In Vivo Reprogramming of hTERT by Trans-splicing Ribozyme to Target Tumor Cells
Authors: Seung-Hee Hong, Jin-Sook Jeong, Yoon-Jong Lee, Haeng-Im Jung, Kyoung-Sook Cho, Chang-Min Kim, Byung-Su Kwon, Bruce A Sullenger, Seong-Wook Lee, In-Hoo Kim
Journal: Molecular Therapy (2007)

We have developed and validated a new tumor-targeting gene therapy strategy based upon the targeting and replacement of human telomerase reverse transcriptase (hTERT) RNA, using a trans-splicing ribozyme. By constructing novel adenoviral vectors harboring the hTERT-targeting trans-splicing ribozymes with the downstream reporter gene (Ad-Ribo-LacZ) or suicide gene (Ad-Ribo-HSVtk) driven by the cytomegalovirus (CMV) promoter, we demonstrated that this viral system selectively marks tumor cells expressing hTERT or sensitizes tumor cells to prodrug treatments. We confirmed that Ad-Ribo-LacZ successfully and selectively delivered a ribozyme that performed a highly specific trans-splicing reaction into hTERT-expressing cancer cells, both in vitro and in a peritoneal carcinomatosis nude mouse model. We also determined that the hTERT-specific expression of the suicide gene in the Ad-Ribo-HSVtk, and treatment with the corresponding prodrug, reduced tumor progression with almost the same efficacy as the strong constitutive CMV promoter-driven adenovirus, both in cancer cell lines and in nude mouse HT-29 xenografts. These observations provide the basis for a novel approach to cancer gene therapy, and demonstrate that trans-splicing ribozymes can be employed as targeting anti-cancer agents which recognize cancer-specific transcripts and reprogram them, thereby combating cancerous cells. We have developed and validated a new tumor-targeting gene therapy strategy based upon the targeting and replacement of human telomerase reverse transcriptase (hTERT) RNA, using a trans-splicing ribozyme. By constructing novel adenoviral vectors harboring the hTERT-targeting trans-splicing ribozymes with the downstream reporter gene (Ad-Ribo-LacZ) or suicide gene (Ad-Ribo-HSVtk) driven by the cytomegalovirus (CMV) promoter, we demonstrated that this viral system selectively marks tumor cells expressing hTERT or sensitizes tumor cells to prodrug treatments. We confirmed that Ad-Ribo-LacZ successfully and selectively delivered a ribozyme that performed a highly specific trans-splicing reaction into hTERT-expressing cancer cells, both in vitro and in a peritoneal carcinomatosis nude mouse model. We also determined that the hTERT-specific expression of the suicide gene in the Ad-Ribo-HSVtk, and treatment with the corresponding prodrug, reduced tumor progression with almost the same efficacy as the strong constitutive CMV promoter-driven adenovirus, both in cancer cell lines and in nude mouse HT-29 xenografts. These observations provide the basis for a novel approach to cancer gene therapy, and demonstrate that trans-splicing ribozymes can be employed as targeting anti-cancer agents which recognize cancer-specific transcripts and reprogram them, thereby combating cancerous cells. IntroductionRecent gene therapy for the treatment of cancer has focused on the development of novel strategies to enhance both efficacy and safety. Gene targeting may represent an attractive approach to that end, given that the objective of gene therapy is the eradication of cancer cells without harming normal cells.1McCormick F Cancer gene therapy: fringe or cutting edge?.Nat Rev Cancer. 2001; 1: 130-141Crossref PubMed Scopus (314) Google Scholar In the effort to achieve this, three significant approaches have been attempted: transcriptional targeting, transduction, and exploitation of cancer-associated cellular pathways.2Wu L Johnson M Sato M Transcriptionally targeted gene therapy to detect and treat cancer.Trends Mol Med. 2003; 9: 421-429Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar However, the ideal of precise and effective cancer cell-specific targeting remains a significant challenge.3Tarner IH Müller-Ladner U Fathman CG Targeted gene therapy: frontiers in the development of 'smart drugs'.Trends Biotechnol. 2004; 22: 304-310Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar In this study, we evaluated a new tumor-targeting gene therapy tool that utilizes a trans-splicing ribozyme, and we explored the efficacy of a recombinant adenovirus encoding for a ribozyme as a potential therapeutic agent against human cancers.The self-splicing group I intron from Tetrahymena thermophila mediates the trans-splicing of an exon ligated at its 3′-end onto a targeted and separate 5′ exon RNA both in vitro4Been MD Cech TR One binding site determines sequence specificity of Tetrahymena pre-rRNA self-splicing, trans-splicing, and RNA enzyme activity.Cell. 1986; 47: 207-216Abstract Full Text PDF PubMed Scopus (165) Google Scholar and in bacteria5Sullenger BA Cech TR Ribozyme-mediated repair of defective mRNA by targeted, trans-splicing.Nature. 1994; 371: 619-622Crossref PubMed Scopus (218) Google Scholar and mammalian cells.6Jones JT Lee SW Sullenger BA Tagging ribozyme reaction sites to follow trans-splicing in mammalian cells.Nat Med. 1996; 2: 643-648Crossref PubMed Scopus (105) Google Scholar These trans-splicing ribozymes have been used for correcting defective transcripts that cause human genetic or malignant diseases.7Lan N Howrey RP Lee SW Smith CA Sullenger BA Ribozyme-mediated repair of sickle beta-globin mRNAs in erythrocyte precursors.Science. 1998; 280: 1593-1596Crossref PubMed Scopus (183) Google Scholar,8Phylactou LA Darrah C Wood MJ Ribozyme-mediated trans-splicing of a trinucleotide repeat.Nat Genet. 1998; 18: 378-381Crossref PubMed Scopus (122) Google Scholar,9Rogers CS Vanoye CG Sullenger BA George AL Functional repair of a mutant chloride channel using a trans-splicing ribozyme.J Clin Invest. 2002; 110: 1783-1789Crossref PubMed Scopus (74) Google Scholar,10Watanabe T Sullenger BA Induction of wild-type p53 activity in human cancer cells by ribozymes that repair mutant p53 transcripts.Proc Natl Acad Sci USA. 2000; 97: 8490-8494Crossref PubMed Scopus (117) Google Scholar,11Shin KS Sullenger BA Lee SW Ribozyme-mediated induction of apoptosis in human cancer cells by targeted repair of mutant p53 RNA.Mol Ther. 2004; 10: 365-372Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar Ribozyme-mediated trans-splicing has also been used in the induction of selective cytotoxicity in target cells.12Ayre BG Köhler U Goodman HM Haseloff J Design of highly specific cytotoxins by using trans-splicing ribozymes.Proc Natl Acad Sci USA. 1999; 96: 3507-3512Crossref PubMed Scopus (44) Google Scholar Recently, we showed that the trans-splicing ribozyme specifically and selectively replaces viral transcripts with a new RNA that exerts anti-viral effects by recognizing the viral-specific RNA, and catalyzing the in-frame fusion of desired RNA sequences.13Ryu KJ Kim JH Lee SW Ribozyme-mediated selective induction of new gene activity in hepatitis C virus internal ribosome entry site-expressing cells by targeted trans-splicing.Mol Ther. 2003; 7: 386-395Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar Moreover, we developed a ribozyme that reprogrammed human telomerase reverse transcriptase (hTERT) RNA to selectively induce transgene activity in cancer cells expressing hTERT, thereby demonstrating that trans-splicing ribozymes can serve as anti-cancer agents.14Kwon BS Jung HS Song MS Cho KS Kim SC Kimm K et al.Specific regression of human cancer cells by ribozyme-mediated targeted replacement of tumor-specific transcript.Mol Ther. 2005; 12: 824-834Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar We selected hTERT-targeting trans-splicing ribozyme because it is expressed at high levels in most malignant tumors, but is not detected in normal postmitotic cells.15Shay JW Bacchetti S A survey of telomerase activity in human cancer.Eur J Cancer. 1997; 33: 787-791Abstract Full Text PDF PubMed Scopus (2369) Google Scholar,16Shay JW Wright WE Telomerase: a target for cancer therapeutics.Cancer Cell. 2002; 2: 257-265Abstract Full Text Full Text PDF PubMed Scopus (294) Google Scholar Thus far, however, there has been no proof of the efficacy of ribozyme-mediated trans-splicing in inducing therapeutic genes in vivo, in a tumor-specific manner.In this context, we evaluated the feasibility of using an adenoviral vector encoding for an hTERT-targeting trans-splicing ribozyme as a tumor-targeting gene therapy tool. Targeting the 5′-untranslated region of hTERT messenger RNA, we assessed the efficacy and specificity of the trans-splicing reaction in hTERT+ and hTERT− cells. We also attempted to determine whether reporter gene activity could be induced selectively in a peritoneal carcinomatosis mouse model by systemic delivery of the adenoviral vector, and whether the ribozyme would trigger selective cytotoxicity in hTERT-expressing cells. We also analyzed the therapeutic effect of the adenovirus on mice implanted subcutaneously with hTERT-expressing human cancer cells.ResultsAd-Ribo-LacZ induces reporter gene expression selectively in hTERT+ cell linesBy evaluating telomerase activity, we confirmed hTERT+ in HT-29, SNU-407, HepG2, SNU-398, and SBC-5 cell lines, and hTERT− in an SKLU-1 cell line. Telomerase activity was highest in SBC-5 cells and relatively high in the other hTERT+ cell lines when compared with the commercial positive control. However, no telomerase activity was detected in the SKLU-1 cells (Figure 1a). We then assessed whether new gene activity could be induced selectively in hTERT+ cells by Rib21AS-3′exon-LacZ ribozyme, which trans-splices the β-galactosidase gene (lacZ) onto the hTERT transcript and allows for the monitoring of β-galactosidase expression. We observed high β-galactosidase levels in cells infected with Ad-LacZ, regardless of hTERT status. In contrast, Ad-Ribo-LacZ infection conferred elevated β-galactosidase activity only in the hTERT+ cells (Figure 1b). Moreover, Ad-Ribo-LacZ was observed to induce β-galactosidase activity in hTERT+ cells almost as efficiently (80–90%) as Ad-LacZ control virus did. The histological analysis of β-galactosidase activity verified the selectivity of Ad-Ribo-LacZ with regard to transgene expression. When infected with Ad-LacZ, all tested cell lines expressed β-galactosidase and were visualized by 5-bromo-4-chloro-3indoyl N-acetyl-β-d-galactosaminide (X-gal) staining. However, Ad-Ribo-LacZ was observed to induce β-galactosidase expression only in hTERT+ cells, and not in the hTERT− SKLU-1 cells (Figure 1c). This indicates that Ad-Ribo-LacZ induces transgene expression both efficiently and selectively, in an hTERT-dependent manner.In order to determine whether ribozyme-mediated transgene expression induces the trans-splicing reaction with hTERT RNA at the predicted site, we infected hTERT+ HT-29 and hTERT− SKLU-1 cells with various multiplicity of infection (MOI) of Ad-Ribo-LacZ, and then analyzed β-galactosidase expression by reverse transcriptase-polymerase chain reaction (PCR). We detected trans-spliced molecules (TSMs) only in the hTERT+ cells and not in the hTERT− cells, even though ribozymes (represented as lacZ RNA production) were present in the hTERT− cells (Figure 1d). Furthermore, the quantity of TSM produced correlated with the quantity of transfected Ad-Ribo-LacZ. Sequence analysis of the amplified fragment confirmed that the Ad-Ribo-LacZ correctly targeted hTERT's uridine, at position 21, and inserted lacZ RNA at its 3′exon (Supplementary Figure S1).We also observed reduced telomerase activity in the hTERT+ cells, as a consequence of the trans-splicing reaction of Ad-Ribo-LacZ. Since telomerase activity depends on hTERT RNA expression, endogenous telomerase activity should be reduced by Ad-Ribo-LacZ transfection. In order to test this, we assayed telomerase activity in the HT-29 cells following infection with Ad-Ribo-LacZ and Ad-LacZ. As expected, Ad-Ribo-LacZ infection reduced telomerase activity in a dose-dependent manner (10, 20, 23, and 33% reduction at MOI of 1, 10, 50, and 100, respectively). In contrast, Ad-LacZ infection did not effect a reduction in telomerase activity (Figure 1e). Collectively, these results clearly indicate that the Ad-Ribo-LacZ-mediated trans-splicing reaction is highly specific for its target sequence, and that the ribozyme in Ad-Ribo-LacZ (Rib21AS-3′exon-LacZ) effectively executes the cleavage/trans-ligation activity as designed.Transgene induction by Ad-Ribo-LacZ in hTERT-expressing tumor is selective in vivo as wellIn view of the fact that Rib21AS-3′exon-LacZ selectively induced transgene expression in target RNA-expressing cells in vitro, we tested whether an adenovirus harboring the ribozyme also functions similarly in vivo. In a preliminary study, we systemically administered Ad-Ribo-LacZ or Ad-LacZ (1 × 109 plaque forming units) to female Balb/c mice by intravenous injection via tail veins, killed them 2 days later, and examined various tissues. Using PCR analysis with vector-specific primers, we detected high levels of Ad-Ribo-LacZ DNA in the liver, significantly lower levels in the lung, adipose tissue, and spleen, and barely detectable levels in other tissues (Supplementary Figure S2a). We further analyzed transgene expression by histochemical analysis (X-gal staining). Interestingly, mice injected with Ad-Ribo-LacZ evidenced little or no reporter gene activity, even in virus-laden tissues. We did, however, observe diffuse and extensive β-galactosidase expression in the liver and distinct cortical glomerular expression in the kidneys of Ad-LacZ-transfected mice (Supplementary Figure S2b).Next, we tested whether the ribozyme-harboring adenovirus had the potential for tumor-targeted anti-cancer gene therapy in a colorectal adenocarcinoma–derived peritoneal carcinomatosis model. Ten days after peritoneal carcinomatosis induction, we randomly grouped mice and injected them with Ad-Ribo-LacZ (n = 5), Ad-LacZ (n = 5), or phosphate-buffered saline (PBS) (n = 3). We injected 1 × 109 plaque forming units of virus in 400 μl PBS intraperitoneally three times at 2-day intervals. Two days after the final virus injection, we killed the mice. Prior to analysis, we randomly selected one mouse from each virus-injected group and examined its abdominal organs macroscopically after X-gal staining for lacZ expression. We determined that Ad-Ribo-LacZ induced lacZ expression in tumor nodules, but not in other organs. In contrast, in the mice treated with Ad-LacZ, multifocal X-gal staining was observed in tumor nodules as well as on the exteriors of the liver and gastroenteric serosa (Figure 2a). However, the X-gal staining appeared heterogeneous in this case, suggesting that viruses tend to spread in a non-homogeneous manner after intraperitoneal administration.17Lechanteur C Princen F Lo Bue S Detroz B Fillet G Gielen J et al.HSV-1 thymidine kinase gene therapy for colorectal adenocarcinoma-derived peritoneal carcinomatosis.Gene Ther. 1997; 4: 1189-1194Crossref PubMed Scopus (24) Google Scholar We then analyzed the non-tumor-containing tissues of the mice remaining in each group for lacZ expression. In mice injected with Ad-LacZ, we observed β-galactosidase expression on the surfaces of the liver, stomach, and esophagus. In Ad-Ribo-LacZ-treated mice, we detected no β-galactosidase expression (Figure 2b). We also conducted PCR in order to verify the efficacy of transgene expression. We found that intraperitoneal viral introduction into mouse abdominal organs successfully delivered viruses, as determined by transgene expression. However, no TSM was detected in non-tumorous mouse organs, even in the Ad-Ribo-LacZ injected group, thereby indicating that the trans-splicing reaction specifically targeted hTERT only in vivo (Figure 2c). We observed no significant differences between individuals within groups.Figure 2Selective induction of transgene expression by Ad-Ribo-LacZ in mouse cancer models. (a) β-galactosidase expression pattern detected by X-gal staining in toto in the peritoneal cavities of mice with established peritoneal carcinomatosis, intraperitoneally injected with Ad-Ribo-LacZ or Ad-LacZ. Arrows indicate tumor nodules. (b) Representative histochemical findings of transgene expression patterns in abdominal organs. Histologic findings (hematoxylin and eosin (H&E), ×40) and β-galactosidase expression (X-gal, ×40) are shown in non-tumor tissues from Ad-Ribo-LacZ or Ad-LacZ-injected peritoneal carcinomatosis model mice. (c) Tissue distribution and transgene expression patterns of injected viruses in non-tumor tissues of mice with established peritoneal carcinomatosis. The distribution of injected viruses (Lane R, Ad-Ribo-LacZ; Lane L, Ad-LacZ), transgene expression or ribozyme production, and the production of trans-spliced molecule detected in various tissues by polymerase chain reaction (PCR) for the inverted terminal repeats (ITR) region, reverse transcriptase-PCR (RT-PCR) for lacZ RNA (LacZ RNA), and trans-spliced molecule (TSM), respectively. Tissues from phosphate-buffered saline (PBS)-injected mice (Lane P) served as a negative control. The RT-PCR product of mouse glyceraldehyde-3-phosphate dehydrogenase (mGAPDH) served as an internal control. (d) Representative histochemical findings in developed tumor nodules. Histologic findings (H&E, ×40), β-galactosidase expression (X-gal, ×40), and human telomerase reverse transcriptase (hTERT) expression (Anti-hTERT, ×100) are shown in tumor nodules from Ad-Ribo-LacZ- or Ad-LacZ-injected mice with peritoneal carcinomatosis. (e) Presence and transgene expression patterns of injected viruses in tumor nodules; analysis similar to the one described in c. Each lane represents a tumor nodule from an individual mouse. The endogenous human GAPDH (hGAPDH) served as an internal control.View Large Image Figure ViewerDownload Hi-res image Download (PPT)We then analyzed at least three tumor nodules from each group to see whether differences existed in the transgene expression patterns. We observed β-galactosidase expression in the developed tumor nodules of mice injected with Ad-Ribo-LacZ or Ad-LacZ. Furthermore, the Ad-Ribo-LacZ-treated mice, when compared against the Ad-LacZ- or PBS-treated mice, had markedly lower hTERT expression in tumor nodules (Figure 2d). PCR analysis detected viral inverted terminal repeats and lacZ RNA in tumor nodules infected with Ad-Ribo-LacZ and Ad-LacZ, suggesting that the viruses efficiently dispersed within the tumor nodules. Using reverse transcriptase-PCR we determined whether the Rib21AS-3′exon-LacZ specifically targeted hTERT+ tumor nodules. As in the in vitro targeting studies with the HT-29 cells, we detected correctly targetedTSMs only in tumor nodules transfected with Ad-Ribo-LacZ (Figure 2e). Moreover, rapid amplification of cDNA end reverse transcriptase-PCR analysis revealed that all of the trans-splicing products generated in the tumors of mice injected with Ad-Ribo-LacZ (20 clones) resulted only from reactions with the hTERT RNA (data not shown). These results demonstrate that adenovirus-mediated trans-splicing reactions by hTERT-specific ribozymes both effectively and specifically induce transgene expression in vivo.Efficacy and specificity of suicide gene therapy using trans-splicing ribozymeWe constructed a recombinant adenovirus, Ad-Ribo-HSVtk, for therapeutic use. In order to determine whether Ad-Ribo-HSVtk induces herpes simplex virus thymidine kinase (HSVtk) gene expression in the presence of hTERT RNA, we exposed HT-29 and SKLU-1 cells to different MOI of Ad-Ribo-HSVtk, Ad-HSVtk, or Ad-Mock, and assayed for [3H]fluoroganciclovir (PCV) cellular uptake, a radio-labeled HSVtk substrate. Rib21AS-3′exon-HSVtk mediated the uptake of [3H]PCV in HT-29 cells approximately 20% less efficiently than Ad-HSVtk did. In contrast, SKLU-1 showed approximately 80% lower [3H]PCV uptake following Ad-Ribo-HSVtk infection (Figure 3a). Additionally, in order to determine whether adenovirus-mediated transduction with Rib21AS-3′exon-HSVtk renders hTERT+ cells sensitive to ganciclovir (GCV), we treated HT-29 and SKLU-1 cells with varying amounts of Ad-HSVtk, Ad-Ribo-HSVtk, or Ad-Mock, and evaluated cell survival after adding GCV (final concentration of 20 μmol/l). As expected from our results with Ad-Ribo-LacZ, Ad-Ribo-HSVtk caused a dose-dependent reduction in cell survival, to approximately the same degree as Ad-HSVtk did. However, Ad-Ribo-HSVtk had only a minimal effect on hTERT− SKLU-1 cells (Figure 3b). To assess whether the cytotoxicity was dependent on GCV concentration, we conducted the same experiment using a fixed dose of adenovirus and varying GCV concentrations (Supplementary Figure S3). Ad-Ribo-HSVtk killed hTERT+ HT-29 cells but not hTERT− SKLU-1 cells, whereas Ad-HSVtk was cytotoxic, independent of hTERT status. These in vitro results demonstrate that Ad-Ribo-HSVtk induces HSVtk in an hTERT RNA-dependent manner, and sensitizes hTERT+ HT-29 cells to GCV.Figure 3Therapeutic efficacy and specificity of Ad-Ribo-HSVtk in vitro and in vivo. (a) Thymidine kinase activity of Ad-Ribo-HSVtk, Ad-HSVtk, or Ad-Mock at various multiplictiy of infection (MOI). HT-29 and SKLU-1 cells were infected with adenoviruses and exposed with media containing [3H]PCV. Radioactivity levels were then measured. For each of the uptake studies, at least two experiments were carried out, with three simultaneous samples in each experiment. Data were expressed as the net accumulation of probe in dpm cells/dpm medium/μg total protein ± SD. (b) Efficacy and specificity of the tumor-killing activity of Ad-Ribo-HSVtk in vitro. hTERT+ HT-29 and hTERT− SKLU-1 were transfected with Ad-Ribo-HSVtk, Ad-HSVtk, or Ad-Mock at various MOI. After 5 days, cell viability was determined by MTS assay. Error bars indicate the mean values ± SD for triplicate assays. (c) Antitumor efficacy of Ad-Ribo-HSVtk in vivo. The tumor xenografts, approximately 140 mm3 in size and composed of hTERT+ HT-29 cells, were injected with Ad-Ribo-HSVtk/GCV (n = 12), Ad-HSVtk/GCV (n = 12), Ad-Mock/GCV (n = 9) or PBS/PBS (n = 9). Tumor size was measured on alternate days. Error bars indicate the mean values ± SD.View Large Image Figure ViewerDownload Hi-res image Download (PPT)We then assessed the therapeutic effects of Ad-Ribo-HSVtk in vivo, using tumor xenografts established with hTERT+ HT29 cells in nude mice. After the tumors became palpable, we intratumorally injected viruses with either Ad-Ribo-HSVtk or Ad-HSVtk (treatment groups), and Ad-Mock or PBS (controls), and then administered GCV or PBS intraperitoneally every day for 10 days. Thirty days after the viral injections, the tumor volumes (in cubic millimeters) were 610.40 ± 296.54 for the Ad-HSVtk/GCV group; 744.24 ± 279.38 for the Ad-Ribo-HSVtk/GCV group; 1,482.10 ± 205.67 for the Ad-Mock/GCV group; and 1,628 ± 421.19 for the PBS/PBS group (Figure 3c). As is evident, tumor growth was reduced significantly in treatment groups, as compared to controls (analysis of variance; P < 0.0001). Interestingly, we detected no statistically significant differences in the tumor growth inhibitory effects of Ad-HSVtk/GCV and Ad-Ribo-HSVtk/GCV (analysis of variance; P = 0.12). These results indicate that the hTERT-specific therapeutic adenovirus Ad-Ribo-HSVtk is as potent as Ad-HSVtk in suppressing tumor growth.DiscussionThe ability of therapeutic genes to selectively eradicate rapidly proliferating cancer cells, without exerting toxic effects on normal cells, determines the success or failure of gene therapy in cancer.2Wu L Johnson M Sato M Transcriptionally targeted gene therapy to detect and treat cancer.Trends Mol Med. 2003; 9: 421-429Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar,3Tarner IH Müller-Ladner U Fathman CG Targeted gene therapy: frontiers in the development of 'smart drugs'.Trends Biotechnol. 2004; 22: 304-310Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar,18Dong JY Woraratanadharm J Gene therapy vector design strategies for the treatment of cancer.Future Oncol. 2005; 1: 361-373Crossref PubMed Scopus (10) Google Scholar Herein, we present a gene therapy strategy against cancer with specificity and efficacy based on the concept that the group I trans-splicing ribozyme, which targets hTERT transcripts expressed in cancer cells, would recognize its own substrates, switch them into "suicide" gene transcripts, and promote cell death. For the ribozyme delivery system, we selected adenovirus, which has been used in several in vitro and in vivo pre-clinical analyses.19Kanerva A Hemminki A Modified adenoviruses for cancer gene therapy.Int J Cancer. 2004; 110: 475-480Crossref PubMed Scopus (117) Google Scholar,20Cascalló M Alemany R Adenovirus-mediated gene transfer to tumor cells.Methods Mol Biol. 2004; 246: 121-138PubMed Google Scholar,21Ghosh SS Gopinath P Ramesh A Adenoviral vectors: a promising tool for gene therapy.Appl Biochem Biotechnol. 2006; 133: 9-29Crossref PubMed Scopus (106) Google Scholar,22St George JA Gene therapy progress and prospects: adenoviral vectors.Gene Ther. 2003; 10: 1135-1141Crossref PubMed Scopus (269) Google Scholar We constructed novel adenoviral vectors harboring hTERT-targeting ribozymes with downstream lacZ (Ad-Ribo-LacZ) or HSVtk (Ad-Ribo-HSVtk) driven by a cytomegalovirus (CMV) early promoter. We analyzed the Ad-Ribo system for effective and specific delivery in animal models. By monitoring reporter gene activity, we verified that this viral system successfully delivers a ribozyme that performs a highly specific trans-splicing reaction with hTERT pre-messenger RNA, both in vitro and in vivo. We observed the reduction of hTERT and hTERT-dependent β-galactosidase expression in various cancer cell lines and mouse tumor models with hTERT+ HT-29 cells. No β-galactosidase was expressed in non-tumor tissues from non-tumor bearing or mouse cancer models transfected with Ad-Ribo-LacZ. Moreover, trans-spliced products were not detected in non-tumor tissues in the Ad-Ribo-LacZ-treated mice. Notably, no mis-spliced products were generated in the tumor nodules of mice injected with the ribozyme-encoding adenovirus. These results indicate the in vivo target specificity of the trans-splicing ribozyme.We also evaluated the efficacy of this adenoviral system. Telomerase activity was reduced in a viral dose-dependent manner when Ad-Ribo-LacZ was transfected into the HT-29 cells. Telomerase inhibition, however, reached a plateau at 100 MOI of virus administered in vitro, with approximately 30% reduction in telomerase activity. Interestingly, there were no significant differences in the antitumor effects of Ad-Ribo-HSVtk and Ad-HSVtk, thereby suggesting that 10–30% of delivered tk gene activity is sufficient for effective tumor killing. In addition, we determined that reporter gene activity in hTERT+ cells infected with Ad-Ribo-LacZ, was only slightly (10–20%) lower than those infected with Ad-LacZ, and the effects were independent of internal telomerase activities. These results indicate that the efficacy of Ad-Ribo-LacZ is almost as high as Ad-LacZ in cells expressing target RNA. The efficacy of the adenoviral transfer, however, may influence the therapeutic effects of this system. As shown in the peritoneal carcinomatosis model, whole abdominal organs were not evenly transfected by Ad-LacZ, and trans-spliced products were detected only in 66% of tumor nodules transfected with Ad-Ribo-LacZ. This may reflect either the inefficacy of intraperitoneal delivery17Lechanteur C Princen F Lo Bue S Detroz B Fillet G Gielen J et al.HSV-1 thymidine kinase gene therapy for colorectal adenocarcinoma-derived peritoneal carcinomatosis.Gene Ther. 1997; 4: 1189-1194Crossref PubMed Scopus (24) Google Scholar or organ tropisms of the adenovirus. However, in view of the fact that the efficacy of viral delivery and promoter activity were indistinguishable between Ad-LacZ and Ad-Ribo-LacZ, the efficiency of trans-splicing ribozymes is unlikely to be a major limitation for the application of this technique to human anti-cancer therapy. Concerns regarding the utilization of adenoviral vectors for clinical tumor treatment include the existence of preexisting immunity against adenovirus in the majority of human populations, and the rapid immune response to the vector after initial injection.23Bangari DS Mittal SK Current strategies and future directions for eluding adenoviral vector immunity.Curr Gene Ther. 2006; 6: 215-226Crossref PubMed Scopus (138) Google Scholar The development of immune response limits the use of repeated inoculations of the adenoviral vector, as we employed in this study. Thus, strategies to circumvent the shortcomings of adenovirus, such as serotype switching,24Vogels R Zuijdgeest D van Rijnsoever R Hartkoorn E Damen I de Béthune MP et al.Replication-deficient human adenovirus type 35 vectors for gene transfer and vaccination: efficient human cell infection and bypass of preexisting adenovirus immunity.J Virol. 2003; 77: 8263-8271Crossref PubMed Scopus (399) Google Scholar may be required for an eventual successful approach to cancer therapy with ribozyme-encoding adenoviral vectors.Most importantly, we showed that the hTERT-specific delivery of the HSVtk suicide gene and treatment with the corresponding prodrug (GCV) reduced tumor progression with almost the same efficacy as was observed with the strong constitutive CMV promoter-driven adenovirus, both in cancer cell lines and nude mouse HT-29 xenografts. These results indicate that ribozymes, in an adenovirus system, confer specificity without reducing viral transfection efficacy and transgene expression, even in an in vivo model. One frequently used approach in targeted gene therapy for cancer involves the use of adenovirus harboring a tissue/tumor