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839 The Use of Third Generation Oncolytic HSV 1 Expressing Luciferase for Demonstration of Real Time Biodistribution and Dynamics Molecular Therapy Volume 18, Supplement 1, May 2010 Copyright © The Am[.]
CANCER-ONCOLYTIC VIRUSES III X12-V2), rQnestin34.5-Vstat120 showed increased viral replication (25.8-fold (p=0.0065), 21-fold (p=0.0018), and 817-fold (p=0.0110) respectively), and increased glioma cytotoxicity (88.7% (p=0.0004), 90.6% (p=0.0001), and 76.8% (p=0.0014)) relative to HSVQ infected glioma cells In contrast little or no signicant increase in virus replication and cytotoxicity was observed in glioma cells with low nestin expression (T98G and Gli36∆5), or in primary non transformed human endothelial cells (nestin negative) Collectively these results indicated nestin specic increased viral replication and cytotoxicity of rQnestin34.5-Vstat120 Further we have confirmed early and efficient secretion of Vstat120 in glioma cells infected with rQnestin34.5-Vstat120 The functionality of Vstat120 produced by rQnestin34.5-Vstat120 was veried by its ability to inhibit endothelial cell migtration and tube formation in vitro To test its antitumor efcacy was mice with subcutaneous U251T3 glioma (150-250 mm3) were injected with PBS, rQnestin34.5 (transcriptionally driven OV), rQVstat120 (double attenuated expressing Vstat120 OV), or rQnestin34.5-Vstat120 (transcriptionally driven and Vstat120 expressing OV) Mice were evaluated for tumor growth A signicant increase in anti-tumor effect was observed between rQnestin34.5-Vstat120, and rQnestin 34.5, (p=0.0198) and between rQnestin34.5-Vstat120, and rQVstat120 (p= 0.0429) We are now in the process of testing therapeutic efcacy of rQnestin34.5-Vstat120 in intracranial tumor model MRI imaging of mice bearing intracranial tumors has revealed increased antitumor efcacy of rQnestin34.5-Vstat120 against intracranial gliomas This study has led to the development of a novel transcriptionally retargeted and “armed” oncolytic virus 837 Determining Adenovirus Titers Following Extraction of the Virus from Tumors in the Presence of Anti-Adenovirus Antibodies Debanjan Dhar,1 Karoly Toth,1 William S M Wold.1 Molecular Microbiology & Immunology, Saint Louis University, St Louis, MO Oncolytic adenovirus (Ad) vectors are being investigated as possible therapies for human cancer These vectors are generally injected into the tumor, and multiple injections are usually necessary for the vector to be effective When the vector is injected into a naïve immunocompetent animal, an immune response is mounted against the vector Immune effector cells and antibodies (Ab) inltrate the site of injection Determining the Ad vector titer present inside the tumor or any other tissue is challenging since the neutralizing Ab (NAb) could affect the results Even if the NAbs are physically separated from the infectious vector within the tumor microenvironment, as soon as the tumor is disrupted in order to titer the vector, the NAbs could bind to the vector and neutralize its infectivity We have shown previously that Syrian hamsters are a good model to evaluate oncolytic Ad5-based vectors Normal tissues of hamsters are permissive for Ad5, and hamsters are immunocompetent In the current study, we used hamsters to determine the best way to determine the infectious vector titer from an immunocompetent animal and also from animals having preexisting immunity against the vector We also investigated whether the infectious vectors that are extracted from tumors and titered in a standard TCID-50 assay are intracellular within the tumor microenvironment or are extracellular trapped in the tumor architecture As control we used hamsters that were immunosuppressed before the vector was injected into the tumor and were kept immunosuppressed throughout the study Tumors were isolated at various time points, chopped into small pieces, and separated into three different tubes The tumor in rst tube was homogenized by bead-beating, freeze thawed, and sonicated to release the intracellular vector, and then titered by TCID-50 assay The second tube was treated with collagenase-dispase to isolate single cells from the tumor The cells were washed and treated with Proteinase K to Molecular Therapy Volume 18, Supplement 1, May 2010 Copyright © The American Society of Gene & Cell Therapy get rid of the NAb, sonicated, and assayed for infectious vector Since high amounts of Proteinase K are detrimental to the cell, the optimal concentration of Proteinase K was determined in a separate experiment so that Proteinase K affected the extracellular NAb and not the tumor cell and the intracellular vector We found that most of the vector recovered from the tumor is intracellular We also found that Collagenase-dispase along with Proteinase K treatment or extensive washing is an effective method to eliminate NAb during vector extraction from tumors, allowing accurate measurement of infectious vector titers in a tumor 838 Abstract Withdrawn 839 The Use of Third Generation Oncolytic HSV-1 Expressing Luciferase for Demonstration of Real-Time Biodistribution and Dynamics Yihan Wu,1 Yasushi Ino,2 Tomoki Todo.2 Department of Neurosurgery, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan; 2Department of Neurosurgery and Translational Research Center, The University of Tokyo, Tokyo, Japan Background G47∆ is a third generation oncolytic herpes simplex virus type (HSV-1) containing deletions in the γ34.5 and α47 genes and an inactivating LacZ insertion in the UL39 (ICP6) gene Previous studies have shown that G47∆exhibits an efcient oncolytic activity in a variety of tumor cells, yet minimal toxicity in normal tissues In order to optimize the route of administration and dosing for various types of cancer, it is essential to assess the in vivo distribution and the time course of viral infection in relation to efcacy Conventionally, biodistribution studies required sacricing and obtaining tissue samples from animals at different time points after virus administration In this study, we newly constructed a third generation oncolytic HSV-1 expressing luciferase, and used it to demonstrate a real time biodistribution of the virus in a non-invasive fashion Methods A third generation, armed oncolytic HSV-1 termed T-luc was constructed by inserting the CMV promoter-driven, luciferase gene into the G47∆backbone, using the bacterial articial chromosomemediated, recombinant HSV-1 construction system (T-BAC system) After limiting dilutions three times, the nal virus clone was selected and the correct construction conrmed by Southern blot analyses In vitro studies on cytopathic effect and viral replication ability were performed using Vero and Neuro2a (murine neuroblastoma) cells In vivo studies were performed using A/J mice bearing syngeneic Neuro2a tumors or athymic mice bearing U87MG (human malignant glioma) tumors The in vivo luciferase expression, reecting the fresh infection by T-luc, was measured at different time points after intratumoral or intravenous administration with T-luc using the in vivo imaging system (IVIS, Xenogen) Results The in vitro cytopathic effect and replication capability of T-luc were comparable to those of G47∆ in both cell lines tested The in vivo antitumor efcacy of T-luc was also similar to that of G47∆ when tested in A/J mice bearing subcutaneous Neuro2a tumors When T-luc was injected intratumorally into subcutaneous Neuro2a tumors, the local luciferase expression was detected up to day In athymic mice with subcutaneous U87MG tumors, intratumoral injections with T-luc resulted in local expression of luciferase for more than 14 days The T-luc infection was not detected from any other organs at any time point after an intratumoral administration Contrarily, an intravenous administration with T-luc in animals with subcutaneous tumors resulted in a high expression of luciferase in the liver and the tail (the injection site) as well as in tumors In contrast with the luciferase expression in tumors which gradually increased by day 7, the luciferase expression in the liver rapidly vanished by the next day of viral injection The luciferase expression in intracranial S323 CANCER-ONCOLYTIC VIRUSES III Neuro-2a tumors was detectable up to day Conclusion The newly constructed, luciferase-expressing HSV-1, T-luc, is a useful tool for studying biodistribution and dynamics of oncolytic HSV-1 840 Highly Attenuated Vaccinia Virus as a Potential Oncolytic Agent for Cancer Virotherapy Takafumi Nakamura,1,2 Minoru Kidokoro,3 Mina Hikichi,1 Hisatoshi Shida,4 Hideaki Tahara.1 Institute of Medical Science, Tokyo University, Tokyo, Japan; 2PRESTO, Japan Science and Technology Agency, Kawaguchi, Japan; 3National Institute of Infectious Diseases, Musashimurayama, Japan; 4Institute for Genetic Medicine, Hokkaido University, Sapporo, Japan A highly attenuated LC16m8 (m8) smallpox vaccine has been licensed in Japan because of its extremely low neurovirulence prole, which was administrated to >100,000 infants where it induced levels of immunity similar to that of the originating Lister (LO) strain, without any serious side effects from 1973 to 1975 The m8 was indirectly isolated from LO through intermediate strains, such as LC16mO (mO) The m8, a variant that forms small-sized pocks, is a direct descendant of mO, which itself is a clone that forms mediumsized pocks, isolated from LO based on its temperature sensitivity The m8 has lost the function of B5R as the result of a single base deletion in the ORF, which encodes a 42-kDa glycoprotein that is essential for formation of extracellular enveloped virus (EEV) Since EEV is critical for cell-to-cell and long-range spread of the virus, the B5R dysfunction results in a dramatic reduction of EEV, and as a consequence, the virus produces small plaques in vitro and is highly attenuated in vivo We evaluated the oncolytic potential of m8∆, which is more genetically stable virus by deleting B5R from m8 that spontaneously generates B5R+ revertants (Kidokoro M et al., PNAS, 2005), for a variety of different tumor cell lines To investigate relationship between B5R and oncolytic activity, we also constructed m8B5R virus from m8∆ by introducing the complete B5R cloned from mO The m8∆ was able to more efciently lyse A549, BxPC-3 and Caco-2 cells than Huh-7, MDA-MB-231 and SKNAS cells in culture However, the cytolytic activity of m8∆ was much lower than that of mO in all kinds of tumors The reduced oncolytic activity was fully restored by the expression of B5R, as shown in the cells infected with m8B5R Nevertheless, the m8∆ demonstrated in vivo oncolytic activity in intraperitoneal xenografts of BxPC-3 cells stably expressing luciferase Seven days after tumor implantation, mice received single i.p injection of mO or m8∆ (105, 106, or 107 pfu per mouse) In vivo tumor growth was monitored noninvasively by bioluminescence imaging after luciferin addition to the treated SCID mice The bioluminescence imaging on day 11 after treatment revealed that there was signicant inhibition of tumor growth in both mO and m8∆-treated groups, compared with mock therapy group Quantication of this signal indicated that there was no signicant difference of tumor volume reduction among mice given 105 pfu and mice given mO or m8∆ at 10- to 100-fold higher input multiplicities On the other hand, all mice treated with mO were dead or sacriced on days 14 to 21 after treatment because of weight loss and pock lesions on their tails, paws and in oral cavities In contrast, m8∆ did not induce these viral toxicities, however tumor regrowth was observed from day 22 post-treatment Our study demonstrated that m8∆ has the potential of more selective and safer oncolytic agent than parental virus mO, although it may be necessary to enhance the antitumor activity S324 841 Enhanced Tumor Killing by a Mortalin Targeting Modied Oncolytic Adenovirus Jung-Sun Lee,1 Ji Young Yoo,1 Jihoon Ryu,1 Tomoko Yaguchi,2 Sunil C Kaul,2 Renu Wadhwa,2 Chae-Ok Yun.1 Brain Korea21 Project for Medical Sciences, Institute for Cancer Research, Yonsei University College of Medicine, Seoul, ShinchonDong, Seodaemun-Gu, Korea; 2Advanced Industrial Science & Technology, Advanced Industrial Science & Technology, Higashi, Tsukuba, Ibaraki, Japan Oncolytic adenoviruses (Ads) offer an effective cancer therapeutic tool with several advantages including wide host cell permeability, high transduction efciency, safety, tumor selectivity, non-invasiveness, high genetic modifiability and high level of expression of the integrated transgenes Armed oncolytic Ad in which the therapeutic efcacy of virus is enhanced by their coupling with cytotoxic, antiangiogenic or anti-vascular gene products have gained importance as these engage additional mechanisms for tumor cell killing In this study, we selected mortalin, a stress chaperone that is tightly involved in human carcinogenesis, constructed a mortalin-targeting oncolytic Ad (mot-Ad) and examined its therapeutic potential in vitro and in vivo We demonstrate that the mot-Ad has selective cytotoxicity for human cancer cells in vitro Retrovirus- mediated overexpression of mortalin protected the cells against mot-Ad, conrming that mortalin silencing was the real cause of cancer cell death While mortalin overexpression enhanced malignant properties of cancer cells in breast xenograft models, mot-Ad elicited enhanced anti-tumor effect Immuno-histochemical examination of the tumors showed that the mot-Ad caused enhanced apoptosis and suppression of microvessel formation Since mortalin is upregulated in a large variety of tumors, mot-Ad is proposed as a candidate cancer therapeutic agent 842 Increased Oncolytic Ability of a Double RGD-Modied Adenovirus in Ovarian Cancer Lena J Gamble,1 Qiana L Matthews,1,2 Anton V Borovjagin,3 David T Curiel.1 Department of Pathology, The University of Alabama at Birmingham, Birmingham, AL; 2Center for Aids Research, The University of Alabama at Birmingham, Birmingham, AL; 3School of Dentistry, Institute of Oral Health Research, The University of Alabama at Birmingham, Birmingham, AL Ovarian cancer is a leading cause of gynecological cancer mortality in Western countries Attempts to address the urgent need for effective anti-tumor treatments for ovarian cancer patients include development of oncolytic virotherapy agents targeted specically to ovarian cancer cells One major hindrance to effective virotherapy has been suboptimal transduction of ovarian cancer cells due to the sparse presence of the adenovirus receptor on cancer cells Addition of an Arg-Gly-Asp (RGD) motif to the viral binding protein, ber, increases viral ability to bind and transduce ovarian cancer cells This RGD modication has been added to the HI-loop domain of ber in a virus designed to conditionally replicate in cancer cells, Ad5.∆24RGD The safety of Ad5.∆24RGD has been tested in a Phase I clinical trial Our goal was to improve this anti-cancer agent by further increasing viral infectivity for ovarian cancer cells To this end we created a doublemodied virus that incorporates an additional RGD modication at a separate capsid locale, protein IX (pIX) We have shown that the double-modied virus, Ad5.pIXRGD.fRGD, increases ovarian cancer transduction over both of the singly modied Ad5.pIXRGD and Ad5.fRGD viruses Based on these data, we hypothesized that a conditionally replicative version of this virus would more efciently kill tumor cells than either of the single-modied virus controls Using standard molecular biology techniques we added an RGD-4C sequence to the pIX portion of Ad5.∆24.fRGD to yield, Ad5.∆24 pIXRGD.fRGD Preliminary data indicate that the newly created double modied virus incorporates the RGD motif on both the ber Molecular Therapy Volume 18, Supplement 1, May 2010 Copyright © The American Society of Gene & Cell Therapy ...CANCER -ONCOLYTIC VIRUSES III Neuro-2a tumors was detectable up to day Conclusion The newly constructed, luciferase -expressing HSV- 1, T-luc, is a useful tool for studying biodistribution and dynamics. .. MDA-MB-2 31 and SKNAS cells in culture However, the cytolytic activity of m8∆ was much lower than that of mO in all kinds of tumors The reduced oncolytic activity was fully restored by the expression of. .. temperature sensitivity The m8 has lost the function of B5R as the result of a single base deletion in the ORF, which encodes a 42-kDa glycoprotein that is essential for formation of extracellular enveloped