260 The Effect of E1B 19kDa Adenoviral Protein in Combination with Radiation Therapy Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy S102 CANCER TA[.]
CANCER - TARGETED GENE THERAPY I at a time In CT26, CAR-deficient murine colon cancer cell lines, the expression level of transgenes, is dramatically increased when we transfected adenoviruses harboring transgenes such as luciferase or GFP in conjugated with NCC-K1 The effect of NCC-K1 is also repeated in mouse subcutaneous tumor model using CT26 cells like in cell lines Furthermore, we could find the dramatic effect on tumor growth inhibition in mouse subcutaneous tumor model when we treated adenovirus harboring thymidine kinase as a suicide gene therapy, implying the successful gene delivery by NCC-K1 At that time, it is possible to monitor the localization of adenoviruses at regular time intervals using MRI Our findings suggest that, in the future, NCC-K1 is very easy and worthy for successful viral gene delivery and noninvasive in vivo MRI tracking of targeted gene therapy simultaneously 258 Relationship of Genetic Change and Efficacy of Viral Therapy in the Treatment of Gliomas Takahito Yazaki,1,2 Ryuichi Kanai,2 Takeshi Kawase.2 Neurosurgery, International Univ of Health and Welfare, Tokyo, Japan; 2Neurosurgery, School of Medicine, Keio University, Tokyo, Japan Although HSV-1 mutants possessing deletions in both copies of the γ134.5 gene have been proven safe through a number of animal experiments and clinical trials, their therapeutic efficacy was also markedly reduced In order to overcome this situation, we concentrated on the use of tumor-specific promoter to express ICP34.5 selectively in malignant glioma cells As a molecular marker for malignant glioma, we focused on the neural RNA binding protein, Musashi1 We created, via homologous recombination, a novel HSV-1 vector termed KeM34.5, which expresses ICP34.5 under the transcriptional control of Musashi1gene promoter (P/musashi1) Cytotoxicity mediated by KeM34.5 was significantly enhanced in human glioma cell lines, resulting in about log increase of viral yield, compared to its parental vector G207 We studied in this time, whether genetic change as 1p/19q loss in gliomas is correlated with therapeutic efficacy of KeM34.5 in vivo glioma model This virus showed a much higher therapeutic efficacy in the gliomas with 1p/19q loss, while maintaining the genetically intact gliomas These results suggest that the oncolytic HSV-1 expressing ICP34.5 under the transcriptional control of Musashi1 gene promoter can be a promising therapeutic agent for the treatment of malignant glioma with 1p/19q loss 259 The Effects of Radiation on Adenoviral Transgene Expression in Cancer Cells Petri Nokisalmi,1 Maria Rajecki,1 Laura Ahtiainen,1 Vincenzo Cerullo,1 Mikko Tenhunen,2 Sari Pesonen,1 Akseli Hemminki.1 Cancer Gene Therapy Group, Molecular Cancer Biology Program & Transplantation Laboratory & HUSLAB & Finnish Institute for Molecular Medicine, University of Helsinki and Helsinki University Central Hospital, Helsinki, Finland; Department of Oncology, Helsinki University Central Hospital, Helsinki, Finland Genetically modified adenoviruses have potential to kill cancer cells by oncolysis due to viral infection per se, by anti-cancer transgene expression and by activation of immunological responses Adenoviruses have been safe in clinical cancer trials However, gene therapy with adenoviruses alone is often not effective enough to eradicate advanced tumors A common clinical practice is to combine several treatment modalities in order to reach improved outcomes Radiotherapy provides accurate and precise modes to treat different cancer types and is widely combined with surgery and chemotherapy Combination of cancer gene therapy with adenoviruses and S102 radiotherapy could lead to increased tumor control perhaps without an increase in toxicity due to a non-overlapping side effect profile The knowledge of interactions and potential synergy mechanisms is important when adenoviruses are combined to traditional treatments and treatment protocols are optimized Methods and results: We studied the effect of radiation on five (Ad5cmv-luc, Ad5mdr-luc, Ad5ala-luc, Ad5cox2-luc and Ad5vegf-luc) promoters in PC-3MM2 prostate cancer cells We show that Gy radiation dose increased transgene expression with all promoters Furthermore, FACS analysis of prostate cancer cells infected with green fluorescence protein (GFP) expressing Ad5GL and Ad5pk7GL viruses showed up to 8-fold increase in GFP expression To study the effect of radiation treatment on cellular proteins, we analyzed GFP expression also in two cancer cell lines stably expressing GFP: M4A4-LM3 breast cancer and LNM35-eGFP lung cancer cell line Cellular GFP expression was increased in both cell lines in a same manner as expression of transgenic GFP Moreover, radiation increased total mRNA levels up to 2.5-fold in M4A4-LM3-cells These results imply that radiation generally activates cancer cells to produce more protein including viral proteins Finally we studied the effect of different radiation doses on luciferase expression in M4A4-LM3-cells infected with Ad5luc 0.5 Gy dose increased the amount of luciferase 1.6-fold and highest 2.4fold increase was achieved with 12 Gy dose Conclusions: Radiation increased adenoviral transgene expression regardless of transgene, transgene promoter or radiation dose Radiation also increased total mRNA-levels and cellular proteins in cancer cells These results imply that radiation can be utilized to increase the potency of adenoviral gene therapy due to general activation of gene expression/protein production, including therapeutic viral proteins 260 The Effect of E1B 19kDa Adenoviral Protein in Combination with Radiation Therapy Pyung-Hwan Kim,1 Jaesung Kim,2 Ji Young Yoo,1 A-Rum Yoon,2 In-Wook Kim,1 Hye Jin Choi,1,3 Jin Sil Seong,4 Chae-Ok Yun,1,2 Joo-Hang Kim.1,3 Graduate Program for Nanomedical Science, Institute for Cancer Research, Yonsei Cancer Center, Yonsei University College of Medicine, Seoul, Republic of Korea; 2Brain Korea 21 Project for Medical Sciences, Institute for Cancer Research, Yonsei Cancer Center, Yonsei University College of Medicine, Seoul, Republic of Korea; 3Department of Internal Medicine, Yonsei University College of Medicine, Seoul, Republic of Korea; 4Department of Radiation Oncology, Yonsei University College of Medicine, Seoul, Republic of Korea Radiation therapy is a mainstay for anti-tumor therapeutic regimen for a variety of tumor types It triggers tumor cell apoptotic pathway via eliciting direct DNA damage or indirectly by inducing the formation of oxygen radicals In efforts to augment the anti-tumor efficacy of oncolytic adenovirus (Ad), we have examined the effect of E1B 19kDa, a strong anti-apoptotic protein present in oncolytic Ad, in combination with radiation therapy An enhanced cancer cell killing response was observed for both E1B 55kDa-deleted Ad (Ad-∆E1B55) and E1B 19kDa- and E1B 55kDa-deleted Ad (Ad-∆E1B19/55) in combination with radiotherapy than single treatment alone Notably, the highest cytotoxicity was observed in cancer cells treated with Ad-DE1B19/55 and irradiation Significant induction of apoptosis was noted in cancer cells treated with Ad-∆E1B19/55 and irradiation as demonstrated by flow cytometry and TUNEL assays Moreover, enhanced levels of p53, phospho-p53, phospho-Chk1, phospho-Chk2, PI3K, phospho-AKT and cleavage of PARP and caspase-3 were observed when cells were treated with Ad-∆E1B19/55 plus radiation, demonstrating that E1B 19kDa protein present in Ad-∆E1B55 may play a critical role in blocking the effect of irradiation-induced apoptosis Significant therapeutic benefit also translated in vivo when oncolytic Ads and radiation were combined Moreover, tumors treated Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy CANCER - TARGETED GENE THERAPY I with Ad-∆E1B19/55 plus radiation showed large areas of necrosis and abundant apoptosis with induction of p53 In relative comparison, combination of Ad-∆E1B19/55 and radiation was superior over that of Ad-∆E1B55 and radiation combination treatment Overall, this study presents strong therapeutic rationale for combination of radiation therapy and E1B 19kDa-deleted oncolytic Ad 261 Cells Adenovirus Infection of Epithelial Cancer Robert Strauss,1 Pavel Sova,1 Ying Liu,1 ZongYi Li,1 Sari Pesonen,2 Akseli Hemminki,2 Pascal Fender,3 Andre Lieber.1 Medicine, University of Washington, Seattle, WA; 2Cancer Gene Therapy Group, University of Helsinki, Helsinki, Finland; 3Institut de Biologie Structural, Grenoble, France The majority of solid cancers are of epithelial origin A hallmark of epithelial cells are tight and adherens junctions Tight and adherens junctions seal intercellular spaces and significantly limit the perfusion of anti-tumor agents such as drugs, antibodies, and immune cells within the tumor, thus severely diminishing the efficacy of such therapeutic modalities The epithelial phenotype of cancer cells also represents a barrier to infection with commonly used adenoviruses that target CD46 or the coxsackie-adenovirus receptor (CAR), due to trapping of these receptors in tight and adherens junctions which explains, in part, why these serotypes were inefficient in cancer gene therapy We found however, that specific human species B adenoviruses, namely Ad3, Ad7, Ad11, and Ad14 (AdB-group 2) that use a yet unknown receptor (receptor X), which is different from CAR and CD46, efficiently infect epithelial cancer cells This makes these serotypes potential tools for virotherapy of cancer as well as for gene transfer into normal epithelial tissue In addition, these serotypes are important pathogens, exemplified by recent outbreaks of a highly pathogenic new Ad14 stain So far, our studies on mechanisms of AdB-group infection of epithelial cells have shown: i) These Ads use at least two binding moieties on the cell membrane The C-terminal part of the Ad fiber (the fiber knob) binds to sulfated carbohydrate chains of heparin-sulfate proteoglycans (HSPG) This Ad3knob – interaction with heparin sulfate glucosaminoglycans (HSGAGs) allows for subsequent high affinity attachment and/or access to receptor X, whereby receptor X is either the protein part of the HSPG that interacts with the Ad3 knob or an independent non-HSPG protein ii) Ad binding to epithelial cells involves both the Ad3 fiber and the Ad3 penton or a composite fiber-penton moeity formed in Ad3 virions iii) During virus replication, Ad pentons and fibers selfassemble in dodecahedra (PtDd) formed through interaction of 12 penton bases with protruding fibers These PtDd are thought to disturb tight junctions, thus favoring lateral virus spreading iv) Recombinant PtDd (produced in insect cells) can enter epithelial cancer cells and can faciliate uptake of Ads (and potentially other tumor agents) for which tight and adherens junctions represent a barrier Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy 262 Serotype Chimeric and Fiber Mutated Adenovirus Ad5/19p-HIT for Targeting Renal Cancer and Untargeting the Liver Iulia Diaconu,1,2 Laura Denby,3 Sari Pesonen,1,2 Vincenzo Cerullo,1,2 Gerd J Bauerschmitz,4 Kilian Guse,1,2 Maria Rajecki,1,2 João D Dias,1,2 Kimmo Taari,5 Anna Kanerva,1,2,6 Andrew H Baker,3 Akseli Hemminki.1,2 Cancer Gene Therapy Group, Molecular Cancer Biology Program & Transplantation Laboratory & Haartman Institute & Finnish Institute for Molecular Medicine, University of Helsinki, Helsinki, Finland; 2HUSLAB, Helsinki University Central Hospital (HUCH), Helsinki, Finland; 3British Heart Foundation Glasgow Cardiovascular Research Center, University of Glasgow, Glasgow, United Kingdom; 4Department of Obstetrics and Gynecology, Heinrich-Heine University, Düsseldorf, Germany; 5Department of Urology, Helsinki University Central Hospital (HUCH), Helsinki, Finland; 6Department of Obstetrics and Gynecology, Helsinki University Central Hospital (HUCH), Helsinki, Finland Despite some recent advances, patients with advanced renal cell carcinoma (RCC) cannot usually be cured Alteration of the natural tropism of adenoviruses may permit more specific gene transfer to target tissues The aim of this study was to utilize novel targeting moieties for adenoviral gene therapy of RCC Previous work in rats suggested that utilization of Ad5/19p (Ad5 capsid with Ad19p fiber) with kidney vascular targeting moieties HTTHREP (HTT), HITSLLS (HIT) and APASLYN (APA) placed into the fiber knob might be useful for targeting kidney vasculature Therefore, we sought to investigate the utility of Ad5/19p variants for gene delivery to human RCC cell lines, clinical samples and orthotopic murine models of metastatic RCC Six different human RCC cell lines were infected with Ad5/19p variants but only Ad5/19p-HIT showed increased transduction, and only in one cell line Thus, we analyzed human normal and cancerous kidney specimens fresh from patients, which might better mimic the three dimensional architecture of clinical tumors and found that Ad5/19p-HIT showed transduction levels similar to Ad5 In mice, we found that intraperitoneal and intravenous Ad5/19p-HIT transduced tumors at levels comparable to Ad5, while intratumoral Ad5/19p-HIT was even superior to Ad5 Liver tropism was significantly reduced in comparison to Ad5 Improvements in tumor to liver transduction ratios suggested that Ad5/19p-HIT may be promising for systemic gene delivery to kidney tumors 263 An Efficient Targeted Gene Therapy Using Brain Tumor-Specific Promoter Toshio Yawata,1 Eri Ishida,1 Yu Kawanishi,1 Masakazu Tamura,1 Keiji Shimizu.1 Neurosurgery, Kochi Medical School, Nankoku, Kochi, Japan Despite many efforts to develop effective therapy, the outcome of malignant glioma remains poor Gene therapy for this disease using retroviral vector is attractive, because the virus can infect only mitotic cells Previously, we reported the eradication of mouse glioma by retroviral-mediated gene therapy In this study, a tumor-specific targeting system was studied to develop the effective and safe gene therapy We searched for genes expressing at high frequency in brain tumors but not in normal human astrocyte (NHA) among cancer testis antigen (CTA) genes MAGEA3 and SSX4 were identified as tumor-specific genes The promoter of both genes was cloned into luciferase reporter vector and the activity was measured in glioma, telomerase-immortalized fibroblast and normal human astrocyte cells The SSX4 promoter but not MAGEA3 showed the tumorspecific activity The minimal promoter of SSX4 was defined as a 256 bp fragment upstream of transcriptional start site In order to define a useful tumor-specific promoter for targeting in context of retroviral-mediated gene therapy, the SSX4 promoter were used to S103 ... TARGETED GENE THERAPY I with Ad-? ?E1B1 9/55 plus radiation showed large areas of necrosis and abundant apoptosis with induction of p53 In relative comparison, combination of Ad-? ?E1B1 9/55 and radiation. .. high affinity attachment and/or access to receptor X, whereby receptor X is either the protein part of the HSPG that interacts with the Ad3 knob or an independent non-HSPG protein ii) Ad binding... Gene Therapy Group, University of Helsinki, Helsinki, Finland; 3Institut de Biologie Structural, Grenoble, France The majority of solid cancers are of epithelial origin A hallmark of epithelial