- Email: [email protected]
790. Monitoring of Transgene Expression In Vivo Via Exosomes Shed from Transduced Cells into the Bloodstream
OTHER DNA VIRUSES (n=5). One mouse per group was euthanized and necropsied on days 3 and 8 after virus administration while the remaining mice were followed for 90 days. Mice were regularly bled and examined for the presence of MV-NIS viremia. No clinical symptoms or pathological changes at autopsy were observed. Immunological analysis showed antimeasles antibodies peaking by day 54 after virus treatment. CONCLUSIONS: MV-NIS has significant in vitro oncolytic activity against both androgen-sensitive and androgen-insensitive prostate cancer cell lines and allows in vivo viral localization by non-invasive imaging. Intraprostatic MV-NIS administration was nontoxic in measles replication susceptible transgenic mice pointing to the safety of the approach. The therapeutic effect of IT MV-NIS treatment can be further enhanced by I-131 administration. Experiments to assess the potential added benefit of combined radiovirotherapy following IV injection of MV-NIS are currently underway. This study was supported by the Prostate SPORE grant (P50 CA 091956).
790. Monitoring of Transgene Expression In Vivo Via Exosomes Shed from Transduced Cells into the Bloodstream
Johan Skog,1 Ryan Lessard,1 Leonora Balaj,1 Xandra O. Breakefield.1 1 Neurology, Massachusetts General Hospital, Charlestown, MA. Glioblastomas, as well as other tumors and normal cells release exosomes (microvesicles) containing mRNA, miRNA and proteins. These exosomes can be taken up by normal host cells in the vicinity, such as microvascular endothelial cells in the case of tumors, but are also released into the blood. These membrane-encapsulated portions of the tumor cell transcriptome can serve as a source of biomarkers to monitor endogenous mutational profiles and gene expression profiles of solid tumors through a blood sample. These microvesicles can also function as endogenous vectors generated by tumor cells to modify the genetic content of normal cells in their environment. We were able to show that tumor cells transduced with an expression cassette encoding an exogenous mRNA for a reporter protein was incorporated into these microvesicles and that the microvesicle-delivered message was translated in recipient endothelial cells. Another potential use is to monitor transgene expression following vector delivery in vivo in a non-invasive way. mRNAs expressed from the vector are incorporated into the exosomes and released into the circulation where the levels can be quantified through a blood sample. A sequence-tag introduced into the 3-prime UTR will be specifically detected using qRT-PCR, eliminating the need for potentially immunogenic reporter proteins. The protective membrane of the exosomes serves to prevent immune recognition or degradation of encoded proteins and mRNAs, respectively, thus allowing assessment of the level of expression of the transgene in tumor/tissues in vivo over time non-invasively.
Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy
Other DNA Viruses 791. Vasculostatin Expression Mediated by Oncolytic HSV Inhibits Tumor Growth and Angiogenesis
Jayson J. Hardcastle,1,2,5 Kazuhiko Kurozumi,1,5,7 Nina Dmitrieva,1,5 Martin P. Sayers,1,3,5 Sarwat Ahmad,4,5 Peter Waterman,6 Ralph Weissleder,6 E. A. Chiocca,1,5 Balveen Kaur.1,5 1 Dardinger Laboratory for Neuro-Oncology and Neurosciences, The Ohio State University, Columbus, OH; 2Integrated Biomedical Sciences Graduate Program, The Ohio State University, Columbus, OH; 3Undergraduate Major in Biomedical Science, The Ohio State University, Columbus, OH; 4College of Medicine, The Ohio State University, Columbus, OH; 5Department of Neurological Surgery, James Comprehensive Cancer Center and The Ohio State University Medical Center, Columbus, OH; 6Center for Molecular Imaging Research, Massachusetts General Hospital, Charlestown, MA; 7Department of Neurological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan. Brain angiogenesis inhibitor 1 (BAI1), is a seven transmembrane G protein coupled receptor expressed primarily in normal brain. We have previously shown that the extracellular fragment of BAI1 is proteolytically processed at a conserved G protein coupled receptor proteolytic cleavage site (GPS), resulting in the release of secreted Vasculostatin (Vstat120). Stable expression of Vstat120 in multiple glioma cell lines results in a poorly vascularized tumors with slower subcutaneous and intracranial tumor growth. However, the therapeutic efficacy of Vstat120 delivery into established tumors has not been studied. In order to test this we created a dually armed oncolytic HSV-1 (OV) which can destroy tumors by cancer cell specific replication and lysis and also expresses Vsta120. Glioma cells infected with RAMBO (Rapid Anti-angiogensis Mediated By Oncolytic virus), expressed secreted Vstat120 as early as four hours post infection. Vstat120 did not interfere with RAMBO’s ability to replicate and lyse in multiple glioma cell lines. Treatment of cultured microvascular endothelial cells with conditioned medium derived from glioma cells infected with RAMBO reduced endothelial cell migration and tube formation (P=0.0005 and P=0.0184 respectively) in vitro. Treatment of viral infected glioma matrigel angioreactors also showed a significant decrease in angiogeneisis (P=0.007) in vivo compared to control OV (HSVQ) treated angioreactors. Mice bearing intracranial gliomas treated with RAMBO demonstrated a doubling in the median survival compared to the control HSVQ treated mice (26 days and 56 days, respectively, P=0.0021, n=5/group). Treatment of mice bearing subcutaneous glioma with RAMBO also resulted in a doubling of median survival with 50% of the animals showing a complete response (P<0.05). A statistically significant reduction in tumor perfusion (FMT analysis) and microvessel density (CD31 staining) was observed in glioma tumors treated with RAMBO. This is the first study describing the therapeutic efficacy of Vsta120 gene delivery in established tumors.
S303
790. Monitoring of Transgene Expression In Vivo Via Exosomes Shed from Transduced Cells into the Bloodstream
Johan Skog,1 Ryan Lessard,1 Leonora Balaj,1 Xandra O. Breakefield.1 1 Neurology, Massachusetts General Hospital, Charlestown, MA. Glioblastomas, as well as other tumors and normal cells release exosomes (microvesicles) containing mRNA, miRNA and proteins. These exosomes can be taken up by normal host cells in the vicinity, such as microvascular endothelial cells in the case of tumors, but are also released into the blood. These membrane-encapsulated portions of the tumor cell transcriptome can serve as a source of biomarkers to monitor endogenous mutational profiles and gene expression profiles of solid tumors through a blood sample. These microvesicles can also function as endogenous vectors generated by tumor cells to modify the genetic content of normal cells in their environment. We were able to show that tumor cells transduced with an expression cassette encoding an exogenous mRNA for a reporter protein was incorporated into these microvesicles and that the microvesicle-delivered message was translated in recipient endothelial cells. Another potential use is to monitor transgene expression following vector delivery in vivo in a non-invasive way. mRNAs expressed from the vector are incorporated into the exosomes and released into the circulation where the levels can be quantified through a blood sample. A sequence-tag introduced into the 3-prime UTR will be specifically detected using qRT-PCR, eliminating the need for potentially immunogenic reporter proteins. The protective membrane of the exosomes serves to prevent immune recognition or degradation of encoded proteins and mRNAs, respectively, thus allowing assessment of the level of expression of the transgene in tumor/tissues in vivo over time non-invasively.
Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy
Other DNA Viruses 791. Vasculostatin Expression Mediated by Oncolytic HSV Inhibits Tumor Growth and Angiogenesis
Jayson J. Hardcastle,1,2,5 Kazuhiko Kurozumi,1,5,7 Nina Dmitrieva,1,5 Martin P. Sayers,1,3,5 Sarwat Ahmad,4,5 Peter Waterman,6 Ralph Weissleder,6 E. A. Chiocca,1,5 Balveen Kaur.1,5 1 Dardinger Laboratory for Neuro-Oncology and Neurosciences, The Ohio State University, Columbus, OH; 2Integrated Biomedical Sciences Graduate Program, The Ohio State University, Columbus, OH; 3Undergraduate Major in Biomedical Science, The Ohio State University, Columbus, OH; 4College of Medicine, The Ohio State University, Columbus, OH; 5Department of Neurological Surgery, James Comprehensive Cancer Center and The Ohio State University Medical Center, Columbus, OH; 6Center for Molecular Imaging Research, Massachusetts General Hospital, Charlestown, MA; 7Department of Neurological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan. Brain angiogenesis inhibitor 1 (BAI1), is a seven transmembrane G protein coupled receptor expressed primarily in normal brain. We have previously shown that the extracellular fragment of BAI1 is proteolytically processed at a conserved G protein coupled receptor proteolytic cleavage site (GPS), resulting in the release of secreted Vasculostatin (Vstat120). Stable expression of Vstat120 in multiple glioma cell lines results in a poorly vascularized tumors with slower subcutaneous and intracranial tumor growth. However, the therapeutic efficacy of Vstat120 delivery into established tumors has not been studied. In order to test this we created a dually armed oncolytic HSV-1 (OV) which can destroy tumors by cancer cell specific replication and lysis and also expresses Vsta120. Glioma cells infected with RAMBO (Rapid Anti-angiogensis Mediated By Oncolytic virus), expressed secreted Vstat120 as early as four hours post infection. Vstat120 did not interfere with RAMBO’s ability to replicate and lyse in multiple glioma cell lines. Treatment of cultured microvascular endothelial cells with conditioned medium derived from glioma cells infected with RAMBO reduced endothelial cell migration and tube formation (P=0.0005 and P=0.0184 respectively) in vitro. Treatment of viral infected glioma matrigel angioreactors also showed a significant decrease in angiogeneisis (P=0.007) in vivo compared to control OV (HSVQ) treated angioreactors. Mice bearing intracranial gliomas treated with RAMBO demonstrated a doubling in the median survival compared to the control HSVQ treated mice (26 days and 56 days, respectively, P=0.0021, n=5/group). Treatment of mice bearing subcutaneous glioma with RAMBO also resulted in a doubling of median survival with 50% of the animals showing a complete response (P<0.05). A statistically significant reduction in tumor perfusion (FMT analysis) and microvessel density (CD31 staining) was observed in glioma tumors treated with RAMBO. This is the first study describing the therapeutic efficacy of Vsta120 gene delivery in established tumors.
S303