1104 Identification of Pretreatment Agents To Enhance the Adenovirus Infection of Uro Epithelium Molecular Therapy �������� ��� ���� ���������������� �������� ��� ������®������������ �!����� ����"� ��[.]
CANCER TARGETED GENE THERAPY III Ad vectors containing luciferase (Adflt-1Luc) or E-coli βgalactosidase (Adflt-1LacZ) reporter genes under the control of the flt-1 promoter to evaluate the transductional activity of this promoter in SCCHN cell lines in vitro Ad vectors with these reporter genes under the control of cytomegalovirus (CMV) promoter were used as control vectors (AdCMVLuc, AdCMVLacZ) RT-PCR and flow cytometry analyses showed significant transcriptional and translational activity of the flt-1 gene in SCCHN cells, whereas flt1 promoter activity was relatively high in SCCHN cells, very low activity was observed in the flt-1 negative control cell lines, HeLa and normal human keratinocyte, NHEK cells In the analysis with AdfltLuc or AdCMVLuc, the average relative percentage of flt-1 promoter activity compared to that of the CMV promoter in SCCHN cells was 32.2%, ranging from 6.76% in FaDu cells to 55.9% in SCC29 cells Whereas the relative flt-1 promoter activity in the flt1 negative cell lines was 0.098% and 0.136% in HeLa and NHEK, respectively β-Gal expression was detected in each SCCHN cell line infected with AdfltLacZ or AdCMVLacZ; however, HeLa cells infected with Adflt-1LacZ did not express β-Gal These results indicate that flt-1 promoter activity is relatively efficient and specific in SCCHN cells, making it an attractive candidate TSP for SCCHN cells Driving therapeutic genes or conditionally replicative Ads with the flt-1 promoter may be useful for the gene therapy of SCCHN 1102 Evaluation of Reproductive Toxicity of Adenovirus p53 Following Intraperitoneal Administration in Mice Kyung Hee Sohn,1 Soon Sun Kim,1 Seung Jun Kwack,1 Rhee Da Lee,1 Hyun Joo Lee,1 Yong Huck Won,1 Seung Hoon Lee,2 Kui Lea Park,1 Gyu Seek Rhee.1 Reproductive and Developmental Toxicology, National Institute of Toxicological Research, Seoul, Republic of Korea; 2Molecular Therapy center, Sungkyungkwan University, Seoul, Republic of Korea Reproductive and developmental toxicology in the setting of gene therapy is a relatively new area of investigation but is a central safety issue for the field An important safety concern of gene therapy products is the distribution of vector beyond the target organ This is particularly important if vector distributes to gonads, raising the possibility of inadvertent germ-line transmission In addition, for indications such as prostate cancer and ovarian cancer, the proximity of the point of viral administration to organs of the reproductive system raises concerns regarding inadvertent germline transmission of genes carried by the virus To evaluate the reproductive and developmental toxicity of in vivo adenovirus mediated gene transfer, we studied the biodistribution and potential germ-line transmission of p53-expressing adenovirus (Ad-CMVp53) Both male and female Balb/c mice were injected with 1x108 pfu of Ad-CMV-βgal or Ad-CMV-p53 DNA and RNA extracted from major organs including gonadal tissues were analyzed for vector sequences and expression The PCR analysis showed that there were detectable vector sequences in liver, kidney, spleen, seminal vesicle, epididymis, prostate, ovary, and uterus The RT-PCR analysis showed that Ad-CMV-βgal or Ad-CMV-p53 viral RNA were present in spleen, prostate and ovary Vector-administered female and male mice were mated and their offspring were evaluated for germ-line transmission of the adenoviral vector The PCR analysis showed no evidence of germ-line transmission, although vector sequences were detected in DNA extracted from gonadal tissues We have also established a ovarian tumor model of human ovarian cancer in the nude mouse Together, we conclude that the risk of the inadvertent germline transmission of vector sequences following intraperitoneal injection of adenovirus is extremely low, although vector distributed to gonadal tissues Molecular Therapy Vol 7, No 5, May 2003, Part of Parts Copyright ® The American Society of Gene Therapy 1103 Potent Antitumoral Efficacy of a Novel Replicative Adenovirus CNHK300 Targeted to the Telomerase-Positive Cancer Cells Changqing Su,1 Jonsthon Sham,2 Huibin Xue,1 Mengchao Wu,1 Qijun Qian.1,2 Labortory of Viral and Gene Therapy, Eastern Hepatobiliary Surgical Hospital, Shanghai, China; 2Department of Clinical Oncology, Hong Kong University, Hong Kong, Cape Verde Telomerase, a ribonucleoprotein complex mainly composed of the RNA component (hTR) and the telomerase reverse transcriptase (hTERT), plays a crucial role in cellular immortalization and oncogenesis More detailed studies suggested that almost various human primary cancers (>85%) exhibit telomerase activity, but normal tissues not Thus telomerase may be a promising target for cancer gene therapy Based on our knowledge that hTERT component is a key determinant of human telomerase activity, we constructed a novel replicative adenovirus CNHK300, in which hTERT promoter was introduced three extra E-boxes downstream of the promoter core sequence and used to regulate E1a gene involved in adenoviral replication Luciferase assay showed that introducing extra E-boxes into hTERT promoter is beneficial to decreasing the promoter activity in normal cells but does not influence its strong activity in cancer cells Experiments in vitro and in vivo demonstrated that, CNHK300 can selectively target to hTERT-positive cancer cells and replicate in them, then result in oncolytic or antitumoral effect, but not affect the normal cells CNHK300 is superior to ONYX-015 in aspects of selective replication and oncolytic or antitumoral effect These studies suggested that the hTERT promoter-controlled, cancer-selective, replication-competent adenovirus CNHK300 is an ideal vector system for targeted cancer gene therapy 1104 Identification of Pretreatment Agents To Enhance the Adenovirus Infection of UroEpithelium Nagarajan Ramesh,1 Eric Memarzadeh,1 Melinda VanRoey,1 Virginia Rojas,1 David Frey,1 De-Chao Yu.1 Cell Genesys, Inc, Foster City, CA Adenovirus has been used widely as an in vitro and in vivo gene transfer vector for the purpose of gene therapy CG8840 is a conditionally replication competent oncolytic adenovirus for the treatment of superficial bladder cancer Intravesicular therapy of refractory superficial bladder cancer employing an adenovirus would allow for local administration and efficient delivery of virus This would reduce the systemic exposure and could allow for repeat treatment of the bladder tumor The glycosaminoglycan (GAG) layer on the surface of the bladder urothelium has been shown to act as a nonspecific antiadherence factor and may be an important roadblock to efficient infection of the bladder urothelium by adenoviruses Enhanced infection of bladder urothelium has been achieved in animal models by pretreatment of the bladder or by using specialized adenovirus formulations to disrupt the GAG In order to allow for efficient infection of the urothelium with the oncolytic viruses, we have identified a class of compounds that could be used as a pretreatment agent Preliminary experiments have shown that, at low concentrations, pretreatment with these compounds result in enhanced infection of mice bladder urothelium (>90%) by a replication-defective adenovirus expressing the beta galactosidase gene Of the compounds that were identified, pretreatment with oxychorosene or dodecyl-β D maltoside (DDM) allowed for significant enhancement in the infection of bladder epithelium by adenovirus In a murine model, pretreatment of bladder for with DDM at a concentration of 0.05% lead to >90% transduction of the bladder urothelial layer Similar levels of transduction were S425 CANCER TARGETED GENE THERAPY III observed even when only 109 viral particles were instilled into the bladder Only the uppermost epithelial layer was infected by adenovirus and there was no infiltration of the virus into the inner layers The integrity of the epithelial layer was not affected following pretreatment and virus infection Enhancement of adenoviral infection following pretreatment of bladder with DDM and oxycholorosene was also observed in a rat model Further work is in progress to understand the mechanism of action of these chemicals along with their compatibility for formulation with the adenovirus Results from these studies will also be presented 1105 Dominant Negative Effect of Mutant Rad50 Sensitizes Cisplatin-Based Chemotherapy for Human Head and Neck Cancer Guoli Shi,1 Guoyan Li,1 Cara Lang,1 Shuzhen Yu,1 Bert O’Malley,1,2 Daqing Li.1,2 Otolaryngology-Head & Neck Surgery, University of Maryland School of Medicine, Baltimore, MD, United States; 2Greenebaum Cancer Center, University of Maryland School of Medicine, Baltimore, MD, United States Chemotherapy plays an important role in the neoadjuvant or adjuvant setting and management of advanced and metastatic disease for the treatment of head and neck squamous cell carcinoma (HNSCC) Cisplatin, a DNA-damaging agent, is one of the most promising single chemotherapuetic agents used against HNSCC to date However, the clinical success of Cisplain is compromised if tumor cells become resistant by various mechanisms Enhanced DNA double-strand break (DSB) repair could be one of the primary causes for development of resistance in tumor cells to cisplatin which induce DNA DSBs or crosslinks Recently, a protein complex made up of hMre11, hRad50 and Nbs1 (MRN) has been shown to play a critical component of the cellular response to the repair of DNA DSBs The present study investigates if the expression of mutant Rad50 by a dominant negative adenoviral construct in tumor cells inhibits the function of MRN and increases sensitivity to Cisplatin-based chemotherapy against human HNSCC Human HNSCC cell lines were used in all experiments A dominant negative recombinant adenovirus containing a gene coding for the mutant Rad50 protein under transcriptional control of a cytomegalovirus promoter (Ad-Rad50) was constructed Control adenovirus DL312 with E1a region deletion was obtained from Dr Tom Shenk, Princeton University Cisplatin was purchased form Bristol-Myers Squibb Co Expression of adenovirus-mediated Rad50 was confirmed by Q-RT-PCR Cell growth curves were determined by MTT Q-RT-PCR analysis confirmed the expression of mutant Rad50 expression after Ad-Rad50 gene transfer in HNSCC cells In multiple trials, cell growth curves were seen to flatten and decline in combined Ad-Rad50 and Cisplatin treated group as compared to the control groups in which cellular division continued at a brisk pace Our current study indicates that alteration of DNA repair may provide a novel approach to enhance sensitization of HNSCC to chemotherapy and support the potential application of Ad-Rad50 in combination of cisplatin to treat advanced and metastatic HNSCC 1106 Survey of Coxsackie-Adenovirus Receptor (CAR) Expression in the Non-Neoplastic GenitoUrinary Tract: Implications for Gene Therapy Using Adenoviral Vectors Jeff Simko,1 Vivian Weinberg,1 Peter R Carroll,1 Frank McCormick,1 Katherine A Rauen.1 Comprehensive Cancer Center, University of California San Fransisco, San Fransisco, CA, United States Adenoviral-based gene therapies are dependent on infectivity of the target tissue through the coxsackie-adenovirus receptor, CAR, which is the primary adenoviral receptor Its presence is a determining factor in efficient adenoviral infection CAR serves as the site for attachment of human adenovirus via the fiber protein to the target cell membrane CAR is found in the tight junctions between epithelia cells and its physiologic function is believed to be important in cellcell interaction and adhesion We have recently demonstrated that metastatic prostate carcinoma expresses high levels of CAR, especially relative to primary tumors confined to the prostate (Rauen et al., Cancer Research 62: 3812 2002) While studies of CAR membrane expression levels in other tumors of the genito-urinary (GU) tract is ongoing, we have evaluated the benign epithelia of the male GU system to obtain a baseline of CAR expression in these tissues The rationale for the study is several fold: to examine the normal physiologic expression of this cell adhesion molecule so as to provide a baseline for comparing relative CAR expression levels between normal and tumor tissue, to understand how much virus may be absorbed by the benign tissue component during therapy and to know whether toxicity of normal tissues may be a serious consideration due to possible infectivity We evaluated CAR expression in normal formalin-fixed, paraffin-embedded archival tissues of the male GU system which included prostate (n=33), kidney (n=15), ureter (n=9), urinary bladder (n=12), seminal vesicle (n=6), urethra (n=8) and testis (n=10) The intensity of CAR staining for the normal specimens were grouped into four categories: no staining/background of negative controls (0), weak staining detectable above background (1+), moderate staining (2+), and intense staining (3+) All prostate, bladder, kidney, ureter and testis tissues demonstrated strong 2-3+ staining Reduced staining (1 – 2+) was present in all seminal vesicle and urethral specimens These results indicate that the intrinsic expression level of CAR throughout the epithelia of the GU tract is high, suggesting that these tissues are potential targets for infectivity 1107 Novel Strategies for CAR-Independent Adenovirus Targeting of Gliomas Winan J Van Houdt,1,3 Joel N Glasgow,1,2 Hongju Wu,1,2 Xiaosheng Lei,1,2 Martine L M Lamfers,3,4 Clemens M F Dirven,4 David T Curiel,1,2 Yosef S Haviv.1,2 Division of Human Gene Therapy, Departments of Medicine, Pathology, and Surgery; 2Gene Therapy Center, University of Alabama at Birmingham, Birmingham, AL; 3Department of Medical Oncology, Division of Gene Therapy; 4Department of Neurosurgery, VUMC, Amsterdam, Netherlands Malignant gliomas are resistant to radiotherapy and chemotherapy Because of the high mortally rates in patients with malignant glioma, new treatment strategies are required Therefore, gene therapy approaches have been undertaken, to the end of introducing novel mechanisms of glioma cell killing Adenoviral vectors (Ads), based on serotype (Ad5), have been widely used for gene delivery in different cancer types However, a limiting factor for the utility of Ad5 vectors for cancer gene therapy is their critical dependence on the membrane expression of the primary Ad receptor, the coxsackie and adenovirus receptor (CAR) Most tumor cells, including most glioma cells, express CAR at low levels Thus, the efficacy of Ad5 S426 Molecular Therapy Vol 7, No 5, May 2003, Part of Parts Copyright ® The American Society of Gene Therapy ... virus into the inner layers The integrity of the epithelial layer was not affected following pretreatment and virus infection Enhancement of adenoviral infection following pretreatment of bladder... limiting factor for the utility of Ad5 vectors for cancer gene therapy is their critical dependence on the membrane expression of the primary Ad receptor, the coxsackie and adenovirus receptor (CAR)... made up of hMre11, hRad50 and Nbs1 (MRN) has been shown to play a critical component of the cellular response to the repair of DNA DSBs The present study investigates if the expression of mutant