500 Replication Competent Retrovirus Vector Mediated Suicide Gene Therapy Achieves Significant Therapeutic Efficacy Against Human Malignant Mesothelioma Xenografts Molecular Therapy Volume 18, Supplem[.]
CANCER-ONCOLYTIC VIRUSES II 498 Susceptibility of Neuroblastoma Primary Tumor-Initiating Cells to Oncolytic Herpes Simplex Virus Is Determined by Nectin-1 Expression Pin-Yi Wang,1 Mark A Currier,1 Margaret H Collins,2 Betsy A DiPasquale,2 Loen Hansford,3 David Kaplan,3,4 Sean Lawler,5 E Antonio Chiocca,5 Timothy P Cripe.1 Division of Hematology/Oncology, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH; 2Division of Pathology, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH; Molecular Genetics, University of Toronto, Toronto, ON, Canada; Cell Biology, The Hospital for Sick Children, Toronto, ON, Canada; 5Department of Neurological Surgery, The Ohio State University, Columbus, OH Neuroblastoma is the most common extracranial solid tumor in children Although patients with high-risk disease can usually achieve remission with conventional therapies, disease relapse is common and long-term survival is less than 40% Bone marrow metastases are enriched for tumor-initiating cells (TICs; Hansford et al., Cancer Research 67:11234, 2007), consistent with the cancer stem cell hypothesis We previously showed that neuroblastoma cell lines are killed by oncolytic Herpes simplex virus (oHSV) infection (Parikh et al., Ped Blood Cancer 44:469, 2005) and sought to determine the susceptibility of neuroblatoma TICs to oHSV While NB12 and NB122R cells were relatively susceptible to HSV transduction and cytolysis, NB88R2 cells were resistant Resistance correlated with low expression of the known major HSV-1 receptor, nectin-1, and could be partially reversed by exogenous nectin-1 expression Xenograft tumors derived from these TICs retained delity of nectin-1 expression: tumors derived from NB12 cells were positive while those from NB88R2 were negative Although direct intratumoral oHSV injection efcacy studies are still underway to conrm a differential response to virus therapy, our data suggest that nectin-1 expression may be a predictor of response to oHSV Using a tissue microarray to screen ∼90 cases of primary neuroblastoma, we found a majority of cases expressed nectin-1, suggesting that overall neuroblastoma is a reasonable target disease for oHSV-based therapy Interestingly, numerous primary glioblastoma neurosphere cultures were all found to be highly susceptible to oHSV infection but showed very low or absent nectin-1 expression These cells were positive for nectin-2 and nectin-3, suggesting that HSV-1 might utilize different cellular entry receptors to infect peripheral versus central nervous system tumors Our novel ndings raise questions about HSV-1 receptor expression on cancer cells that may represent barriers to virus spread within a tumor and may be predictive of susceptibility to oncolytic HSV therapy 499 Activity of Oncolytic Measles Virus Strains Against Brain Tumor Stem Cells Cory Allen,1 Mark A Schroeder,1 Jann N Sarkaria,1 Mark J Federspiel,1 Stephen J Russell,1 Evanthia Galanis.1 Mayo Clinic College of Medicine, Rochester, MN Glioblastoma multiforme (GBM) is the most common primary brain tumor in adults and has a dismal prognosis despite multimodality treatment Given the resistance of brain tumor stem cells (BTSC) to chemotherapy and radiation therapy, their eradication is thought to be essential for long term glioma remission We have previously demonstrated that measles virus (MV) derivatives have signicant activity against glioma lines and orthotopic xenografts and are currently being tested in a phase I trial in recurrent GBM patients We sought to evaluate the antitumor activity of MV derivatives against GBM BTSC METHODS/RESULTS: Primary GBM xenografts, passaged in the anks of nude mice, were excised, mechanically disrupted with a sterile blade and grown at 37°C in a humidied environment containing 5% CO2 in NeuroCult media (Stemcell Molecular Therapy Volume 18, Supplement 1, May 2010 Copyright © The American Society of Gene & Cell Therapy Technologies, Vancouver, BC, CA) GBM6, GBM12 and GBM44 neurospheres were expanded in this media and subsequently half of each were further passaged in DMEM containing 10% FBS to serve as differentiated controls These were further examined for the expression of the BTSC markers CD133, Nestin and ATF-5, expression of the differention markers GFAP and beta -III-tubulin and expression of the MV receptor CD46, by immunouorescence Cytopathic effect (CPE) of MV against both BTSC and differentiated controls was examined in vitro using trypan blue exclusion assays Cells were infected with the MV strains MV-NIS or MV-CEA (MOI 0.1 and 1) and the total number of surviving cells was counted at days 2, 4, 6, 8, &16 post infection and compared against uninfected controls BTSC expressed high levels of the MV receptor CD46 Both BTSC and corresponding differentiated cells were effectively killed at a comparable rate even at low multiplicity of infection (MOI 0.1) One-step viral growth curves showed similar growth kinetics of oncolytic MV strains in BTSC and their differentiated counterpart To examine the impact of MV infection on BTSC survival in vivo, GBM 44 BTSC were exposed to MV at an MOI of 10 in vitro and subsequently implanted into the right caudate nucleus of nude mice with uninfected BTSC serving as controls A signicant prolongation of survival in mice implanted with infected BTSC was observed (p=0.0483) In addition, in vivo therapy experiments were performed in GBM6 and GBM12 BTSC derived orthotopic xenografts in nude mice Signicant prolongation of survival was observed in animals treated orthotopically with MV (1e6 and 1.8e6 respectively) for both GBM6 and GBM12 BTSC xenografts as compared to UV inactivated virus control treated animals (GBM6 p=0.0021, GBM12 p=0.0416) CONCLUSION: Measles virus derivatives have signicant antitumor activity against brain tumor derived stem cells in vitro and in vivo Supported by the Mayo Brain SPORE CA 108961 500 Replication-Competent Retrovirus Vector-Mediated Suicide Gene Therapy Achieves Signicant Therapeutic Efcacy Against Human Malignant Mesothelioma Xenografts Yoshiko Kawasaki,1 Norie Yamaoka,1 Nobuyuki Terada,2 Haruki Okamura,1 Noriyuki Kasahara,3 Shuji Kubo.1 Laboratory of Host Defenses, Institute for Advanced Medical Sciences, Hyogo College of Medicine, Nishinomiya, Hyogo, Japan; 2Department of Pathology, Hyogo College of Medicine, Nishinomiya, Hyogo, Japan; 3Department of Medicine, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA Purpose: Replication-competent retrovirus (RCR) vectors have been shown to achieve signicantly enhanced tumor transduction efciency and therapeutic efcacy in various cancer models We and others have previously engineered RCR vectors for highly efcient delivery of suicide genes, and as the virus is intrinsically incapable of infecting post-mitotic normal cells, retrovirus spread after intratumoral injection is highly restricted to tumor tissue, particularly in immunocompetent hosts In the present study, we hypothesized that RCR vector-mediated suicide gene therapy could be effectively applied to the treatment of malignant mesothelioma, a highly aggressive tumor with poor prognosis Methods: To evaluate the utility and efciency of RCR vectors for gene delivery to mesothelioma cells, RCR-GFP vector, expressing the green uorescent protein (GFP) marker gene, was rst tested on a panel of human malignant mesothelioma and non-malignant transformed mesothelial cell lines in vitro Transduction efciency and replicative spread of the RCR vector over time was monitored by ow cytometry and in vivo imaging Next, to evaluate the potential of RCR vector-mediated suicide gene therapy for this malignancy, we employed RCR-yCD, expressing the yeast cytosine deaminase (yCD) suicide gene Following administration of the prodrug S193 CANCER-ONCOLYTIC VIRUSES II 5-uorocytosine (5FC), cytotoxicity against RCR-yCD infected malignant mesothelioma cells was assessed in vitro by Alamar blue assay, and tumor growth inhibition effects in vivo were assessed in both subcutaneous xenograft tumor and disseminated peritoneal tumor models of malignant mesothelioma Results: Even at multiplicities of infection (MOI) as low as 0.01, RCR vectors successfully infected and efciently replicated in human malignant mesothelioma cell lines, as compared to nonmalignant transformed mesothelial cells In mice with pre-established subcutaneous tumor xenografts, the RCR-GFP showed robust spread throughout entire tumor masses by Day 12 after intratumoral administration of x 104 total infectious units per 100 µl inoculum Notably, no RCR infection was detectable in adjacent normal tissue RCR-yCD showed efcient transmission of the suicide gene associated with replicative spread of the virus, resulting in efcient killing of malignant mesothelioma cells in a 5FC-dose dependent manner in vitro After intratumoral injection of RCR-yCD followed by intraperitoneal administration of 5FC prodrug, RCR vectormediated suicide gene therapy achieved signicant inhibition of subcutaneous tumor growth, and signicantly prolonged survival in the disseminated peritoneal model of malignant mesothelioma Conclusions: These data indicate that RCR vector-mediated suicide gene therapy may represent a highly useful new treatment strategy for malignant mesothelioma 501 Oncolytic Adenovirus with Somatostatin Motif for Selective Infection of Neuroendocrine Tumors Di Yu,1 Justyna Leja,1 Berith Nilsson,1 Magnus Essand.1 Clinical Immunology, Uppsala University, Uppsala, Sweden We have produced an oncolytic serotype adenovirus, Ad[CgAE1A-miR122], where the human chromogranin A (CgA) promoter and miR122 target sequences control E1A gene expression It selectively replicates in and kills neuroendocrine cells, including neuroblastoma and carcinoid cells Neuroendocrine tumors (NETs) contain a high density of homogeneously distributed somatostatin receptors (SSTR) on their surface and somatostatin analogues, such as octreotide, are used for tumor imaging and treatment In order to target SSTRs and increase the infectious capacity for NETs, we have introduced a cyclic loop with four amino acids (FWKT) from octreotide into either the HI-loop of the ber knob or the hexon hypervariable region (HVR) of the capsid We investigate binding properties of GFP-expressing adenoviral vectors with FWKT motif in the ber knob Ad[CMVGFP]HIFWKT and HVR-5 region Ad[CMV-GFP]HVR5FWKT The vectors were evaluated in various NET cell lines as well as in freshly isolated carcinoid cells and normal hepatocytes Both Ad[CMV-GFP] HIFWKT and Ad[CMV-GFP]HVR5FWKT infect SSTR-positive, CAR-negative neuroendocrine tumor cells with greater efcacy than Ad[CMV-GFP] Furthermore, Ad[CMV-GFP]HVR5FWKT can detarget factor X binding and neutralization antibody binding to the virus capsid and hopefully prolong the systematic circulation time We conclude that modication of the capsid with the FWKT motif can be benecial for adenovirus-based NET therapy 502 Silencing a p53 Inhibitor in Cancer Cells Increases Killing Potency of Oncolytic Adenovirus Christie Vermeulen,1 Nikki Tol,1 Winald R Gerritsen,1 Victor W van Beusechem.1 Dept Medical Oncology, VU University Medical Center, Amsterdam, Netherlands Virotherapy of cancer using oncolytic adenoviruses has shown promise in both preclinical and clinical settings The potency of oncolytic adenoviruses depends on their replication efciency in cancer cells and their capacity to destroy these cells by oncolysis S194 Previously, we found that the oncolytic potency of adenoviruses was enhanced by expressing tumor suppressor p53 from the virus genome Not unexpectedly, the effect was minimal in cancer cells expressing high levels of the p53 negative regulator human double minute (HDM2) By abrogating the interaction between HDM2 and p53 the oncolytic adenovirus potency was elevated This suggested that oncolytic adenoviruses could be made more effective by suppressing p53 inhibitor expression in cancer cells We envision that this could be done by incorporating a gene silencing cassette into the oncolytic adenovirus genome To develop such next generation viruses, the p53 inhibitor expression prole in cancer cells should be determined and it should be conrmed that silencing these inhibitors increases oncolytic potency In the present study, we screened derivatives of U2OS and A549 cancer cells carrying a p53 reporter construct for 18 putative p53 inhibitors using siRNA In U2OS cells, several molecules including HDM2 were found to inhibit p53 activity In contrast, in A549 cells considerable p53 activation was observed only upon silencing the SYVN1 gene SYVN1 encodes synoviolin, an ER-resident ubiquitin ligase that is highly expressed in rheumatoid arthritis synoviocytes Interestingly, functional screening for inhibition of adenovirusinduced cell death by the 18 putative p53 inhibitors in A549 cells also revealed only SYVN1 Using three independent cell viability assays (i.e., CellTiter-Blue conversion, BCA protein assay and microscopic cell counting), we found that silencing SYVN1 reproducibly and selectively increased adenovirus killing potency Adenovirus late gene expression, as tested using a virus with endogenous MLP-driven GFP expression, was not signicantly affected Our results establish that SYVN1 silencing empowers adenovirus-mediated killing of A549 cells without affecting early events in the virus life cycle They also show that endogenous p53 reactivation in cancer cells correlates with oncolytic adenovirus potency Therefore, our ndings suggest that knowledge on p53 inhibitor expression in cancer could be exploited to develop a new class of potent oncolytic adenoviruses 503 Safety of Intravenous Ad5-Mda-7, a Potential Anti-Metastatic Agent in Gynecologic Cancer Kenneth H Kim,1 Meredith A Preuss,1 Minghui Wang,1 Anand C Annan,1 Justin A Barnes,1 Devanand Sarkar,2 Steven Grant,2 Paul Dent,2 Paul B Fisher,2 Ronald D Alvarez,1 David T Curiel.1 University of Alabama, Birmingham, AL; 2Virginia Commonwealth University, Richmond, VA Objective: Mda-7 is a cytokine protein with many antitumoral properties that is highly conserved across multiple species; its progressive loss of expression has been shown to correlate with cancer progression Preclinical trials involving its use within an adenoviral viral vector Ad5-mda-7, have shown cancer-cell-selective apoptosis induction, and enhancement of cancer-selective cytotoxicity paired with adjuvant therapy in various solid tumors This study evaluated safety prole after IV injection of AdCMVmda-7, and characterizes its expression and secretion from the liver, in anticipation of ongoing evaluation as an anti-metastasis agent Methods: Two cohorts of mice were treated with doses of 2x10^10 vp/mL of either Ad5-mda-7 (5 mice/cohort) or Ad-CMV.CEA (3 mice/cohort) via tail vein injection, while a third control group (2 mice/cohort) remained uninjected On study days 0, 1, 3, 7, 14, and 21, blood samples were taken from each cohort of mice for chemistry panel analysis Animals were then euthanized and the following organs were removed from each animal for histopathological examination Immunohistochemistry was performed to evaluate mda-7 and CEA tissue expression in harvested organs Results: On immunohistochemistry analysis, mda7 expression within the liver was noted to increase, peaking on day and then returning to normal Similar mda-7 expression patterns were also noted within the kidney, also returning to normal after day There was minimal to no mda-7 expression in other organs At the Molecular Therapy Volume 18, Supplement 1, May 2010 Copyright © The American Society of Gene & Cell Therapy ... RCR vectormediated suicide gene therapy achieved signicant inhibition of subcutaneous tumor growth, and signicantly prolonged survival in the disseminated peritoneal model of malignant mesothelioma. .. mesothelioma Conclusions: These data indicate that RCR vector- mediated suicide gene therapy may represent a highly useful new treatment strategy for malignant mesothelioma 501 Oncolytic Adenovirus with Somatostatin... tumor models of malignant mesothelioma Results: Even at multiplicities of infection (MOI) as low as 0.01, RCR vectors successfully infected and efciently replicated in human malignant mesothelioma