- Email: [email protected]
958. Sleeping Beauty-Mediated Gene Therapy for Colorectal Cancer
CANCER-TARGETED GENE THERAPY: OTHER VIRUSES AND NEW APPROACHES 956. Imaging of Radiation-Inducible Promoters Using a Naturally Secreted Luciferase from the Marine Copepod Gaussia princeps Christian E. Badr,1,2,4 Pierre Zalloua,4 Xandra O. Breakefield,1,2,3 Bakhos A. Tannous.1,2,3 1 Molecular Neurogenetics Uit, Massachusetts General Hospital, Boston, MA; 2Neuroscience Program, Harvard Medical School, Boston, MA; 3Center for Molecular Imaging Research, Massachusetts General Hospital, Boston, MA; 4Cellular and Molecular Biology Unit, American University of Beirut, Beirut, Lebanon. Ionizing Radiation (IR)-inducible promoters used as genetic switches for cancer therapy are promising tool for controlling therapeutic gene expression. Here we describe the use of a naturally secreted luciferase from the marine copepod Gaussia princeps (Gluc) for monitoring the activity and induction levels of different IRsensitive promoters. The following promoters were tested: wildtype p53-activated fragment 1 (WAF1, 2.4 kb); early growth response factor (Egr-1, 550 bp); four tandem repeats of the transcriptional factor nuclear factor-kB (4NF-kB, 400 bp); and a combination of both Egr-1 and 4NF-kB promoters. These promoters were cloned upstream of the humanized Gluc cDNA in a plasmid containing the eGFP gene under the control of an immediate-early herpes simplex virus type-1 promoter. Vero2-2 (African green monkey kidney cells) were transfected with each of these constructs and irradiated at different doses of g-rays. Activity of these promoters and their response to radiation was monitored overtime by taking an aliquot of the cell-free conditioned medium, adding coelenterazine, the Gluc substrate, and measuring bioluminescence. At-least twofold induction was detected with each promoter as observed by bioluminescence imaging of Gluc. This system can be used to monitor, in real-time, the activity as well as the effectiveness of radiationinducible promoters to drive the expression of toxic genes for cancer therapy.
957. Effect of Ligand Binding Affinity and Receptor Density in Relation to Biology of Retargeted Viruses Kosei Hasegawa,1 Takafumi Nakamura,1 James D. Marks,2 Stephen J. Russell, Kah-Whye Peng. 1 Molecular Medicine Program, Mayo Clinic College of Medicine, Rochester, MN; 2Department of Anesthesia, University of California San Francisco, San Francisco, CA. The measles virus display platform technology is highly flexible and can accommodate insertions of single-chain antibodies (scFvs) into the viral attachment protein without significant compromise to virus infectivity and cytopathic effects of cell fusion. Using the measles display platform, we investigated the role of ligand affinity and receptor density in the biology of retargeted viruses and sought to evaluate the role of these parameters in modulating efficiency of virus-cell fusion and cell-to-cell fusion. Fully retargeted measles viruses that interact exclusively with the Her-2/neu (Her-2) receptor were used. A panel of 6 viruses displaying scFvs with affinities for Her-2 in the range of 10-6 to 10-11 M was generated. All viruses were rescued and infected cells via the Her-2 receptor with different efficiencies and in a manner that is dependent on receptor density on the target cells. Syncytia were undetectable with the low affinity (10-6 and 10-7 M) scFvs but became apparent above the threshold of 10-8 M. Viruses displaying the high affinity scFvs can overcome the limitation imposed by low receptor density and were significantly more efficient in inducing intercellular fusion in MDA-MB-231 cells that express only 3000 copies of Her-2/cell. While these 6 viruses produced equivalent cell associated virions, release of viral Molecular Therapy Volume 13, Supplement 1, May 2006 Copyright The American Society of Gene Therapy
progeny from the infected cells was progressively poorer with increase in scFv affinities, indicating that there is an optimal affinity for target receptors. Potential differences in kinetics of virus entry and cell fusion are currently being evaluated in conjunction with in vivo targeting studies. Results in this work have important implications for the selection of the target tumor antigen and the design of future generations of retargeted measles viruses.
958. Sleeping Beauty-Mediated Gene Therapy for Colorectal Cancer Lalitha R. Belur,1 Tavanna Buske,1 Brent Sorensen,2 Kelly Podetz-Peterson,1 Daniel Saltzman,2 Perry B. Hackett,1 R. Scott McIvor.1 1 Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, MN; 2Pediatrics, University of Minnesota, Minneapolis, MN. The Sleeping Beauty (SB) transposon system combines the advantages of non-viral plasmid based gene transfer with an integrative ability, thus leading to long term expression of the transgene. Our goal is to use the SB transposon system to deliver antiangiogenic and immunostimulatory genes, and to test their antitumor effectiveness in an animal model of colorectal cancer metastases to the liver. We have assembled a series of reporter transposons that have been engineered to contain transcriptional regulatory elements known to be upregulated for expression in the tumor cell environment. These elements include the CEA promoter, COX-2 promoter and the survivin promoter. These constructs were tested for tumor cell specific expression by transfection into a variety of cell types, including mouse CT26 colon carcinoma cells, mouse MRC5 fibroblast cells, and human 293T embryonic kidney cells. A control plasmid with luciferase under transcriptional regulation of the ubiquitous hybrid CAGS promoter, known to provide a high level of gene expression in the liver and other tissues, was included as a positive control. In transient transfection analyses, we found that the CEA and COX-2 promoters were transcriptionally active in the fibroblast (MRC5) cell line, even though the level of luciferase expression was lower than in the tumor cell line. In contrast, luciferase expression transcriptionally regulated by the survivin promoter was at background levels in the normal MRC5 cell line, but significantly elevated in the CT26 tumor cell line. The survivin promoter is highly active during embryonic and fetal development and consequently, we found very high luciferase expression in 293T cells (human embryonic kidney cell line). From these initial studies, it appears that survivin is the most optimal tumor specific promoter, with basal expression in normal cells in combination with a high level of expression in the CT26 tumor cell line. Therapeutic trangenes under transcriptional regulation of the survivin promoter will be assembled and tested for expression and growth suppression of tumor cells both in vitro and in vivo. We have created a colorectal tumor cell line that stably expresses luciferase (CT26L), thus facilitating the monitoring of tumor development and growth over time, by in vivo imaging of luciferase expression. The CT26/luciferase cell line was injected intrasplenically into Balb/c mice, followed by splenectomy. Luciferase expression was determined at 7, 12 and 16 days post tumor injection using the Xenogen in vivo imaging system, to ascertain tumor growth. In animals injected with 1 x 106 cells, tumors were clearly detectable by in vivo imaging at day 7, with the luciferase bioluminescent signal increasing steadily over time. Mice were sacrificed at day 16 and livers were examined in order to correlate bioluminescent signal with tumor size. Animals similarly bearing CT26L tumors will be co-injected with therapeutic transposons along with a source of SB transposase to evaluate their effect on the emergence and regression of liver metastases, work that is currently in progress. S369
957. Effect of Ligand Binding Affinity and Receptor Density in Relation to Biology of Retargeted Viruses Kosei Hasegawa,1 Takafumi Nakamura,1 James D. Marks,2 Stephen J. Russell, Kah-Whye Peng. 1 Molecular Medicine Program, Mayo Clinic College of Medicine, Rochester, MN; 2Department of Anesthesia, University of California San Francisco, San Francisco, CA. The measles virus display platform technology is highly flexible and can accommodate insertions of single-chain antibodies (scFvs) into the viral attachment protein without significant compromise to virus infectivity and cytopathic effects of cell fusion. Using the measles display platform, we investigated the role of ligand affinity and receptor density in the biology of retargeted viruses and sought to evaluate the role of these parameters in modulating efficiency of virus-cell fusion and cell-to-cell fusion. Fully retargeted measles viruses that interact exclusively with the Her-2/neu (Her-2) receptor were used. A panel of 6 viruses displaying scFvs with affinities for Her-2 in the range of 10-6 to 10-11 M was generated. All viruses were rescued and infected cells via the Her-2 receptor with different efficiencies and in a manner that is dependent on receptor density on the target cells. Syncytia were undetectable with the low affinity (10-6 and 10-7 M) scFvs but became apparent above the threshold of 10-8 M. Viruses displaying the high affinity scFvs can overcome the limitation imposed by low receptor density and were significantly more efficient in inducing intercellular fusion in MDA-MB-231 cells that express only 3000 copies of Her-2/cell. While these 6 viruses produced equivalent cell associated virions, release of viral Molecular Therapy Volume 13, Supplement 1, May 2006 Copyright The American Society of Gene Therapy
progeny from the infected cells was progressively poorer with increase in scFv affinities, indicating that there is an optimal affinity for target receptors. Potential differences in kinetics of virus entry and cell fusion are currently being evaluated in conjunction with in vivo targeting studies. Results in this work have important implications for the selection of the target tumor antigen and the design of future generations of retargeted measles viruses.
958. Sleeping Beauty-Mediated Gene Therapy for Colorectal Cancer Lalitha R. Belur,1 Tavanna Buske,1 Brent Sorensen,2 Kelly Podetz-Peterson,1 Daniel Saltzman,2 Perry B. Hackett,1 R. Scott McIvor.1 1 Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, MN; 2Pediatrics, University of Minnesota, Minneapolis, MN. The Sleeping Beauty (SB) transposon system combines the advantages of non-viral plasmid based gene transfer with an integrative ability, thus leading to long term expression of the transgene. Our goal is to use the SB transposon system to deliver antiangiogenic and immunostimulatory genes, and to test their antitumor effectiveness in an animal model of colorectal cancer metastases to the liver. We have assembled a series of reporter transposons that have been engineered to contain transcriptional regulatory elements known to be upregulated for expression in the tumor cell environment. These elements include the CEA promoter, COX-2 promoter and the survivin promoter. These constructs were tested for tumor cell specific expression by transfection into a variety of cell types, including mouse CT26 colon carcinoma cells, mouse MRC5 fibroblast cells, and human 293T embryonic kidney cells. A control plasmid with luciferase under transcriptional regulation of the ubiquitous hybrid CAGS promoter, known to provide a high level of gene expression in the liver and other tissues, was included as a positive control. In transient transfection analyses, we found that the CEA and COX-2 promoters were transcriptionally active in the fibroblast (MRC5) cell line, even though the level of luciferase expression was lower than in the tumor cell line. In contrast, luciferase expression transcriptionally regulated by the survivin promoter was at background levels in the normal MRC5 cell line, but significantly elevated in the CT26 tumor cell line. The survivin promoter is highly active during embryonic and fetal development and consequently, we found very high luciferase expression in 293T cells (human embryonic kidney cell line). From these initial studies, it appears that survivin is the most optimal tumor specific promoter, with basal expression in normal cells in combination with a high level of expression in the CT26 tumor cell line. Therapeutic trangenes under transcriptional regulation of the survivin promoter will be assembled and tested for expression and growth suppression of tumor cells both in vitro and in vivo. We have created a colorectal tumor cell line that stably expresses luciferase (CT26L), thus facilitating the monitoring of tumor development and growth over time, by in vivo imaging of luciferase expression. The CT26/luciferase cell line was injected intrasplenically into Balb/c mice, followed by splenectomy. Luciferase expression was determined at 7, 12 and 16 days post tumor injection using the Xenogen in vivo imaging system, to ascertain tumor growth. In animals injected with 1 x 106 cells, tumors were clearly detectable by in vivo imaging at day 7, with the luciferase bioluminescent signal increasing steadily over time. Mice were sacrificed at day 16 and livers were examined in order to correlate bioluminescent signal with tumor size. Animals similarly bearing CT26L tumors will be co-injected with therapeutic transposons along with a source of SB transposase to evaluate their effect on the emergence and regression of liver metastases, work that is currently in progress. S369