732. Defining Tropism of Oncolytic Vectors by Protease Availability: Measles Viruses Selectively Fusing Matrix-Metalloproteinase Expressing Cells

CANCER-TARGETED GENE THERAPY: TARGETING OF NON-ADENOVIRAL VECTORS in the control irradiated cells). To determine the effect of c-Met antisense gene therapy in the in vivo orthotopic tumor model, C57BL/6J mice were injected intratracheally with 3LL lung tumor cells. Beginning one day later and continuing for the next three days, the mice were injected I.V. with c-Met antisense plasmid liposome complexes. The mice were irradiated 48 hours after tumor implantation to 18 Gy to the pulmonary cavity. Mice injected with the antisense c-Met plus 18 Gy had improved survival compared to mice injected with the 3LL cells alone plus 18 Gy of irradiation. Optimization of the delivery of the c-Met antisense plasmid liposome complexes may facilitate radiosensitization of lung cancer.

730. Cancer Specific Promoters for Gene Therapy of Small Cell Lung Cancer Nina Pedersen,1 Mikkel W. Pedersen,1 Michael S. Lan,2 Thomas T. Poulsen,1 Hans S. Poulsen.1 1 Department of Radiation Biology, Section 6321, National University Hospital, Copenhagen, Denmark; 2Department of Pediatrics, Research Institute for Children, Children’s Hospital, Louisiana State University Health Sciences Center, New Orleans, LA. For small cell lung cancer (SCLC) there is currently no satisfactory treatment, wherefore development of novel modalities, such as gene therapy, is highly in demand. As SCLC is a disseminated disease, treatment must be administered systemically and therefore requires absolute targeting to the cancer cells. One way to achieve targeting is by expressing the therapeutic gene from highly active, cancer specific promoters. We have identified a number of genes highly and specifically expressed in SCLC (Pedersen et al., Cancer Res 63, p1943, 2003) and are analysing the regulatory regions of these genes to identify promoters which can be used for targeting. The insulinoma-associated 1 (INSM1), which is normally exclusively expressed during early embryonic development, has been found highly re-activated in neuroendocrine tumours. The human INSM1 promoter has been shown to confer correct spatial and temporal expression in transgenic mice (Breslin, MB et al., J Biol Chem 278: 38991, 2003). We find that this promoter can confer very high and SCLC specific gene expression. When regulating expression of the Herpes Simplex Virus Thymidine Kinase (HSVTK) gene combined with ganciclovir treatment, the expression level from this promoter is sufficient to mediate cell death specifically in SCLC cell lines with INSM1 expression with no effect on other cell lines. Preliminary in vivo experiments with xenografted tumours expressing HSV-TK from the INSM1 promoter show significant tumour regression after ganciclovir treatment. We have therefore demonstrated that the activity of the INSM1 promoter is sufficient for gene therapy approaches and that this promoter confers the cancer specificity needed for systemic treatment. In addition, we have designed a novel cloning system utilising the Cre recombinase –loxP system for easy transfer of cloned promoter regions to regulate expression of a variety of reporter and therapeutic genes without the use of restriction enzymes. This system facilitates the cloning of promoter regions and we are currently screening the regulatory regions from other of the potential genes highly and specifically expressed by SCLC.

Molecular Therapy Volume 11, Supplement 1, May 2005 Copyright  The American Society of Gene Therapy

731. Completely Elimination of Xenograft SW620 Cancer by Combined Tumor Targeting MnSOD and Trail Genes Yan-hong Zhang,1,2 Jin-fa Gu,2 Li-li Zhao,2 Yi-gang Wang,1 Jin-hui Wang,2 Ling-feng He,2 Wei-guo Zou,3 Qi-Jun Qian,1,4 Cheng Qian,1,5 Xin-yuan Liu.1,2 1 Gene Therapy, Xinyuan Institute of Medicine and Biotechnology, School of Life Sciences, Zhejiang University of Sci-Tech and Zhejiang Province Center for Gene Therapy, Hangzhou, Zhejiang, China; 2Gene Therapy, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chineses Academy of Sciences, Shanghai, China; 3The Scripps Research Institute, La Jolla, CA; 4Gene Therapy, Lab of Gene and Virus Therapy, Eastern Hepatobiliary Surgery Hospital, Second Military Medical University, Shanghai, China; 5Gene Therapy, Division of Hepatology and Gene Therapy, School of Medicine, Centro de Investigación Médica Aplicada (CIMA), University of Navarra, Pamplona, Spain. MnSOD is a latent tumor suppressor gene. In order to studies the therapeutic effect and mechanisms of MnSOD action, the tumorspecific replication-competent adenovirus pZD55 (E1B55 KD deleted adenovirus) was constructed and two human genes were inserted into it to form ZD55-MnSOD and ZD55-Trail. After studying, it was found the first time that the antitumor effect of ZD55-MnSOD is about 1000 fold than that of Ad-MnSOD. Further more by the combination of ZD55-MnSOD and ZD55-Trail, which was called before as “Targeting Dual Gene-ViroTherapy” strategy, then all the established xenograft SW620 solid tumor could be completely eliminated. This is an excellently date for the tumor killing effect of combined ZD55-MnSOD and ZD55-Trail, which was due to the compensative and synergic effect between ZD55MnSOD and ZD55-Trail and also due to their apoptosis when assayed by apoptotic method, such as DNA fragmentation, FACS or TUNEL. The overexpression of MnSOD accumulated the generation of H2O2, which could arrest cell cycle and induce apoptosis. The overexpressed Bax could result from the overexpression of MnSOD and Trail. The MnSOD could induced the releasing of AIF and cyto C, and then caspase-9 and caspase-3 were cleaved separately, while Trail could induced the cleavage of caspase-8. These results suggest that the antitumor efficiency of ZD55-MnSOD and the combination of ZD55-MnSOD with ZD55Trail were all mediated by apoptosis.

732. Defining Tropism of Oncolytic Vectors by Protease Availability: Measles Viruses Selectively Fusing Matrix-Metalloproteinase Expressing Cells Christoph Springfeld,1 Veronika von Messling,1 Christian Buchholz,2 Roberto Cattaneo.1 1 Molecular Medicine Program and Virology and Gene Therapy Track, Mayo Clinic College of Medicine, Rochester, MN; 2Medical Biotechnology, Paul Ehrlich Institute, Langen, Germany. Measles virus (MV) is oncolytic: wild type MV infection occasionally induces lymphoma regression in humans, and the MV vaccine strain has been used to eliminate different types of human cancer xenografts in mice. Towards enhancing the MV specificity for different tumor types we are operating at different levels, including selective viral activation by tumor-specific matrix metalloproteinases (MMPs). We report here the engineering of a recombinant MV which fusion (F) protein is selectively cleaved and activated by a MMP. The F protein of wild type and vaccine MV is produced as an inative precursor F0 that is cleaved into an aminoterminal F2 subunit and a membrane-anchored F1 subunit bearing a

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GENE REGULATION: OVERCOMING OBSTACLES TO GENE TRANSFER AND EXPRESSION fusion peptide. MV F0 is activated intracellularly by furin, but we have previously engineered a F protein activated by the extracellular protease trypsin (J. Gen. Virol. 81, 441-9, 2000). RESULTS. We produced MV F proteins that contain a MMP six residue cleavage site either in addition to or in exchange for the furin oligobasic cleavage site. When these proteins are co-expressed with the MV attachment protein (hemagglutinin) in MMP-secreting cells (HT1080), these cells form multinucleated syncytia. Control cells (Vero) that do not express MMPs do not support syncytia formation. Recombinant MV expressing the MMP-activated F proteins in place of the furin-activated F protein were rescued. These viruses propagated in HT1080 but not Vero cells: activated viral particles produced in HT1080 cells infect Vero cells but the infection remains restricted to single cells. When the MMP inhibitor GM 6001 is added to the media fusion of HT1080 cells was prevented. The amino-terminal sequence of MMP-cleaved F1 subunits is currently being sought. PERSPECTIVES. We show here that the tropism of oncolytic vectors can be defined by the availability of tumor-specific proteases. For virotherapy MMP-activatable F proteins can be combined with 1) attachment protein allowing cell entry through cancer-specific glycoproteins (J. Virol. 75:2087-96, 2001; J. Virol. 78, 302-13, 2004; Nat. Biotechnol. 22:331-6, 2004) 2) inactivation of the host control evasion proteins V and C (Devaux et al., this Abstract book; von Messling and Cattaneo, this Abstract book) and 3) additional expression of suicide genes and modulators of the tumor immune response (Mol. Ther. 9:S224, 2004; J. Gen. Virol. 80:101-6, 1999). Towards producing viruses with customized cleavage by different MMPs we are testing the efficacy of alternative MMP cleavage sites that have been functionally selected on tumor cells using a retroviral protease substrate library approach (Gene Ther. 10, 137080, 2003).

GENE REGULATION: OVERCOMING OBSTACLES TO GENE TRANSFER AND EXPRESSION 733. The Human XIST Gene Promoter Prevents Silencing of an Integrated Reporter Gene Michael R. Greene,1 Christopher H. Lowrey.1 Pharmacology, Dartmouth Medical School, Hanover, NH.

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Epigenetic silencing and position dependent expression are longstanding problems which continue to limit the development of gene replacement therapy. As a strategy to overcome this problem we have tested the ability of the human XIST (X inactivation-specific transcript) gene promoter to overcome epigenetic silencing. The XIST gene is one of a relatively small cohort of genes which are expressed from the inactive X chromosome in mammalian cells containing multiple copies of the X chromosome. The product of this gene is an untranslated structural RNA which coats the selected redundant X chromosome(s) prior to the covalent modification of histones and methylation of CpG islands and the resultant silencing of the majority the genes on the chromosome. Continued expression of the XIST gene in this highly repressive environment is required to maintain the chromosome in an inactive state. The region of the proximal promoter of the XIST gene on the inactive X chromosome has been shown to retain an active chromatin structure. Based on these findings we hypothesized that the XIST gene promoter might be able to resist the epigenetic changes which lead to transgene silencing in therapeutic applications. To test this idea, we subcloned a 315 base pair sequence of 5’ untranslated region of XIST known to contain the minimal promoter sequence upstream of an enhanced GFP reporter gene in a pUC-based plasmid which also contained a neomycin resistance gene. The same plasmid, with a CMV promoter, served as our control vector. The MEL cell line (mouse S284

erythroleukemia) is often used as a first screen for vectors with potential for use in therapeutic gene transfer to erythroid cells because genes transferred into these cells are frequently silenced. The plasmids were electroporated into MEL cells and then grown in media containing G418. Individual colonies were selected, grown to confluence, G418 removed and then maintained in non-selective culture. Flow cytometry was used to determine the percentage of cells in each clonal population which were expressing GFP above the level of the untransduced MEL cell line. Statistical analyses of results were performed using the t-test. 16 XIST and 13 CMV clones were available for analysis at the start of the experiment (time 0). 11/13 CMV clones and 12/16 XIST clones were initially expressing GFP. Of the clones which were expressing GFP, the average percentages of positive cells were higher for those with the XIST promoter (63% vs. 41%, p= 0.015). Expression was reanalyzed after 6 weeks of culture without G418 selection. At this time point, the average percentage of GFP expressing cells was much higher for the XIST clones (57% vs. 19%, p=0.00008) and when analyzed for silencing, XIST clones were expressing at an average of 90% of their time 0 levels vs. 46% for the CMV clones (p=.0008). These results indicate that the XIST promoter is resistant to silencing in our model and is a candidate for further development and mechanistic studies.

734. Association of DNA Polymerase µ with Recombinant Adeno-Associated Viral Vectors David C. Brooks,2 Fayaz R. Khazi,1 Katherine A. High.1 Hematology, Howard Hughes Medical Institute and The Children’s Hospital of Philadelphia, Philadelphia, PA; 2University of Pennsylvania, Philadelphia, PA.

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Recombinant adeno associated viral (rAAV) vectors have been successfully used for efficient delivery and stable transgene expression in animal models and clinical trials. Transduced vector genomes primarily persist as extra-chromosomal concatamers, with a low frequency of integration into host chromosomes. Investigations into possible mechanisms involved in vector genome integration and analyses of integration junctions reveal actively transcribed regions of chromosomes to be the hotspots for vector integration via a proposed nonhomologous end joining (NHEJ) mechanism of DNA repair (Nakai et.al 2003). Although the primary sensors of DNA damage, Ku, ATM and DNA-PK have been implicated in rAAV integration, there is limited information about the involvement of proteins that are directly involved in NHEJ. A recently discovered mammalian DNA polymerase µ (Polµ) has been shown to be an important component of the NHEJ ligation complex and to have a functional interaction with Ku-DNA complex. In this study, we investigated the effect of rAAV on induction of Polµ expression and the possible interaction with vector genomes. HEK293 cells transduced with rAAV2 at an MOI of 20,000 were incubated at 37°C for 0, 0.5, 1, 3, 6, 12 and 24 hours. Total proteins were harvested at each time point and the lysates were analyzed by Western blotting using anti-Polµ antibody. The results showed a nine-fold increase in Polµ expression in cells incubated for 12h post transduction. A three fold increase in expression was observed at the 3h time point. The 0.5 and 1hr treatments did not show a significant increase over basal levels. Expression levels dropped down to 2 fold at the 24h time point. Further, fluorescent microscopy revealed mobilization of GFPtagged Polµ from the cytoplasm to the perinuclear region at 3h post transduction. Foci, characteristic of a response to DNA damage, were distributed around the nucleus at 3h which were then localized into the nucleus at 6h and 12h post transduction. The Polµ signal was diminished but localized in the nucleus at 24h post transduction. The maximum intensity of the signal was observed at 6h in the nucleus. Polµ-GFP maintained a diffused appearance in untransduced Molecular Therapy Volume 11, Supplement 1, May 2005 Copyright  The American Society of Gene Therapy