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Hypoxia-targeted and pharmacologically controlled luciferase expression in vivo. Six groups of mice bearing tumors were injected either into the tumors-groups A, B and C (A) or into muscle-groups D, E and F (B)-with different plasmid combinations. Half of the mice were treated with doxycycline (+) and half were not (x). A total of 10, 9, 10, 10, 9, and 10 tumors in groups Ax, A+, Bx, B+, Cx, and C+, respectively, 8, 6, 6, 6, 6, and 4 muscles in groups Dx, D+, Ex, E+, Fx and F+, respectively, were analyzed. The points and the histograms represent the individual and mean luciferase activities in each group. The results for the Bx and Cx groups are statistically different from those for other groups of tumors (Ax, A+, B+ and C+) according to the Mann-Whitney U-test ( p<0.05). The differences between group Fx and other muscle groups (Dx, Ex, D+, E+ and F+) are also significant
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The combination of physiologically and pharmacologically controlled elements may provide a means to ensure both the regulation and the safety of transgene expression--two major goals in gene therapy.
A two-gene modulation system was developed that uses the following three levels of control: (i) the hypoxia-responsive element directing the transcrip...
Context in source publication
Context 1
... test whether hypoxia in a solid tumor might specifically stimulate somatic transgene expression in vivo by using the presently described regulation system, plasmids were transferred in a Lewis lung carcinoma (3LL) and in leg muscle. Tumor-bearing mice were divided into six groups: in groups A and D, mice were injected with the reporter plasmid pTRE-luc alone; in groups B and E, mice were injected with 12A-hH104 and pTRE-luc; in groups C and F, mice were injected with pCMV-tTA and pTRE-luc. In groups A, B and C, DNA was transferred in tumors, whereas in groups D, E and F, DNA was transferred in muscle. For each group, half of the mice were treated with doxycycline and half were not. Two days after the transfer, the mice were killed, muscles and tumors were excised, and luciferase activities were determined. As shown in Figure 4, in the absence of doxycyline, luciferase was detected above the non-induced level (groups A and D, pTRE-luc alone) in groups B, C and F but not in group E. When the transactivator expression was under the control of the CMV promoter (groups C and F), the mean luciferase activity was 37-fold (range 20-80-fold) and 61-fold (range 20-98-fold) higher than background levels in tumors and muscles, respectively. When transactivator expression depended upon HREs and the Hif1a destabi- lizing element (groups B and E), the mean luciferase activity was 19-fold (range 1-99-fold) and 0.5-fold (range 0.3-0.7-fold) higher than background levels in tumors and muscles, respectively, in the absence of doxycycline. It is noteworthy that in group B, the results were much more heterogeneous than in other groups. Doxycycline efficiently blocked the activity of the transactivator in tumors and muscles, and inhibited luciferase production irrespective of the expression system (groups B, C, ...
Citations
... Hypoxia-inducible factor 1 HIF-1 is a transcription factor that regulates multiple genes involved in angiogenesis, invasion, metastasis, apoptosis and cellular metabolism (Bussink et al. 2003;Harada et al. 2005;Liu et al. 2005;Tatum 2006;Lehmann et al. 2009;Carlin et al. 2010;Hall and Giaccia 2011;Sun et al. 2011;Mimeault and Batra 2013;Lee et al. 2014;Li et al. 2015). HIF-1 is upregulated in hypoxic conditions (pO 2 < 20 mmHg) (Semenza 2000;Payen et al. 2001 Viola et al. 2008;Yeom et al. 2008;Ebbesen et al. 2009;Sun et al. 2011;Mimeault and Batra 2013;Lee et al. 2014;Li et al. 2015). HIF-1 is a heterodimer that consist of two subunits; HIF-1a and HIF-1b. ...
... HIF-1 is a heterodimer that consist of two subunits; HIF-1a and HIF-1b. HIF-1a is oxygen sensitive, and under hypoxic conditions HIF-1a is not degraded due to the activation of HIF-1 (Semenza 2000;H€ ockel and Vaupel 2001;Payen et al. 2001;Semenza 2001;Serganova et al. 2004;Liu et al. 2005;Kang et al. 2006;Serganova et al. 2006;Tatum 2006;Lungu et al. 2007;Viola et al. 2008;Yeom et al. 2008;Lehmann et al. 2009;Carlin et al. 2010;Lu and Kang 2010;Cassavaugh and Lounsbury 2011;Sun et al. 2011;Lee et al. 2014;Li et al. 2015). The accumulation of HIF-1 triggers a pathway that promotes the transcription of multiple genes, including CA IX, GLUT-1 and vascular endothelial growth factor (VEGF) (Bussink et al. 2003;Serganova et al. 2004;Wen et al. 2004;Liu et al. 2005;Tatum 2006;Dubois et al. 2007;Russell et al. 2009;Carlin et al. 2010;Sun et al. 2011;Bao et al. 2012;Lee et al. 2014;Dubois et al. 2015;Li et al. 2015). ...
... One method is to label transgenes and introduce them to hypoxic cells so they bind to HIF-1 (Sun et al. 2011). Luciferase is a bioluminescent enzyme that has been bound to transgenes, one common gene being HRE (hypoxia responsive element) reporter gene (Shibata et al. 2000;Payen et al. 2001;Harada et al. 2005;Kang et al. 2006;Yeom et al. 2008;Dubois, Lieuwes, et al. 2009;Lehmann et al. 2009;O'Neill et al. 2010;Snoeks et al. 2010;Cassavaugh and Lounsbury 2011;Kircher et al. 2011;Sun et al. 2011;Dubois et al. 2015). The biomarker is injected intravenously where it will bind to HIF-1 via the binding site HRE Harada et al. 2005;Lehmann et al. 2009;Dubois et al. 2015;Tafreshi et al. 2016). ...
Purpose
Tumors exhibit areas of decreased oxygenation due to malformed blood vessels. This low oxygen concentration decreases the effectiveness of radiation therapy, and the resulting poor perfusion can prevent drugs from reaching areas of the tumor. Tumor hypoxia is associated with poorer prognosis and disease progression and is therefore of interest to preclinical researchers. Although there are multiple different ways to measure tumor hypoxia and related factors, there is no standard for quantifying spatial and temporal tumor hypoxia distributions in preclinical research or in the clinic. This review compares imaging methods utilized for the purpose of assessing spatio-temporal patterns of hypoxia in the preclinical setting. Conclusions: Imaging methods provide varying levels of spatial and temporal resolution regarding different aspects of hypoxia, and with varying advantages and disadvantages. The choice of modality requires consideration of the specific experimental model, the nature of the required characterisation and the availability of complementary modalities as well as immunohistochemistry.
... Optical imaging has had an important role to play in evaluating hypoxia, especially in biopsy specimens. With the introduction of transgenes with the hypoxia responsive element as promoter sequences coupled to reporter genes, e.g., luciferase reporter gene [90,91] or green fluorescent protein (GFP) [92], a number of modalities have been developed to allow HIF-1 activity to be directly measured. Of these, a HIF-1-dependent promoter-regulated luciferase reporter gene, shown to produce a 100-fold increased luciferase response to hypoxia, has been used to evaluate anti-hypoxia therapy for its efficacy in animals [93]. ...
Measurement of tumor hypoxia is required for the diagnosis of tumor and the evaluation of therapeutic outcome. Currently, invasive and noninvasive techniques being exploited for tumor hypoxia measurement include polarographic needle electrodes, immunohistochemical (IHC) staining, magnetic resonance imaging (MRI), radionuclide imaging (positron emission tomography [PET] and single-photon emission computed tomography [SPECT]), optical imaging (bioluminescence and fluorescence), and hypoxia imaging endoscopy. This review provides a summary of the modalities available for assessment of tissue oxygenation as well as a discussion of current arguments for and against each modality, with a particular focus on noninvasive hypoxia imaging with emerging agents and new imaging technologies intended to detect molecular events associated with tumor hypoxia.
... Thus, the level of that gene transcription is proportional to the light intensity produced by the luciferase.So far, a few bioluminescent imaging methods have been developed to image hypoxia via measuring HIF-1 transcriptional activity. Payen et al. first proposed the idea using HRE sequences coupled with reporter genes, such as luciferase or GFP, to enable imaging HIF-1 transcriptional activity(Payen et al., 2001).In 2004, Serganov et al. demonstrated in vivo small animal imaging of HIF-1 transcriptional activity using GFP/tk reporter under control of 8×HREs(Serganova et al., 2004). ...
Tumor growth often outpaces its vascularization, leading to development of a hypoxic tumor microenvironment. In response, an intracellular hypoxia survival pathway is initiated by heterodimerization of hypoxia-inducible factor (HIF)-1α and HIF-1β, which subsequently upregulates the expression of several hypoxia-inducible genes, promotes cell survival and stimulates angiogenesis in the oxygen-deprived environment. Hypoxic tumor regions are often associated with resistance to various classes of radio- or chemotherapeutic agents. Therefore, development of HIF-1α/β heterodimerization inhibitors may provide a novel approach to anti-cancer therapy. To this end, a novel approach for imaging HIF-1α/β heterodimerization in vitro and in vivo was developed in this study. Using this screening platform, we identified a promising lead candidate and further chemically derivatized the lead candidate to assess the structure-activity relationship (SAR). The most effective first generation drug inhibitors were selected and their pharmacodynamics and anti-tumor efficacy in vivo were verified by bioluminescence imaging (BLI) of HIF-1α/β heterodimerization in the xenograft tumor model. Furthermore, the first generation drug inhibitors, M-TMCP and D-TMCP, demonstrated efficacy as monotherapies, resulting in tumor growth inhibition via disruption of HIF-1 signaling-mediated tumor stromal neoangiogenesis.
... Under hypoxic conditions, accumulated HIF-1 binds to the hypoxia responsive element (HRE) and triggers the recruitment of co-activators essential for transcriptional activation [31]. Several elegant methods have been developed to directly measure HIF-1 activity by the introduction of transgenes with HREs as promoter sequences coupled to reporter genes such as luciferase [32,33] or GFP [34]. Another example is that heat-shock protein promoters, particularly HSP70 promoters, have been commonly used for gene therapy strategies because they are both heat-inducible and efficient [35]. ...
Molecular imaging is a newly emerged multiple disciplinary field that aims to visualize, characterize and quantitatively measure biological processes at cellular and molecular levels in humans and other living systems. A reporter gene is a piece of DNA encoding reporter protein, which presents as a readily measurable phenotype that can be distinguished easily from the background of endogenous protein. After being transferred into cells of organ systems (transgenes), the reporter gene can be utilized to visualize transcriptional and posttranscriptional regulation of gene expression, protein-protein interactions, or trafficking of proteins or cells in living subjects. Herein, we review previous classification of reporter genes and regroup the reporter gene based imaging as basic, inducible and activatable, based on the regulation of reporter gene transcription and post-translational modification of reporter proteins. We then focus on activatable reporters, in which the signal can be activated at the posttranslational level for visualizing protein-protein interactions, protein phosphorylation or tertiary structure changes. The applications of several types of activatable reporters will also be summarized. We conclude that activatable reporter imaging can benefit both basic biomedical research and drug development.
... An alternative is to combine pharmacological and pathophysiological regulation in a single system. A good example of this was reported by Payen et al. [39], where they placed the expression of the tetracycline transactivator molecule rtTA under the control of a hypoxia-regulated promoter and expressed luciferase in a second vector from a Ptet. In this arrangement, luciferase expression is co-dependent on both hypoxia and Dox. ...
The aim of this study was to construct a promoter containing DNA motifs for an endogenous transcription factor associated with inflammation along with motifs for pharmacological regulation factors. We demonstrate in transfected cells that expression of a gene of interest is induced by hypoxic conditions or through pharmacological induction, and also show pharmacological repression. In vivo studies utilised electroporation of plasmid to mouse paws, a delivery method shown to be effective by bioluminescence imaging. For gene therapy, the promoter was used to drive expression of IL-1Ra in a paw inflammation model with therapeutic effect observed which was further enhanced when the promoter was additionally induced with a pharmacological activator. One of the most important observations from this study was that promoter induction by hypoxia or inflammation could be prevented by the pharmacological repressor in the absence of doxycycline. These studies demonstrate that hybrid promoters enable pharmacological adjustment to the pathophysiological level of gene expression and, importantly, that they allow termination of gene expression even in the presence of pathophysiological stimuli.
... Autoimmune anemia has also been described due to the appearance of neutralizing antibodies against endogenous Epo (20,31). Preliminary in vivo studies of the regulation of Epo secretion by oxygen seem promising, but few studies have been carried out in this field (28,32,33). Regulation of Epo secretion by doxycycline is Figure 10. ...
The effectiveness of the caspase-9-based artificial "death switch" as a safety measure for gene therapy based on the erythropoietin (Epo) hormone was tested in vitro and in vivo using the chemical inducer of dimerization, AP20187. Plasmids encoding the dimeric murine Epo, the tetracycline-controlled transactivator and inducible caspase 9 (ptet-mEpoD, ptet-tTAk and pSH1/Sn-E-Fv'-Fvls-casp9-E, respectively) were used in this study. AP20187 induced apoptosis of iCasp9-modified C2C12 myoblasts. In vivo, two groups of male C57BI/6 mice, 8-12 weeks old, were injected intramuscularly with 5 microg/50 g ptet-mEpoD and 0.5 microg/50 g ptet-tTAk. There were 20 animals in group 1 and 36 animals in group 2. Animals from group 2 were also injected with the 6 microg/50 g iCasp9 plasmid. Seventy percent of the animals showed an increase in hematocrit of more than 65% for more than 15 weeks. AP20187 administration significantly reduced hematocrit and plasma Epo levels in 30% of the animals belonging to group 2. TUNEL-positive cells were detected in the muscle of at least 50% of the animals treated with AP20187. Doxycycline administration was efficient in controlling Epo secretion in both groups. We conclude that inducible caspase 9 did not interfere with gene transfer, gene expression or tetracycline control and may be used as a safety mechanism for gene therapy. However, more studies are necessary to improve the efficacy of this technique, for example, the use of lentivirus vector.
... In addition, DT-A/ODD exhibited a comparable inhibitory activity of protein synthesis with DT-A in vitro, indicating that fusion of the ODD domain to DT-A does not affect its translation inhibitory activity. The ODD domain of HIF-1a has been implicated to be useful as a hypoxia switch, limiting the production of a fusion protein in normoxia while stabilizing it in hypoxia (43). For example, ODDcaspase-3 fusion protein is shown to be specifically stabilized and activated in hypoxic cells (44). ...
Tumor cells in hypoxic areas of solid tumors are resistant to conventional chemotherapy and radiotherapy and thus are obstacles of cancer therapy. We report here the feasibility of applying hypoxia-regulated expression of diphtheria toxin A (DT-A) for killing hypoxic tumor cells. The expression vector was constructed to express DT-A fused with hypoxia-inducible factor-1alpha (HIF-1alpha) oxygen-dependent degradation (ODD) domain under the control of vascular endothelial growth factor gene promoter and contain erythropoietin mRNA-binding protein (ERBP)-binding sequence downstream of the DT-A/ODD sequence. In vitro ubiquitination assay showed that DT-A/ODD, but not DT-A, was ubiquitinated as efficient as HIF-1alpha under normoxic conditions in a von Hippel-Lindau- and oxygen-dependent manner. DT-A/ODD exhibited a comparable translation inhibitory activity to DT-A. ERBP-binding sequence was effective in stabilizing mRNA under hypoxic conditions in various cell types. Transfection of the vector expressing DT-A/ODD into high-metastatic Lewis lung carcinoma (3LL) A11 cells resulted in induction of apoptosis independently of hypoxia, probably due to its extreme toxicity. However, transfection of the vector expressing attenuated DT-A(W153F)/ODD or DT-A(H21A)/ODD resulted in a hypoxia-dependent induction of apoptosis. Liposomal gene transfer of the vector encoding DT-A(W153F)/ODD induced apoptosis in hypoxic, but not in normoxic, areas of solid tumors established by A11 variant cells with higher resistance to hypoxia-induced apoptosis and inhibited the growth of hypoxic tumors established by 3LL-P29 cells. These results suggest that hypoxia-regulated expression of attenuated DT-A(W153F)/ODD fusion protein is potentially of use for killing hypoxic tumor cells with minimizing the damage to normoxic normal tissues.
... Electrotransfer has also proved to be a valuable tool for the study of gene regulation, such as the tetracycline system, which requires at least co-transfection of two plasmids in the same cell [6]. For instance, new regulation systems of potential interest are novel doxycycline transactivators in a gene switch system [93] and a system based on hypoxiaresponsive element and tetracycline transactivators [94]. Finally, electrotransfer is a promising tool to screen for optimized secreted protein design, as has been shown in the case of TNF-α receptor variants (C. ...
In vivo electrotransfer is a physical method of gene delivery in various tissues and organs, relying on the injection of a plasmid DNA followed by electric pulse delivery. The importance of the association between cell permeabilization and DNA electrophoresis for electrotransfer efficiency has been highlighted. In vivo electrotransfer is of special interest since it is the most efficient non-viral strategy of gene delivery and also because of its low cost, easiness of realization and safety. The potentiality of this technique can be further improved by optimizing plasmid biodistribution in the targeted organ, plasmid structure, and the design of the encoded protein. In particular, we found that plasmids of smaller size were electrotransferred more efficiently than large plasmids. It is also of importance to study and understand kinetic expression of the transgene, which can be very variable, depending on many factors including cellular localization of the protein, physiological activity and regulation. The most widely targeted tissue is skeletal muscle, because this strategy is not only promising for the treatment of muscle disorders, but also for the systemic secretion of therapeutic proteins. Vaccination and oncology gene therapy are also major fields of application of electrotransfer, whereas application to other organs such as liver, brain and cornea are expanding. Many published studies have shown that plasmid electrotransfer can lead to long-lasting therapeutic effects in various pathologies such as cancer, blood disorders, rheumatoid arthritis or muscle ischemia. DNA electrotransfer is also a powerful laboratory tool to study gene function in a given tissue.
... Quatre systèmes de régulation pharmacologique sont en développement : ceux régulés par la tétracycline [16], par le RU486 [17], par le stéroïde ecdysone ou ses analogues [17], et par la rapamycine et ses analogues [18]. Payen et al. [19] ont proposé une régulation à la fois physiologique (par la pression en O 2 ), et pharmacologique (par la tétracycline). Rinsch et al. [20] ont proposé une régulation fondée uniquement sur la pression en O 2 . ...
Aims. – Draw up the inventory of various methods able to increase the oxygen transport by the blood and present methods and strategy of detection.Current knowledge. – Maximal oxygen uptake is the major performance limiting factor in endurance sports. Sophisticated training methods have been developed to increase this variable. On the other hand, attempts have been made to improve maximal oxygen uptake by artificial means: blood doping, administration of human recombinant erythropoietin (rhu-Epo) and, probably, by the use of a new class of therapeutic agents: the artificial oxygen carriers. All these substances and methods are prohibited by the International Olympic Committee. But, until now, the detection of the misuse of these compounds is a problem: there is no detection method for blood doping, the current test method for rhu-Epo can do false negative cases and no screening methods are performed for oxygen carriers.Points of views and plans. – Despite the emergence of the oxygen carriers, in fact easy to detect, the use of r-HuEpo seems the most efficient practice. Today there is a test to detect abuse of r-HuEpo, that’s why the cheats should have recourse to red blood cells transfusion. However concomitant injections of r-HuEpo and GH or IL-3 should use least doses and so to make the detection more difficult. Although indirect methods will be refined, this approach will likely insufficient by itself. A careful definition of an individual’s hematologic profile, the so-called “hematologic passport”, should form the basis for a more successful application of any indirect method and should disturb recourse to doping.
... 81 An attractive alternative is the introduction of transgenes with hypoxic response elements (HREs) as promoter sequences coupled to reporter genes such as GFP (green fluorescent protein) 82,83 or luciferase. 84,85 GFP synthesis is an energetic process, which could be hindered under hypoxia conditions. Likewise, bioluminescence accompanying action of luciferase on luciferin requires adenosine triphosphate (ATP) and O 2 , but reports suggest that even under exceedingly low pO 2 , sufficient oxygen remains to reveal hypoxia. ...
This chapter discusses the measurement of changes in tumor oxygenation. The oxygen pressure determinations could be of great clinical value and they are also vital to many laboratory investigations of new drugs and studies of tumor development. Blood oxygen level dependent (BOLD) contrast proton nuclear magnetic resonance (NMR) facilitates rapid interrogation of vascular oxygenation, and is particularly appropriate for examining dynamic responses to interventions. Both spectroscopic and imaging approaches have been applied to tissue oxygen pressure measurements depending on the available signal to noise. It appears that uptake and distribution efficiency vary with tumor type, but in general, maximum signal is detected from the tumor periphery corresponding with regions of greater perfusion. The various aspects of fluorocarbon relaxometry using echo planar imaging for dynamic oxygen mapping (FREDOM) are elaborated. The results suggested FREDOM as a valuable tool for assessing the dynamic time course of interventions to provide clear insight into the mode of action of therapeutic approaches and aid in the high-throughput screening of new drugs, such as vascular targeting and antiangiogenic agents.
