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ONCOLYTIC VIRUS AND SUICIDE GENE THERAPY FOR CANCER drug concentration is difficult to obtain in the vicinity of the tumor since GCV does not cross readily the blood brain barrier We have previously reported a major improvement by using TK30, a much more potent mutant of TK: TK30-expressing cells (TK30 cells) were killed more efficiently than TK cells (10- to 100-fold) and the BE was strongly increased too The use of 5-fluorouracil (FU) has been shown by others to enhance the efficacy of the TK/GCV strategy FU is a thymidylate synthase inhibitor which acts by decreasing the pools of thymidine, the major competitor for GCV phosphorylation In this study, we have investigated the potential of FU to interact with GCV on four different human (SKI1, SKMG4, U87 and U251) and three rat (C6, F98 and 9L) TK- or TK30 glioma cells METHODS: The various cell lines were retrovirally infected to stably express the mutant TK30 or the wild-type TK Drug sensitivities were determined by their median inhibitory concentration (IC50) using conventional MTT proliferation assays BE experiments with either TK30 or TK were realized by mixing ratios of infected and non-infected cells The multiple drug-effect analysis of Chou-Talalay was used to qualify the interaction between GCV and FU RESULTS: As calculated by the Chou-Talalay method, at least an additive effect of the drugs was noted in proliferation experiments with all glioma TK30 cells In BE experiments carried out with SKI1 and SKMG4 cells, potentiation and/or synergism were observed and the synergism was stronger in TK- than in TK30 cells Nevertheless, the IC50 (GCV) was still much lower with TK30- vs TK cells in the presence of FU (by a factor of 35 and 126 for SKI1 and SKMG4, respectively) Interestingly, except for U251 cells, a strong synergistic effect was also found when non-infected cells alone were treated with GCV + FU, a phenomenon unreported until now The mechanisms of this antitumor effect as well as in vivo studies are presently under investigation CONCLUSION: Based on these data, the local delivery of GCV + FU associated with TK30 gene transfer should be considered to optimize the TK/GCV strategy by efficiently eliminating both infected and more importantly non-infected glioma cells In vivo experiments will address the antitumoral efficacy of this system and also the possible loco-regional toxic side effects of the drug combination CANCER TARGETED GENE THERAPY II 745 Combination of OncoVEX with Chemotherapy for Cancer Treatment Ziqun Han,1 Jennifer Hu,2 Binlei Liu,1 Michael Robinson,1 Ruth Branston,1 Charles Coombes,2 Robert Coffin.1 BioVex Ltd, Abingdon, Oxon, United Kingdom; 2Cancer Cell Biology, Hammermsith Hospital Campus, Imperial College of Science and Medicine, London, United Kingdom OncoVEX is an oncolytic version of HSV with several improvements as compared to oncolytic versions of HSV previously tested in the clinic These improvements are (i) OncoVEX is derived from a clinical isolate of HSV with enhanced anti-tumour potency as compared to previously used laboratory strains (ii) tumour selective replication is enhanced via increased expression of US11 in addition to deletion of ICP34.5 (iii) ICP47, which usually blocks antigen presentation in HSV infected cells, is deleted A version of this virus additionally encoding the gene for GM-CSF is currently in a Phase I clinical trial in a number of tumour types This virus is designed to provide a direct anti-tumour effect by tumour selective virus replication and also to induce an anti-tumour immune response enhanced by the expression of GM-CSF It is anticipated that in routine clinical practice oncolytic viruses are likely to be combined with standard therapies such as chemo- or radiotherapy The work S288 reported here assesses the anti-tumour effects of OncoVEX in combination with a number of first line chemotherapeutic agents (e.g taxol and cisplatin) in in vitro models Work in vivo is underway Results so far demonstrate that OncoVEX treatment of tumour cells either prior to, concurrently with, or after addition of the drug provides enhanced drug sensitivity and that the chemotherapeutic agents tested not reduce the effectiveness of the oncolytic effect Initial conclusions are therefore that combination of OncoVEX with anti-cancer drug therapy may be beneficial for cancer treatment 746 Effective Therapy of Metastatic Ovarian Cancer with an Oncolytic Herpes Simplex Virus Incorporating Two Membrane-Fusion Mechanisms Mikihito Nakamori,1 Xinping Fu,1 Feng Meng,1 Aiwu Jin,1 Lihua Tao,1 Robert C Bast, Jr.,2 Xiaoliu Zhang.1 Center for Cell and Gene Therapy, Baylor College of Medicine, Houston, TX; 2M.D.Anderson Cancer Center, The University of Texas, Houston, TX Replication-competent herpes simplex virus (oncolytic HSV) holds considerable promise for treating malignant solid tumors including ovarian cancer However, the potency of the virus needs improvement if its full clinical potential is to be realized In our previous studies, we showed that incorporation of membrane fusion capability into an oncolytic HSV, either by screening for a syncytial HSV mutant after random mutagenesis or by inserting a hyperfusogenic glycoprotein from gibbon ape leukemia virus (GALV.fus) into the viral genome, can significantly increase the antitumor capability of the virus We recently constructed a newer version of fusogenic oncolytic HSV by incorporating both membrane fusion mechanisms into a single virus In vitro characterization of this doubly fusogenic oncolytic HSV (Synco-2D) showed that this virus produces a distinctive syncytial phenotype, leading to a significantly increased tumor cell killing ability, compared with that of a nonfusogenic virus When injected directly into the abdominal cavity of mice bearing human ovarian cancer xenografts, Synco-2D eradicated all tumor masses in 75% of the animals, whereas no animals in the conventional oncolytic HSV treated group was tumorfree Our results indicate that this newly generated fusogenic oncolytic HSV is a promising candidate for clinical testing against advanced ovarian cancer 747 Comparaison of K-Ras Antisense Oligonucleotides and siRNAs Strategies in Human Pancreatic Cancer Amor Hajri, Hazare Nouari, Severine Wack, Marc Aprahamian Tumor Biology and Gene Therapy Dept., IRCAD-INSERM U375, Strasbourg, France Human pancreatic cancer is an extremely aggressive and incurable carcinoma because of its poor sensitivity to chemotherapy and/or radiotherapy Point mutations of the K-ras gene are detected in > 90% of human pancreatic cancers and may play an important role in tumorigenesis Antisense therapeutics with phosphorothioate oligonucleotides (PS ODN) and short interfering RNAs (siRNA) targeting specific gene expression represent a promising strategy for pancreatic cancer treatment The aim of the present work was to address a comparative study concerning the antitumor activity of synthetic PS-ODN and siRNA antisense targeting specific k-ras mutation (k-Ras mut12) The siRNA was delivered as a synthetic 19-22 bases and as a recombinant vector for stable expression The antitumor activity was evaluated (i) in vitro, in cell culture with different human pancreatic cancer cell lines (BxPc3, Panc1, Capan1) The endogenous Ras expression was determined by RT-PCR and Western-blotting Antiproliferative and cytotoxic effects were evaluated by [3H] thymidine incorporation and MTT tests (ii) In Molecular Therapy Vol 7, No 5, May 2003, Part of Parts Copyright © The American Society of Gene Therapy CANCER TARGETED GENE THERAPY II vivo, antisense (PS-ODN) and siRNAs were delivered by electroporation in pancreatic tumors xenografts into athymic mice The tumor growth was evaluated by tumor volume measurement during weeks Our results demonstrate that the antisense PSODN targeted specific k-ras mutation oncogene transcripts and reduced its expression The antitumor effect observed in vitro and in vivo was less than 50% The siRNAs appear to be more efficient with a long lasting effect in cell culture The best results were obtained with the recombinant vector expressing siRNA (k-Ras mut12) which induced an efficient and specific down-regulation and persistent oncogene expression The tumor growth of the pancreatic cancer cells (k-Ras mut12) targeted with specific siRNA was dramatically inhibited in vitro as well as in vivo In conclusion; these results indicate that the siRNA strategy is more efficient than antisense oligonucleotides and should be more investigated as a novel rationale for the targeting of pancreatic cancer therapy as effective as bcl-xl RNAi constructs Through BCL-XL downregulation via siRNA vectors, we have demonstrated a significantly improved response to CDDP in NSCLC in vitro These findings could have significant clinical applicability 748 siRNA Vectors Targeted to BCL-XL Significantly Reduce BCL-XL Expression and Enhance Response to Chemotherapy in NonSmall Cell Lung Adenocarcinoma Xiaobo Cao,1 Jonathan Daniel,1 Mustafa K Ozvaran,1 Steven Miller,1 Roy W Smythe.1 Thoracic and Cardiovescular Surgery, University of Texas M.D.Anderson Cancer Center, Houston, TX Introduction: An anti-apoptotic member of the bcl-2 gene family, bcl-xl, is strongly expressed in human non-small cell lung adenocarcinoma (NSCLC) specimens and cultured cell lines Previously, we have demonstrated that down-regulation of BCLXL protein expression can enhance chemosensitivity in NSCLC cell lines RNA interference (RNAi) has emerged as an effective tool for inhibiting gene expression in a specific fashion Effective inhibition of targeted RNA by siRNAs expressed from an RNA polymerase III vector based on an H1 or U6 promoter has been demonstrated The purpose of this study is to evaluate the effectiveness of hairpin siRNA directed against bcl-xl by using an H1 promoter based expression vector to downregulate BCL-XL expression and also to determine if this effect enhances response to conventional chemotherapy in vitro Methods: The human NSCL cell line H1299 was maintained in culture and utilized for these experiments Five expression vectors were generated by inserting five pairs of 64-nt oligonucleotides, each containing a 19-nt target which is included in both sense and antisense orientation, separated by a 9-nt spacer sequence H1299 was then transfected with these siRNA vectors using lipofectamine 2000 Seventy-two hours later, RNA and cell lysates were collected and BCL-XL expression was evaluated by quantitative RT-PCR and Western blot Additional members of the bcl-2 family, BAX, BAK and BCL-2, were also evaluated by Western blot and Super array A colorimetric assay (XTT) was used to assess cellular viability 72 hours following exposure of cells to siRNA and cisplatin (CDDP) Apoptosis was measured by Annexin V staining Results: Among the five siRNA vectors generated, two constructs were able to significantly decrease BCL-XL at the level of transcription (evaluated by RT-PCR) and protein expression (evaluated by Western blot) Expression of BAX, BAK, and BCL2 were unchanged Decreased BCL-XL expression led to apoptosis in H1299 cells (evaluated by Annexin V staining) The percentage of cells which stained positive were as follows: Control vector group – 12%, 30uM cisplatin group – 18%, BCL-XL siRNA vector group – 40%, and cisplatin plus BCL-XL siRNA vector group – 70% XTT assay demonstrated H1299 was sensitized to cisplatin after bcl-xl siRNA vectors were transfected Conclusion: siRNA vectors directed at bcl-xl demonstrate significant and specific reduction in BCL-XL protein levels in NSCLC Molecular Therapy Vol 7, No 5, May 2003, Part of Parts Copyright © The American Society of Gene Therapy 749 Transcriptional Targeting of Tumors with a Novel Tumor Specific Promoter-The Survivin Gene Promoter Zeng B Zhu,1 Bao G Lu,1 Ming H Wang,1 Lioudmila Kaliberova,1 Feng Z Li,3 Angel A Rivera,1 Parameshwar J Mahasreshti,1 Charles A Leath III,2 Sharmila K Makhija,2 David T Curiel.1 Division of Human Gene Therapy, University of Alabama at Birmingham, Birmingham, AL, United States; 2Department of Obatetrics & Gynecology, University of Alabama at Birmingham, Birmingham, AL, United States; 3Department of Pharmacology & Therapeutics, Roswell Park Institute, Buffalo, NY, United States Survivin, a novel member of the IAP (inhibitor of apoptosis) protein family, appears to be over-expressed in common cancers but not in corresponding normal adult tissues We hypothesized that the survivin promoter might be an excellent candidate to deliver transgenes to tumor cells Advantages of tumor-specific expression of transgenes include limitation of toxicity of therapeutic genes, such as herpes simplex virus thymidine kinase, and the potential for creation of a tumor-selective replicate-competent oncolytic virus To test this hypothesis, we measured the transcriptional activity of the survivin gene promoter in different tumor cell lines, including pancreatic cancer, breast cancer, ovarian cancer, and melanoma We also tested this promoter to see if it exhibited a “liver off” status in vivo using a mouse model A recombinant adenovirus (reAdGL3BSurvivin) was constructed by inserting a luciferase reporter gene and the survivin promoter, which drives the reporter expression, into the deleted E1 region of adenovirus type5 The tumor cell lines were infected with reAdGL3BSurvivin, and a human fibroblast and a mammary epithelial cell line were used as normal controls A reAdGL3BCMV, containing the CMV promoter, was used as a positive promoter control to normalize the luciferase levels To test the distribution of promoter activity, in vivo, a dose of 5x108 pfu of reAdGLB3Survivin or reAdGL3BCMV was injected intravenously into mice The major organs were harvested two days post-injection, and the luciferase activities were determined in major organs Luciferase assays revealed that the survivin promoter induced relatively high luciferase gene transcription in several cell lines including breast cancer, MDA-MB-361, ovarian cancer, OVCAR3, and melanoma, SK-Mel-28, 14.4%, 11%, and 16.8% of the activity of the CMV promoter, respectively The survivin promoter activities S289 ... promoter in different tumor cell lines, including pancreatic cancer, breast cancer, ovarian cancer, and melanoma We also tested this promoter to see if it exhibited a “liver off” status in vivo using... Park Institute, Buffalo, NY, United States Survivin, a novel member of the IAP (inhibitor of apoptosis) protein family, appears to be over-expressed in common cancers but not in corresponding... chemotherapy in vitro Methods: The human NSCL cell line H1299 was maintained in culture and utilized for these experiments Five expression vectors were generated by inserting five pairs of 64-nt oligonucleotides,