354 Effect of tumor necrosis factor-related apoptosis-inducing ligand (trail) gene therapy combined with chemotherapeutic administration on suppression of multiple hepatic metastasis

Category 4e: Molecular and Cellular Biology: Gene Transfer/Gene Therapy developed a novel tool in gene transfer technology based on cell permeable HBV-nucleocapsids loaded with non-viral or viral nucleic acids. Cell permeability is mediated by fusion of the cell permeable TLM peptide to HBcAg. TLM-core particles, lacking the C-terminal basic stretch, were isolated from an eubacterial expression system. The purified particles were dissociated, the HBcAg-monomers were combined with the plasmid DNA to be packaged and reassociated. Assembled nucleocapsids were separated from unassembled proteins by gelfiltration. The proper particular structure of reassembled nucleocapsids was confirmed by electron microscopy, the loading with DNA by taqman PCR. Moreover, an eukaryotic expression system for packaging of nucleic acids into virus like particles (VLPs) was established. Sf21 cells were triple infected by baculovirus coding for the HBV polymerase, the core-TLM fusion protein and the packaging construct (harboring two encapsidation signals and a reporter gene). The cell permeable nucleocapsids were isolated by a combination of gel filtration and sucrose gradient centrifugation. Taqman PCR revealed that about one reporter genome molecule is packaged into one nucleocapsid. The cell permeability of the E.coli- and of the Sf21 cell derived TLM-capsids was demonstrated by confocal microscopy. The capacity of these particles for gene transfer was shown by using SHBs and eGFP as marker genes. These results indicate that cell permeable HBV-derived nucleocapsids loaded with nucleic acid provide a novel tool for safe and efficient gene transfer.

354 EFFECT OF TUMOR NECROSIS FACTOR-RELATED APOPTOSIS-INDUCING LIGAND (TRAIL) GENE THERAPY COMBINED WITH CHEMOTHERAPEUTIC ADMINISTRATION ON SUPPRESSION OF MULTIPLE HEPATIC METASTASIS

M. Ishii 1 , M. Iwai 1 , Y. Harada 1 , J. Imanishi 2 , O. Mazda 2 , T. Okanoue 1 . 1 Department of Molecular Gastroenterology and Hepatology, Graduate School of Medicine, Kyoto Prefectural University of Medicine, Kyoto, Japan; 2 Department of Microbiology, Graduate School of Medicine, Kyoto Prefectural University of Medicine, Kyoto, Japan Background/Aims: TRAIL induces apoptosis in various malignant cells but not in most normal cells. Here we evaluated the therapeutic efficacy of hydrodynamics-based TRAIL gene therapy against multiple hepatic metastasis. Combination therapy of TRAIL gene and actinomycin D (ACD) was also examined. Methods: (1) Recombinant TRAIL (rTRAIL) was added to the culture medium of CT-26 (murine colon carcinoma) in presence or absence of ACD. To assess proliferation and apoptosis of the cells in vitro, the tetrazolium assay and flowcytometric analysis were performed. (2) The plasmid DNA encoding the extracellular domain of human TRAIL fused with a signal sequence (pGEG.TRAIL) was constructed. To establish liver metastasis, CT-26 cells were injected into the spleen of BALB/c mice (day 0). On days 1 and 8, mice received hydrodynamics-based transfection with pGEG.TRAIL or pGEG.4 (empty plasmid). ACD was injected intraperitoneally. The number of hepatic metastatic nodules was counted on day 14. Results: (1) rTRAIL alone failed to kill CT-26 cells in culture, while rTRAIL plus ACD treatment induced apoptosis in a dose dependent manner. (2) The number of metastatic nodules was 63.8±31.5 (untreated), 49.4±21.0 (pGEG.4 plus ACD), 52.3±32.7 (pGEG.TRAIL alone) nd 14.8±17.3 (pGEG.TRAIL plus ACD). The pGEG.TRAIL plus ACD treatment significantly suppressed the liver metastasis in comparison with the untreated and PGEG.4 plus ACD group (p=0.015 and 0.047). Conclusion: These results suggest that the combination therapy of hydrodynamics-based TRAIL gene transfer and n anticancer drug administration is an efficient means for suppression of metastatic liver tumors.

107

355 IN-VIVO ADENOVIRUS MEDIATED RNA INTERFERENCE OF MULTIDRUG RESISTANCE PROTEIN-2 (MRP-2/ABCC2/CMOAT)

I. Narvaiza, M. Vera, N. Razquin, M. Zaratiegui, J. Prieto, P. Fortes. Gene Therapy Unit. School of Medicine. Fundacion Para La Investigacion Médica Aplicada (FIMA). University of Navarra, Pamplona, Spain RNA interference (RNAi) is a natural process, originally described in Caenorhabditis elegans and plants, initiated by the cleavage of long double stranded RNA (dsRNAs) by the ribonuclease III Dicer generating short interfering RNAs (siRNAs). siRNAs induce selective degradation of mRNAs that are homologous to their sequence. In mammalian cells, RNAi interference can be achieved by transfection of chemically synthesized siRNAs or short-hairpin RNA (shRNAs) precursor expressing plasmids. In-vivo gene silencing of endogenous and exogenous genes has been proven by hydrodynamics injection of both shRNAs and synthetic siRNAs, or by using shRNA expressing viral vectors such as lentivirus or adenoviruses. We have constructed first generation recombinant adenoviruses encoding different shRNA against murine multidrug resistance protein 2 (MRP2/ABCC2/cMOAT) (AdshMRPs). Mutations in MRP2 gene are responsible for Dubin-Johnson syndrome, a hereditary disease characterized by hyperbilirubinemia. AdMRP infected murine hepatocellular carcinoma cells (Hepa 1-6 cell line) have decreased MRP2 mRNA levels as quantified by quantitative-PCR. In addition, we are able to detect siRNAs targeting MRP2 mRNA by primer extension experiments in AdMRP2 infected Hepa 1-6 cells. In-vivo experiments in C57BL/6 mice revealed the presence of antisense siRNAs against MRP2 in liver extracts seven days after AdMRP intra-venous (i.v.) injection. Furthermore, AdMRP i.v. administration in mice resulted not only in basal serum hyperbilirubinemia, but also in significant impairment of synthetic bilirubin uptake after bilirubin i.v. administration. Our results demonstrate that adenoviral vectors are efficient tools for shRNA delivery into the liver, allowing the targeted silencing of an endogenous mRNA that result in transient function inhibition.

356 POTENTIAL OF SEMLIKI FOREST VIRUS (SFV) VECTORS FOR THE GENE THERAPY OF TUMORS THAT CAN PRODUCE METASTASES IN LIVER

J.R. Rodriguez-Madoz, E. Casales, J. Prieto, C. Smerdou. Medical School Fundacion Para La Investigacion Medica Aplicada (FIMA) Universidad De Navarra, Pamplona, Spain Two SFV based vectors expressing murine interleukin-12 (IL-12) have been developed and their antitumoral efficacy has been tested in a mouse model of colon carcinoma that frequently produces liver metastases. In SFV-IL-12 vector, genes coding for IL-12 subunits were cloned under a single viral subgenomic promoter using an IRES between them. SFVenhIL-12 vector carries each gene fused to the SFV capsid translation enhancer, under independent viral subgenomic promoters. Recombinant SFV viral particles were produced and IL-12 expression was tested in supernatants of BHK infected cells by ELISA. Biologically active IL-12 was expressed at high levels from both vectors with a 9-fold increase in the case of SFV-enhIL-12. Single tumor nodules were implanted in the flank of C57BL/6 mice by subcutaneous injection of MC38 colon carcinoma cells and treated with a single intratumoral injection of SFV-IL-12, SFV-enhIL12, and SFV-LacZ or saline as controls. With 108 viral particles of SFV-IL12 or SFV-enhIL-12 more than 80% of treated mice experienced complete tumor regression with long-term tumor-free survival. However, when lower doses of vector were used SFV-enhIL-12 was more efficient than SFV-IL12 in inducing antitumoral responses. In addition, all mice that rejected the tumors showed specific protection against tumor rechallenge. CTL assays and depletion studies showed a CD8+ T cell dependent response. In animals treated with IL-12 expressing vectors high cytokine levels were found within tumors at 24 h. postinjection but decreasead along time until day 6. A similar result was found in serum although with lower cytokine levels.