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[.]
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 Department of Surgery, University of Minnesota, Minneapolis, MN; 2Laboratory 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 fiber modified 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 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 (fig 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 (fig 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 S232 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 Pharmaceutics, University of Florida, Gainesville, FL; 2M D Anderson Cancer Center, Houston, TX; 3Vical Incorporated, San Diego, CA Developing an efficient 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 verified 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 FGF2PEG-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 efficient gene delivery system A xenograph model of human glioma (U87-MG cells) tumors in nude/nude mice was built by subcutaneously injecting × 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 sacrificed, 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