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638 Telomerase Targeted Oncolytic Adenovirus in Combination with Chemotherapy Elicits Enhanced Cytopathic and Anti Tumor Effects in Head Neck Squamous Cell Carcinoma targeted CRAD Cytotoxic effects of[.]
targeted CRAD Cytotoxic effects ofAd5/35-TERTand Ad5-TERT were compared in in vitro experiments using various primary HNSCC cell lines The results indicate that Ad5/35- TERT is up to 500 % more efficient in killing primary HNSCC cells than Ad5-TERT Preliminary results from in vivo experiments using a subcutaneous HNSCC model suggest similarly improved efficacy without an increase in toxicity These results suggest that Ad5/35-TERT presents an attractive approach for development ofclinically applicable therapies for head and neck cancer 636 Development of Prostate-Specific Conditional Replication-Competent Adenovirus for Prostate Cancer Gene Therapy Yi Lu; Guimin Chang; Jun Zhang.' Pathology, Medicine and Urology, University ofTennessee Health Science Center, Memphis, TN I Three different prostate specific promoters, prostate specific antigen (PSA), probasin (PB) and mouse mammary tumor virus long terminal repeat (MMTV), were examined and compared for their activity and specificity to express a reporter gene in prostate and nonprostate cells both in vitro and in vivo The best prostate specific promoter (PSA) was used to generate a conditional , replication-competent adenovirus (AdPSAE I), in which the adeno viral E I region was under the control of the PSA promoter, in an effect to target the prostate for cancer gene therapy The anti-prostate tumor efficacy and specificity of AdPSAE l-mediated cytotoxicity were examined in vitro and in vivo in prostate and nonprostate cancer models In vitro at multiplicity of infection (moi) of I, AdPSAE I effectively killed the human prostate cancer cclliines PPC-I and LNCaP, but had no effect on nonprostate cancer cells ineluding the human bladder cancer cell line RT4, human breast cancer cell line MCF-7, and rat gliosarcoma cell line 9L As a control, an adenovirus expressing the l3-galactosidase transgene under the control of the same PSA promoter (AdPSAlacZ) was used in parallel in all experiments The in vivo tissue-specific expression driven by this PSA promoter was examined in a xenograft tumor model Intratumoral injection ofAdPSAlacZ resulted in PSA promoter-driven expression of lacZ in xenograft tumors in nude mice derived from human prostate cancer PPC-l cells, but not in tumors derived from human bladder cancer RT4 cells Intratumoral injection ofAdPSAE I effectively inhibited in vivo growth of xenograft PPC-I prostate tumors compared to the untreated or AdPSAlacZ treated tumors Conversely, intratumoral injection of AdPSAEI had no effect on the growth ofxenograft RT4 bladder tumors when compared to the untreated control group These results indicate that prostate-targeted conditional replication-competent adenoviruses may hold a promise in gene therapy for prostate cancer 637 Targeted Cancer Gene Therapy Using AAV2-Mediated Anti-Angiogenesis Approaches Keerang Park,' Young-Hwa Cho,' Wun-Jae Kim,' Wongi Seol,' Eui-Sic Cho,4 Yeun-Ju Kim,! Sung-Ha Cho,2 Hee-Jong Kim; Bong-Su Kang,' Won-Jin Ji.' 'Juseong Gene Therapy R&D Center, Juseong College, Cheongwon-Gun, Chung-Buk, Republic ofKorea; lCollege ofMedicine and Institute for Tumor Research, Chungbuk National University, Cheongju, Chung-Buk; Republic ofKorea; 'Insttuue for Brain Science and Technology, lnje University; Pusan, Republic ofKorea; "School of Dentistry; Chonbuk National University, Chonju, Chon-Buk; Republic ofKorea For several years, wc have intensively focused on developing a potential anti-angiogenic cancer gene therapy using an AAV2-mediated gene delivery system due to its high transduction efficiency in various human cancer cell lines Among the tested rAAV2 vectors , S244 a mixture of antisense VEGF isoform A (rAAV2-AShVEGF-A), soluble VEGF R-I (rAAV2-TShVEGFR-I), and soluble VEGF R-2 (rAAV2-TShVEGFR-2) receptors showed higher anti-cancer therapeutic efficacy than paelitaxel in the nude mice implanted with NCI-H460 Although AAV2 was able to infect human cancer cell lines with great efficiency, it could also transduce other cell types, which may cause undesirable side effects In the present study, to avoid undesired expression in normal tissue and to accomplish tumor-specific expression of a therapeutic gene, we replaced the CMV promoter in rAAV2-CMV-GFPwith the promoters ofa protein regulator ofcytokinesis I (PRC I), ribonuclease reductase (RRM2), baculoviral lAP repeat-containing (BIRC5), hypoxia responsive clement (HRE) , human telomerase reverse transcriptase (hTERT), and 3E-hTERT-TPL to establish rAAV2-PRC I-GFp, rAAV2-RRM2GFr, rAAV2-BIRC5-GFP, rAAV2-3HRE-GFP, rAAV2-hTERT-GFP, and rAAV2-3E-hTERT-TPL-GFP vector These rAAV vectors containing tumor-specific promoters were examined to compare promoter activities expressed in normal cells and cancer cell lines The results demonstrated that PRCI, RRM2, and BIRCS promoters showed cancer-specific expression and their promoter activities were highly comparable to that of the CMV promoter In contrast, the promoter activities of3HRE, hTERT, and 3E-TERT-TPL were quite low and similar to that of promoter-less vectors In conclusion, our data suggest that a rAAV vector containing PRC I, RRM2, or BIRCS promoter may be used for targeted cancer gene therapy and could be applied for cancer-specific expressions ofAAV2-mediated antiangiogenesis approaches (This study was supported by the grant from the Ministry of Science and Technology (M I053404000505N3404-00511) , Seoul, and the Chung-Buk Pioneering Bioindustry R&D Grant, Chung-Buk, Korea ) 638 Telomerase-Targeted Oncolytic Adenovirus in Combination with Chemotherapy Elicits Enhanced Cytopathic and Anti-Tumor Effects in Head & Neck Squamous Cell Carcinoma Pyung-Hwan Kim,':' JinSun Kim,2.J Min Jung Kim,2.J O.-Jun Kwon.s-' Joo-Hyuk Sohn,3 Chae-Ok Yun;·J Joe-hang Kim.J I KOSEF through National Core Research Center for Nanomedical Technology, l'onsei Cancer Center, l'onsei University College ofMedicine, Seoul, Republic ofKorea; lBrain Korea 21 Project for Medical Science , l'onsei Cancer Center; Yonsei University College ofMedicine, Seoul, Republic ofKorea; Jlnsilutefor Cancer Research, Yonsei Cancer Center; l'onsei University College of Medicine, Seoul, Republic ofKorea The head and neck squamous cell earcinoma(HNSCC) affcets an estimated 500,000 patients annually worldwide At present , the combination therapy of chemotherapeutic agents, cisplatin and 5fluorouracil (5-FU), has been used as a standard therapy for advanced HNSCC Gene attenuated replication competent adenoviruses are being developed as novel anti-tumor therapeutics Clinical trials with the ONYX-O IS (EI B 55kDa-deleted) adenovirus in combination with chemotherapeutic agents (cisplatin, 5-FU) have demonstrated a synergistic antitumor effect However, most ofreplicating adenoviruses used in combination therapy to date have their £1 B 19kDa gene intact Because the E IB 19 protein is an anti-apoptotie agent, adenoviruses retaining their EI B 19kDa gene may interfere with the mode of action of some chemotherapeutic agents , resulting in attenuating the overall potency ofthese combination treatment In our previous report, EI B 19kDa-deleted adenovirus Ad-mTERT-A 19, which encode the EIA gene driven by the uniquely modified mhTERT promoter, elicited enhanced viral replication and cytopathic effects in a cancer cell-specific manner For the aim of improving the therapeutic efficacy for HNSCC cancer, we investigated the value of different combinations ofvarious chemotherapeutic agents Molecular Therapy Volume 15 Supplement I, \by 2007 Copyright © '111C AmericanSocietyo f Gene TIICr.lpr combined with Ad-mTERT-819 Wc assessed thc cytotoxic activity of each combination in four different I-lNSCC cancer cell lines, utilizing MIT assay Our results show that the combination therapy with Ad-mTERT-8l9 and chemotherapeutic agents led to improved cytotoxic effect compared with a virus or chemotherapeutic agents alone treatment The induction ofapoptosis was also syncrgistically increased in cancer cells treated with Ad-mTERT-819 and chemotherapeutic agents , demonstrated by FACS analysis and TUNEL assay Furthermore, combination therapy with Ad-mTERT-819 and chemotherapeutic agents exhibited superior anti-tumor effect in FaDu head and neck tumor in vivo compared with either agent alone Overall, this study presents strong evidence for a synergistic effect when treatment of Ad-mTERT-819 is combined with chemotherapeutic agents, 639 Irradiation Prior to Adenovirus Treatment Increases Transgene Expression in Prostate and Breast Cancer Cells Petri Nokisalmi,' > Maria Rajccki.P Tanja Hakkaraincn.P Mikko Tcnhunen.l Akseli Hemminki.l-' /Haartman Institute & Molecular Cancer Biology Program, University ofHelsinki, Helsinki, Finland; 2Department ofOncology, Helsinki University Central Hospital, Helsinki, Finland Radiotherapy is a widely used modality to treat different cancer types It has been proposed that radiotherapy could sensitize cancer cells to adenovirus infection and vice versa, and therefore useful interactions are possible Modem radiotherapy provides accurate and precise modes to target radiation into the tumor while reducing the dose in surrounding healthy tissues Genetically modified adenoviruses can also targcr tumor cells but with distinct mechanisms In principle, the combination of adenovirus mediated cancer gene therapy and radiotherapy could lead to increased tumor control, oncolysis and decreased side effects Evaluation of the possible mechanisms of the putative synergy is important when treatment protocols are planned Methods and results: We studied the effect of irradiation on gene transfer with various capsid modified or type adcnoviruses to prostate and breast cancer cells Moreover, the effect of irradiation on CAR- , HSPG-, aVp3-, «vps- and CD46expression levels was assessed with FACS Lucifcrase expression levels were systematically increased by Gy irradiation, regardless ofthe cell line or virus concentration In general, approximately 87 % mean RLU increase was observed when compared to non-irradiated cells In contrast, Gy irradiation did not alter CAR-, HSPG, aVp3-, aVp5- and CD46-expression levels, suggesting that the increase in transgene expression levels are due to other factors These responses might bc related to enhanced cell metabolism, increased transgcnc mRNA production or increased protein production in general, all of which arc being analyzed and data will be presented Further, in vivo experiments are in progress Conclusions: Radiation increases adenoviral transgene expression in tumor cell lines but this is not due to increased virus receptor expression 640 Evaluation of Biodistribution and Safety of DWP418, Relaxin Expressing Oncolytic Adenovirus Therapeutics Kyunghyun Min,' Jaemook Choi,' Kinam Kim,' Joohang Kim,' Chaeok Yun,' Yoewook Koh,' I-lyunsoo Kim ' /Biotechnology Research Center, Daewoong Pharmaceutical Co., Ltd , Yongin, Kyunggi -do, Korea; 2}{)Jjsei Cancer Center; Yonsei University College ofMedicine, Seoul, Korea To improve cancer cell specificity and the strength of the wild type hTERT promoter, the modified hTERT promoter was generated by our own recombinant biotechnology Furthermore, to increase viral spreading efficiency and stimulate an apoptotic effect in cancer cells, Relaxin gene was inserted into the E3 gene region In the in vitro and in vivo experiments, DWP418, a novel oncolytic adenovirus, demonstrated potent antitumoral efficacy The toxicological evaluation ofDWP418 included single, repeated dose general toxicity and biodistribution studies in mice A single subcutaneous administraion ofDWP418 at dose levels of3 x 10'11, I X1011 or x 1011 vplmouse followed by a 14-day observation period was shown no treatmentrelated toxicity during this study DWP418 was administered once weekly in mice for or 13 consecutive weeks by subcutaneously at doses of x IO'", X 1011 or x 1011 vp/mouse There were no toxicologically-relevant ehangcs in the hematology parameters and clinical biochemistry examination Therefore the NOAEL (No-Observed-Adverse-EITect-Level) was considered to x 1011 vp/mouse and I x 1011 vp/mouse, respectively Also , host genome integration assays were conducted in a week study DWP418 was not integrated in the host genome A single subcutaneous administration ofDWP418 at dose level of3 x 10 10 vp/mouse followed by a 56-day observation period was not associated with any overt toxicity Biodistribution data indicated there was no systemic exposure, as detection was limited to the injection site Taken together, these findings demonstrate that DWP418 is a safe and novel candidate for the treatment of human malignancies We have a plan to start Phase I study of DWP418 administered intratumorally to patients with recurrent head and neck cancer in Korea 641 Triple Helix-Forming Oligonucleotides Specifically Reduces ATM Transcription, Translation, and Radiosensitivity in Human Glioma Cells Cfm-Chiao we, Chi-Shiun Chiang /Department ofBiomedical Engineering and Environmental Sciences, National Tsing Hua University; Hsinchu, Taiwan The loss ofATM (ataxia telangiectasia-mutated) gene leads to oxidative damage, AT disease, and the sensitivity to radiation damage Recognition ofB-DNA by oligonucleotides that form triple helices is a unique method to specifically recognize and inhibit thc functions of target double-stranded DNA sequence This study explored the relationship between atm expression and radiosensitivity of human glioma cell, U87, by TFO antigene therapy To achieve this, 10-17 mer oligonucleotides were designed to suppress atm gene expression of U87 The results showed that stable intercellular triplexes were formed under physiologic condition The melting point of the triplex was 47.5 "C, indicating that triplex can be stable fonncd within cells Time course analysis of gene inhibition was studied by RT-PCR, Western blot, and now cytometry, ATM gene expressions in both RNA and protein levels were successfully interfered by the oligonucleotides The suppression ofATM gene expression was associated increase radiosensitivity Cell survival assay showed that alb ratio was changed from 0.00 I to 2.543-5.994 with primary change on a value This indicates thatATM mainly affects the repair capacity of radiation-induced double strands breaks This is further supported by the finding thatATM-sileneed cells had reduced SLDR ability Taken together, this study indicates these TFO sequences might be an eligible approach for the development of ATM radiogene therapy Key wonk Ataxia telangiectasia-mutated; glioma; triplex forming oligonucleotide; sublethal damage; radiosensitivity * Corresponding author DWP418 is a novel oncolytic adenovirus engineered to target and kill cancer cells expressing high levels oftelomerase Telomerase is an attractive target for tumor cell specificity because close to 90% of tumors have telomcrase activity, whereas most normal cells not Molecular Therapy Volume 15.Supplement I ~br Copyright © The American Soc iety o f Gene Therapy 2007 S245 ... modified adenoviruses can also targcr tumor cells but with distinct mechanisms In principle, the combination of adenovirus mediated cancer gene therapy and radiotherapy could lead to increased tumor. .. recombinant biotechnology Furthermore, to increase viral spreading efficiency and stimulate an apoptotic effect in cancer cells, Relaxin gene was inserted into the E3 gene region In the in vitro and. ..combined with Ad-mTERT-819 Wc assessed thc cytotoxic activity of each combination in four different I-lNSCC cancer cell lines, utilizing MIT assay Our results show that the combination therapy with