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271. Downregulation of ErbB3 Expression by Adenovirus Expressing ErbB3 Specific shRNA Enhances Antitumor Efficacy through Apoptosis Induction
CANCER - TARGETED GENE THERAPY I and internalization into target cells must be incorporated; (2) the gene delivery system must be inert against nonspecific interactions with the biological environment such as blood components and nontarget cells. Minicircles are a novel form of supercoiled DNA molecule for nonviral gene transfer, which have neither bacterial origin of replication nor antibiotic resistance gene. Minicircles are superior to conventional plasmids due to its capability of mediating long-lasting and high level of gene expression. In this study, folate-PEG (F-PEG) was attached on the poly(ethylenimine) (PEI)surface to obtain efficiency and specificity of gene delivery and minicircle-endostatin is encapsulated to obtain persistent and high-level transgene expression in vivo for antitumor gene therapy. We developed this carrier system and evaluated the effects of nanoparticle/minicircle(mc) complex in folate-receptor(FR) positive (HeLa and Skov-3) cells and FR-negative (HepG2,A431) cells in vitro and on xenografts in vivo. Reporter (GFP, luciferase) assay showed that FA-PEG-PEI/mc complexes had significantly higher transgene activity than that of FA-PEG-PEI/mc. Significant specificity and transfection efficiency was exhibited in FAtargeted cells. These results indicated that FA-PEG-PEI /minicircle complex might be a promising candidate for gene delivery with the characteristics of better biocompatibility, potential biodegradability, and relatively high gene transfection efficiency compared to viral vectors.
270.
Abstract Withdrawn
271. Downregulation of ErbB3 Expression by Adenovirus Expressing ErbB3 Specific shRNA Enhances Antitumor Efficacy through Apoptosis Induction
Ji Young Yoo,1 Hye Won Shin,1 Joo-Hang Kim,1 Chae-Ok Yun.1 1 Brain Korea 21 Project for Medical Sciences, Institute of Cancer Research, Yonsei Cancer Center, Yonsei University College of Medicine, Seoul, Republic of Korea.
Human epidermal growth factor receptor-3 (ErbB3) is overexpressed mainly in breast cancer, and abberant signaling of ErbB3 causes the progression and metastasis of breast cancer. Therefore, targeted inhibition of ErbB3 signaling would be a promising approach for breast cancer therapy. In this study, we examined the effect of the ErbB3 downregulation via adenovirus-mediated siRNA expression in breast cancer. In order to determine whether ErbB3 could be a potential target for the therapy, we generated an ErbB3-specific shRNA-expressing recombinant adenovirus, Ad-∆E1-shErbB3, to downregulate ErbB3 and evaluated knockdown efficacy of ErbB3. Ad-∆E1-shErbB3 inhibited ErbB3 expression with effectiveness, and remarkably enhanced cytotoxic effect up to 83.5% in breast cancer cell lines tested. Propidium iodide (PI) staining and TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling) assay showed that Ad-∆E1-shErbB3-mediated cytotoxicity was largely driven by apoptosis. Interestingly, it was observed dramatically increased cleavage of cellular apoptosis-related enzymes caspase-3 and poly(ADP-ribose) polymerase (PARP) in cells treated with Ad∆E1-shErbB3, while not with control Ad, Ad-∆E1. In vivo studies also showed that Ad-∆E1-shErbB3 enhances antitumor effect in xenograft model of MDA-MB-231 human breast cancer. In addition, survival rates of Ad-∆E1-shErbB3-treated mice were also significantly increased. Further, consistent with its expected mechanism of action, Ad-∆E1-shErbB3 was shown to greatly reduce the level of ErbB3 in tumor. It was also revealed from the in vivo TUNEL assay that the apoptosis in Ad-∆E1-shErbB3-trasduced tumor cells significantly are originated along the border between necrotic and viable areas of the tumors. These results demonstrate the targeted downregulation of ErbB3 by adenovirus-mediated RNA interference can lead breast cancer cells selectively to apoptosis, and similar approaches can be applied to other cancer cells for targeted therapies.
272. Oncolytic Adenovirus Expressing Granulocyte Macrophage Colony-Stimulating Factor (GM-CSF) for Treatment of Cancer
Vincenzo Cerullo,1 Iulia Diaconu,1 Matteo Ugolini,1 Petri Nokisalmi,1 Sari Pesonen,1 Elina Haavisto,1 Akseli Hemminki.1,2 1 Molecular Cancer Biology, University of Helsinki, Helsinki, Finland; 2Finnish Institute for Molecular Medicine, Helsinki, Finland. Gene therapy with adenoviruses is a promising modality for the treatment of advanced cancer refractory to the other therapies. However, it is becoming clear that, virus replication and the consequent cell death alone, is not sufficient to eradicate tumors. Potentiation and redirection of the tumor-specific immune response is a promising approach. Dendridic cells (DC) are optimal antigen presenting cell to present tumor antigen epitopes to CD8+ and CD4+ T cells. Exogenous antigens can be loaded onto MHC class I (MHC I) for “cross-presentation” to CD8+ T cell, which is believed to occur after the phagocytosis of apoptotic cells. Unfortunately the frequency of activated CD8+ T cell is often too low to mount an effective response against the tumor. Different strategies can be used to enhance the T cell response towards the tumor: A) Recruiting DCs to the tumor site to augment peptide loading onto MHC I; B) specifically activate DCs at the tumor site to increase their expression of co-stimulatory molecules enhancing cross-priming and T cell activation rather than S106
Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy
270.
Abstract Withdrawn
271. Downregulation of ErbB3 Expression by Adenovirus Expressing ErbB3 Specific shRNA Enhances Antitumor Efficacy through Apoptosis Induction
Ji Young Yoo,1 Hye Won Shin,1 Joo-Hang Kim,1 Chae-Ok Yun.1 1 Brain Korea 21 Project for Medical Sciences, Institute of Cancer Research, Yonsei Cancer Center, Yonsei University College of Medicine, Seoul, Republic of Korea.
Human epidermal growth factor receptor-3 (ErbB3) is overexpressed mainly in breast cancer, and abberant signaling of ErbB3 causes the progression and metastasis of breast cancer. Therefore, targeted inhibition of ErbB3 signaling would be a promising approach for breast cancer therapy. In this study, we examined the effect of the ErbB3 downregulation via adenovirus-mediated siRNA expression in breast cancer. In order to determine whether ErbB3 could be a potential target for the therapy, we generated an ErbB3-specific shRNA-expressing recombinant adenovirus, Ad-∆E1-shErbB3, to downregulate ErbB3 and evaluated knockdown efficacy of ErbB3. Ad-∆E1-shErbB3 inhibited ErbB3 expression with effectiveness, and remarkably enhanced cytotoxic effect up to 83.5% in breast cancer cell lines tested. Propidium iodide (PI) staining and TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling) assay showed that Ad-∆E1-shErbB3-mediated cytotoxicity was largely driven by apoptosis. Interestingly, it was observed dramatically increased cleavage of cellular apoptosis-related enzymes caspase-3 and poly(ADP-ribose) polymerase (PARP) in cells treated with Ad∆E1-shErbB3, while not with control Ad, Ad-∆E1. In vivo studies also showed that Ad-∆E1-shErbB3 enhances antitumor effect in xenograft model of MDA-MB-231 human breast cancer. In addition, survival rates of Ad-∆E1-shErbB3-treated mice were also significantly increased. Further, consistent with its expected mechanism of action, Ad-∆E1-shErbB3 was shown to greatly reduce the level of ErbB3 in tumor. It was also revealed from the in vivo TUNEL assay that the apoptosis in Ad-∆E1-shErbB3-trasduced tumor cells significantly are originated along the border between necrotic and viable areas of the tumors. These results demonstrate the targeted downregulation of ErbB3 by adenovirus-mediated RNA interference can lead breast cancer cells selectively to apoptosis, and similar approaches can be applied to other cancer cells for targeted therapies.
272. Oncolytic Adenovirus Expressing Granulocyte Macrophage Colony-Stimulating Factor (GM-CSF) for Treatment of Cancer
Vincenzo Cerullo,1 Iulia Diaconu,1 Matteo Ugolini,1 Petri Nokisalmi,1 Sari Pesonen,1 Elina Haavisto,1 Akseli Hemminki.1,2 1 Molecular Cancer Biology, University of Helsinki, Helsinki, Finland; 2Finnish Institute for Molecular Medicine, Helsinki, Finland. Gene therapy with adenoviruses is a promising modality for the treatment of advanced cancer refractory to the other therapies. However, it is becoming clear that, virus replication and the consequent cell death alone, is not sufficient to eradicate tumors. Potentiation and redirection of the tumor-specific immune response is a promising approach. Dendridic cells (DC) are optimal antigen presenting cell to present tumor antigen epitopes to CD8+ and CD4+ T cells. Exogenous antigens can be loaded onto MHC class I (MHC I) for “cross-presentation” to CD8+ T cell, which is believed to occur after the phagocytosis of apoptotic cells. Unfortunately the frequency of activated CD8+ T cell is often too low to mount an effective response against the tumor. Different strategies can be used to enhance the T cell response towards the tumor: A) Recruiting DCs to the tumor site to augment peptide loading onto MHC I; B) specifically activate DCs at the tumor site to increase their expression of co-stimulatory molecules enhancing cross-priming and T cell activation rather than S106
Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy