479 Transduction and Oncolytic Efficacy Profile of RCAd11pGFP, a Potent and Selective Oncolytic Adenovirus 11p Vector Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Socie[.]
ONCOLYTIC ADENOVIRUS VECTORS with adenovirus This carrier cell-mediated transfer of adenovirus enabled repetitive infections After the induction of antiadenoviral CTL responses by immunization of adenovirus, administration of carrier cells infected with a replication-competent adenovirus induced complete tumor regression Re-injection of ovarian tumor was rejected in the mice after the induction of complete tumor regression Acute toxicity and distribution test were carried out to inject carrier cells into ovarian tumor in nude mice at once Chronic toxicity test was carried out to inject carrier cells into rabbits at times for weeks Acute toxicity test did not show any lethal side effects in nude mice In distribution test, single injection of carrier cells into ovarian tumor induced the peak levels of cells and oncolytic virus in tumors on the next day but did not show any significant levels of cells and oncolytic virus in tumors and other organs of nude mice 14 days after injection Chronic toxicity test, injections of carrier cells did not show any significant toxicity in rabbits 477 Inhibition of Breast Tumor Angiogenesis and Metastasis by Modulating VEGF Expression Via Adenoviral-Mediated p16’s Interaction with HIF1α Jun Zhang,1 Andrew Lu,1 Liyuan Li,1 Yi Lu.1 Pathology and Medicine, University of Tennessee Health Science Center, Memphis, TN One effective approach to suppress malignant tumor progression is to block tumor angiogenesis Vascular endothelial growth factor (VEGF) plays a pivotal role in tumor angiogenesis Because the degree of tumor malignancy directly correlates with the expression of VEGF, but inversely correlates with the expression of tumor suppressor gene p16, we examined whether restoration of p16 in breast cancer cells would modulate VEGF expression To facilitate induction of p16 expression, a recombinant adenovirus expressing p16 (AdRSVp16) was generated and used to transduce breast cancer cells Adenoviral-mediated p16 expression downregulated VEGF gene expression in breast cancer cells, inhibited breast cancer cell-induced angiogenesis by a dorsal air sac model in mice, and suppressed breast tumor metastasis in a spontaneous metastasis animal model Moreover, p16 appears to bind directly to hypoxia inducible factor1α (HIF-1α), the transcription factor for the VEGF gene promoter, inside the cells at the protein level by both co-immunoprecipitation and colocalization assays Taken together, these results demonstrated that p16 modulates VEGF expression and inhibits tumor-induced angiogenesis and metastasis The binding between p16 and HIF-1α protein may alter HIF-1α’s ability to transactivate VEGF expression This study reveals a less explored function of p16, namely, p16’s anti-angiogenesis function by its interaction with HIF-1α and modulation of VEGF gene expression The dual function of p16’s anti-angiogenesis and its well-known anti-proliferation should warrant p16 gene transfer as an effective gene therapy strategy for suppressing breast cancer growth and metastasis 478 Enhanced Delivery of mda-7/IL-24 Using a Serotype Chimeric Ad.5/3 Improves Therapeutic Outcome in Low CAR Prostate Cancer Cells Rupesh Dash,1 Devanand Sarkar,1,3 Zhao-zhong Su,1 Nichollaq Vozhilla,1 Adly Yacoub,2 Paul Dent,2 Igor Dmitriev,4 David T Curiel,4 Paul B Fisher.1,3 Department of Human and Molecular Genetics, Virginia Commonwealth University, School of Medicine, Richmond, VA; Biochemistry and Molecular Biology2, Virginia Commonwealth University, School of Medicine, Richmond, VA; 3Institute of Molecular Medicine and the VCU Massey Cancer Center, Virginia Commonwealth University, School of Medicine, Richmond, VA; Gene Therapy Center, University of Alabama in Birmingham, Birmingham, AL Prostate cancer is the most common cancer and the second leading cause of cancer-related deaths in men in the US The therapeutic options for patients with prostate cancer include radiotherapy and cytotoxic chemotherapeutic agents Despite a palliative benefit, none of these approaches engender a long-term beneficial effect on the overall survival of patients Gene therapy is being scrutinized as a potential therapeutic for prostate cancer because of accessibility to gene delivery and lack of survival benefit of the prostate gland Serotype adenovirus (Ad5) is routinely used as a vector for transgene delivery However, infectivity of Ad5 is dependent on coxsackie-adenovirus receptors (CAR), which is problematic since many tumor types show a reduction in this receptor in vivo thereby limiting therapeutic gene transduction Serotype chimerism is one approach to circumvent CAR deficiency and this strategy has been used to generate an Ad5/3 recombinant virus that infects cancer cells via Ad3 receptors in a CAR-independent manner In this report the enhanced transgene delivery and efficacy of Ad5/3 recombinant virus was evaluated using a novel tumor suppressor gene melanoma differentiation associated gene-7/interleukin-24 (mda-7/IL-24) in a low CAR prostate cancer line MDA-7/IL-24, a secreted cytokine with cancer-selective apoptosis-inducing properties profoundly inhibits prostate cancer cell growth Our data demonstrate that in low CAR human prostate cancer cells (PC-3) a recombinant Ad5/3 virus delivering mda-7/IL-24 (Ad.5/3-mda-7) is more efficacious than Ad5.mda-7 in infecting tumor cells, expressing MDA-7/IL-24 protein, and inducing apoptosis an killing tumor cells in vitro and in vivo in a nude mice xenograft model This data argues that Ad.5/3mda-7, through enhanced infectivity and by producing more MDA-7/ IL-24 protein than Ad5.mda-7, will be a superior therapeutic virus for cancer-gene therapy Considering the fact that Ad5.mda-7 has demonstrated significant objective responses in a Phase I clinical trial for solid tumors, it is anticipated that Ad.5/3-mda-7 should exert profound and enhanced therapeutic benefit in patients with prostate and other cancers (Supported by P01 R0104177) 479 Transduction and Oncolytic-Efficacy Profile of RCAd11pGFP, a Potent and Selective Oncolytic Adenovirus 11p Vector Jim Silver, Ya-Fang Mei* Virology, Clinical Microbiology, Umeå University, Umeå, Sweden Human adenovirus 11 prototype (Ad11p) is one of species B:2 adenoviruses, causing urinary tract infection, uses CD46 as a primary receptor that is up-regulated on human tumor cells Defective species B adenovirus vectors have been reported; however, there is limited information about the efficacy of the replicating oncolytic species B adenovirus vectors We have developed a replication competent (RC) Ad11pGFP vector and report here the biological and oncolytic profile of RCAd11pGFP in colon carcinoma cells This highly potent vector plays a key role in oncolytic effect meanwhile colon carcinoma cells adversely affect the vector replication and subsequent Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy S185 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 ... tumor transduction and antitumor efficacy and make repeated dosing of oncolytic adenovirus more feasible To create Ad3-hTERT-E 1A, we placed the Ad3 E 1A region under an hTERT We found that Ad3-hTERT-E 1A. .. 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... 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