638 telomerase targeted oncolytic adenovirus in combination with chemotherapy elicits enhanced cytopathic and anti tumor effects in head neck squamous cell carcinoma

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

HN-SCC 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

pres-ents 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.'

I Pathology, Medicine and Urology, University ofTennessee

Health Science Center, Memphis, TN.

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,

replica-tion-competent adenovirus (AdPSAE I), in which the adenoviralE 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

adeno-virus expressingthe 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

Intra-tumoral 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-JaeKim,'WongiSeol,'

Eui-Sic Cho,4 Yeun-Ju Kim,!Sung-Ha Cho,2Hee-Jong Kim;

Bong-Su Kang,' Won-Jin Ji.'

'Juseong Gene Therapy R&D Center, Juseong College ,

Cheong-won-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

ofKo-rea; "School ofDentistry; 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-medi-ated gene delivery system due to its high transduction efficiency in

various human cancer cell lines Among the tested rAAV2 vectors,

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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 therapeuticgene, we replaced the CMV promoter in rAAV2-CMV-GFPwith the promoters ofa protein regulator ofcytokinesis I (PRC I), ribonuclease reductase 2 (RRM2), baculoviral lAP repeat-containing 5 (BIRC5), hypoxia responsive clement (HRE),human telomerase reverse transcriptase (hTERT), and 3E-hTERT-TPL to establish rAAV2-PRC I-GFp, rAAV2-RRM2-GFr, 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

highlycomparable 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 containingPRC I, RRM2, or BIRCS promoter may be used for targeted cancer gene therapy and could

be applied for cancer-specific expressions ofAAV2-mediated anti-angiogenesis approaches.(This study was supported by the grant from the Ministry of Science and Technology (M I0534040005-05N3404-00511),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.JO.-Jun Kwon.s -'Joo-Hyuk Sohn,3 Chae-Ok Yun;·JJoe-hang Kim.J

I KOSEF through National Core Research Center for Nanomedi-cal Technology, l'onsei Cancer Center, l'onsei University Coll ege

ofMedicine, Seoul, Republic ofKorea ; lBrain Korea 21 Project for Medical Science , l'onsei Cancer Center; Yonsei University College ofMedicine, Seoul, Republic ofKorea; Jlnsilutefor Can-cer Research , Yonsei Cancer Center; l'onsei University College of Medicine, Seoul, Republic ofKorea.

The head and neck squamous cell earcinoma(HNSCC) affcets

an estimated 500,000patients annually worldwide At present,the combination therapy of chemotherapeutic agents, cisplatin and 5-fluorouracil (5-FU), has been used as a standard therapy for advanced HNSCC Gene attenuated replication competent adenoviruses are being developed as novelanti-tumor therapeutics.Clinical trials with the ONYX-OIS(EI B 55kDa-deleted) adenovirus in combination with chemotherapeutic agents (cisplatin,5-FU) have demonstrated

a synergistic antitumor effect However, most ofreplicating adeno-viruses used in combination therapy to date have their£1B 19kDa

gene intact Because the E IB 19 protein is an anti-apoptotie agent, adenoviruses retaining theirEI B 19kDagene may interfere with the mode of action of some chemotherapeutic agents,resulting in a t-tenuating the overall potency ofthese combination treatment In our previous report,EI B 19kDa-deletedadenovirus Ad-mTERT-A19, which encode the EIA gene driven by the uniquely modified m-hTERT 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 ofdifferentcombinations ofvarious chemotherapeutic agents

Molecular Therapy Volum e 15 Supplement I, \b y 2007

Co pyright © '111C A merican Socie tyo f G eneTI ICr.lpr

combined with Ad-mTERT-819 Wc assessed thc cytotoxic

activ-ity 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

chemo-therapeutic 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 tumorin vivocompared with either agent

alone Overall, this study presents strong evidence for a synergistic

effect when treatment of Ad-mTERT-819 is combined with

che-motherapeutic agents,

Increases Transgene Expression in Prostate and

Breast Cancer Cells

Petri Nokisalmi,' >MariaRajccki.PTanjaHakkaraincn.PMikko

Tcnhunen.l Akseli Hemminki.l-'

/Haartman Institute&Molecular Cancer Biology Program,

Uni-versity 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

adeno-viruses can also targcr tumor cells but with distinct mechanisms

Inprinciple,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

5 adcnoviruses to prostate and breast cancer cells Moreover, the

effect of irradiation on CAR-,HSPG-, aVp3-,«vps-and

CD46-expression levels was assessed with FACS Lucifcrase CD46-expression

levels were systematically increased by 8 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,8 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

of DWP418, Relaxin Expressing Oncolytic

Adenovirus Therapeutics

Kyunghyun Min,' Jaemook Choi,' Kinam Kim,' JoohangKim ,'

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

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 do not

Molecular Therapy Vo lume 15.S upplement I ~br 20 07

C opyright © TheAm erican Soc ietyo fGen e Therapy

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 apoptoticeffectin cancer cells, Relaxin gene was inserted into the E3 gene region In thein vitro

and in vivoexperiments,DWP418, a novel oncolytic adenovirus, demonstrated potent antitumoral efficacy The toxicological evalua-tion ofDWP418 included single,repeated dose general toxicity and biodistribution studies in mice A single subcutaneous administraion ofDWP418 at dose levels of3 x10'11,IX1011or 3 x1011 vplmouse

followed by a 14-day observation period was shown no treatment-related toxicity during this study DWP418 was administered once weekly in mice for 4 or 13 consecutive weeks by subcutaneously

at doses of 3 x IO'",1X 1011or 3 x1011vp/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 3 x 1011

vp/mouse and I x 1011vp/mouse,respectively Also,host genome integration assays were conducted in a 4 week study DWP418 was not integrated in the host genome A single subcutaneous administra-tion ofDWP418 at dose level of3 x 1010vp/mouse followed by a

56-dayobservation period was not associated with any overt toxic-ity 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

Specifically Reduces ATM Transcription, Translation, and Radiosensitivity in Human Glioma Cells

Cfm-Chiaowe,Chi-Shiun Chiang

/Department ofBiomedical Engineering and Environmental Sci-ences, National Tsing Hua University; Hsinchu, Taiwan.

The loss ofATM (ataxia telangiectasia-mutated) gene leads to oxi-dative 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 betweenatmexpression and radiosensitivity of human

gliomacell, U87, by TFO antigene therapy To achieve this, 10-17 mer oligonucleotides were designed to suppressatmgene expres-sion 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 expres-sions 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 thatalbratio 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-sileneedcells had reduced SLDR ability Taken together,this study indicates these TFO sequences might be an eligible approach for the development of ATM radio-gene therapy Key wonkAtaxia telangiectasia-mutated;glioma; triplex forming oligonucleotide; sublethal damage; radiosensitivity

*Corresponding author

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