EXPERIMENTAL and MOLECULAR MEDICINE, Vol. 39, No. 6, 722-732, December 2007 Gene expression responses in vivo by human telomerase reverse transcriptase (hTERT)-targeting trans-splicing ribozyme Min-Sun Song1, Jin-Sook Jeong2, Kyung-Sook Cho3 ery of the ribozyme. In conclusion, this study demon- and Seong-Wook Lee1,4 strates that an hTERT-specific RNA replacement ap- proach using trans-splicing ribozyme represents a po- 1Department of Molecular Biology tential modality to treat cancer. Institute of Nanosensor and Biotechnology Dankook University Keywords: gene therapy; microarray analysis; RNA, Yongin 448-701, Korea catalytic; TERT protein, human; trans-splicing 2Department of Pathology Medical Center for Cancer Molecular Therapy Dong-A University College of Medicine Introduction Busan 602-103, Korea 3 Telomerase is a multicomponent ribonucleoprotein Research Institute and Hospital located within the nucleus that synthesizes the National Cancer Center repetitive nucleotide sequence forming the telo- Goyang 411-764, Korea meres at the end of chromosomes (Morin, 1989). 4 Corresponding author: Tel, 82-31-8005-3195; Telomerase activity appears to stabilize telomeres Fax, 82-31-8005-4058; E-mail, [email protected] and allow the possibility of cellular immortality (Zhu et al., 1999). Of note, telomerase activity is highly Accepted 15 October 2007 activated in 80-90% of malignant tissues and immortal cell lines (Kim et al., 1994) and the acti- Abbreviations: HCC, hepatocellular carcinoma; hTERT, human vity is modulated mainly by the telomerase reverse telomerase reverse transcriptase; PEPCK, phosphoenolpyruvate transcriptase (TERT), the catalytic subunit of the carboxykinase; PL, Ad-PEPCK-LacZ; PRL, Ad-PEPCK.Ribo-LacZ; telomerase. Therefore, TERT is a useful target for TS, trans-splicing the development of anti-cancer agent. The group I ribozyme from Tetrahymena thermo- phila was previously demonstrated to trans-splice Abstract an exon that is attached to its 3' end onto a separate 5' exon RNA, not only in vitro (Been and A trans-splicing ribozyme which can specifically re- Cech, 1986) but also in E. coli (Sullenger and program human telomerase reverse transcriptase Cech, 1994) and mammalian cells (Jones et al., (hTERT) RNA was previously suggested as a useful 1996). We, as well as other groups, have shown agent for tumor-targeted gene therapy. In this study, that these group I-based trans-splicing ribozymes we evaluated in vivo function of the hTERT-targeting can revise mutant transcripts that are associated trans-splicing ribozymes by employing the molecular with several human genetic or malignant diseases analysis of expression level of genes affected by the (Lan et al., 1998; Phylactou et al., 1998; Watanabe ribozyme delivery into peritoneal carcinomatosis mice and Sullenger, 2000; Rogers et al., 2002; Shin et model. To this effect, we constructed adenoviral vector al., 2004). Moreover, we demonstrated that trans- splicing ribozymes could selectively and speci- encoding the specific ribozyme. Noticeably, more than fically induce therapeutic gene activities in specific four-fold reduction in the level of hTERT RNA was ob- RNA-expressing cells through specific replacement served in tumor nodules by the systemic infection of of the target transcript (Ryu et al., 2003). Accor- the ribozyme-encoding virus. Such hTERT RNA knock- dingly, we developed human TERT (hTERT) RNA- down in vivo induced changes in the global gene ex- replacing trans-splicing ribozyme that can stimulate pression profile, including the suppression of specific the hTERT-dependent cytotoxicity in tumor cells genes associated with anti-apoptosis including bcl2, (Kwon et al., 2005). Furthermore, we recently and genes for angiogenesis and metastasis. In addi- showed that adenoviral vector harboring the ribo- tion, specific trans-splicing reaction with the targeted zyme can either selectively mark tumor cells or hTERT RNA took place in the tumors established as selectively induce suicide gene activity in the peritoneal carcinomatosis in mice by systemic deliv- tumors, thereby regressing the tumors with the Gene expression profile in vivo by trans-splicing ribozyme 723 Figure 1. Scheme of ribozyme-mediated selective induction of new RNA coupled with reduction of target RNA by targeted trans-splicing of hTERT mRNA. A ribo- zyme can recognize hTERT RNA at ac- cessible uridine residue by base pairing to the sequence through its internal guide sequence. The ribozyme then re- moves the sequence downstream of the target site and replaces it with a 3' exon that encodes a lacZ RNA sequence in this study. prodrug treatment, in hTERT-expressing tumor Culture Collection. The cells were maintained in xenografts (Hong et al., 2007). These observations Eagle minimal essential medium, supplemented indicate that the trans-splicing ribozyme can be with 10% heat-inactivated FBS (Join Bio-Innova- used as anti-cancer agents that reprogram cancer- tion, Seoul, Korea), 50 U/ml penicillin G, and 50 specific transcripts to frustrate the cancerous cells. µg/ml streptomycin (Sigma, St. Louis, MO) at 37oC The hTERT-specific trans-splicing ribozyme will in a humidified atmosphere of 5% CO2. be more effective as anti-cancer tool because it can induce therapeutic transgene activity selec- tively in the telomerase-positive cancer cells and Generation of recombinant adenoviral vectors can simultaneously reduce the hTERT RNA level We constructed hTERT-specific trans-splicing ribo- in the cells (Figure 1). In this study, to evaluate the zyme under the control of a phosphoenolpyruvate dual activity of the ribozyme and to analyze gene carboxykinase (PEPCK) promoter as previously expression responses upon hTERT RNA reduction described (Song and Lee, 2006). The expression by the ribozyme in vivo, we observed any altera- of the specific ribozyme harboring diphtheria toxin tions of the gene expression profiles of tumor A or HSVtk with the PEPCK promoter was shown nodules in mice with intraperitoneal carcinomatosis to be the most effective and specific with regard to after systemic delivery of the hTERT-targeting ribo- the retardation of the growth of hTERT+ liver zyme. cancer cells, as compared with other liver-specific promoters (Song and Lee, 2006). Recombinant adenovirus vector encoding the PEPCK promoter- Materials and Methods derived hTERT-targeting ribozyme flanked by the cDNA for LacZ (β-galactosidase) as 3' exon Cell lines (Figure 2A) was constructed using the in vivo The Hep3B cells (human hepatocellular carcinoma homologous recombination technique in bacteria cell line) were purchased from the American Type (BJ5183) with a type 5 adenoviral vector backbone 724 Exp. Mol. Med. Vol. 39(6), 722-732, 2007 Figure 2. Characterization of recombi- nant adenovirus encoding the hTERT- specific ribozyme. (A) The PEPCK.Ribo- LacZ cassette was inserted into the E1 region of an Adenovirus type 5 backbone by homologous recombination in BJ5183. The resultant vector was transfected into 293 cells, and the recombinant viruses, Ad-PEPCK.Ribo-LacZ, were generated. (B) Each clone of recombinant viral vec- tor generated by homologous recombi- nation in BJ5183 cells was analyzed with NotI. Eight bands of restriction DNA frag- ments were then shown in the recombi- nant vectors as indicated with arrows. (C) PCR analysis of lacZ (280 bp) or ITR (210 bp) DNA from clone number 24 or 26 of the recombinant virus genomic DNA generated from 4th or 5th super- natant of the viral infected 293 cells. (D) RT-PCR analysis of GAPDH (210 bp) and lacZ (280 bp) RNA from Hep3B cells infected with clone number 24 or 26 of Ad-PEPCK.Ribo-LacZ. Mo represents mock-infected cells. (E) β-galactosidase activity of lysates from Hep3B cells in- fected with each clone of Ad-PEPCK. Ribo-LacZ. in which the E1 and E3 region have been deleted BJ5183 cells with AdenoVectorTM. The recombi- (pAdenoVator ∆E1/E3 backbone E1/E3 deleted nant vectors generated by homologous recombina- Ad5 genome, AdenoVectorTM, Qbiogene, Irvine, tion in BJ5183 cells were isolated, analyzed with CA). Briefly, a SpeI/BstBI fragment containing the restriction enzymes, and linearized with PacI. The PEPCK promoter-Rib21AS-LacZ sequence was linearized vectors were then transfected into 293 inserted into the pAdenoVator-CMV5-IRES-GFP cells and the produced recombinant adenoviruses shuttle vector. To construct recombinant virus were isolated by three rounds of plaque purifica- vector, the shuttle plasmid was cleaved with PmeI. tion. Recombinant viruses were amplified using 293 The linearized fragment was cotransformed into cells, purified and concentrated by VivapureⓇ Gene expression profile in vivo by trans-splicing ribozyme 725 AdenoPACKTM 100 (Sartorius AG, Edgewood, NY). microscope. To analyze specific transgene induc- Recombinant adenovirus was titered by deter- tion, we injected 1 × 109 pfu Ad-PEPCK-LacZ or mination of the TCID50 (tissue culture infectious Ad-PEPCK.Ribo-LacZ in PBS (total volume, 100 dose for 50% of the cells). We designated the µl) intraperitoneally three times with two day inter- recombinant adenovirus encoding for the ribo- val after the carcinomatosis establishment. Two zyme with lacZ under the control of the PEPCK days after the last virus injection, the established promoter as Ad-PEPCK.Ribo-LacZ. An adenovirus tumor tissues were removed. with lacZ driven by the PEPCK promoter (Ad- PEPCK-LacZ) was employed as a control. Viral stocks were stored in 10% sucrose and kept at RNA analysis ࠏ80oC until use. Following tumor dissection, total RNA was ex- tracted using Tri-Reagent according to the manu- facturer's protocol (Molecular Research Center Molecular analysis of recombinant virus Inc., Cincinnati, OH). After re-suspension in nu- Recombinant viral vector generated by homolo- clease-free water, the RNA was quantified by UV gous recombination in BJ5183 cells was confirmed spectroscopy (Bio-Rad, Hercules, CA), and quali- by digestion with NotI. For the analysis of recombi- fied on a 2100 Bioanalyzer (Agilent, Palo Alto, CA). nant viral genomic DNA, viral supernatant gene- To assess the reduction level of the hTERT RNA rated in 293 cells were isolated, and the presence in vivo by the ribozyme, hTERT cDNA was am- of lacZ and ITR gene was assessed using PCR.