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Antitumor Activity of AAV-2 Vector Encoding Modified TRAIL Gene
CANCER TUMOR SUPPRESSOR GENE AND APOPTOSIS 950. FasL- Over-Expressing T Lymphocytes as Effector Cells in Anti-Prostate Cancer Gene Therapy Julie C. Bielawski,1 Daniel H. Fowler,2 Jeffrey A. Medin.1,3 Department of Medical Biophysics, University of Toronto, Toronto, ON, Canada; 2Experimental Transplantation and Immunology Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD; 3Division of Experimental Therapeutics, Ontario Cancer Institute, Toronto, ON, Canada. 1
Prostate cancer is the second most prevalent cancer in men, with limited effective treatment options, especially for metastatic disease. We propose to address this problem by developing enhanced Fasmediated apoptosis as a treatment modality for prostate cancer. It has been shown that Fas expression on some prostate tumor lines, including the murine RM-1 line, can be up-regulated by a variety of treatment regimens. In this study, primary T cells and functionallydefined subsets of these T cells will be engineered to over-express Fas and will be introduced into tumor-bearing mice in order to interact with normal or up-regulated Fas receptor on prostate tumors. To do this, three recombinant onco-retroviruses have been produced which engineer over-expression of FasL. The first carries only the FasL gene, while the second contains cDNA for a modified, non-cleavable form of FasL (ncFasL) with potentially reduced liver toxicity. The third, a bicistronic construct, includes the cDNA for c-FLIP, an inhibitor of the Fas-mediated apoptotic pathway, in addition to ncFasL. c-FLIP is included to prevent self-killing of transduced, FasL-expressing T cells. The constructs have been packaged in ecotropic E86 and amphotropic FLYRD18 cells and shown to direct production of FasL as well as produce infectious virions, as measured by staining with anti-FasL antibody and flow cytometric analysis. Assays have confirmed that the FasL and ncFasL produced by infected cells is able to mediate killing of Fas-expressing Jurkat cells, and is thus functional. Transduction of primary murine C57BL/6 pan-T cells has been optimized. Irradiation of several tumor cell lines, both murine and human, has shown this to be a plausible method to increase Fas expression. The ability of interferons and chemotherapeutic drugs, including mitoxantrone, to increase tumor Fas expression will also be examined. Experiments are underway in tumor-bearing mice to assess the efficiency of killing by these modified T cells, and to compare the effectiveness of the three viral constructs. We expect to see effective activation of death pathways, leading to tumor destruction. Finally, the relative abilities of the type 1 versus type 2 T lymphocytes as delivery vehicles for this treatment will be compared. This approach may provide a noninvasive treatment for prostate cancer and may also be extended to other malignancies such as breast cancer.
951. Antitumor Activity of AAV-2 Vector Encoding Modified TRAIL Gene Jinsang Yoo,1 Seeyoung Choi,1 Kyung-Sun Hwang,1 Won-Kyung Cho,1 Cho-Rok Jung,1 Dong-Soo Im.1 1 Gene Therapy Research Unit, Korea Research Institute of Bioscience and Biotechnology, Daejeon, Korea. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis in a variety of cancer cell lines, but is relatively nontoxic to normal cells. Adeno-associated virus (AAV) vectors have a number of advantages including the potential sustained expression of transgenes. Here, we investigated potential usefulness of the TRAIL gene for systemic cancer therapy using AAV-2 vector. We constructed a plasmid encoding modified human TRAIL protein composed of signal peptide, an isoleucine zipper sequence, and active domain of TRAIL (pTR-PLF-ITRAD), which allows the TRAIL gene product to be released from the cells. TRAIL-sensitive cells were killed by transfection with pTR-PLF-ITRAD or a plasmid S364
encoding full-length TRAIL (pTR-TRAIL), indicating that the plasmids produce bioactive TRAIL proteins. The culture media from TRAIL-resistant cells transfected with pTR-PLF-ITRAD, but not pTR-TRAIL induce apoptosis in a number of cancer cell lines, indicating that the modified TRAIL gene product, but not fulllength TRAIL is released into the culture media. AAV-2 vector producing the modified TRAIL protein (AAV-2/PLF-ITRAD) inhibited tumor growth in A549 lung tumor-bearing nude mice. Our results suggest that AAV-2/PLF-ITRAD may be useful for systemic cancer gene therapy, provided that TRAIL is not toxic to normal cells.
952. Delivery of Tissue Inhibitor of Metalloproteinase 3 Inhibits Growth of Lung Cancer Tumours In Vivo Katherine M. Finan,1 Greg Hodge,2 Ann M. Reynolds,1 Sandra J. Hodge,1 Mark D. Holmes,1 Andrew H. Baker,3 Paul N. Reynolds.1 1 Thoracic Medicine, Royal Adelaide Hospital, Adelaide, SA, Australia; 2Haematology, Womens and Childrens Hospital, Adelaide, SA, Australia; 3Cardiovascular and Medical Sciences, University of Glasgow, Glasgow, United Kingdom. Lung cancer causes more cancer deaths than cancers related to colon, breast and prostate combined. More primary lung cancers are now diagnosed in former smokers than current smokers and this trend is set to continue. Despite advances in chemotherapeutic agents current treatment strategies are not effective enough in reducing the burden of disease, producing survival benefit or improving quality of life. There is no doubt that new treatment strategies are necessary to improve the outlook from this disease. Gene therapy exploits our expanding knowledge of the molecular basis of cancer using a number of cancer modifying techniques. The extracellular matrix (ECM) provides the scaffolding for all cells and is the environment in which all tissues and cells exist. In order for tissues to change their form the ECM must be degraded and the tissue remodelled. Matrix Metalloproteinases (MMPs) are the proteins that regulate ECM functions. Through a variety of complex mechanisms they control cell differentiation, proliferation and migration. The Tissue inhibitors of Metalloproteinases (TIMPs) are a family of multifunctional proteins named for their ability to inhibit MMPs. TIMP 3 also has the unique ability to directly induce apoptosis in certain tumour types. Hypothesis: In vitro delivery of AdCMVTIMP-3 induces apoptosis and cell death in lung cancer cells, that AdCMVTIMP-3 has a significant bystander effect and in vivo delivery of AdCMVTIMP-3 to implanted subcutaneous A549 tumours reduces tumour growth. Aims: To demonstrate the in vitro and in vivo apoptotic and cell killing effects of AdCMVTIMP-3. Methods: Lung cancer cells A549 were plated at known concentrations and then infected with adenoviral vectors carrying the genes for TIMP1, -2 and 3. Changes consistent with apoptosis were observed at 4866 hours post infection. A variety of flow cytometric analyses including Annexin V, Propidium Iodide, 7AAD and Single Stranded DNA confirm that AdCMVTIMP-3 induces apoptosis and cell death. Elevated Caspace, p53, BAX/Bcl2 and Fas levels show activation of the caspase cascade that regulates programmed cell death. MTT assays confirm reductions in cell numbers and demonstrate the significant bystander effects of AdCMVTIMP-3. In vivo analysis of A549 tumours implanted subcutaneously in nude mice shows delivery of AdCMVTIMP-3 inhibits tumour growth. TUNEL analysis of paraffin embedded tissue sections of these tumours shows evidence of increased numbers of apoptotic cells when compared to uninjected controls and those injected with control virus. Conclusion: Delivery of AdCMVTIMP-3 induces apoptosis and cell death in lung cancer cells and this can be demonstrated in vitro and in vivo. Molecular Therapy Volume 9, Supplement 1, May 2004
Copyright The American Society of Gene Therapy
Prostate cancer is the second most prevalent cancer in men, with limited effective treatment options, especially for metastatic disease. We propose to address this problem by developing enhanced Fasmediated apoptosis as a treatment modality for prostate cancer. It has been shown that Fas expression on some prostate tumor lines, including the murine RM-1 line, can be up-regulated by a variety of treatment regimens. In this study, primary T cells and functionallydefined subsets of these T cells will be engineered to over-express Fas and will be introduced into tumor-bearing mice in order to interact with normal or up-regulated Fas receptor on prostate tumors. To do this, three recombinant onco-retroviruses have been produced which engineer over-expression of FasL. The first carries only the FasL gene, while the second contains cDNA for a modified, non-cleavable form of FasL (ncFasL) with potentially reduced liver toxicity. The third, a bicistronic construct, includes the cDNA for c-FLIP, an inhibitor of the Fas-mediated apoptotic pathway, in addition to ncFasL. c-FLIP is included to prevent self-killing of transduced, FasL-expressing T cells. The constructs have been packaged in ecotropic E86 and amphotropic FLYRD18 cells and shown to direct production of FasL as well as produce infectious virions, as measured by staining with anti-FasL antibody and flow cytometric analysis. Assays have confirmed that the FasL and ncFasL produced by infected cells is able to mediate killing of Fas-expressing Jurkat cells, and is thus functional. Transduction of primary murine C57BL/6 pan-T cells has been optimized. Irradiation of several tumor cell lines, both murine and human, has shown this to be a plausible method to increase Fas expression. The ability of interferons and chemotherapeutic drugs, including mitoxantrone, to increase tumor Fas expression will also be examined. Experiments are underway in tumor-bearing mice to assess the efficiency of killing by these modified T cells, and to compare the effectiveness of the three viral constructs. We expect to see effective activation of death pathways, leading to tumor destruction. Finally, the relative abilities of the type 1 versus type 2 T lymphocytes as delivery vehicles for this treatment will be compared. This approach may provide a noninvasive treatment for prostate cancer and may also be extended to other malignancies such as breast cancer.
951. Antitumor Activity of AAV-2 Vector Encoding Modified TRAIL Gene Jinsang Yoo,1 Seeyoung Choi,1 Kyung-Sun Hwang,1 Won-Kyung Cho,1 Cho-Rok Jung,1 Dong-Soo Im.1 1 Gene Therapy Research Unit, Korea Research Institute of Bioscience and Biotechnology, Daejeon, Korea. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis in a variety of cancer cell lines, but is relatively nontoxic to normal cells. Adeno-associated virus (AAV) vectors have a number of advantages including the potential sustained expression of transgenes. Here, we investigated potential usefulness of the TRAIL gene for systemic cancer therapy using AAV-2 vector. We constructed a plasmid encoding modified human TRAIL protein composed of signal peptide, an isoleucine zipper sequence, and active domain of TRAIL (pTR-PLF-ITRAD), which allows the TRAIL gene product to be released from the cells. TRAIL-sensitive cells were killed by transfection with pTR-PLF-ITRAD or a plasmid S364
encoding full-length TRAIL (pTR-TRAIL), indicating that the plasmids produce bioactive TRAIL proteins. The culture media from TRAIL-resistant cells transfected with pTR-PLF-ITRAD, but not pTR-TRAIL induce apoptosis in a number of cancer cell lines, indicating that the modified TRAIL gene product, but not fulllength TRAIL is released into the culture media. AAV-2 vector producing the modified TRAIL protein (AAV-2/PLF-ITRAD) inhibited tumor growth in A549 lung tumor-bearing nude mice. Our results suggest that AAV-2/PLF-ITRAD may be useful for systemic cancer gene therapy, provided that TRAIL is not toxic to normal cells.
952. Delivery of Tissue Inhibitor of Metalloproteinase 3 Inhibits Growth of Lung Cancer Tumours In Vivo Katherine M. Finan,1 Greg Hodge,2 Ann M. Reynolds,1 Sandra J. Hodge,1 Mark D. Holmes,1 Andrew H. Baker,3 Paul N. Reynolds.1 1 Thoracic Medicine, Royal Adelaide Hospital, Adelaide, SA, Australia; 2Haematology, Womens and Childrens Hospital, Adelaide, SA, Australia; 3Cardiovascular and Medical Sciences, University of Glasgow, Glasgow, United Kingdom. Lung cancer causes more cancer deaths than cancers related to colon, breast and prostate combined. More primary lung cancers are now diagnosed in former smokers than current smokers and this trend is set to continue. Despite advances in chemotherapeutic agents current treatment strategies are not effective enough in reducing the burden of disease, producing survival benefit or improving quality of life. There is no doubt that new treatment strategies are necessary to improve the outlook from this disease. Gene therapy exploits our expanding knowledge of the molecular basis of cancer using a number of cancer modifying techniques. The extracellular matrix (ECM) provides the scaffolding for all cells and is the environment in which all tissues and cells exist. In order for tissues to change their form the ECM must be degraded and the tissue remodelled. Matrix Metalloproteinases (MMPs) are the proteins that regulate ECM functions. Through a variety of complex mechanisms they control cell differentiation, proliferation and migration. The Tissue inhibitors of Metalloproteinases (TIMPs) are a family of multifunctional proteins named for their ability to inhibit MMPs. TIMP 3 also has the unique ability to directly induce apoptosis in certain tumour types. Hypothesis: In vitro delivery of AdCMVTIMP-3 induces apoptosis and cell death in lung cancer cells, that AdCMVTIMP-3 has a significant bystander effect and in vivo delivery of AdCMVTIMP-3 to implanted subcutaneous A549 tumours reduces tumour growth. Aims: To demonstrate the in vitro and in vivo apoptotic and cell killing effects of AdCMVTIMP-3. Methods: Lung cancer cells A549 were plated at known concentrations and then infected with adenoviral vectors carrying the genes for TIMP1, -2 and 3. Changes consistent with apoptosis were observed at 4866 hours post infection. A variety of flow cytometric analyses including Annexin V, Propidium Iodide, 7AAD and Single Stranded DNA confirm that AdCMVTIMP-3 induces apoptosis and cell death. Elevated Caspace, p53, BAX/Bcl2 and Fas levels show activation of the caspase cascade that regulates programmed cell death. MTT assays confirm reductions in cell numbers and demonstrate the significant bystander effects of AdCMVTIMP-3. In vivo analysis of A549 tumours implanted subcutaneously in nude mice shows delivery of AdCMVTIMP-3 inhibits tumour growth. TUNEL analysis of paraffin embedded tissue sections of these tumours shows evidence of increased numbers of apoptotic cells when compared to uninjected controls and those injected with control virus. Conclusion: Delivery of AdCMVTIMP-3 induces apoptosis and cell death in lung cancer cells and this can be demonstrated in vitro and in vivo. Molecular Therapy Volume 9, Supplement 1, May 2004
Copyright The American Society of Gene Therapy