop metastasis after treatment, suggesting that functional regions, CTAR-1 and CTAR-2, which mediate supplementary therapy is required. most LMP1-involved signal transduction. CTAR-1 as- Epstein–Barr virus (EBV) is a human herpes virus, sociates with TNF receptor-associated factors (TRAF),8 which infects human B lymphocytes, T lymphocytes, while CTAR-2 interacts with the TNF receptor-associated and epithelial cells. EBV is closely associated with death domain protein, TRADD.9 Through interactions NPC, and at least two viral genes are expressed in tumor with TRAF and TRADD, LMP1 induces NF-kB and cells. One of these is the latent membrane protein 1 JNK activation.10–13 Furthermore, LMP1 triggers NF-kB activation by inducing the degradation of IkB and facilitating the translocation of NF-kB molecules into Received December 2, 2002. 14 Address correspondence and reprint requests to: Dr Yu-sun Chang, the nucleus. This results in the activation of target genes Graduate Institute of Basic Medical Sciences, Chang-Gung through association of p50/p65 NF-kB molecules with the University, 259 Wen-Hwa Ist Road, Kwei-shan, Taoyuan, Taiwan NF-kB-binding motif located within the upstream reg- 333, Republic of China. E-mail: [email protected] ulatory region of promoters. Electroporation-mediated and EBV LMP1-regulated gene therapy Y-h Hsieh et al 627 We previously reported a therapeutic strategy using binding motif and ligated into SpeI- and XbaI-digested LMP1-induced NF-kB activation to induce the HSVTK/ pBluescript II KS (+) (Stratagene, USA). The resulting GCV prodrug system.15 The HSVtk gene in the earlier plasmid was designated pBluescript-2kB. The pBlue- study was expressed from the HIVLTR sequence contain- script-4kB plasmid containing four copies of the kB- ing two copies of the NF-kB motif, and activated by binding motif was generated by the ligation of XmaI- and NPC-derived LMP1 (NLMP1).16 Cells expressing both SacI-digested pBluescript-2kBtoSpeI- and ScaI-digested NLMP1 and HSVtk genes were highly sensitive to GCV pBluescript-2kB. Similarly, plasmid pBluescript-6kB con- treatment, and tumors induced in nude mice regressed. To taining six copies of the kB binding motif was generated develop a therapeutic program for the treatment of EBV- by the ligation of XmaI- and SacI-digested pBluescript- associated diseases, we constructed a transcriptional 4kBtoSpeI- and ScaI-digested pBluescript-2kB. Plasmid cassette containing ED-L1E, a promoter that is preferen- pBluescript-8kB containing eight copies of the kB-binding tially activated in NPC cells, and copies of the NF-kB motif was generated by ligation of XmaI- and SacI- motif that specifically enhance HSVtk expression by digested pBluescript-4kBtoSpeI- and ScaI-digested LMP1. Experiments with CT-26 tumors in syngenic Balb/ pBluescript-4kB. The kB-containing luciferase reporter c mice revealed that the best combination involved the constructs were generated by insertion of DNA fragments direct delivery of GCV and a therapeutic cassette from the four pBluescript-kB plasmids containing 2, 4, 6, containing six copies of the NF-kB motif and ED-L1E or 8 binding motifs, respectively, into pEDL1E-Luc.16 to LMP1-positive tumors by in vivo electroporation. The p2kB-EDL1E-Luc plasmid was generated by insert- ing two copies of the kB-binding motif obtained from SpeI- and XmaI-digested pBluescript-2kB into the EagI B Materials and methods site ( 520 bp upstream of the ED-L1E promoter) of the ED-L1E promoter of the EBV LMP1 gene.16 Similar Cell lines methods were used to generate p4kB-EDL1E-Luc, p6kB- CT-26 is a murine colon adenocarcinoma cell line EDL1E-Luc, and p8kB-EDL1E-Luc. Plasmids p2kB- syngenic to BALB/c mice. NPCTW02 derived from EDL1E-tk and p6kB-EDL1E-tk were generated by NPC tumor was generously provided by Dr CT Lin insertion of an B0.8 kb SmaI–BglII fragment (containing (National Taiwan University, Taiwan). C33A (ATCC two or six copies of the NF-kB-binding motif and ED- HTB31) is a human HPV-negative cervical carcinoma cell L1E promoter) of p2kB-EDL1E-Luc and p6kB-EDL1E- line. Cells were cultured in Dulbecco’s modified Eagle’s Luc into ClaI–BglII-digested pHSV-106 (GibcoBRL). 15 medium supplemented with 10% fetal bovine serum The pLTR-tk plasmid is described by Wu et al. (FBS) (GibcoBRL). LMP1-positive CT-26 cell clones (CT-26-NLMP1) were established by transfection of 2 mg DNA transfection NLMP1-expressing vector, pT7(E)17 and a neo-contain- Plasmid DNA was prepared using Nucleobond kits ing vector, pSV2neo (Clontech, Palo Alto, CA, USA), (Macherey-Nagel, Germany). For DNA transfection, followed by selection with G418 (500 mg/ml). 1 Â 105 cells in six-well plates were transfected with 2 mg To generate an LMP1-positive cell clone that also of plasmid DNA by lipofectaminet reagent (GibcoBRL). expresses the HSVtk gene, CT-26-NLMP1 cells were co- Cells were harvested 48 hours after transfection and transfected with 1 mg pBabe-Hygro and 10 mgp6kB- examined using the luciferase activity assay described EDL1E-tk (see ‘‘Plasmid construction’’). Cells were below. selected using G418 and hygromycin, and maintained in DMEM medium supplemented with 10% FBS, 500 mg/ml G418 and 200 mg/ml hygromycin. Tumor induction and growth monitoring Mice were subcutaneously inoculated with 1 Â 105 CT 26- Experimental animals NLMP-1 cells in a total volume of 50 ml. When the tumor 3 Male 6–8-week-old BALB/c mice were purchased from reached a measurable size (30–50 mm ), mice were the National Laboratory Animal Breeding and Research randomly divided into groups of five to seven. Tumor Center of National Science Council, ROC (Nangang, growth was monitored three times a week with a caliper, Taipei, Taiwan). Animals were maintained at the Animal as soon as treatment commenced. Tumor volume was 2 Care Center of Chang-Gung University. calculated using the formula: V ¼ a Â b /2, where V represents volume (mm3), a is the maximum tumor 19 Plasmid construction diameter, and b is the minimum tumor diameter. Plasmids containing the kB binding motif were generated as follows: two copies of the motif were constructed by Western blotting annealing two complementary oligonucleotides corre- Cells were washed twice with PBS and collected. sponding to the binding sequence located on HIV- Following centrifugation at 1500 g for 5 minutes, cell LTR.18 Briefly, two NF-kB motif-containing primers pellets were resuspended and lysed in lysis buffer (10 mM (primer 1, 50-CTAGTCCCCAGCGGAAAGTCCCTT Tris-HCl, pH 7.5, 1 mM phenylmethylsulfonyl fluoride, GT-30 and primer 2, 50-CTAGACAAGGGACTTTCCG and 1% Triton X-100) by sonication. Supernatants were CTGGGGA-30) were annealed to generate 2 Â NF-kB collected by centrifugation at 12,000 g for 10 minutes, and

Cancer Gene Therapy Electroporation-mediated and EBV LMP1-regulated gene therapy Y-h Hsieh et al 628 protein concentration was measured with the Bradford 470 mM luciferin, and 530 mM ATP) was mixed with the assay (BioRad). Protein samples (200 mg) were resolved same amount of protein from the supernatant and the on SDS-polyacrylamide gels, transferred to nitrocellular relative light unit (RLU) was measured using an Auto- membranes (Hybond) and analyzed using S12 mono- lumat LB953 (Berthold, Germany). clonal antibody against LMP-17 and antibody against To determine the luciferase activity in muscle, DNA actin (Santa Cruz). Immunoreactive protein bands were was delivered by in vivo electroporation as described visualized with an enhanced chemiluminescence detection above. Mice were killed by cervical dislocation at 1, 2, or kit (Santa CruzBiotechnology Inc.). 30 days after electroporation, and the treated region was To detect LMP1, tumor tissues were minced and homogenized in 100 ml PBS in liquid nitrogen. Protein digested with collagenase and trypsin for 1 hour. Sus- concentration and luciferase activity of lysates were pended cells were collected and cultured in 500 mg/ml measured. The RLU was determined using an Autolumat G418-containing medium. Western blotting was per- LB953 (Berthold, Germany). formed as described above.

Intramuscular DNA injection and in vivo Thymidine kinase activity assay electroporation At 2 days after plasmid DNA injection, mice were killed To deliver DNA into muscle tissue, 50 mg closed circular by cervical dislocation. The required portions of muscle plasmid DNA in 50 ml sterile injectable saline was injected were collected and homogenized in 300 ml lysis buffer into the left and right hind limb tibial muscle, using an (10 mM Tris-HCl, pH 7.5, 10 mM KCl, 2 mM MgCl2, insulin syringe (BD Pharmigen). Following plasmid DNA 1 mM ATP, 10 mM NaF, 0.5% NP-40 and 10% glycerol) injection, muscle was pulsed in vivo with a T820 square- using liquid nitrogen. Supernatants were clarified by wave electroporator (BTX, San Diego), which was fitted centrifugation at 12,000 g for 10 minutes, and quantitated with an array of 0.5 cm diameter genetrodes. Six pulses using the Bradford assay (Bio-Rad). Muscle lysate (50 mg) were delivered at a frequency of 1 Hz, with a pulse length was mixed with 50 ml reaction mixture containing 0.16 M of 50 ms and 100 V. Tris-HCl, pH 7.5, 0.14 mM albumin, 12.6 mM creatine phosphate, 11.2 U/ml creatine phosphokinase, 10 mM 3 Intratumoral DNA injection and in vivo electroporation [8- H] ganciclovir (GCV) (1000 cpm/pmol), 2 mM ATP, 2 mM MgCl2, 7.5 mM NaF, and 1.6 mM DTT. The Mice were anesthetized with pentobarbital sodium. An mixture was incubated at 371C for 30 minutes. A 30 ml established subcutaneous tumor with a volume of 30– 3 sample was spotted onto a Whatman DE-81 filter, washed 50 mm was injected percutaneously with 50 mg closed three times with 95% alcohol and dried. Radioactivity on circular plasmid DNA in 50 ml sterile injectable saline, the filters was measured by a liquid scintillation counter, using an insulin syringe equipped with a 27-gauge needle LS-500OTD (Beckman, Fullerton, CA). (BD Pharmigen). Following plasmid DNA injection, the tumor was pulsed with a T820 square-wave electroporator (BTX, San Diego), which was fitted with an array of six 0.5 cm diameter needle electrodes. For optimization of in GCV sensitivity assay vivo electroporation condition, specified voltage and pulse For a cell survival assay, CT-26-NLMP1-6–kB-tk cells conditions were used (Fig 4 legend). The most efficient (104) were plated into 96-well microplates per well. After condition was that with one 50 ms pulse delivered at a 18 hours, the regular medium was replaced with fresh frequency of 1 Hzand 150 V. medium containing increasing concentrations (10, 50, 100, For intratumoral delivery of plasmid DNA, plasmids 200, 300, 500, and 1000 mg/ml) of GCV in duplicate. The with or without GCV were delivered into the tumor by in percentage cell survival was determined after 4 days using vivo electroporation, as described previously. Adminis- the Cell Titer 96 AQueous Nonradioactive Cell Prolifera- tration was performed five times every other day and tion Assay kit (Promega), according to the manufacturer’s tumor growth was monitored until day 20. instructions. Cell absorption in each well was measured with an MR5000 microwell plate reader (DYNATCH Luciferase assay Laboratory, Chantilly, VI) at 450 nm. The percentage of cell survival was calculated using the equation: % cell After DNA transfection (48 hours), cells were washed, survival ¼ (absorption of GCV-treated cells/absorption of lysed in 100 ml of lysis buffer (25 mM Tris-phosphate, pH control cells) Â 100%. 7.8, 2 mM 1,2-diaminocyclohexane- N,N,N0,N0-tetraacetic acid, 10% glycerol, Triton X-100, and 2 mM DTT), and mixed by vortexing. The supernatant was collected by centrifuging at 12,000 g for 10 minutes, and the protein Statistical analysis concentration estimated using the Bradford assay (Bio- Statistical evaluations of the qualitative variables were Rad) in a spectrophotometer (SHIMADZU UV-160A) at performed using the Fisher’s exact probability test. The OD595. Luciferase assay buffer (100 ml) (20 mM tricine, mean volume of tumors in the control group, minus 1.07 mM (MgCO3)4Mg(OH)2 Á 5H2O, 2.67 mM MgSO4, 2 Â SD (standard deviation), was used as the cutoff value. 0.1 mM EDTA, 33.3 mM DTT, 270 mM coenzyme A, Statistical significance was defined at Po.05.

Cancer Gene Therapy Electroporation-mediated and EBV LMP1-regulated gene therapy Y-h Hsieh et al 629 Results Activation of custom-made kB motif-containing transcriptional cassettes by LMP1 in human carcinoma cells To determine whether the genetically engineered transcriptional cassettes containing the specified copies of the NF-kB-binding motif and epithelial-specific EBV promoter (ED-L1E) were inducible by LMP1, the LMP1- expressing vector was cotransfected with transcriptional cassettes into NPCTW02 and C33A cells, respectively. The activity of promoter constructs was determined by the luciferase assay. In NPCTW02 cells, the 2kB-containing cassette and promoter-only construct (pEDL1E-luc) were slightly activated in the presence of LMP1, whereas the 6kB-containing cassette was induced B20-fold (Fig 1a). LMP1 activated the 4kB- and 8kB-containing cassettes by B12- and B15-fold, respectively. Similar results were obtained with C33A cells, except that the 2kB-containing construct was activated Bthree-fold, compared to pEDL1E (Fig 1b). Consequently, we selected the 6kB-containing cassette for future studies.

Activation of the custom-made kB motif-containing transcriptional cassettes by LMP1 in CT-26 carcinoma cells In order to establish a model that permits in vivo testing of this transcription cassette in a mouse carcinoma model, murine CT-26 that induces tumors in syngenic Balb/c mice was used to establish cell clones stably expressing NLMP1. As demonstrated in Fig 2, ED-L1E alone was Figure 1 NLMP1-mediated kB activation in transient transfection moderately elevated (Bfour-fold), while the 2kB-contain- B experiments. NLMP1-mediated activation of EDL1E-Luc constructs ing cassette was activated seven-fold. The 4-, 6- and without and with two, four, six, and eight copies of the kB-binding 8kB-containing cassettes were activated B13-, B20-, and motif in transient transfection experiments. LMP1-expressing vector, B12-fold, respectively, by NLMP1. Similar to parental pT7(E), and reporter plasmids were cotransfected into NPCTW02 (a) CT-26 cells, CT-26-NLMP1 induced tumor formation, and C33A (b) cells, following which luciferase activity was and palpable tumors appeared around 5–7 days following determined. Reporter plasmid pEDL1E-Luc (ED-L1E) contains only initial inoculation. Expression of NLMP1 in CT-26- the ED-L1E promoter sequence, p2kB-EDL1E-Luc (2kB) contains NLMP1 cells and corresponding tumors was shown by two copies of the NF-kB-binding motif, p4kB-EDL1E-Luc (4kB) Western blot using anti-LMP1 antibody S12 (Fig 2b). contains four copies of the NF-kB-binding motif, p6kB-EDL1E-Luc (6kB) contains six copies of the NF-kB-binding motif, and p8kB- EDL1E-Luc (8kB) contains eight copies of the NF-kB-binding motif. Construction of reporter plasmids was described in the Materials and GCV sensitivity test in vitro methods. The degree of activation was determined by dividing the luciferase value from cells co-transfected with the NLMP1-expres- To evaluate HSV-TK/GCV toxicity, five cell clones stably sing vector pT7(E) and indicated promoter constructs with that from expressing both NLMP-1 and p-6 kB-EDL1E-TK were cells cotransfected with NLMP1 and pGL2-basic (vector control). established. One of these displaying the strongest expres- Data are the average of at least three independent transfection sion of HSVtk was selected for the GCV sensitivity test. experiments performed in duplicate. SD is signified by vertical bars. Cells (104) were plated on the medium without or with increasing doses (10, 50, 100, 200, 300, 500, and 1000 mg/ ml) of GCV. After 4 days, the percentage of living cells was measured using the MTT assay. As shown in Fig 3a, approximately 30% of the cells were killed at 10 and Effect of GCV on CT-26-NLMP1-EDL1E-tk tumor growth 50 mg/ml GCV. At 100 mg/ml GCV, B70% of the cells To determine the effect of GCV on tumor growth in vivo, were killed. No cells survived, following treatment with CT-26-NLMP1-EDL1E-tk cells were inoculated via GCV at concentrations of 200 mg/ml or higher. The results s.c. injection. GCV (100 mg/kg body weight) or saline strongly indicate that CT-26 cells expressing both LMP-1 was introduced i.p. at days 2, 4, 6, 8, and 10, respec- and HSVTK are highly sensitive to GCV. tively. Tumor-free mice were scored everyday.

Cancer Gene Therapy Electroporation-mediated and EBV LMP1-regulated gene therapy Y-h Hsieh et al 630

Figure 3 GCV sensitivity test in vitro and in vivo.(a) Effect of GCV on cell survival. CT-26-NLMP1-6kB-EDL1E-tk cells (CT-26 cells stably expressing NLMP1 and containing 6kB-EDL1E-tk; closed bars) or CT-26-NLMP1 cells (CT-26 cells stably expressing NLMP1; open bars) were exposed to increasing doses of GCV (10, 50, 100, Figure 2 Characterization of CT-26-NLMP1-expressing cells. (a) 200, 300, 500, and 1000 mg/ml) and cell viability was determined by Evaluation of NF-kB activation in CT-26-NLMP1-expressing cells. the MTT assay after 4 days. Data shown are the average of three NLMP1-expressing CT-26 and parental cells were transfected with independent experiments. SD is represented by vertical bars. (b) pED-L1E (0) or kB-binding motif-containing plasmids, and the Effect of GCV on tumor growth in CT-26-NLMP1-6kB-EDL1E-tk activities of promoters were determined using the luciferase activity. cells. Cells were inoculated via s.c. injection. GCV (100 mg/kg body ‘‘C’’ is the vector control of pED-L1E. Data shown are the average of weight) or saline was introduced i.p. at days 2, 4, 6, 8, and 10, at least three independent transfection experiments performed in respectively. A total of 10 mice were included in each group and duplicate. SD is signified by vertical bars. (b) NLMP1 expression in tumor-free mice were scored everyday until day 13. CT-26-NLMP1 cells and tumor tissue derived from CT-26-NLMP1 cells. Total lysates were prepared from CT-26-NLMP1 cells or tumor tissues. Expression of NLMP1 was examined by Western blot analysis, using S12 monoclonal antibody specific for LMP1. Cell Transgene expression in tissues by in vivo lysates from B95-8 cells were used as the positive control and those electroporation from parental CT-26 were negative for LMP1. To establish an in vivo electroporation protocol, mice muscles were injected with increasing amounts (10, 25, 50, 75, and 100 mg) of pCMV-Luc in a total volume of 50 ml, As depicted in Figure 3b, 60% of the mice (six out of 10) with or without in vivo electroporation. To monitor were tumor-free at day 3, while 10% (one out of 10) was transgene expression, luciferase activity was determined tumor-free at day 4, and none was tumor-free at day 5 in on days 1, 2, and 30. As shown in Fig 4a, transgene the group treated with saline. In contrast, GCV-treated activity was detectable in muscles injected with as low as mice remained tumor-free until day 6. Four out of 10 10 or 25 mg pCMV-Luc, 1 day after in vivo electropora- (40%) mice displayed palpable tumors at day 7, six out of tion. At 50 mg or higher concentrations, elevated activity 10 (60%) at day 9, and seven out of 10 at day 11 (1 day was observed, which was not dose-dependent. The highest after termination of GCV treatment). On day 13, the last activity was detected at day 2 after injection. Transgene mouse developed tumor. Our results collectively suggest expression at day 30 was still detected as B40% of that that GCV treatment attenuates tumor formation in detected at day 2 at the original injection site (those syngenic mice. injected with 50 and 100 mg, respectively).

Cancer Gene Therapy Electroporation-mediated and EBV LMP1-regulated gene therapy Y-h Hsieh et al 631

Figure 5 In vivo assay of the therapeutic HSVtk/GCV system. TK activity was determined in muscle tissues of mice after electroporation of a combination of plasmids containing HIV-LTR-tk, p2kB- EDL1E-tk, and p6kB-EDL1E-tk, with or without the LMP1-expressing vector (pT7(E)). ‘‘C’’ is the vector control of tested promoter- containing plasmids. The data are an average of at least three independent in vivo electroporation experiments.

these in vivo conditions, transgene activity was similar to that expressed in muscle (data not shown).

Thymidine kinase activity in vivo To ensure that HSVTK functions in mouse tissues and to determine the most suitable HSVtk gene expression cassette for this study, p2kB-EDL1E-tk and p6kB- EDL1E-tk plasmid DNA were injected into muscles of mice by in vivo electroporation, with or without the NLMP1-expressing vector pT7(E). Thymidine kinase activity was assayed at 48 hours following in vivo Figure 4 Analysis of luciferase gene expression via in vivo electroporation. As shown in Fig 5, tissues injected with B electroporation. (a) In vivo expression of luciferase in muscle p6kB-EDL1E-tk displayed three-fold higher TK activ- tissues. Increasing concentrations (10, 25, 50, 75, and 100 mg) of ity than those treated with p2kB-EDL1E-tk, and Btwo- pCMV-Luc in a volume of 50 ml were injected into the left and right fold higher activity than previously examined HIVLTR- limb tibial muscles of four mice (n ¼ 8 sites for each treatment or tk.15 control group). Luciferase activity was detected at 1, 2, and 30 (50 and 100 mg only) days after DNA transfer by in vivo electroporation. In vivo effect of a combination of GCV and kB-mediated (b) Optimization of in vivo electroporation conditions for luciferase TK activation on LMP1-expressing tumor growth expression in tumor tissues. pCMV-Luc (100 mg) in a volume of 50 ml was injected into CT-26-derived tumors (n ¼ 6 mice for each To determine the most efficient method of transporting treatment or the control group) at the specified voltage and pulse therapeutic agents, p6kB-EDL1E-tk and GCV were conditions. Luciferase activity was determined in lysates of tumor delivered into CT-26-NLMP1 tumor tissues, either via tissues, 48 hours after in vivo electroporation. Luciferase activity in vivo coelectroporation or in vivo electroporation of measured in muscle tissues was included as the control. p6kB-EDL1E-tk and i.p. injection of GCV. Treatment was performed five times every other day until day 10. Since the method of delivery of transgenes is different Notably, no palpable tumors were detected in the between muscle and tumor tissues, pCMV-Luc at a coelectroporation group (Fig 6a). The tumor volume concentration of 2 mg/ml in a total volume of 50 ml was was measured at day 20 following the initial injection. As used to determine the optimal conditions for in vivo shown in Fig 6b, tumor growth was significantly electroporation of tumors. The results summarized in suppressed in the group coinjected with p6kB-EDL1E-tk Figure 4b indicate that the most efficient conditions and GCV (100 mg in a volume of 50 ml) when measured at (represented by the highest luciferase activity) were day 10. Tumor growth in mice electroporated in vivo with electroporation at 150 V with 1 electric pulse. Under p6kB-EDL1E-tk and intraperitoneal delivery of GCV

Cancer Gene Therapy Electroporation-mediated and EBV LMP1-regulated gene therapy Y-h Hsieh et al 632 (100 mg/kg body weight) was moderately reduced (aver- Tumors examined in Groups I–IV were derived from CT- age tumor volume of B150 mm3 at day 10, compared to 26-NLMP1 cells. Mice in Group I were injected with both B300 mm3 in untreated controls). Using Fisher’s exact p6kB-EDL1E-tk and GCV; Group II was inoculated with probability test, a statistical significance of Po.001 was p6kB-EDL1E-tk; Group III with GCV only; while Group obtained. The data suggest that the concomitant electro- IV was inoculated with 0.9% NaCl solution. Mice of poration of both therapeutic agents leads to significant Groups V and VI were derived from CT-26 cells with suppression of tumor growth. vector controls. Group V animals were injected with The efficacy of the LMP1-mediated kB-modulated p6kB-EDL1E-tk only, while Group VI mice were injected therapeutic strategy was further investigated using coelec- with 0.9% NaCl solution. In vivo electroporation was troporation of p6kB-EDL1E-tk and GCV. CT-26- performed every other day for 10 days. As shown in NLMP1 and parental CT-26-induced tumors with Figure 6c, animals intratumorally injected with both palpable size (30–50 mm3) were treated as described p6kB-ED-L1E-tk and GCV (Group I) remained tumor- above, and the mass of tumors was measured at day 20 free at 10 days following initial treatment. Tumors following initial inoculation. In this study, mice were reappeared, but at a more moderate rate (average of divided into six groups, each comprising six animals. tumor volume o200 mm3 at day 20). NLMP1 tumors injected with GCV (Group III), p6kB-EDL1E-tk only (Group II), or 0.9% NaCl solution (Group IV) displayed no retardation of tumor growth, with tumor volumes ranging from 1200 to 2000 mm3. Similarly, no growth retardation was detected in parental CT-26-derived tumors, following treatment with p6kB-EDL1E-tk and GCV (Group V) or with saline (Group VI). Tumor growth was significantly inhibited in Group I animals (Po.01). Thus, the treatment regimen with p6kB- EDL1E-tk and GCV inhibited tumor growth, suggesting the potential to treat LMP1-positive tumors.

Discussion

We previously reported that transcriptional regulation of LMP1-mediated NF-kB activation is a selective therapeutic strategy for EBV-associated diseases. 15 In this study, we improved the system by custom-designing a transcriptional cassette containing an epithelial-specific

Figure 6 Evaluation of electroporation of the NLMP1-mediated transcriptional cassette and GCV on tumor growth. (a,b) Comparison of therapeutic application methods. Tumors induced in mice were injected with 50 mg of therapeutic DNA by in vivo electroporation. GCV (100 mg/kg body weight) was introduced concomitantly, either by electroporation (co-EP; n ¼ 7) or intraperitoneally (EP+i.p.; n ¼ 5). Control animals (n ¼ 5) did not receive any treatment. Tumor volume was measured at day 10 after the initial inoculation. The horizontal bar indicates the mean value. The growth of subcutaneous CT-26- NLMP1 tumors was significantly inhibited, compared with the control (Po.001, Fisher’s exact test). (c) Effect of coinjection of the transcription cassette and GCV on CT26-NLMP1and CT-26 tumors. Balb/c mice were inoculated with 1x105 CT-26-NLMP1 or CT-26 cells to induce tumors. Mice were divided into six groups, each comprising six animals. CT-26-LMP1-induced tumors became palpable at B6 days following inoculation, and were injected intratumorally with p6kB-EDL1E-tk, GCV (100 mg/kg body weight), or both by in vivo electroporation. Tumor volume was determined using a caliper. CT- 26-induced tumors were injected with or without both therapeutic agents. The data show tumor sizes at days 10 and 20 after inoculation in Balb/c mice. Tumor volume at day 20 was statistically analyzed. The growth of CT-26-NLMP1 tumors (Group I) was significantly inhibited (Fisher’s exact test, Po.01), compared with that in the other five groups (II–IV).

Cancer Gene Therapy Electroporation-mediated and EBV LMP1-regulated gene therapy Y-h Hsieh et al 633 promoter, ED-L1E, and copies of the NF-kB motif that cassette containing the 6kB motif is the most efficient. It is are specifically activated by LMP1. Following incorpora- additionally worth noting that the transcriptional cassette tion of this cassette into the HSVTK/GCV prodrug established in this study is superior to HIV-LTR, since the system, we observed that the EBV ED-L1E promoter with activity of p6kB-EDL1E-tk was higher than that of six copies of the kB-binding motif effectively and pHIVLTR-tk, which contains two copies of the NF-kB specifically controlled the growth of LMP1-expressing motif (Fig 5). tumors delivered intratumorally via in vivo electropora- Numerous methods have been used to deliver ther- tion. This approach resulted in the regression of LMP-1- apeutic DNA into model animals. Among these, direct positive, but not LMP-1-negative tumors, in a syngenic delivery of therapeutic genes to tumors has several mouse model. advantages over intravenous injection of naked DNA or Direct delivery of a regulated therapeutic gene to viral infection. This is due to the fact that many human or targeted cells is the ultimate goal of this gene therapy rodent tumors cannot be infected efficiently with adeno- program. LMP1 activates HIV-LTR-regulated HSVtk viral vectors due to lack of proper receptors. On the other gene expression via two copies of the NF-kB-binding hand, gene transfer by in vivo electroporation, a procedure motif, and tumors expressing LMP1 and HSVTK are that involves DNA injection followed by application of sensitive to GCV treatment in nude mice.15 However, electric fields, is effective for introducing DNA into HIV-LTR may not be an ideal promoter to direct the mouse muscle,26 skin,27 rat liver,28 and tumors.19,29 expression of therapeutic genes, as it may integrate into Earlier data show that use of in vivo electroporation the genome and result in the activation of cellular genes enhances plasmid DNA uptake in tumor tissue, resulting that are initially in an inactivated state. To circumvent in expression within the tumor.30–33 Furthermore, in vivo this problem, we replaced HIV-LTR with EBV ED-L1E, electroporation is a safe and nontoxic delivery system.34 an upstream promoter of the LMP1 gene.16 ED-L1E of This has been used for the efficient delivery of plasmid EBV is a relatively weak promoter that is preferentially DNA encoding chemotherapeutic agents, such as cyto- activated in EBV-positive NPC tumors and cell lines,16 kine IL12, resulting in tumor regression, long-term animal but is not active in human B lymphocytes. This is survival, and resistance to challenge.35 In this study, important, since unregulated activation of NF-kB activity delivery of the therapeutic cassette by in vivo electropora- in normal human B lymphocyte may result in strong side tion into LMP1-expressing tumors also showed a effects. therapeutic effect. The best results were obtained upon To enhance specifically the expression of the therapeu- coinjection of two therapeutic agents, specifically, plasmid tic HSVtk gene, copies of the NF-kB motif were inserted DNA of the transcriptional cassette and GCV. In this set upstream of the transcription initiation site of ED-L1E. of experiments, animals remained healthy in appearance, LMP1 did not enhance ED-L1E promoter activity, unless and two out of six mice completely recovered, indicating copies of the NF-kB binding motif were added to the that in vivo electroporation of DNA and GCV does not transcriptional cassette, suggesting that the transcrip- cause side effects. tional cassette is activated via LMP1-mediated NF-kB No significant effects are expected from a thera- activation. This was further demonstrated in the syngenic peutic transgene (HSVtk), if the percentage of the mouse model, where coinjection of the custom-made NF- transduced tumor tissue is below 5%. The efficiency kB-mediated transcription cassette and GCV via in vivo of transgene delivery by in vivo electroporation in this electroporation into LMP1-expressing tumors suppressed study should be at least higher than 5%, since lucife- tumor growth. A transcriptionally regulated approach has rase activity was significantly high at day 2 and still also been reported for the treatment of other cancers, detectable at day 30 following treatment. Thus, in vivo such as the a-fetoprotein promoter selectively used in gene electroporation of naked DNA seems a reasonably therapy for hepatoma, tyrosinase promoter in melanoma, feasible approach. carcinoembryonic antigen promoter in adenocarcinoma, The GCV drug is safe for use, and the HSVtk/GCV Myc-Max responsive elements in small-cell lung cancer, prodrug system has been tested clinically for cancer hTERT promoter-induced tumor-specific Bax gene, and treatment. Phase I/II clinical trials of HSVtk/GCV caspase-8 expression.20–25 therapy report that a number of patients achieved partial To develop the most suitable transcriptional cassette, or complete clinical remission of tumors.36–38 Preclini- various copies of the NF-kB motif were examined for cally, tumor-specific killing using the HSVtk/GCV system efficient activation by LMP1. A dose-dependent effect has been demonstrated in a number of solid tumors, was observed at two, four, and six copies of the kB motif. whereby cellular cytotoxicity is induced following GCV A synthetic transcriptional cassette containing the ED- administration in animal models. HSVTK converts GCV L1E promoter and six copies of the enhancer NF-kB to a monophosphorylated form, which is then converted motif displayed strongest promoter activity induced by to a toxic triphosphate by cellular kinases. GCV tripho- LMP-1. Interestingly, ED-L1E with eight copies of the kB sphate interferes with DNA synthesis, leading to cell motif exhibited lower activity than that with six copies of death. Studies in animal models demonstrate that HSVtk/ the motif in three cell lines tested (human NPCTW02 and GCV treatment exerts antitumor effects in various C33A, murine carcinoma CT-26). Since transcriptional cancers.15,19, 39–41 activation involves the recruitment of coactivators, this Direct injection of GCV concurrent with the therapeu- suggests that the local structure of the transcriptional tic transcriptional cassette appears to have an advantage

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