608 in vivo anti tumor effect of fusion plasmid hp19ARF TAT using targeted cationic lipid system

608 In Vivo Anti Tumor Effect of Fusion Plasmid hp19ARF TAT Using Targeted Cationic Lipid System Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy S2[.]

Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy

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CANCER - TARGETED GENE THERAPY II

607 Anti TGF-β Pancreatic Cancer Gene

Therapy with Infectivity Enhanced Adenovirus

Expressing Soluble TGF-β Receptor II

Shigenori Hoshino,1 Yasuo Adachi,2 Eric Brown,1 Selwyn M

Vickers,1 Masato Yamamoto.1

1 Department of Surgery, University of Minnesota, Minneapolis,

MN; 2 Laboratory of Immune Regulation, Wakayama Medical

University, Osaka, Japan.

In the normal circumstances, Transforming growth factor (TGF)-β

serves as a tumor suppressor and prevents tumorigenesis However,

in some advanced phase of carcinomas including pancreatic cancer

exhibiting mutations in TGF-β signaling pathway, TGF-β switches

its role to tumor promoter In such cases, tumor itself secrets

TGF-β and uses this cytokine in order to condition the environment

for tumor growth, increased metastasis via enhanced motility,

resistance to chemotherapeutic agents, and suppression of host

immunosurveillance Here we show the effect of TGF-β signaling

blockade expressed with fi ber modifi ed adenovirus (Ad5/3) This

vector (Ad5/3sTβRII) encodes soluble extracellular domain of human

TGF-β receptor type II fused to the Fc fragment of human IgG1

Many pancreatic cancer cell lines do secret TGF-β by themselves

and this supposed to work for tumor progression in both autocrine

and paracrine manner First, we analyzed the effect of the vector to

cell viability Infection of Ad5/3sTβRII dramatically suppressed the

cellular viability of pancreatic cancer cell lines including Panc-1,

AsPC-1 compared to control vector expressing fuciferase (fi g 1)

In some cell lines, TGF-β is known to trigger a phenotypic change

called epithelial-mesenchymal transition (EMT), representing

more aggressive feature (e.g tumor invasion, metastatic character,

chemo resistance) When we added conditioned medium from the

cells infected Ad5/3sTβRII, TGF-β1-induced E-cadherin (epithelial

marker) suppression and vimentin (mesenchymal marker) induction

was suppressed (fi g 2) In the wound healing assay, conditioned

medium from the cells infected Ad5/3sTβRII inhibited the induction

of cell motility to disclose the scratched wound induced by TGF-β1

In aggregate, the TGF-β Receptor II expressing infectivity enhanced

adenoviral vector (Ad5/3sTβRII) would be one of the potent gene

therapy strategies for pancreatic cancers

608 In Vivo Anti-Tumor Effect of Fusion Plasmid hp19ARF-TAT Using Targeted Cationic Lipid System

Guoqin Niu,1 Wouter H P Driessen,2 Sean M Sullivan,3 Jeffrey A Hughes.1

1 Pharmaceutics, University of Florida, Gainesville, FL; 2 M D Anderson Cancer Center, Houston, TX; 3 Vical Incorporated, San Diego, CA.

Developing an effi cient and safe strategy to introduce a therapeutic gene into target cells in vivo is a key issue in cancer gene therapy

A fusion gene, coding for hp19ARF-TAT, was used for cancer gene therapy, which contained a secretory signal sequence and a membrane permeability domain This TAT peptide domain may shuttle the cytotoxic domain into non-transfected cells, increasing the bystander effect Gene expression was verifi ed in vitro using U87-MG cells by Western Blot, and hp19ARF-TAT demonstrated cell killing effect The cell killing could be increased when the cells were co-treated with Portland anti-trypsin protein, a furin inhibitor, which inhibited the cytotoxic peptide degradation The gene induced U87-MG cell apoptosis after transfection was demonstrated by TUNEL staining The targeting peptide with the sequence of MQLPLAT, which has been shown to bind to FGF2 receptor, conjugated with a phospholipid, was used for targeting a lipid-based gene delivery vehicle to U87-MG cells The cationic lipid mixture was composed of 1-(N4-spermine)-2, 3-dilaurylglycerol carbamate : lyso-phosphatidylcholine : glycerol monooleate : oleic acid (10:13:51:26 mol/mol, GL89/LXS) The luciferase gene expression was two-fold higher with the FGF2-PEG-GL89/LXS than by GL89/LXS, and the cell killing effect of hp19ARF-TAT delivered by FGF2-PEG-GL89/LXS was 3-fold higher than that by GL89/LXS Both results indicated that FGF2-PEG-GL89/ LXS was an effi cient gene delivery system A xenograph model of human glioma (U87-MG cells) tumors in nude/nude mice was built

by subcutaneously injecting 2 × 106 U87-MG cells/100µl/mouse Animals were injected 50 µg plasmid/150 µl/mouse/dose lipoplex (GL89/DNA =2:1, mole ratio) at the 4th and 11th day of post tumor cells implanted via tail vein Mice body weight and tumor size was measured everyday after the treatment At the 15th day of post-tumor cells implanted, mice were sacrifi ced, and tumors were collected and weighed Compared to PBS group, FGF2-targeted plasmid treatment group had a reduced tumor weight of 64.3% ± 29.3% (p<0.05, n=6), and non-targeted plasmid treatment group was 54.3 ± 35.3% (p<0.05, n=6) When compared to PVC1157/GL89/LXS non-coding vector control group, FGF2-targeted group had a tumor weight inhibition

of 60.2% ± 32.7% (p<0.05, n=6), and non-targeted lipoplex was 49.0 ± 39.3% (p>0.05, n=6) RT-PCR and Western-Blot of tumor homogenization results indicated that there was gene expression in

Molecular Therapy Volume 17, Supplement 1, May 2009

CANCER - TARGETED GENE THERAPY II

tumors after 48 h of systemic administration of the lipoplex There

was no difference in the mice body weight of post-treatment among

the control and gene treatment groups In conclusion, hp19ARF-TAT

was a cytotoxic gene and could be a promising brain cancer gene

treatment when using an effi cient targeted delivery system, such as

FGF2-PEG-GL89/LXS

609 Leukemia-Specifi c siRNA Delivery by

Immunonanoplexes Consisting of Anti-CD43

Minibody Conjugated to Oligo-9 Arg-Peptides

Yeon Kyung Lee,1 Keun Sik Kim,2 Jung Seok Kim,1 Jin Ee Baek,1

Sang Il Park,1 Hwa Yon Jung,1 Sang Soon Yoon,3 Kyeong Cheon

Jung,4 Hyung Geun Song,5 Yong Serk Park.1

1 Biomedical Laboratory Science, Yonsei University, Wonju,

Gangwon, Korea, Republic of; 2 Biomedical Sciences, Youngdong

University, Youngdong, Chungbuk, Korea, Republic of;

3 Development and Manufacturing, DINONA INC, Iksan, Chonbuk,

Korea, Republic of; 4 Pathology, Seoul National University College

of Medicine, Seoul, Korea, Republic of; 5 Pathology, Chungbuk

National University College of Medicine, Cheongju, Chungbuk,

Korea, Republic of.

Recently siRNAs have been considered as one of the more

promising new classes of anticancer drugs in development The clinical

applications of siRNA therapeutics are, however, hindered by poor

intracellular uptake, limited blood stability and non-specifi c delivery

To address these problems, we developed a 9R-immunonanoplex

for siRNA delivery using JL-1 minibody (T leukemia cell-specifi c

anti-CD43 minibody) for effective siRNA delivery to leukemic cells

First of all, the JL-1 minibody was conjugated to oligo-9-arginine

peptide for specifi c recognition of leukemic cells Self-assembling of

the minibody-9R conjugates and siRNA at appropriate molar ratios of

protein to nucleic acid was able to produce stable immunonanoplexe

particles The anti-CD43 immunonanoplexes were able to deliver

siRNA specifi cally to leukemic cells (CEM and Jurkat), but not to

control cancer cells (H9) According to FACS analysis, siRNAs

delivered by the anti-CD43 immunonanoplex particles were rapidly

taken up and then escaped from the endosomal process in 2 hr The

transfection effi ciency of the optimized anti-CD43 immunonanoplexes

varied depending on the concentration of CD43 molecules expressed

on leukemic cell surface; higher siRNA transfer to CEM cells than to

Jurkat cells This facilitated siRNA transfer was presumably due to

specifi c recognition of leukemic cells via JL-1 minibody and effi cient

intracellular uptake via modifi ed 9-Arg peptide These results suggest

that the anti-CD43 immunonanoplex is a powerful siRNA delivery

system for human T-cell leukemias therapies

610 Preferential Anti-Tumoral Activity Via

Genetically Modifi ed Adeno-Associated Virus

Serotype 5

HanSaem Lee,1 Ji Yun Kim,1 Won Il Lee,1 Sung Jin Kim,1

Jeonghyun Ahn,1 Min Ji Ko.1

1 Departments of Microbiology, Research Institute for

Biomacromolecules, University of Ulsan College of Medicine,

Seoul, Korea, Republic of.

Recombinant adeno-associated virus serotype 5 (rAAV5) can be

useful in gene therapy, including caner gene therapy owing to its

long-term gene expression with minimal toxicity However, its broad

tissue tropism is one of major drawbacks, hindering preferential

gene transfer to tumor tissues In the present study, we attempted

to accomplish selective gene transfer to human cancer cells by

genetically modifying rAAV5 capsids For this, we modifi ed VP1

capsid protein of rAAV5 by specifi cally inserting peptide sequences

recognizing cancer surface markers, such as integrins, sialyl Lewis X

(sLeX), and tenascin C (TnC) All eight modifi ed rAAV5 vectors could

be produced as effi ciently as parental rAAV5 The mutant vectors could be readily purifi ed by conventionally CsCl2 gradient Moreover, rAAV5-RGD targeting integrins could transduce a variety of human cancer cells, depending on newly-introduced target recognition motif

In addition, we could successfully introduce another cancer targeting motif called TnC by the identical strategy When HSV-TK gene was transferred to cells by the re-targeted rAAV5 vectors, cytotoxicity was clearly observed in the presence of ganciclovir Thus, the results suggested that the target-specifi c genetic modifi cation of rAAV5 is possible and that these genetically-modifi ed rAAV5 vectors could be

utilized to achieve tumor targeting in vivo Further improvement of

re-targeted rAAV5 should allow these vectors to be practically utilized

for gene-based cancer treatment Key words: cancer gene therapy,

recombinant adeno-associated virus serotype 5, virus tropism, capsid modifi cation, integrin, tenascin C * Heuiran Lee: corresponding author; † HS and JY contributed this study equally

611 Potential Role of Human Telomerase Reverse Transcriptase (hTERT) as Target for Virotherapy in Human Gastrointestinal Stromal Tumors (GISTs)

Fuminori Teraishi,1,2 Yuuri Hashimoto,2 Yasuo Urata,3 Toshiyoshi Fujiwara,2 Tadashi Horimi.1

1 Department of Gastroenterological Surgery, Kochi Health Sciences Center, Kochi, Japan; 2 Center for Gene and Cell Therapy, Okayama University Hospital, Okayama, Japan; 3 Oncolys BioPharma, Inc., Tokyo, Japan.

Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal tumors of the gastrointestinal tract, and an immunohistochemical marker c-KIT has greatly assisted pathologists Although advanced GISTs usually cause local recurrence and metastasis to other organ, therapeutic inhibition of c-KIT by the small-molecule inhibitor imatinib gives objective responses in about 50% of patients Despite the therapuetic success of imatinib, some patients express c-KIT oncoproteins that are resistant to imatinib-mediated inhibition The majority of malignant tumors express telomerase activity, whereas most normal cells do not hTERT is the one of the major component associated with telomerase activity in humans The purposes of this study were to examine the expression

of hTERT in human GISTs and establish the novel strategy with oncolytic adenovirus for treatment of advanced GISTs We fi rstly examined hTERT expression in a series of 9 gastric GISTs We found that most gastric GISTs (89%) expressed hTERT while its expression had little correlation with their malignant potential and prognosis We then tested the effect of OBP-401 (Telomelysin-GFP) that expresses the green fl uorescent protein (GFP) reporter gene under the control

of the cytomegalovirus promoter in the E3 region to monitor viral distribution, in human GIST882 (imatinib-sensitive), human GIST48 (imatinib-resistant) cells, and rat GIST GIST-DR cells OBP-401 could replicate and effectively induced oncolysis in human and rat GIST cell lines after 72-120 hours We further designed the in vivo experiments to detect subcutaneous tumors and peritoneal dissemination of GIST cells The established GIST-DR tumors were treated with OBP-401 subcutaneously or intraperitoneally After 5 days, GFP-positive tumors were detected in GIST-DR subcutaneous tumor model and peritoneal dissemination model Our data indicates hTERT could be a useful diagnostic marker and a critical target for oncolytic virotherapy in GISTs Further studies including in vivo experiments about antitumor effect of OBP-401 against imatinib-resistant cells are warranted in order to explore its potential clinical applications