501. Oncolytic Adenovirus with Somatostatin Motif for Selective Infection of Neuroendocrine Tumors

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 efciently 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 1 x 104 total infectious units per 100 µl inoculum. Notably, no RCR infection was detectable in adjacent normal tissue. RCR-yCD showed efcient transmission of the suicide gene associated with replicative spread of the virus, resulting in efcient 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 signicant inhibition of subcutaneous tumor growth, and signicantly 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 1 Clinical Immunology, Uppsala University, Uppsala, Sweden.

We have produced an oncolytic serotype 5 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) 5 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 efcacy 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 modication of the capsid with the FWKT motif can be benecial 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 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 efciency 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 prole in cancer cells should be determined and it should be conrmed 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 signicantly 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 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 prole 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 3 and then returning to normal. Similar mda-7 expression patterns were also noted within the kidney, also returning to normal after day 3. 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