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770. Evaluation of Different Tissue Specific Promoters for Cervical Cancer Gene Therapy
CANCER TARGETED GENE THERAPY II different tumor cell lines as described above, with human fibroblasts and a mammary epithelial cell line as normal controls. The activity of the CXCR4 promoter in cell lines was determined by luciferase assays. A reAdGL3BCMV, containing the CMV promoter, was used as a positive promoter control to normalize the luciferase levels. For in vivo studies, a dose of 5x108 pfu of reAdGL3BCXCR4 or reAdGL3BCMV was injected intravenously into mice. The major organs were harvested two days post-injection, and the luciferase activities were determined from major organs Luciferase assays revealed that the CXCR4 promoter demonstrated relatively high transcriptional activity in a breast cancer cell line, MDA-MB-361, and an ovarian cancer cell line, SKOV3ip1, 65% and 20% compared to the CMV promoter, respectively. However, after infection with reAdGL3BCXCR4, both normal cell lines had activities less than 5% luciferase expression compared to that induced by reAdGL3BCMV. Interestingly, the in vivo testing demonstrated the luciferase activity induced by the CXCR4 promoter in liver was only 0.32% (“liver off”) of that produced by the CMV promoter. The CXCR4 promoter appears to be a tumor specific-promoter in some tumor cell lines with a very low predicted in vivo expression based on the data of “tumor on” and “liver off” in the mouse model. The data suggested that the CXCR4 promoter is a good candidate for use in transcriptional targeting in cancer gene therapy, especially in breast and ovarian cancers.
770. Evaluation of Different Tissue Specific Promoters for Cervical Cancer Gene Therapy Daniel T. Rein,1 Dirk M. Nettelbeck,1 Masato Yamamoto,1,2,3 Koichi Takayama,4 Yasuo Adachi,1 Shannon D. Barker,1 David T. Curiel.1,2,3 1 Division of Human Gene Therapy; 2Departments of Medicine, Pathology and Surgery; 3The Gene Therapy Center, University of Alabama at Birmingham, Birmingham, AL; 4Research Institute for Diseases of the Chest, Kyushu University, Fukuoka, Japan. Carcinoma of the uterine cervix is the third most common cancer, affecting women worldwide. Although the prognosis of patients with cervical cancer has improved dramatically due to the success of systematic screening programs, the disease remains a major health problem. Approximately 30% of patients have locally advanced disease at the time of primary diagnosis. Patients with advanced-, recurrent- or metastatic-disease have a poor chance of cure. Gene therapy is a novel approach in these patients and adenoviral (Ad) vectors are promising candidates due to their ability to transduce both dividing and non dividing cells, ease of manipulation and their capability of producing high titers. However, the tropism of adenoviruses is promiscuous and the natural tropism to normal liver cells can lead to severe side effects when the virus is administered systemically in a therapeutic approach. One effective way to direct transgene expression to specific tissues or tumors is the use of tissue-specific-promoters (TSPs). In the context of adenovirus mediated cancer gene therapy it is rational to choose a TSP, which is highly expressed in the tumor but has potentially low activity in the liver. However, TSPs for cervical cancer have never been systematically explored. Therefore we have investigated several promoters, which are known to be turned “off” in normal tissues such as the liver, and are hypothesized to be overexpressed in cervical cancer. Candidate cervical cancer-specific TSPs include promoters of the genes for secretory leukoprotease inhibitor (SLPI), Cyclooxygenase-2 (COX-2), Midkine (MK), vascular endothelial growth factor receptor type 1 (flt-1) and vascular endothelial growth factor (VEGF). To evaluate the specific gene expression of the different promoters in the context of cervical cancer, we constructed a panel of E1-deleted adenoviruses that express luciferase under the control of the promoters of interest. In a first step we infected different established cervical cancer cell lines (HeLa, SiHa, C33a, Molecular Therapy Vol. 7, No. 5, May 2003, Part 2 of 2 Parts Copyright © The American Society of Gene Therapy
Caski). The non-specific cytomegalovirus (CMV) promotor was used as a control. Luciferase activity was measured using standard luminometry techniques and normalized with the results obtained with AdCMVluc. The results show highest activity for the MK and VEGF promoter in all cell lines tested. The promoter activity in SiHa and C33a cells is 8-12% of activity seen with CMV promoter. Caski and HeLa show lower expression for all viruses (approximately 1% of activity seen with CMV promoter) but MK and VEGF again show the highest activity. Both promoters have previously demonstrated no activity in cells which have been shown not to express the MK or VEGF gene respectively. The results show that the MK and VEGF promoters are active in cervical cancer cell lines. On the basis of these data conditionally replicative adenoviruses containing the MK and VEGF promoter are being used to demonstrate the efficiency and specificity in cervical cancer cell lines as as well as corresponding control cell lines. In light of data obtained to date, we believe that the MK and VEGF promoters can be successfully employed as a TSPs for gene therapy or viral oncolysis targeted to cervical cancer.
771. Dual Hypoxia and Radiation Responsive α to Tumours Promoters for the Delivery of TNFα Naomi Chadderton,1 Rachel L. Cowen,1 Kaye J. Williams,1 Ian J. Stratford.1 1 Department of Pharmacy, University of Manchester, Manchester, United Kingdom. Of the current therapeutic agents used in anticancer strategies TNFα holds the most promise given the broad range and strength of its activity. However, despite demonstrating potent anti-tumour effects in animal models, human trials employing systemic administration of recombinant TNFα have been limited by doselimiting toxicities. Consequently, gene therapy strategies using tissue specific or inducible promoters are in development to control this systemic toxicity. Regulation by the radiation-sensitive Early Growth Response 1(Egr-1) promoter is at the most advanced stage of development. However, controlling expression of TNFα using radiation alone limits its application to tumour cells within the radiation field and precludes targeted expression in disseminated disease. We propose a refinement of this approach through the development of novel, synthetic enhancer/promoter elements that show dual specificity, being responsive to both radiation and hypoxia. Incorporating hypoxia responsive elements (HREs) will enable therapeutic gene expression in tumour cells both at the site of radiotherapy and in distant metastases containing foci of oxygendeprived cells with the ultimate aim of enabling the safe systemic administration of TNFα. We generated a chimeric enhancer that incorporated pentamers of the radiation responsive elements isolated from the Egr-1 gene (serum response element, SRE) and the Epo HRE. When expressed in a minimal viral (SV40) promoter context, this construct yielded maximum expression in response to anoxia. The weakened HRE mediated response at increasing oxygen concentrations was rescued by additional treatment with 5 Gy of radiation. In preliminary studies, an additional benefit of the incorporation of the SRE into this construct was a reduction in the basal expression observed in aerobic cells, which currently limits the use of a number of HRE constructs with directly cytotoxic gene products. However, when these studies were expanded the attenuation of unwanted aerobic expression was cell line dependent. This necessitated the development of an alternative vector that we have based upon the promoter of the hypoxia responsive carbonic anhydrase IX (CA9) gene. The HRE within this gene is situated 4bp from the transcription start site. We exploited this precise positioning and through rational configuration studies have generated a novel CA9-based promoter that affords robust hypoxic expression (30-fold greater than the native CA9 S297
770. Evaluation of Different Tissue Specific Promoters for Cervical Cancer Gene Therapy Daniel T. Rein,1 Dirk M. Nettelbeck,1 Masato Yamamoto,1,2,3 Koichi Takayama,4 Yasuo Adachi,1 Shannon D. Barker,1 David T. Curiel.1,2,3 1 Division of Human Gene Therapy; 2Departments of Medicine, Pathology and Surgery; 3The Gene Therapy Center, University of Alabama at Birmingham, Birmingham, AL; 4Research Institute for Diseases of the Chest, Kyushu University, Fukuoka, Japan. Carcinoma of the uterine cervix is the third most common cancer, affecting women worldwide. Although the prognosis of patients with cervical cancer has improved dramatically due to the success of systematic screening programs, the disease remains a major health problem. Approximately 30% of patients have locally advanced disease at the time of primary diagnosis. Patients with advanced-, recurrent- or metastatic-disease have a poor chance of cure. Gene therapy is a novel approach in these patients and adenoviral (Ad) vectors are promising candidates due to their ability to transduce both dividing and non dividing cells, ease of manipulation and their capability of producing high titers. However, the tropism of adenoviruses is promiscuous and the natural tropism to normal liver cells can lead to severe side effects when the virus is administered systemically in a therapeutic approach. One effective way to direct transgene expression to specific tissues or tumors is the use of tissue-specific-promoters (TSPs). In the context of adenovirus mediated cancer gene therapy it is rational to choose a TSP, which is highly expressed in the tumor but has potentially low activity in the liver. However, TSPs for cervical cancer have never been systematically explored. Therefore we have investigated several promoters, which are known to be turned “off” in normal tissues such as the liver, and are hypothesized to be overexpressed in cervical cancer. Candidate cervical cancer-specific TSPs include promoters of the genes for secretory leukoprotease inhibitor (SLPI), Cyclooxygenase-2 (COX-2), Midkine (MK), vascular endothelial growth factor receptor type 1 (flt-1) and vascular endothelial growth factor (VEGF). To evaluate the specific gene expression of the different promoters in the context of cervical cancer, we constructed a panel of E1-deleted adenoviruses that express luciferase under the control of the promoters of interest. In a first step we infected different established cervical cancer cell lines (HeLa, SiHa, C33a, Molecular Therapy Vol. 7, No. 5, May 2003, Part 2 of 2 Parts Copyright © The American Society of Gene Therapy
Caski). The non-specific cytomegalovirus (CMV) promotor was used as a control. Luciferase activity was measured using standard luminometry techniques and normalized with the results obtained with AdCMVluc. The results show highest activity for the MK and VEGF promoter in all cell lines tested. The promoter activity in SiHa and C33a cells is 8-12% of activity seen with CMV promoter. Caski and HeLa show lower expression for all viruses (approximately 1% of activity seen with CMV promoter) but MK and VEGF again show the highest activity. Both promoters have previously demonstrated no activity in cells which have been shown not to express the MK or VEGF gene respectively. The results show that the MK and VEGF promoters are active in cervical cancer cell lines. On the basis of these data conditionally replicative adenoviruses containing the MK and VEGF promoter are being used to demonstrate the efficiency and specificity in cervical cancer cell lines as as well as corresponding control cell lines. In light of data obtained to date, we believe that the MK and VEGF promoters can be successfully employed as a TSPs for gene therapy or viral oncolysis targeted to cervical cancer.
771. Dual Hypoxia and Radiation Responsive α to Tumours Promoters for the Delivery of TNFα Naomi Chadderton,1 Rachel L. Cowen,1 Kaye J. Williams,1 Ian J. Stratford.1 1 Department of Pharmacy, University of Manchester, Manchester, United Kingdom. Of the current therapeutic agents used in anticancer strategies TNFα holds the most promise given the broad range and strength of its activity. However, despite demonstrating potent anti-tumour effects in animal models, human trials employing systemic administration of recombinant TNFα have been limited by doselimiting toxicities. Consequently, gene therapy strategies using tissue specific or inducible promoters are in development to control this systemic toxicity. Regulation by the radiation-sensitive Early Growth Response 1(Egr-1) promoter is at the most advanced stage of development. However, controlling expression of TNFα using radiation alone limits its application to tumour cells within the radiation field and precludes targeted expression in disseminated disease. We propose a refinement of this approach through the development of novel, synthetic enhancer/promoter elements that show dual specificity, being responsive to both radiation and hypoxia. Incorporating hypoxia responsive elements (HREs) will enable therapeutic gene expression in tumour cells both at the site of radiotherapy and in distant metastases containing foci of oxygendeprived cells with the ultimate aim of enabling the safe systemic administration of TNFα. We generated a chimeric enhancer that incorporated pentamers of the radiation responsive elements isolated from the Egr-1 gene (serum response element, SRE) and the Epo HRE. When expressed in a minimal viral (SV40) promoter context, this construct yielded maximum expression in response to anoxia. The weakened HRE mediated response at increasing oxygen concentrations was rescued by additional treatment with 5 Gy of radiation. In preliminary studies, an additional benefit of the incorporation of the SRE into this construct was a reduction in the basal expression observed in aerobic cells, which currently limits the use of a number of HRE constructs with directly cytotoxic gene products. However, when these studies were expanded the attenuation of unwanted aerobic expression was cell line dependent. This necessitated the development of an alternative vector that we have based upon the promoter of the hypoxia responsive carbonic anhydrase IX (CA9) gene. The HRE within this gene is situated 4bp from the transcription start site. We exploited this precise positioning and through rational configuration studies have generated a novel CA9-based promoter that affords robust hypoxic expression (30-fold greater than the native CA9 S297