482 Replicating Adenoviruses That Harbors the ADP Protein and the Sodium Iodide Symporter Protein (NIS) under the Control of the Major Late Adenovirus Promoter Molecular Therapy Volume 17, Supplement[.]
ONCOLYTIC ADENOVIRUS VECTORS release We found that the transduction efficiency of RCAd11pGFP is correlated to the degree of expression of integrins rather than to CD46 molecules, since there was an excess of CD46 molecules expressed on the colon carcinoma cells studied Cell viability and proliferation assays indicated that RCAd11p vector and Ad11p wt viruses were indistinguishable Interestingly, RCAd11pGFP showed efficiently cell-killing activities in T84 and HT-29 cells, which also expressed relatively high levels of carcinoma embryonic antigen (CEA) family molecules In vivo experiments revealed a significant growth inhibition of the T84 and HT29 tumors in the xenograft nude mouse group treated with RCAd11p vector or Ad11pwt in comparison with the untreated control group Thus, we have demonstrated that RCAd11p vector possesses a potent, selective oncolytic effect on colon carcinoma cells 480 Fully Non-Serotype Oncolytic Adenovirus Based on Serotype Otto Hemminki, Gerd Bauerschmitz, Silvio Hemmi, Sergio Lavilla-Alonso, Iulia Diaconu, Ranee A Desmond, Maija Lappalainen, Kilian Guse, Anna Kanerva, Vincenzo Cerullo, Sari Pesonen, Akseli Hemminki Cancer Gene Therapy Group, University of Helsinki, Helsinki, Finland Oncolytic adenoviruses are a promising experimental approach for treatment of cancers refractory to currently available modalities Even though they have been quite safe in clinical trials, efficacy has been mostly limited So far all published trials have been performed with serotype based viruses Also, all oncolytic adenoviruses tested preclinically have been based on Ad5 The expression level of the Ad5 receptor CAR may be variable in advanced tumors The Ad3 receptor is still unknown but accumulating evidence suggests abundant expression in most tumors Patients treated with adenoviruses develop high titers of neutralizing anti-viral antibodies thus compromising the effect of re-administration We hypothesise that switching the serotype might restore effective tumor transduction and antitumor efficacy and make repeated dosing of oncolytic adenovirus more feasible To create Ad3-hTERT-E1A, we placed the Ad3 E1A region under an hTERT We found that Ad3-hTERT-E1A had good cell killing efficacy in vitro with tumor cell lines representing seven major cancer types, while low toxicity was seen in non-malignant cells and no oncolysis was seen in cells negative for the Ad3 receptor In vivo, the virus had anti-tumor efficacy in three different animal models Although in vitro oncolysis mediated by Ad3-hTERT-E1A occurred slower than with Ad5 or Ad5/3 (Ad3 fiber knob in Ad5) based viruses, in vivo the virus was at least as potent as controls In summary, we report the first non-Ad5 based oncolytic adenovirus, which might be useful for testing in cancer patients with high anti-Ad5 neutralizing antibodies, or previously treated with Ad5 481 A Hypoxia and α-Fetoprotein Dependent Oncolytic Adenovirus Exhibits Specific Killing of AFP Positive Hepatocellular Carcinomas Oh-Joon Kwon,1 Pyung-Hwan Kim,1 Steve Huyn,2 Lily Wu,2 ChaeOk Yun.1 Brain Korea 21 Project for Medical Sciences, Institute for Cancer Research, Yonsei Cancer Center, Yonsei University College of Medicine, Seoul, Korea, Republic of; 2Department of Molecular and Medical Pharmacology, The University of California at Los Angeles, Los Angeles Oncolytic adenoviruses (Ads) are a new modality for cancer therapy and lead to improved efficacy over non-replicating Ads We have previously shown that an E1B19kDa-deleted oncolytic Ad exhibits strong cell killing effect but lacks tumor selectivity To acquire tissue-restricted cytotoxicity, a modified human alphaS186 fetoprotein (hAFP) promoter was used to control the expression of the viral E1A protein To tailor the promoter activity further according to the context of the tumor microenvironment, we introduced either or 12 copies of a hypoxia response element (HRE) upstream of the promoter and compared their transcriptional activities in luciferase expressing plasmids and replication–incompetent Ads Both vector systems confirmed that twelve copies of the HRE (HRE12) promoted higher gene expression levels in AFP positive cells but remained silent in AFP negative cell lines Under hypoxic conditions, gene expression was further augmented both in vitro and in vivo Based on these results, we constructed a series of oncolytic Ads with their E1 gene expression and replication strictly regulated by the chimeric HREs and hAFP promoter To confirm the cytotoxicity and specificity of the oncolytic Ads, cytopathic effect assays and cell viability assays were performed in vitro We showed that the HRE12–AFP promoter controlled E1B19kDa-deleted oncolytic virus, Ad-HRE12/hAFP∆19, exhibited strongest cell killing and tissue selectivity both in normoxic and hypoxic conditions Moreover, Ad-HRE12/hAFP∆19 achieved effective tumoricidal activity in both subcutaneous models and in liver orthotopic models by significantly delaying tumor growth Histological examination of tumor after treatment confirmed that a significant level of viral proteins accumulated near hypoxic areas These results provide clear evidence of the ability of Ad-HRE12/ hAFP∆19 to unleash its tumoricidal activity in an environmental- and tissue-selective manner and thus, support it as an effective therapeutic agent in the treatment of AFP positive liver cancers 482 Replicating Adenoviruses That Harbors the ADP Protein and the Sodium Iodide Symporter Protein (NIS) under the Control of the Major Late Adenovirus Promoter Miguel A Trujillo,1 Julia Davydova,2 Michael J Oneal,3 Elizabeth R Bergert,1 Masato Yamamoto,2 John C Morris.1 Endocrinology, Mayo Clinic Rocheser, Rochester, MN; Department of Surgery, University of Minnesota, Minneapolis, MN; 3Molecular Medicine, Mayo Clinic Rocheser, Rochester, MN The characteristic featured by Adenovirus makes it well suited for gene delivery in human clinical settings All gene therapy approaches depend upon the ability to deliver therapeutic genes to target cells Limited tumor transduction has been identified as the fundamental barrier to effective cancer gene therapy A conclusion that can be drawn from recent virotherapy clinical trials is that multimodal therapy combining virotherapy with chemo- or radiotherapy may be necessary for more complete tumor eradication The Ad5 protein ADP has been shown to enhance viral-mediated cytolysis and thus viral spread The sodium iodide symporter (NIS) directs the uptake and concentration of iodide in thyroid cells This in turn allows for radioiodine imaging and therapy of thyroid cancer To extend the use of NIS-mediated radioiodine therapy to other types of cancer, we have successfully transferred and expressed the sodium-iodide symporter (NIS) gene in prostate, colon, and breast cancer cells both in vivo and in vitro using adenoviral vectors Our working model to improve virotherapy efficiency is the development of conditionally replicating adenoviruses (CRAd) in which the ADP gene and the NIS gene are cloned in tandem at the E3 region and under the control of the MLP (∆E3ADP-NIS) while the E1a gene will be driven by tumor specific promoters This arrangement will also restrict NIS expression to permissive cells To seek proof of principle we constructed the replication competent virus Ad5∆E3ADP-NIS in which the E1a gene remains wild type In vitro results virus showed that infection of the adenoviral replication permissive cell line A549 with Ad5∆E3ADPNIS at 0.1 vp resulted in complete cytolysis Conversely, the Ad5 non-permissive cell line BNL was refractory to the viral cytopathic effect Radio-iodine uptake was assessed Ad5∆E3ADP-NIS showed 125 I uptake selectively in replication permissive A549 cells The signal Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy CHEMICAL AND MOLECULAR CONJUGATES II peak appearance, magnitude, and duration are dependent on the MOI of infection as well as the cytocidal effect caused by viral replication The data indicate that Ad5∆E3ADP-NIS replication, and consequently NIS expression, is stringently restricted to adenovirus permissive cells Having obtained proof of principle of our hypothesis we can now construct transcriptionally regulated tumor-specific CRAds so that multimodal therapies which combine virotherapy with radiotherapy can be developed based on this novel CRAds 483 Inducible Oncolytic Transformation of Replication-Deficient Virus for the Cell-Based Virotherapy Hiroshi Nakashima,1 Yoshinaga Saeki,1 E Antonio Chiocca.1 Neurological Surgery, The Ohio Sate University Medical Center, Columbus, OH Replication-conditional viral mutants promise an alternative treatment modality for malignant gliomas because of their potential to selectively eradicate tumor cells through oncolysis and spread their progeny throughout the tumor tissues Initial data obtained from clinical trials have demonstrated their clinical safety, but not their effectiveness, probably because of the still inefficient delivery, propagation and spread of such viruses in tumor tissues Efficient delivery of oncolytic viruses to tumor regions is desirable due to the diffusely invasive property of malignant gliomas as a major obstacle in their treatments Cell-based delivery of oncolytic viruses has become a promising strategy to target the diffused tumors Some cell types like neural and mesenchymal stem cells have a homing ability to pathological regions including tumors However, oncolytic viral carrier cells may not survive long enough to migrate to scattered tumor regions, because of viral lytic cycle To overcome this limitation, we have developed a novel strategy to improve carrier cell-based virotherapy This new method consists of two parts: 1) transformation from replication-deficient pro-oncolytic virus to replicationcompetent oncolytic virus, 2) inducible viral transformation by tumor microenvironment Our results showed viral gene expression was suppressed in pro-oncolytic virus, but activated by recombinase-based inducer in the infected cells Once pro-oncolytic viruses changed to replication-competent viruses, these induced viruses efficiently propagated in glioma cells compared to non-induced viruses This strategy enables viral carrier cells to survive for a longer period of time, compared to cells carrying general oncolytic viruses We believe that this promising method will enhance the development of cell-based virotherapy Chemical and Molecular Conjugates II 484 Preparation of Polymer-Coated Adenovirus Particles and Their Therapeutic Effects in Mouse Tumor Models Yoshiyuki Koyama,1 Chieko Yoshihara,1 Katsuyuki Hamada.2 Otsuma Women’s University, Tokyo, Japan; 2Ehime University, Toon, Ehime, Japan Recombinant adenovirus has been accepted as a useful gene vector for cancer gene therapy because of its high infection efficiency and capacity for transgene expression in both proliferating and nonproliferating cells Although, in some cases, clinical benefit was reported, transgene efficiency of the virus is not so high as expected from the in vitro experimental data Neutralizing by innate antibodies would be one of the most serious barriers to efficient infection Taking advantage of the anionic surface charge of the virus, coating by polycations has been performed Ionic complexes of the virus having positive surface charge showed enhanced infection efficiency on cultured cells, and in some cases, improved in vivo infectivity However, cationic surface of the viral complexes should invite adverse Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy interaction with proteins or non-target cells On the other hand, DNA/polycation complexes were also known to have uncontrolled interaction with bio-components, diminishing the transfection efficiency in vivo We have developed the protective anionic polymercoating on the DNA complex particles which re-charged the particles to negative The anionic polymer-coated DNA complexes showed very little interaction with blood components, and achieved the high extra-gene expression in tumor after intravenous injection into mice In this study, we coated the adenovirus/polycation complex with anionic polymers to produce novel virus/polymer complexes with negative surface charge They showed relatively higher infectivity on the cultured cells even in the presence of antibodies Moreover, remarkable therapeutic effects of the complexes comprising recombinant oncolytic virus and polymer-coatings were observed in mouse tumor models Structure and the infection-mechanism of these virus-complexes are investigated 485 Biodegradable Polymer-Based Gene Delivery System for Repeat Administration Keiji Itaka,1 Takehiko Ishii,2 Kazunori Kataoka.2 Division of Clinical Biotechnology, CDBIM, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan; 2Department of Materials Science and Engineering, Graduate School of Engineering, The University of Tokyo, Tokyo, Japan Gene therapy is a versatile and cost-effective approach, applicable for many clinical fields including stem cell engineering The safety issue of gene delivery system is acquiring more importance When the repeat administration of functional genes is needed to achieve the cell reprogramming or the differentiation, it is essential to develop highly safe materials that show no toxicity on the cells and tissues even in the cumulative dose In this regard, we developed a polycationic polymer system of (PEG-)PAsp[DET], where the ethylene diamine units (DET) were located at the side chain of poly-aspertic acid (PAsp) Besides excellent transfection efficiency with minimal toxicity, it has been revealed that the efficient cell differentiation was induced by introduction of transcription factors using this system in both in vitro and in vivo experiments The key feature is that the time-dependent toxicity, that may perturb cellular homeostasis during long-term incubation, was minimal, showing little fluctuation on the endogenous gene expression profiles by pharmacogenomic analysis In this study, the biodegradable nature of this system was analyzed in detail PAsp[DET] was rapidly degraded at 37°C, mostly to the state of monomer in a week The degradation was estimated due to the autocatalytic reaction toward the main chain of PAsp What is important is that the degradation products showed almost no cytotoxicity even in an extremely high concentration in the culture medium, nor showed cytokine induction after i.p injection to mice A derivative polymer, PGlu[DET], which had the main chain of polyglutamic acid (PGlu), showed no degradable property even after a long incubation at 37°C Interestingly, while these two polymers showed comparable transgene expressions in many cell lines after a day of transfection, PAsp[DET] specially provided higher expression in a sustained manner for more than several days to primary cells When using ES cells, we achieved efficient induction of cell differentiation by repeat transfection using PAsp[DET] These results indicate that the biodegradable property of PAsp[DET] contributed to the safe and beneficial gene introduction without affecting cellular functions after internalized in the cells This system is promising for therapeutic applications of gene therapy requiring regulated gene expressions S187 ... Society of Gene Therapy interaction with proteins or non-target cells On the other hand, DNA/polycation complexes were also known to have uncontrolled interaction with bio-components, diminishing the. .. Division of Clinical Biotechnology, CDBIM, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan; 2Department of Materials Science and Engineering, Graduate School of Engineering, The. ..CHEMICAL AND MOLECULAR CONJUGATES II peak appearance, magnitude, and duration are dependent on the MOI of infection as well as the cytocidal effect caused by viral replication The data indicate that