Two-Step Transcription Amplification Research Articles

Selective suppression of autocatalytic caspase-3 driven by two-step transcriptional amplified human telomerase reverse transcriptase promoter on ovarian carcinoma growth in vitro and in mice.

The objective of our study was to construct recombinant adenovirus (rAd) AdHTVP2G5-rev-casp3, which expresses autocatalytic caspase-3 driven by human telomerase reverse transcriptase promoter (hTERTp) with a two-step transcription amplification (TSTA) system and investigate its antitumor effects on ovarian cancer in vitro and in vivo. Fluorescent detection was used to detect EGFP expression in various cells. Cell viabilities were determined using the Cell Counting Kit-8 and flow cytometry. RT-PCR and immunoblotting assays were used to detect cellular apoptotic activities. Tumor growth and survival of tumor-bearing mice were studied. The hTERTp-TSTA system showed the strongest activity in hTERT-positive cancer cells when compared with hTERTp and cytomeglovirus promoter (CMVp). In contrast, it showed no activity in hTERT‑negative HUVECs. AdHTVP2G5‑rev-casp3 markedly suppressed the survival of AO cells in a dose-dependent modality with a viability rate of 17.8 ± 3.5% at an MOI of 70, which was significantly lower than that by AdHT-rev-casp3 and Ad-rev-casp3 (rAds which express rev-caspase-3 driven by hTERTp and CMVp, respectively). In contrast, AdHTVP2G5‑rev-casp3 induced little HUVEC death with a viability rate of 92.7 ± 5.2% at the same MOI. Additionally, AdHTVP2G5-rev-casp3 (MOI=70) caused significant apoptosis in AO cells with an apoptotic rate of 42%. The tumor growth suppression rate of AdHTVP2G5-rev-casp3 was 81.52%, significantly higher than that of AdHT-rev-casp3 (54.94%) or Ad-rev-casp3 (21.35%). AdHTVP2G5-rev-casp3 significantly improved the survival of tumor-bearing mice with little liver damage, with a mean survival of 258 ± 28 days. These results showed that AdHTVP2G5-rev-casp3 caused effective apoptosis with significant tumor selectivity, strongly suppressed tumor growth and improved mouse survival with little liver toxicity. It can be a potent therapeutic agent for tumor targeted treatment of ovarian cancer.

Enhanced Incretin Effects of Exendin-4 Expressing Chimeric Plasmid Based On Two-Step Transcription Amplification System with Dendritic Bioreducible Polymer for the Treatment of Type 2 Diabetes

Glucagon-like peptide 1 (GLP-1) agonist, exenxdin-4, is currently being advanced as a promising diabetes remedy via a variety of incretin actions similar with GLP-1. In this study, we investigated an effective anti-diabetic therapy via exendin-4 expressing chimeric plasmid based on two-step transcription amplification (TSTA) system with dendrimer-type bioreducible polymer for more improved incretin-based gene therapy. Arginine-grafted poly (cystaminebisacrylamide-diaminohexane) (ABP)-conjugated poly (amido amine) (PAMAM) dendrimer (PAM-ABP) was used as gene carrier. PAM-ABP/chimeric DNA polyplex was markedly elevated exendin-4 expression in ectopic cells as well as increased insulin production through an enhanced activation of protein kinase K (PKA) induced by up-regulation of exendin-4-stimulated cyclic adenosine monophosphate (cAMP) in pancreatic β-cell. Consistent with these results, intravenous administration of PAM-ABP/chimeric DNA polyplex improved glucoregulotory effects, as well as increased insulin secretion by high expression of exendin-4 in blood in type 2 diabetic mice with no any toxicity. Our exendin-4 system can be attributed to provide a potential diabetes therapeutic agent for improved incretin gene therapy.

Delivery of two-step transcription amplification exendin-4 plasmid system with arginine-grafted bioreducible polymer in type 2 diabetes animal model

Exendin-4, glucagon-like peptide 1 (GLP-1) receptor agonist, is an exocrine hormone, which has potent insulinotropic actions similar to GLP-1 such as stimulating insulin biosynthesis, facilitating glucose concentration dependent insulin secretion, slowing gastric emptying, reducing food intake and stimulating β-cell proliferation. Exendin-4, also, has a longer half-life than GLP-1, due to its resistance to degradation by dipeptidyl peptidase‐IV (DPP-IV). In spite of its many advantages as a therapeutic agent for diabetes, its clinical application is still restricted. Thus, to improve the activity of exendin-4 in vivo, gene therapy system was developed as an alternative method. An exendin-4 expression system was constructed using the two-step transcription amplification (TSTA) system, which is composed of pβ-Gal4-p65 and pUAS-SP-exendin-4 with combining the advantages of signal peptide (SP) in order to facilitate its secretion in ectopic cells or tissue. Arginine-grafted cyctaminebisacrylamide-diaminohexane polymer (ABP) was used as a gene carrier. Increased expression of exendin-4, glucose dependent insulin secretion in NIT-1 insulinoma cells, and high insulin expression in the presence of DPP-IV were evaluated in vitro after delivery of ABP/TSTA-SP-exendin-4. Blood glucose levels in diabetic mice were decreased dramatically from the third day for experimental period after single intravenous administration with ABP/TSTA-SP-exendin-4. The highest insulinotropic effect of exendin-4 was also observed in the ABP/TSTA/SP-exendin-4-treated mice groups, compared with the others groups from the 3rd day after injection. TSTA exendin-4 expression system with SP and ABP polymer has a potential gene therapy for the treatment of type 2 diabetes.

419. Effect of Exendin-4 Expressed by Two-Step Transcription Amplification Plasmid System with Arginine-Grafted Bioreducible Polymer for Type 2 Diabetes

419. Effect of Exendin-4 Expressed by Two-Step Transcription Amplification Plasmid System with Arginine-Grafted Bioreducible Polymer for Type 2 Diabetes

Efficient GLP-1 gene delivery using two-step transcription amplification plasmid system with a secretion signal peptide and arginine-grafted bioreducible polymer

Glucagon-like peptide (GLP-1) encoding dual plasmid (pDNA) system (TSTA (SP-GLP-1)) which is composed of pβ-Gal4-p65 and pUAS-SP-GLP-1 was constructed to improve the production and secretion of expressed GLP-1 by combining the advantages of signal peptide (SP) and two-step transcription amplification (TSTA) system. Its potential for GLP-1 gene delivery system was investigated with employment of arginine-grafted bioreducible polymer (ABP) as a gene carrier. Their polyplexes have about 140nm-sizes and 20mV Zeta-potential values. ABP showed no cytotoxicity contrary to PEI25k. It was found in RT-PCR experiments that TSTA-SP pDNA systems showed increased GLP-1 gene transcription level in comparison with mono pDNA system (pβ-GLP-1). It was also observed in GLP-1 ELISA that GLP-1 secretion level of TSTA (SP-GLP-1) pDNA system was 2.7–3.4 times higher than those of pβ-GLP-1 and 1.5–1.7 times than TSTA (GLP-1). Additionally, 2.5–3.5 folds increased level of GLP-1 secretion was found in ABP gene carrier system in comparison with PEI25k. When transfection medium containing secreted GLP-1 was transferred to NIT-1 insulinoma cells, the highest secretion level of insulin was induced in ABP/TSTA (SP-GLP-1) polyplex medium-treated cells. Therefore, this novel system could be utilized as a safe and efficient GLP-1 gene delivery system for type 2 diabetes therapy.

Longitudinal, Noninvasive Imaging of T-Cell Effector Function and Proliferation in Living Subjects

Adoptive immunotherapy is evolving to assume an increasing role in treating cancer. Most imaging studies in adoptive immunotherapy to date have focused primarily on locating tumor-specific T cells rather than understanding their effector functions. In this study, we report the development of a noninvasive imaging strategy to monitor T-cell activation in living subjects by linking a reporter gene to the Granzyme B promoter (pGB), whose transcriptional activity is known to increase during T-cell activation. Because pGB is relatively weak and does not lead to sufficient reporter gene expression for noninvasive imaging, we specifically employed 2 signal amplification strategies, namely the Two Step Transcription Amplification (TSTA) strategy and the cytomegalovirus enhancer (CMVe) strategy, to maximize firefly luciferase reporter gene expression. Although both amplification strategies were capable of increasing pGB activity in activated primary murine splenocytes, only the level of bioluminescence activity achieved with the CMVe strategy was adequate for noninvasive imaging in mice. Using T cells transduced with a reporter vector containing the hybrid pGB-CMVe promoter, we were able to optically image T-cell effector function longitudinally in response to tumor antigens in living mice. This methodology has the potential to accelerate the study of adoptive immunotherapy in preclinical cancer models.

240. Enhanced In Vivo Transgene Expression by Two-Step Transcription Amplification

240. Enhanced In Vivo Transgene Expression by Two-Step Transcription Amplification

193. Novel Adenoviral Vector Two-Step Transcription Amplification Expression System (TSTA) for Expressing CDK2-Associated Cell Cycle Regulators for Cancer Gene Therapy

193. Novel Adenoviral Vector Two-Step Transcription Amplification Expression System (TSTA) for Expressing CDK2-Associated Cell Cycle Regulators for Cancer Gene Therapy

An efficient GLP-1 expression system using two-step transcription amplification

Glucagon-like peptide 1 (GLP-1) is an insulinotropic protein. It was reported that the continuous infusion of GLP-1 normalized the blood glucose level in type 2 diabetes animal model. However, the short half-life of GLP-1 has limited its application in clinical settings and prompted us to develop a GLP-1 gene therapy system. Our previous results showed that the delivery of pβ-GLP-1 using polyethylenimine (PEI) reduced the blood glucose level effectively. However, the glucose level was not completely normalized. In the present study, the more efficient GLP-1 expression system was developed using two-step transcription amplification (TSTA). To evaluate the TSTA system, pβ-Gal4-p65 and pUAS-Luc were constructed. The pUAS-Luc/pβ-Gal4-p65 system showed the highest transfection efficiency at a 2:1 pUAS-Luc/pβ-Gal4-p65 weight ratio. In addition, the transgene expression by the TSTA system was at least 4 times higher than pβ-Luc. To apply the TSTA system to the GLP-1 expression plasmid, pUAS-GLP-1 was constructed. The pUAS-GLP-1/pβ-Gal4-p65 system showed higher mRNA level than pβ-GLP-1. In addition, the level of GLP-1 by the pUAS-GLP-1/pβ-Gal4-p65 system was more than 4 times higher than pβ-GLP-1. Therefore, the TSTA GLP-1 expression system may be useful to develop gene therapy system for type 2 diabetes.

638. The Optimal Placement and Orientation of the Bipartite Two-Step Transcription Amplification (TSTA) Expression System in Adenoviral Vectors for Prostate Cancer Gene Therapy

Top of pageAbstract A significant challenge for successful gene therapy is to achieve a high-level of specific gene expression. However, tissue-specific promoters are much weaker than strong viral promoters (CMV). We developed a two-step transcriptional activation (TSTA) system to amplify the prostate-specific PSA promoter to levels comparable or higher than CMV while maintaining prostate specificity. The TSTA system consists of two parts, one containing a modified prostate-specific promoter (PSE-BC) driving the expression of a chimeric activator (GAL4-VP16), and a target construct containing five GAL4-binding sites upstream of the gene of interest. Recombinant adenoviral vectors (Ad) harboring the bipartite prostate-specific TSTA expression cassettes in a head-to-head orientation (H-H TSTA) achieve activity exceeding that of CMV, and cell specificity and androgen responsiveness in pre-clinical models, as measured by noninvasive bioluminescence and microPET imaging. Our previous data also indicated that the configuration of the two TSTA components could impact its regulation. Hence, we modified the configuration of the TSTA Ad vectors to further improve their specificity and activity. The new TSTA configurations of activator and reporter cassettes inserted into the same Ad include (1) an opposite tail-to-tail orientation (T-T); (2) separation by |[sim]|20kb (activator in |[Delta]|E3 and reporter in |[Delta]|E1; E1/E3), and (3) separation by a 600-bp insulator sequence (INS). Preliminary characterization comparing to the original H-H TSTA suggests that each configuration exhibits different effects on basal gene expression, activity, and specificity. The maximal androgen-induced activities of T-T, H-H, and INS TSTA vectors are comparable (within a 2-fold difference) and they are slightly higher than E1/E3 ( 2 to 3-fold lower). The most dramatic difference observed is that the E1/E3 vector exhibits extremely low basal activity in absence of androgen compared to the other 3 constructs. Hence, we observed an androgen induction for E1/E3 vector in the 1000-fold range as compared to 10 to 100-fold range for all other constructs. Preliminary data also indicated that this low basal activity of E1/E3 also enhances cell-specificity (i.e. lower activity in non-prostate cells) and detailed characterization is in progress. Our findings support that the orientation and placement of the two components of TSTA in the Ad genome impact the mechanism of transcriptional activation. Further developments of TSTA vectors include bi-directional configurations to direct more effective multi-gene therapy or coupled imaging/therapeutic strategies in a single Ad. Therapies under development include using the E1/E3 for directing expression of a therapeutic gene (p27WT or p27T187A, a mutant more resistant to proteosome degradation) and a firefly luciferase reporter gene in a bidirectional manner. We believe that coupling prostate-targeted gene therapy with non-invasive imaging will afford us a sensitive, real-time assessment of therapeutic activity in the living subject.

1060. An Efficient GLP-1 Expression System Using Two Step Transcription Amplification for Type 2 Diabetes Gene Therapy

Glucagon-like peptide-1 (GLP-1) is an insulinotropic protein. It was reported that the continuous infusion of GLP-1 normalized the blood glucose level in type 2 diabetes animal model. However, the short half-life of GLP-1 has limited its application to clinical settings and prompted us to develop a GLP-1 gene therapy system. Our previous results showed that the delivery of pb-GLP-1 using polyethylenimine (PEI) reduced the blood glucose level effectively. However, the glucose level was not completely normalized. In the present study, the more efficient GLP-1 expression system was developed using two step transcription amplification (TSTA). To evaluate the TSTA system, pb-Gal4-p65 and pUAS-Luc were constructed. The pUAS-Luc/pb-Gal4-p65 system showed the highest transfection efficiency at a 2:1 pUAS-Luc:pb-Gal4-p65 weight ratio. In addition, the transgene expression by the TSTA system was at least 4 times higher than pb-Luc. To apply the TSTA system to the GLP-1 expression plasmid, pUAS-GLP-1 was constructed. The pUAS-GLP-1/pb-Gal4-p65 system showed higher mRNA level than pb-GLP-1. In addition, the level of GLP-1 by the pUAS-GLP-1/pb-Gal4-p65 system was more than 4 times higher than pb-GLP-1. Therefore, the TSTA GLP-1 expression system may be useful to develop gene therapy system for type 2 diabetes.

Molecular engineering of a two-step transcription amplification (TSTA) system for transgene delivery in prostate cancer.

Gene therapy is founded on the concept that tissue-specific promoters can express heterologous genes for molecular imaging or therapeutic applications. The engineering of cell-specific enhancers to improve potency and the development of two-step transcriptional activation (TSTA) approaches have significantly improved the efficacy of transgene expression. Here we combine these technologies to create a robust, titratable, androgen-responsive system targeted to prostate cancer cells. Our "chimeric" TSTA system uses a duplicated variant of the prostate-specific antigen (PSA) gene enhancer to express GAL4 derivatives fused to one, two, or four VP16 activation domains. We targeted the resulting activators to cells with reporter templates bearing one, two, or five GAL4 binding sites upstream of firefly luciferase. We monitored activity via firefly luciferase assays in transfected cell extracts and in live nude mice using a cooled charge-coupled device (CCD) imaging system. In this system, we found that firefly luciferase expression in prostate cancer cells can be varied over an 800-fold range. We also found that a single plasmid bearing the optimized enhancer, GAL4-VP16 derivative, and reporter expressed firefly luciferase at 20-fold higher levels than the cytomegalovirus enhancer. We discuss the implications of this strategy and its application to molecular imaging and therapy.