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Adenoviral-Mediated Neuronal-Specific Overexpression of the Angiotensin AT2 Receptor
CANCER TUMOR SUPPRESSOR GENE AND APOPTOSIS 939. Adenoviral-Mediated Neuronal-Specific Overexpression of the Angiotensin AT2 Receptor Hongwei Li,1 Yongxin Gao,1 Mohan K. Raizada,1 Colin Sumners.1 1 Department of Physiology and Functional Genomics and McKnight Brain Institute, University of Florida, Gainesville, FL. The angiotensin II type 2 receptor (AT2) is a G-protein coupled receptor that can influence a variety of intracellular signaling molecules and cellular functions. The AT2 is highly expressed in neonatal brain and regulates several functions of nerve cells, e.g., ionic fluxes, cell differentiation, axonal regeneration and programmed cell death. However, the physiological roles of the AT2 receptor remain poorly defined. Adenoviral vectors are amongst the most efficient vectors for gene transfer, and have been used to transduce both neurons and non-neuronal cells (astroglia, oligodendroglia, microglia, and endothelial cells) in vitro and in vivo. However, glia are transduced with higher efficiency than neurons. This means that in primary neuronal cultures (which usually contain a small percentage of glia) and in brain in vivo the majority of transgene is expressed in the non-neuronal cells. Therefore, in order to study the functions of AT2 in neurons, and target AT2 expression solely to these cells, we developed a recombinant adenoviral vector Ad5-SYN-AT2-IRESGFP containing both the AT2 gene and an IRES-linked GFP reporter gene, driven by the synapsin 1 (SYN) gene promoter. Primary cortical neuronal cultures, which contain low or undetectable levels of AT2, were transfected with the recombinant Ad5-SYN-AT2-IRES-GFP. Immunocytochemistry, 125SI-AngII receptor binding and real-time RT-PCR were used to identify the expression of AT2 or GFP. The results showed that the high level expression of both AT2 and GFP was restricted exclusively to neurons and that no AT2 overexpression was detected in glial cells. Thus, this vector provides a highly effective means of directing AT2-specific expression in neurons, and as such is an important tool for investigations of its function in the brain and in gene therapy studies involving AT2 receptors in neurons.
β 940. Ribozyme Oligonucleotides Against TGF-β Transferred by Ultrasound Inhibited the Progression of Diseases in Anti-Thy1 Model Naruya Tomita,1 Keita Yamasaki,1 Kei Yamamoto,1 Hiromi Koike,1 Yasuo Kunugiza,1 Toshio Ogihara,2 Ryuichi Morishita.1 1 Division of Clinical Gene Therapy, Osaka University Graduate School of Medicine, Suita, Japan; 2Geriatric Medicine, Osaka University Graduate School of Medicine, Suita, Japan. Antisense as well as ribozyme oligonucleotides (ODN) are often used to inhibit the specific gene espression by targeting to the transcriptional process resulting in the combat against disease processes. On the other hand, TGF-β has been postulated to play an important role in the pathophysiology of renal diseases. In this study, we first examined the inhibitory effects of ribozyme ODN against TGF-β in mesangial cells (MC). Transfer of TGF-β ribozyme ODN into human and rat MC by cationic liposomes significantly decreased mRNA expression and protein production of TGF-β induced by Ang II as compared to the control (Protein production; TGF-β ribozyme: 116 ± 14.2 pg/ml, Control ribozyme: 162 ± 16.7 pg/ml, P<0.01). The examination of FITC-labeled ribozyme ODN transfer by cationic liposomes in MC revealed that ribozyme ODN are mainly located in the cytoplasm. Then, we examined the feasibility of ultrasound method that we established recently to transfer ribozyme ODN into the kidney in vivo. As we expected, FITC-labeled ribozyme ODN was efficiently transferred in glomeruli, interstitial tissues and tubules with this method. Finally, the inhibitory effect of TGF-β ribozyme ODN transferred by ultrasound method in vivo on TGF-β expression and extracellular matrix accumulation in experimental glomerulonephritis, anti-Thy1 S360
model, was examined. TGF-β ribozyme ODN, not control ribozyme ODN, suppressed the TGF-β expression leading to the inhibition of extracellular matrix accumulation in glomeruli significantly. Overall, the present studies demonstrated that our constructed ribozyme ODN against TGF-β can effectively cleavage TGF-β mRNA in MC in vitro and glomeruli in vivo suggesting the feasibility of ribozyme-mediated therapy to treat renal diseases.
CANCER TUMOR SUPPRESSOR GENE AND APOPTOSIS 941. Development of Cell Penetration Domain and Cell Killing Domain Genes for Treatment of Cancer Sean M. Sullivan, Harm J. Knot, Ron Mandel, J. P. Jarajapu, Jacqueline Baltunis. 1 Pharmaceutics, University of Florida, Gainesville, FL; 2 Pharmacology, University of Florida, Gainesville, FL; 3 Neuorscience, University of Florida, Gainesville, FL. Fluorescein labeled peptides derived from HIV-Tat (Tat) and Drosophila antennaepedia homeodomain (ANT) were able to intracellularly label two human glioblastoma cell lines (A172 and T98G) and one rat glioma cell line (RG2). The Tat peptide localized to the cell nuclei whereas the ANT sequence was distributed evenly throughout the cytoplasm. A 14 amino acid sequence derived from P14, a cell cycle suppression protein, was added to the C-terminus of each peptide. Increasing the size of the peptide did not inhibit cell entry and did not change fluorescent staining patterns of cells. It did kill each cell line within 1 hr after adding 10 uM of peptide. Tat, ANT and Exon1 peptides by themselves had no effect on cell viability. A dose response curve yielded an IC50 concentration of 9 uM for the Tat-Exon1. Reversing the domain order reduced the IC50 to 4 uM. Uptake studies showed that addition of Exon1 sequence to the Tat sequence reduced the rate of uptake by half. Reversing the domain order, i.e., Exon-1-Tat, restored the rate of uptake to that observed by Tat alone. To gain a better understanding of the behavior of these peptides in a three dimensional cell system, cell penetration and killing were tested in rat cerebral artery organ cultures. These vessels are maintained under physiological pressure and blood flow. Intraluminal administration of the fluorescent peptides produced cell-staining patterns similar to that observed in the in vitro brain tumor cell lines, with cell staining restricted to the endothelium. Adventitial administration of the Tat and ANT peptides yielded smooth muscle cell labeling of the nucleus and cytoplasm, respectively. No labeling of the endothelium was observed. Addition of the cell-killing domain did not inhibit the ability of the peptides to penetrate into the endothelium; however, response to vasoconstriction was diminished where as smooth muscle cell response was not affected. The Tat-ExonI sequence has been cloned into an expression cassette with an upstream signal sequence and a down stream HA epitope tag. Western blots showed that all three tumor cell lines expressed the peptide. Transfection results further showed a direct correlate between transfection efficiency and cell death. Experiments are directed toward proving the bystander effect mediated by the cell penetration domain in an in vivo rat brain tumor model.
Molecular Therapy Volume 9, Supplement 1, May 2004
Copyright The American Society of Gene Therapy
β 940. Ribozyme Oligonucleotides Against TGF-β Transferred by Ultrasound Inhibited the Progression of Diseases in Anti-Thy1 Model Naruya Tomita,1 Keita Yamasaki,1 Kei Yamamoto,1 Hiromi Koike,1 Yasuo Kunugiza,1 Toshio Ogihara,2 Ryuichi Morishita.1 1 Division of Clinical Gene Therapy, Osaka University Graduate School of Medicine, Suita, Japan; 2Geriatric Medicine, Osaka University Graduate School of Medicine, Suita, Japan. Antisense as well as ribozyme oligonucleotides (ODN) are often used to inhibit the specific gene espression by targeting to the transcriptional process resulting in the combat against disease processes. On the other hand, TGF-β has been postulated to play an important role in the pathophysiology of renal diseases. In this study, we first examined the inhibitory effects of ribozyme ODN against TGF-β in mesangial cells (MC). Transfer of TGF-β ribozyme ODN into human and rat MC by cationic liposomes significantly decreased mRNA expression and protein production of TGF-β induced by Ang II as compared to the control (Protein production; TGF-β ribozyme: 116 ± 14.2 pg/ml, Control ribozyme: 162 ± 16.7 pg/ml, P<0.01). The examination of FITC-labeled ribozyme ODN transfer by cationic liposomes in MC revealed that ribozyme ODN are mainly located in the cytoplasm. Then, we examined the feasibility of ultrasound method that we established recently to transfer ribozyme ODN into the kidney in vivo. As we expected, FITC-labeled ribozyme ODN was efficiently transferred in glomeruli, interstitial tissues and tubules with this method. Finally, the inhibitory effect of TGF-β ribozyme ODN transferred by ultrasound method in vivo on TGF-β expression and extracellular matrix accumulation in experimental glomerulonephritis, anti-Thy1 S360
model, was examined. TGF-β ribozyme ODN, not control ribozyme ODN, suppressed the TGF-β expression leading to the inhibition of extracellular matrix accumulation in glomeruli significantly. Overall, the present studies demonstrated that our constructed ribozyme ODN against TGF-β can effectively cleavage TGF-β mRNA in MC in vitro and glomeruli in vivo suggesting the feasibility of ribozyme-mediated therapy to treat renal diseases.
CANCER TUMOR SUPPRESSOR GENE AND APOPTOSIS 941. Development of Cell Penetration Domain and Cell Killing Domain Genes for Treatment of Cancer Sean M. Sullivan, Harm J. Knot, Ron Mandel, J. P. Jarajapu, Jacqueline Baltunis. 1 Pharmaceutics, University of Florida, Gainesville, FL; 2 Pharmacology, University of Florida, Gainesville, FL; 3 Neuorscience, University of Florida, Gainesville, FL. Fluorescein labeled peptides derived from HIV-Tat (Tat) and Drosophila antennaepedia homeodomain (ANT) were able to intracellularly label two human glioblastoma cell lines (A172 and T98G) and one rat glioma cell line (RG2). The Tat peptide localized to the cell nuclei whereas the ANT sequence was distributed evenly throughout the cytoplasm. A 14 amino acid sequence derived from P14, a cell cycle suppression protein, was added to the C-terminus of each peptide. Increasing the size of the peptide did not inhibit cell entry and did not change fluorescent staining patterns of cells. It did kill each cell line within 1 hr after adding 10 uM of peptide. Tat, ANT and Exon1 peptides by themselves had no effect on cell viability. A dose response curve yielded an IC50 concentration of 9 uM for the Tat-Exon1. Reversing the domain order reduced the IC50 to 4 uM. Uptake studies showed that addition of Exon1 sequence to the Tat sequence reduced the rate of uptake by half. Reversing the domain order, i.e., Exon-1-Tat, restored the rate of uptake to that observed by Tat alone. To gain a better understanding of the behavior of these peptides in a three dimensional cell system, cell penetration and killing were tested in rat cerebral artery organ cultures. These vessels are maintained under physiological pressure and blood flow. Intraluminal administration of the fluorescent peptides produced cell-staining patterns similar to that observed in the in vitro brain tumor cell lines, with cell staining restricted to the endothelium. Adventitial administration of the Tat and ANT peptides yielded smooth muscle cell labeling of the nucleus and cytoplasm, respectively. No labeling of the endothelium was observed. Addition of the cell-killing domain did not inhibit the ability of the peptides to penetrate into the endothelium; however, response to vasoconstriction was diminished where as smooth muscle cell response was not affected. The Tat-ExonI sequence has been cloned into an expression cassette with an upstream signal sequence and a down stream HA epitope tag. Western blots showed that all three tumor cell lines expressed the peptide. Transfection results further showed a direct correlate between transfection efficiency and cell death. Experiments are directed toward proving the bystander effect mediated by the cell penetration domain in an in vivo rat brain tumor model.
Molecular Therapy Volume 9, Supplement 1, May 2004
Copyright The American Society of Gene Therapy