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748. MicroRNA-200a Functions as a Tumor Suppressor in Meningiomas
Small Nucleic Acids in Blood Diseases and Cancer 747. Development of Systemic siRNA Delivery for Tumor with Specifically Tumor Activation System
Hiroto Hatakeyama, Hidetaka Akita, Erika Itoh, Motoi Oishi,2,3 Yukio Nagasaki,2,3 Hideyoshi Harashima.1,2 1 Graduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo, Hokkaido, Japan; 2CREST, Japan Science and Technology, Kawaguchi, Saitama, Japan; 3Tsukuba Research Center of Interdisciplinary Material Science (TIMS), University of Tsukuba, Tsukuba, Ibaraki, Japan. 1,2
1,2
1,2
For development of siRNA therapeutics, systemic delivery of siRNA into specific tissue is major obstacle. Modification of nonviral vehicles with polyethylene glycol (PEG) is useful strategy for delivering the vehicles into tumor tissue via enhanced permeability and retention (EPR) effect. However, the PEGylation affects cellular uptake and following intracellular trafficking, which resulted in significant low level of RNAi activity. To solve these problems such PEG dilemma, we designed cleavable PEG-lipid system (abbrev. as PPD) in response to Matrix Metalloproteinase (MMP) in extracellular space in the tumor tissue(Figure1). Previously, we reported development of our novel non-viral vector, named a multifunctional envelope-type nano device (MEND) modified with PPD (PPD-MEND) as a systemic delivery system of plasmid DNA for tumor[1]. PPD-MEND exhibited the successfully enhancement of transfection activity in tumor tissue after i.v. administration compared with conventional PEG-modified MEND (PEG-MEND).
In this study, we investigated the utility of PPD-MEND as siRNA delivery system. To maximize a systemic stability of MEND, we optimized the modification density of PEG and PPD on the surface of MEND. The MEND modified with PEG and PPD at optimum ratio (PEG/PPD-MEND) is 100 nm in diameter possessing neutral zeta potential, which are desirable physiological characteristics for long systemic circulation and efficient accumulation in tumor via EPR effect. For RNAi study, HT1080 cells expressing luciferase were subcutaneously inoculated into nude mice. PEG/PPD-MEND was able to knockdown more than 70% of luciferase activity in tumor tissue after i.v. administration at a dose of 80 mg of siRNA. In histological observation, most of PEG/PPD-MEND labeled with rhodamine colocalized with tumor cells, which were immunostained by Alexa488-labeled antibody against EGF receptors as a tumor marker. Furthermore, in cytokine study, PEG/PPD-MEND showed no induction of IL-6 and TNF-α. Serum levels of ALT and AST as markers of hepatotoxicity also remained normal value even after i.v. administration of PEG/PPD-MEND. Collectively, PEG/PPD-MEND should be a valuable siRNA delivery system for in vivo tumor and siRNA therapeutics. [1] Hatakeyama, H., et al. (2007). Development of a novel systemic gene delivery system for cancer therapy with a tumor-specific cleavable PEG-lipid. Gene Ther. 14: 68-77. S280
748. MicroRNA-200a Functions as a Tumor Suppressor in Meningiomas
Okay Saydam,1 Yiping Shen,2 Thomas Wurdinger,1 Elvan Boke,3 Bakhos A. Tannous,1 Anat O. Stemmer-Rachamimov,4 James F. Gusella,2 Ming Yi,5 Robert M. Stephens,5 Cornel Fraefel,6 Anna M. Krichevsky,7 Xandra O. Breakefield.1 1 Department of Neurology and Radiology, Molecular Neurogenetic Unit, Massachusetts General Hospital, Harvard Medical School, Boston, MA; 2Department of Neurology, Molecular Neurogenetic Unit, Center for Human Genetic Research, Massachusetts General Hospital, Harvard Medical School, Boston, MA; 3Middle East Technical University, Department of Molecular Biology, Ankara, Turkey; 4Neuro-Oncology Laboratory and Department of Pathology, Massachusetts General Hospital, Harvard Medical School, Boston, MA; 5Advanced Biomedical Computing Center, National Cancer Institute, Bethesda; 6Institute of Virology, University of Zurich, Zurich, Switzerland; 7Department of Neurology, Brigham and Women’s Hospital, Harvard Medical School, Boston. A number of miRNAs are dysregulated in cancer and they can exert critical roles in initiation and progression of various tumors. Meningiomas are intracranial and intraspinal tumors derived from arachnoidal cells associated with the meninges. Although there has been considerable effort to reveal molecular events involved in the development of meningiomas there is no report to date, to our knowledge, on the miRNA signatures of meningiomas. In this study, we carried out high-throughput miRNA expression profiling of human meningiomas using an array which can detect 407 known miRNAs. Twenty miRNAs were found to be significantly dysregulated in benign meningioma tumor samples (both deleted and non-deleted for the merlin locus on chromosome 22) compared with control arachnoidal tissues. Seventeen of these were up-regulated, whereas three were down-regulated. Among these, mir-335, mir-98, and mir181a were overexpressed at highest levels, with mir-200a, mir-373*, and mir-575 down-regulated to the greatest extent, all having greater than 10-fold differences in levels in meningioma samples as compared to arachnoidal tissues. We further investigated the functional consequences of mir-200a down-regulation in meningiomas and showed that overexpression of mir-200a inhibited meningioma cell growth both in culture and in a xenograft tumor model in vivo. Moreover, in these and a larger set of meningioma samples the chromosomal region 1p36 encoding mir-200a was deleted, and this may contribute to mir-200a down-regulation. These findings support a tumor suppressor function for mir-200a in meningiomas, indicating a novel miRNA mediated mechanism underlying meningioma development.
749. Growth Factor-Mediated Induction of Angiomir-1 Regulates Growth Factor Receptor Overexpression on Angiogenic Endothelial Cells
Thomas Wurdinger,1,2,6 Bakhos A. Tannous,1,2 Okay Saydam,1 Johan Skog,1 Stephan Grau,3 Ralph Weissleder,2,4 Xandra O. Breakefield,1,2 Anna M. Krichevsky.5 1 Neurology and Radiology, Massachusetts General Hospital, Harvard Medical School, Boston, MA; 2CMIR, Massachusetts General Hospital, Harvard Medical School, Boston; 3 Neurosurgery, Ludwig-Maximilians-University Munich, Munich, Germany; 4Center for Systems Biology, Massachusetts General Hospital, Harvard Medical School, Boston; 5Neurology, Brigham and Women’s Hospital, Harvard Medical School, Boston; 6 Neuro-Oncology Research Group, Cancer Center Amsterdam, Amsterdam, Netherlands. The formation of new blood vessels by angiogenesis is essential for normal functions and involved in many disease states. Control Molecular Therapy Volume 16, Supplement 1, May 2008 Copyright © The American Society of Gene Therapy
Hiroto Hatakeyama, Hidetaka Akita, Erika Itoh, Motoi Oishi,2,3 Yukio Nagasaki,2,3 Hideyoshi Harashima.1,2 1 Graduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo, Hokkaido, Japan; 2CREST, Japan Science and Technology, Kawaguchi, Saitama, Japan; 3Tsukuba Research Center of Interdisciplinary Material Science (TIMS), University of Tsukuba, Tsukuba, Ibaraki, Japan. 1,2
1,2
1,2
For development of siRNA therapeutics, systemic delivery of siRNA into specific tissue is major obstacle. Modification of nonviral vehicles with polyethylene glycol (PEG) is useful strategy for delivering the vehicles into tumor tissue via enhanced permeability and retention (EPR) effect. However, the PEGylation affects cellular uptake and following intracellular trafficking, which resulted in significant low level of RNAi activity. To solve these problems such PEG dilemma, we designed cleavable PEG-lipid system (abbrev. as PPD) in response to Matrix Metalloproteinase (MMP) in extracellular space in the tumor tissue(Figure1). Previously, we reported development of our novel non-viral vector, named a multifunctional envelope-type nano device (MEND) modified with PPD (PPD-MEND) as a systemic delivery system of plasmid DNA for tumor[1]. PPD-MEND exhibited the successfully enhancement of transfection activity in tumor tissue after i.v. administration compared with conventional PEG-modified MEND (PEG-MEND).
In this study, we investigated the utility of PPD-MEND as siRNA delivery system. To maximize a systemic stability of MEND, we optimized the modification density of PEG and PPD on the surface of MEND. The MEND modified with PEG and PPD at optimum ratio (PEG/PPD-MEND) is 100 nm in diameter possessing neutral zeta potential, which are desirable physiological characteristics for long systemic circulation and efficient accumulation in tumor via EPR effect. For RNAi study, HT1080 cells expressing luciferase were subcutaneously inoculated into nude mice. PEG/PPD-MEND was able to knockdown more than 70% of luciferase activity in tumor tissue after i.v. administration at a dose of 80 mg of siRNA. In histological observation, most of PEG/PPD-MEND labeled with rhodamine colocalized with tumor cells, which were immunostained by Alexa488-labeled antibody against EGF receptors as a tumor marker. Furthermore, in cytokine study, PEG/PPD-MEND showed no induction of IL-6 and TNF-α. Serum levels of ALT and AST as markers of hepatotoxicity also remained normal value even after i.v. administration of PEG/PPD-MEND. Collectively, PEG/PPD-MEND should be a valuable siRNA delivery system for in vivo tumor and siRNA therapeutics. [1] Hatakeyama, H., et al. (2007). Development of a novel systemic gene delivery system for cancer therapy with a tumor-specific cleavable PEG-lipid. Gene Ther. 14: 68-77. S280
748. MicroRNA-200a Functions as a Tumor Suppressor in Meningiomas
Okay Saydam,1 Yiping Shen,2 Thomas Wurdinger,1 Elvan Boke,3 Bakhos A. Tannous,1 Anat O. Stemmer-Rachamimov,4 James F. Gusella,2 Ming Yi,5 Robert M. Stephens,5 Cornel Fraefel,6 Anna M. Krichevsky,7 Xandra O. Breakefield.1 1 Department of Neurology and Radiology, Molecular Neurogenetic Unit, Massachusetts General Hospital, Harvard Medical School, Boston, MA; 2Department of Neurology, Molecular Neurogenetic Unit, Center for Human Genetic Research, Massachusetts General Hospital, Harvard Medical School, Boston, MA; 3Middle East Technical University, Department of Molecular Biology, Ankara, Turkey; 4Neuro-Oncology Laboratory and Department of Pathology, Massachusetts General Hospital, Harvard Medical School, Boston, MA; 5Advanced Biomedical Computing Center, National Cancer Institute, Bethesda; 6Institute of Virology, University of Zurich, Zurich, Switzerland; 7Department of Neurology, Brigham and Women’s Hospital, Harvard Medical School, Boston. A number of miRNAs are dysregulated in cancer and they can exert critical roles in initiation and progression of various tumors. Meningiomas are intracranial and intraspinal tumors derived from arachnoidal cells associated with the meninges. Although there has been considerable effort to reveal molecular events involved in the development of meningiomas there is no report to date, to our knowledge, on the miRNA signatures of meningiomas. In this study, we carried out high-throughput miRNA expression profiling of human meningiomas using an array which can detect 407 known miRNAs. Twenty miRNAs were found to be significantly dysregulated in benign meningioma tumor samples (both deleted and non-deleted for the merlin locus on chromosome 22) compared with control arachnoidal tissues. Seventeen of these were up-regulated, whereas three were down-regulated. Among these, mir-335, mir-98, and mir181a were overexpressed at highest levels, with mir-200a, mir-373*, and mir-575 down-regulated to the greatest extent, all having greater than 10-fold differences in levels in meningioma samples as compared to arachnoidal tissues. We further investigated the functional consequences of mir-200a down-regulation in meningiomas and showed that overexpression of mir-200a inhibited meningioma cell growth both in culture and in a xenograft tumor model in vivo. Moreover, in these and a larger set of meningioma samples the chromosomal region 1p36 encoding mir-200a was deleted, and this may contribute to mir-200a down-regulation. These findings support a tumor suppressor function for mir-200a in meningiomas, indicating a novel miRNA mediated mechanism underlying meningioma development.
749. Growth Factor-Mediated Induction of Angiomir-1 Regulates Growth Factor Receptor Overexpression on Angiogenic Endothelial Cells
Thomas Wurdinger,1,2,6 Bakhos A. Tannous,1,2 Okay Saydam,1 Johan Skog,1 Stephan Grau,3 Ralph Weissleder,2,4 Xandra O. Breakefield,1,2 Anna M. Krichevsky.5 1 Neurology and Radiology, Massachusetts General Hospital, Harvard Medical School, Boston, MA; 2CMIR, Massachusetts General Hospital, Harvard Medical School, Boston; 3 Neurosurgery, Ludwig-Maximilians-University Munich, Munich, Germany; 4Center for Systems Biology, Massachusetts General Hospital, Harvard Medical School, Boston; 5Neurology, Brigham and Women’s Hospital, Harvard Medical School, Boston; 6 Neuro-Oncology Research Group, Cancer Center Amsterdam, Amsterdam, Netherlands. The formation of new blood vessels by angiogenesis is essential for normal functions and involved in many disease states. Control Molecular Therapy Volume 16, Supplement 1, May 2008 Copyright © The American Society of Gene Therapy