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1113. Transcriptionally and Transductionally Targeted Conditionally Replicating Adenovirus for Esophageal Adenocarcinoma
CANCER TARGETED GENE THERAPY III The transduced HUVECs were subjected to an in vitro matrigel tube formation assay by plating on growth factor reduced matrigel in the presence of 100 ng/ml vascular endothelial growth factor and 10% bovine calf serum. Tube formation was analyzed 48 hours posttransduction using normal and fluorescence microscopy. The HUVECs transduced with ADV-hEndo-Angio vector revealed apoptosis induction and nearly complete inhibition of endothelial cell differentiation and lack of any tubular network formation. While the HUVECs transduced with ADV-GFP displayed endothelial cell differentiation, GFP expression and extensive tubular network formation. We next investigated the ability of ADV-hEndo-Angio and ADV-GFP to inhibit angiogenic sprouting from microvascular endothelial cells in an in vitro 3D angiogenesis model using the mouse aortic ring culture. Our results indicate significant reduction in angiogenic sprouting from the aortic rings transduced with ADVhEndo-Angio while extensive sprouting and GFP expression was observed in the case of aortic rings transduced with ADV-GFP. Based upon these results we have initiated animal studies aimed at evaluating the anti-angiogenic and growth inhibition response in an androgen-independent human prostate tumor xenograft nude mouse model. The outcome of these studies would provide new insights into the mechanism of tumor angiogenesis in general and will lead to the development of novel anti-angiogenic gene therapy approaches for prostate cancer in particular.
1113. Transcriptionally and Transductionally Targeted Conditionally Replicating Adenovirus for Esophageal Adenocarcinoma Julia Davydova,1 Long P. Le,1 Gavrikova Tatyana,1 Hidetaka A. Ono,1 Pedro J. Ramirez,1 Minghui Wang,1 Victor Krasnykh,1 David T. Curiel,1 Masato Yamamoto.1 1 Division of Human Gene Therapy, Departments of Medicine, Pathology and Surgery, and the Gene Therapy Center, University of Alabama at Birmingham, Birmingham, AL, United States. Conditionally replicating adenoviruses (CRAds) is a promising and novel approach for treating neoplastic diseases. These agents are designed to replicate specifically in tumor cells, followed by the spread of the viral progeny to neighboring cancer cells. However, in the field of esophageal adenocarcinoma (EAC) transcriptional tumor targeting has been a major challenge due to the absence of an appropriate tumor specific promoter (TSP) with requisite therapeutic index to prevent ectopic transgene expression. Additionally, realization of the full utility of CRAds for EAC is hampered by resistance of the cells to Ad5 infection due to low coxsackie-adenovirus receptor (CAR) expression. In this study, we constracted a TSP based CRAds with a CAR-independent-infection capability to overcome these limitations. We used the cyclooxygenase-2 (COX-2) promoter, which is active in many gastrointestinal (GI) cancers but minimally active in normal tissues, and infectivity enhancement based on fiber modification by incorporating an RGD4C motif into the HI loop (RGD) and replacing of the Ad5 knob region with the Ad3 knob (5/3 chimera). The COX2 promoter profile (-1432/+59: COX-2L) was tested with luciferase (Luc) expression vectors in three EAC cell lines (OE19, OE33, and TE7). All three were COX-2 positive in RT-PCR analysis. The COX-2 promoter showed high promoter activity comparable to CMV in all three EAC cell lines while retaining tumor specificity as indicated by minimal activity in primary cells isolated from normal mouse esophagus and stomach. Subsequent assays for the surface modified vectors demonstrated higher levels of infectivity with the RGD-modified vector and even greater levels with the 5/3 chimeric vector. We incorporated the RGD4C motif and Ad3 knob into fully replicative wild type Ad5 to analyze the effect of infectivity enhancement in the context of virus-cell binding and replicationbased oncolytic efficiency. In both experiments, RGD-modified Ads Molecular Therapy Vol. 7, No. 5, May 2003, Part 2 of 2 Parts Copyright ® The American Society of Gene Therapy
exhibited superior anti-tumor efficacy compared to unmodified Ads while the 5/3 chimeric vectors performed even better. Based on these results, RGD and 5/3 modified CRAds with a COX-2 promoter driven E1 expression cassette were constructed. In the analysis of cytocidal effect, all COX-2 CRAds showed replication and subsequent oncolytic killing in all three EAC cells in various extents, while the COX-2 negative control cell line (BT474) was unaffected. RGD modified CRAds showed enhanced killing relative to unmodified vectors in two out of three EAC cell lines (OE19 and OE33 not in TE7). 5/3 chimera CRAds showed enhanced oncolysis in all three cell lines, and the cytocidal effect was far stronger than that of RGD CRAds. These results indicate that COX-2 CRAds can be transcriptionally targeted to EAC cells and that the infectivity enhancements based on fiber modification (especially 5/3 chimerism) significantly augment the efficacy without hampering selectivity. This modality of combining transcriptional and transductional targeting to increase the therapeutic index should provide a powerful and promising approach for virotherapy of EAC.
1114. Replication of an Adenoviral Vector Controlled by the Human Telomerase Reverse Transcriptase Promoter Causes Tumor-Selective Cell Lysis Nicholas A. Lanson, Jr.,1 Paul Friedlander,2 Paul Schwarzenberger,1 Jay K. Kolls,1 Guoshun Wang.1,3 1 Department of Medicine, Gene Therapy Program, Louisiana State University Health Sciences Center, New Orleans, LA; 2 Department of Medicine, Louisiana State University Health Sciences Center, New Orleans, LA; 3Department of Genetics, Louisiana State University Health Sciences Center, New Orleans, LA. Replication-selective virotherapy holds great promise for the treatment of cancers. Appealing features with regard to this approach include tumor-specific targeting, viral self-spreading in cancer cells, and no cross-resistance to current treatments. Telomerase reactivation is a critical step for tumorigenesis, allowing cancer cells to proliferate indefinitely. Taking advantage of this property, we have generated an adenovirus vector, in which the expression of the adenovirus E1 gene and thus viral replication is under the control of the human telomerase reverse transcriptase (hTERT) promoter. Such a vector, referred to as Ad5-hTERT-E1, replicates in cancer cells specifically. Ad5-hTERT-E1 demonstrated efficient cancer-specific cytolysis in tested human tumor cell lines, including OC8 (ovarian tumor cells), HeLa (cervical adenocarcinoma), Hep G2 (hepatocellular carcinoma), A549 (lung carcinoma), HT-1080 (fibrosarcoma), MCF7 (breast adenocarcinoma) and, SCCLSU1, SCC-4 and -25 (squamous cell carcinomas). In contrast, the same MOIs of Ad5-hTERT-E1 had no effect on primary cultures of normal human fibroblasts, airway epithelial cells or bone marrow mesenchymal stem cells. Moreover, a single injection of Ad5hTERT-E1 (1-5x108 PFU) into pre-established subcutaneous solid tumors (70-180 mm³) in nu/nu mice significantly suppressed tumor growth. Interestingly, this conditionally replicating vector facilitates the replication of E1-deleted anti-tumor adenoviral vector in tumor cells, thus demonstrating synergistic anti-tumor effects in vivo. Combinational injection of Ad5-hTERT-E1 and the E1-deleted Ad5CMV-TK vector resulted in significant tumor suppression and regression. The results suggest that the Ad5-hTERT-E1 adenoviral vector has the potential to develop into a broad-spectrum specific anti-tumor agent for cancer therapy.
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1113. Transcriptionally and Transductionally Targeted Conditionally Replicating Adenovirus for Esophageal Adenocarcinoma Julia Davydova,1 Long P. Le,1 Gavrikova Tatyana,1 Hidetaka A. Ono,1 Pedro J. Ramirez,1 Minghui Wang,1 Victor Krasnykh,1 David T. Curiel,1 Masato Yamamoto.1 1 Division of Human Gene Therapy, Departments of Medicine, Pathology and Surgery, and the Gene Therapy Center, University of Alabama at Birmingham, Birmingham, AL, United States. Conditionally replicating adenoviruses (CRAds) is a promising and novel approach for treating neoplastic diseases. These agents are designed to replicate specifically in tumor cells, followed by the spread of the viral progeny to neighboring cancer cells. However, in the field of esophageal adenocarcinoma (EAC) transcriptional tumor targeting has been a major challenge due to the absence of an appropriate tumor specific promoter (TSP) with requisite therapeutic index to prevent ectopic transgene expression. Additionally, realization of the full utility of CRAds for EAC is hampered by resistance of the cells to Ad5 infection due to low coxsackie-adenovirus receptor (CAR) expression. In this study, we constracted a TSP based CRAds with a CAR-independent-infection capability to overcome these limitations. We used the cyclooxygenase-2 (COX-2) promoter, which is active in many gastrointestinal (GI) cancers but minimally active in normal tissues, and infectivity enhancement based on fiber modification by incorporating an RGD4C motif into the HI loop (RGD) and replacing of the Ad5 knob region with the Ad3 knob (5/3 chimera). The COX2 promoter profile (-1432/+59: COX-2L) was tested with luciferase (Luc) expression vectors in three EAC cell lines (OE19, OE33, and TE7). All three were COX-2 positive in RT-PCR analysis. The COX-2 promoter showed high promoter activity comparable to CMV in all three EAC cell lines while retaining tumor specificity as indicated by minimal activity in primary cells isolated from normal mouse esophagus and stomach. Subsequent assays for the surface modified vectors demonstrated higher levels of infectivity with the RGD-modified vector and even greater levels with the 5/3 chimeric vector. We incorporated the RGD4C motif and Ad3 knob into fully replicative wild type Ad5 to analyze the effect of infectivity enhancement in the context of virus-cell binding and replicationbased oncolytic efficiency. In both experiments, RGD-modified Ads Molecular Therapy Vol. 7, No. 5, May 2003, Part 2 of 2 Parts Copyright ® The American Society of Gene Therapy
exhibited superior anti-tumor efficacy compared to unmodified Ads while the 5/3 chimeric vectors performed even better. Based on these results, RGD and 5/3 modified CRAds with a COX-2 promoter driven E1 expression cassette were constructed. In the analysis of cytocidal effect, all COX-2 CRAds showed replication and subsequent oncolytic killing in all three EAC cells in various extents, while the COX-2 negative control cell line (BT474) was unaffected. RGD modified CRAds showed enhanced killing relative to unmodified vectors in two out of three EAC cell lines (OE19 and OE33 not in TE7). 5/3 chimera CRAds showed enhanced oncolysis in all three cell lines, and the cytocidal effect was far stronger than that of RGD CRAds. These results indicate that COX-2 CRAds can be transcriptionally targeted to EAC cells and that the infectivity enhancements based on fiber modification (especially 5/3 chimerism) significantly augment the efficacy without hampering selectivity. This modality of combining transcriptional and transductional targeting to increase the therapeutic index should provide a powerful and promising approach for virotherapy of EAC.
1114. Replication of an Adenoviral Vector Controlled by the Human Telomerase Reverse Transcriptase Promoter Causes Tumor-Selective Cell Lysis Nicholas A. Lanson, Jr.,1 Paul Friedlander,2 Paul Schwarzenberger,1 Jay K. Kolls,1 Guoshun Wang.1,3 1 Department of Medicine, Gene Therapy Program, Louisiana State University Health Sciences Center, New Orleans, LA; 2 Department of Medicine, Louisiana State University Health Sciences Center, New Orleans, LA; 3Department of Genetics, Louisiana State University Health Sciences Center, New Orleans, LA. Replication-selective virotherapy holds great promise for the treatment of cancers. Appealing features with regard to this approach include tumor-specific targeting, viral self-spreading in cancer cells, and no cross-resistance to current treatments. Telomerase reactivation is a critical step for tumorigenesis, allowing cancer cells to proliferate indefinitely. Taking advantage of this property, we have generated an adenovirus vector, in which the expression of the adenovirus E1 gene and thus viral replication is under the control of the human telomerase reverse transcriptase (hTERT) promoter. Such a vector, referred to as Ad5-hTERT-E1, replicates in cancer cells specifically. Ad5-hTERT-E1 demonstrated efficient cancer-specific cytolysis in tested human tumor cell lines, including OC8 (ovarian tumor cells), HeLa (cervical adenocarcinoma), Hep G2 (hepatocellular carcinoma), A549 (lung carcinoma), HT-1080 (fibrosarcoma), MCF7 (breast adenocarcinoma) and, SCCLSU1, SCC-4 and -25 (squamous cell carcinomas). In contrast, the same MOIs of Ad5-hTERT-E1 had no effect on primary cultures of normal human fibroblasts, airway epithelial cells or bone marrow mesenchymal stem cells. Moreover, a single injection of Ad5hTERT-E1 (1-5x108 PFU) into pre-established subcutaneous solid tumors (70-180 mm³) in nu/nu mice significantly suppressed tumor growth. Interestingly, this conditionally replicating vector facilitates the replication of E1-deleted anti-tumor adenoviral vector in tumor cells, thus demonstrating synergistic anti-tumor effects in vivo. Combinational injection of Ad5-hTERT-E1 and the E1-deleted Ad5CMV-TK vector resulted in significant tumor suppression and regression. The results suggest that the Ad5-hTERT-E1 adenoviral vector has the potential to develop into a broad-spectrum specific anti-tumor agent for cancer therapy.
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