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941. In Vitro Screening of Murine Carcinoma Cell Lines for Testing of Replication-Competent Oncolytic Adenoviruses
CANCER THERAPY its kinetics, transduced ovarian tumor cells were analyzed every 24 hours for 11 days with flow cytometry. When Δ24CMVTGL (MOI 5) was used, the amount of GFP-positive cells increased from initial 7% to 28% and from 8% to 85% in SKOV3.ipI and OV-4 cells, respectively, while in AdTGL transduced cells the presentage of GFP-positive cells remained constant or showed slight decrease. The presentage of GFP-positive cells started to increase 6 and 8 days after transduction in OV-4 cells and SKOV3.ipI cells, respectively. Conclusion: Based on results from MTT-assay and crystal violet staining, rate of virus replication depends on the features of used cell lines ( for example, CAR expression levels). The transgene had no significant influence for virus replication. Our data also revealed that despite the quite low initial transduction efficiency, almost 90% of the cells can be transduced due to replication. Thus, relatively low viral doses are sufficient for achieving high transduction efficiency. In addition, transgene is effectively amplified and delivered to tumor cells, which might be advantageous when combining suicide/prodrug therapy with oncolytic viruses.
940. Combination Therapy of Radiation and Cytotoxic Gene Transfer Using Chimeric Adenovirus Vector for Bladder Cancer Kazumasa Matsumoto,1 Christian T. F. Freund,1 Weiguo Jian,1 Patricia Yotnda,2 Elizabethi A. Olmsted-Davis,2 Alan R. Davis,2 Seth P. Lerner.1 1 Scott Department of Urology, Baylor College of Medicine, Houston, TX, United States; 2The Cell and Gene Therapy, Baylor College of Medicine, Houston, TX, United States. Background: Combination of suicide gene therapy with radiation has potential synergistic antitumor effects. While radiation enhances membrane damage which may facilitate gene transduction from foreign gene expressing cells to neighboring untransduced cells, cytotoxic gene therapy may impair repair of radiation-induced DNA damage. Down-regulation of coxsackie and adenovirus (CAR) has been observed in advanced bladder cancers resulting in decreased transgene expression using CAR-dependent Ad5 vectors. In this study, we evaluate the efficacy of a novel CAR-independent Ad vector in combination radiation and suicide gene therapy for bladder cancer. Methods: A chimeric Ad vector (Ad5/F35) expressing HSV-tk was created by overlap recombination and the Ad35 fiber gene incorporated into the Ad5 backbone. Both Ad5 and Ad5/F35 vectors were transfected in human bladder cancer cell lines T24 (CARnegative) and 5637 (CAR-positive). Suicide gene therapy efficacy was evaluated by exposing cancer cells to various MOI’s of Ad5HSV-tk or Ad5/F35HSV-tk plus ganciclovir (GCV). In preparation for combination therapy, a single layer of tumor cells was exposed to increasing dose of radiation (2.5, 5, 7.5 and 10Gy) and 2.5 Gy selected for use in combination treatment following gene therapy with Ad5/F35HSV-tk. Cell viability was assessed with the MTT assay. Results: Both Ad vectors demonstrated antitumor effects in a dose dependent manner. As compared to Ad5, Ad5/F35HSV-tk was 30-fold more efficacious in achieving antitumor effects in CAR negative T24 cells and 3-fold more efficacious in CAR positive 5637 cells. In both T24 and 5637, combination therapy showed 10fold greater antitumor effect with use of Ad5/F35HSV-tk as compared to gene therapy alone. Conclusions: Use of CAR-independent chimeric Ad5/F35 vector significantly increased antitumor effects of suicide gene therapy. In both CAR-negative and -positive bladder cancer cells, combination radiation and suicide gene therapy demonstrated synergism as compared to single suicide gene monotherapy. S362
941. In Vitro Screening of Murine Carcinoma Cell Lines for Testing of Replication-Competent Oncolytic Adenoviruses Richard J. Hill,1 Gunnel K. Hallden,1 Nick R. Lemoine,1 David H. Kirn.2 1 Molecular Oncology Unit, Cancer Research UK, Imperial College London, Hammersmith Campus, London, United Kingdom; 2Department of Pharmacology, Ocford University Medical School, Oxford, United Kingdom. The development of replication-selective viral mutants as anticancer agents has been hindered by the absence of in vivo murine models with intact immune function. To this end a panel of murine epithelial tumour cell lines were screened in vitro to identify cells that were sensitive to viral infection, could support viral replication and gene expression, and form tumours in an immuno-competent host. Wild type adenovirus type 5 (Ad5) could infect eight out of nine murine tumour cell lines, determined by infection with a nonreplicating, GFP expressing Ad5 mutant followed by FACS analysis 24 h later. The magnitude of GFP expression was similar to that seen with the human epithelial non-small cell lung carcinoma cell line A549 and the lung epithelial small cell carcinoma H460. Cytopathic effect (CPE) following Ad5 infection was demonstrated in seven of the murine cell lines that were infectable. No cytopathic effect was detected following infection with a non-replicating, E1Adeleted mutant virus (dl312). Viral replication was detected 48 h post infection and increased over time with a plateau at 96 to 144 h in all murine cell lines that demonstrated CPE. Replication was determined by limiting dilution assay (TCID50). In two of the murine cell lines viral burst was similar to that observed in the human H460 cells (2000pfu/cell) while four cell lines demonstrated smaller bursts (50-100pfu/cell) and one displayed no detectable viral replication. To establish that early and late viral gene expression was supported in cell lines giving low yields of replicating virus, expression levels of E1A, penton and fiber were compared by immunoblotting. All murine cell lines supporting viral replication expressed equal levels of both early and late viral proteins, comparable to levels in the H460 cells. Murine cell lines that supported viral replication are currently being evaluated for tumour formation in syngeneic mice to determine oncolytic efficacy of viral mutants in the presence of an intact immune system.
942. Oncolytic Adenovirus Expressing GM-CSF and B7.1 Display Potent Antitumor Effects K. J. Choi,1 E. Kim,1 Y. S. Lee,1 J. Kim,1 J. H. Kim,1 C. O. Yun.1 BK21 Project for Medical Sciences, Institute for Cancer Research, Yonsei Cancer Center, Yonsei University College of Medicine, Seoul, Korea. 1
Oncolytic adenoviral vectors are currently being developed as biological anti-cancer therapeutic agents. Coupling the lytic function of oncolytic adenovirus with virus-mediated transgene delivery represents a powerful extension of this treatment. A clear benefit is amplification of the therapeutic gene, as the replicating vector would be able to infect and deliver the therapeutic gene to adjacent cells as well as the cells infected initially. Granulocyte-macrophage colony stimulating factor(GM-CSF) is the most potent stimulator of a specific and long-lasting antitumor immunity and documented its importance for the maturation of antigen presenting cells into stimulators of T cell activation. B7 family has been shown to be an important mechanism of anti tumor response. However, most tumor cells lack expression of costimulatory molecules on their surface, resulting in an escape recognition by the immune system. To increase the potential anti-tumor activity of oncolytic adenovirus, we constructed a E1B 55kDa deleted oncolytic adenoviral vector Y/G/ B, which expresses GM-CSF and B7.1. The expression of GMMolecular Therapy Vol. 7, No. 5, May 2003, Part 2 of 2 Parts
Copyright ® The American Society of Gene Therapy
940. Combination Therapy of Radiation and Cytotoxic Gene Transfer Using Chimeric Adenovirus Vector for Bladder Cancer Kazumasa Matsumoto,1 Christian T. F. Freund,1 Weiguo Jian,1 Patricia Yotnda,2 Elizabethi A. Olmsted-Davis,2 Alan R. Davis,2 Seth P. Lerner.1 1 Scott Department of Urology, Baylor College of Medicine, Houston, TX, United States; 2The Cell and Gene Therapy, Baylor College of Medicine, Houston, TX, United States. Background: Combination of suicide gene therapy with radiation has potential synergistic antitumor effects. While radiation enhances membrane damage which may facilitate gene transduction from foreign gene expressing cells to neighboring untransduced cells, cytotoxic gene therapy may impair repair of radiation-induced DNA damage. Down-regulation of coxsackie and adenovirus (CAR) has been observed in advanced bladder cancers resulting in decreased transgene expression using CAR-dependent Ad5 vectors. In this study, we evaluate the efficacy of a novel CAR-independent Ad vector in combination radiation and suicide gene therapy for bladder cancer. Methods: A chimeric Ad vector (Ad5/F35) expressing HSV-tk was created by overlap recombination and the Ad35 fiber gene incorporated into the Ad5 backbone. Both Ad5 and Ad5/F35 vectors were transfected in human bladder cancer cell lines T24 (CARnegative) and 5637 (CAR-positive). Suicide gene therapy efficacy was evaluated by exposing cancer cells to various MOI’s of Ad5HSV-tk or Ad5/F35HSV-tk plus ganciclovir (GCV). In preparation for combination therapy, a single layer of tumor cells was exposed to increasing dose of radiation (2.5, 5, 7.5 and 10Gy) and 2.5 Gy selected for use in combination treatment following gene therapy with Ad5/F35HSV-tk. Cell viability was assessed with the MTT assay. Results: Both Ad vectors demonstrated antitumor effects in a dose dependent manner. As compared to Ad5, Ad5/F35HSV-tk was 30-fold more efficacious in achieving antitumor effects in CAR negative T24 cells and 3-fold more efficacious in CAR positive 5637 cells. In both T24 and 5637, combination therapy showed 10fold greater antitumor effect with use of Ad5/F35HSV-tk as compared to gene therapy alone. Conclusions: Use of CAR-independent chimeric Ad5/F35 vector significantly increased antitumor effects of suicide gene therapy. In both CAR-negative and -positive bladder cancer cells, combination radiation and suicide gene therapy demonstrated synergism as compared to single suicide gene monotherapy. S362
941. In Vitro Screening of Murine Carcinoma Cell Lines for Testing of Replication-Competent Oncolytic Adenoviruses Richard J. Hill,1 Gunnel K. Hallden,1 Nick R. Lemoine,1 David H. Kirn.2 1 Molecular Oncology Unit, Cancer Research UK, Imperial College London, Hammersmith Campus, London, United Kingdom; 2Department of Pharmacology, Ocford University Medical School, Oxford, United Kingdom. The development of replication-selective viral mutants as anticancer agents has been hindered by the absence of in vivo murine models with intact immune function. To this end a panel of murine epithelial tumour cell lines were screened in vitro to identify cells that were sensitive to viral infection, could support viral replication and gene expression, and form tumours in an immuno-competent host. Wild type adenovirus type 5 (Ad5) could infect eight out of nine murine tumour cell lines, determined by infection with a nonreplicating, GFP expressing Ad5 mutant followed by FACS analysis 24 h later. The magnitude of GFP expression was similar to that seen with the human epithelial non-small cell lung carcinoma cell line A549 and the lung epithelial small cell carcinoma H460. Cytopathic effect (CPE) following Ad5 infection was demonstrated in seven of the murine cell lines that were infectable. No cytopathic effect was detected following infection with a non-replicating, E1Adeleted mutant virus (dl312). Viral replication was detected 48 h post infection and increased over time with a plateau at 96 to 144 h in all murine cell lines that demonstrated CPE. Replication was determined by limiting dilution assay (TCID50). In two of the murine cell lines viral burst was similar to that observed in the human H460 cells (2000pfu/cell) while four cell lines demonstrated smaller bursts (50-100pfu/cell) and one displayed no detectable viral replication. To establish that early and late viral gene expression was supported in cell lines giving low yields of replicating virus, expression levels of E1A, penton and fiber were compared by immunoblotting. All murine cell lines supporting viral replication expressed equal levels of both early and late viral proteins, comparable to levels in the H460 cells. Murine cell lines that supported viral replication are currently being evaluated for tumour formation in syngeneic mice to determine oncolytic efficacy of viral mutants in the presence of an intact immune system.
942. Oncolytic Adenovirus Expressing GM-CSF and B7.1 Display Potent Antitumor Effects K. J. Choi,1 E. Kim,1 Y. S. Lee,1 J. Kim,1 J. H. Kim,1 C. O. Yun.1 BK21 Project for Medical Sciences, Institute for Cancer Research, Yonsei Cancer Center, Yonsei University College of Medicine, Seoul, Korea. 1
Oncolytic adenoviral vectors are currently being developed as biological anti-cancer therapeutic agents. Coupling the lytic function of oncolytic adenovirus with virus-mediated transgene delivery represents a powerful extension of this treatment. A clear benefit is amplification of the therapeutic gene, as the replicating vector would be able to infect and deliver the therapeutic gene to adjacent cells as well as the cells infected initially. Granulocyte-macrophage colony stimulating factor(GM-CSF) is the most potent stimulator of a specific and long-lasting antitumor immunity and documented its importance for the maturation of antigen presenting cells into stimulators of T cell activation. B7 family has been shown to be an important mechanism of anti tumor response. However, most tumor cells lack expression of costimulatory molecules on their surface, resulting in an escape recognition by the immune system. To increase the potential anti-tumor activity of oncolytic adenovirus, we constructed a E1B 55kDa deleted oncolytic adenoviral vector Y/G/ B, which expresses GM-CSF and B7.1. The expression of GMMolecular Therapy Vol. 7, No. 5, May 2003, Part 2 of 2 Parts
Copyright ® The American Society of Gene Therapy