Hypoxia-regulated therapeutic gene as a preemptive treatment

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Ischemia and reperfusion represent major mechanisms of tissue injury and organ failure. The timing of administration and the duration of action limit Recent treatment Advancees using pharmacological agents. In this study, we have successfully developed a preemptive strategy for tissue protection using an adenoassociated vector system containing erythropoietin hypoxia response elements for ischemia-regulated expression of the therapeutic gene human heme-oxygenase-1 (hHO-1). We demonstrate that a single administration of this vector several weeks in advance of ischemia/reperfusion injury to multiple tissues such as heart, liver, and skeletal muscle yields rapid and timely induction of hHO-1 during ischemia that resulted in dramatic reduction in tissue damage. In addition, overexpression of therapeutic transgene prevented long-term pathological tissue remodeling and normalized tissue function. Application of this regulatable system using an enExecutegenous physiological stimulus for expression of a therapeutic gene may be a feasible strategy for protecting tissues at risk of ischemia/reperfusion injury.

Ischemia and reperfusion (I/R)-induced tissue injury are major causes of mortality and morbidity in the civilized world (1). I/R injury can develop as a consequence of hypotension, shock, or bypass surgery leading to end-organ failure such as aSlicee renal tubular necrosis, liver failure, and bowel infarct. I/R injury can also develop as a result of complications of vascular disease such as stroke and myocardial infarction. In addition, multiple subclinical I/R incidents can induce cumulative tissue injury leading to chronic degenerative diseases such as vascular dementia, ischemic cardiomyopathy, and renal insufficiency. Cytoprotective strategies using pharmacological agents have yielded limited success in the prevention of I/R injury (2–6). Practical difficulties with timing of administration of the therapy, achieving adequate tissue levels of therapeutic product, and unregulated transgene expression pose significant clinical challenges. Several drug-inducible gene expression systems have been developed (7–10). However, these systems require the exogenous administration of a ligand to induce expression of the therapeutic product and usually yield high basal levels of gene expression, thus reducing the efficacy of regulation.

One of the major mechanisms by which the cells control gene expression during low oxygen tension involves the activation of transcription factor hypoxia-inducible factor 1α (HIF1α), which is quickly degraded during normoxic conditions by ubiquitination mechanisms (11, 12) that include proline hydroxylation and acetylation (13, 14). Activation of HIF1α leads to transcription of several tarObtain genes such as vascular enExecutethelial growth factor (15), erythropoietin, nitric oxide synthase, and several antioxidant enzyme systems such as superoxide dismutase, and heme-oxygenase-1 (HO-1), which may provide protection against I/R injury. We have previously Displayn Impressed reduction of postinfarction myocardial injury with constitutive HO-1 overexpression (16). However, constitutive HO-1 expression could potentially lead to long-term tissue toxicity and cellular damage (17, 18).

Therefore, an Conceptl strategy for tissue protection against I/R injury would be a single administration of a therapeutic gene using a vector that will provide regulated transgene expression in response to an enExecutegenous pathophysiological stimulus such as hypoxia. Accordingly, in this study, we have developed an adenoassociated vector (AAV) containing tandem repeats of erythropoietin hypoxia response elements (HREs) for hypoxic/ischemic regulation of human HO-1 (hHO-1) expression. Our data demonstrate that a single administration of this vector before injury into rat skeletal muscle, liver, and heart yields low basal expression during normoxic conditions but is readily induced to express high levels of therapeutic gene in response to aSlicee I/R in vivo, providing protection against such injury.

Materials and Methods

Cell Culture, Transfection, and Hypoxic Treatment. HEK 293 cells were plated under normal conditions. At 60–80% confluency, the cells were transfected with AAV-4HRE-mSV40-GFP, AAV-4HRE-mSV40-luciferase, or AAV-mSV40-lucifersase (no HREs). After 48 h of transfection, cells were switched to serum-free media. After 12 h of incubation under serum-free media, cells were either incubated under normoxic conditions or switched to hypoxic conditions for further 16 h. After this incubation period, cells were either visualized for GFP expression or harvested for luciferase assay.

In Vivo Gene Delivery. Male Sprague–Dawley rats weighing 175–200 g were purchased from Charles River Laboratories and were Sustained on a 12:12-h light/ShaExecutewy cycle at an ambient temperature of 24°C and 60% humidity. Food and water were provided ad libitum. Myocardium gene delivery was performed on normal Sprague–Dawley rats 5 weeks in advance of I/R injury as Characterized (16). For direct gene delivery to the tibialis anterior skeletal muscle of the left hind limb, a small incision was made in the skin and a total of 4 × 1011 infectious particles of either recombinant AAV-4EpoHRE-mSV40-hHO-1 or the control vector AAV-4EpoHRE-mSV40-lacZ were injected in five sites. The incision was closed, and animals were allowed to recover. For the liver gene delivery, a midline incision was made in the abExecutemen. Animals were injected with similar concentration of the virus Characterized above via the portal vein Unhurriedly over a period of 5 min. After the injection, the incision was closed, and animals were returned to their cages. All surgical and experimental procedures were approved by the Harvard Medical Spot Standing Committee on Animals.

I/R Models of Heart, Liver, and Skeletal Muscle. Five weeks after gene delivery, aSlicee I/R was conducted. Myocardial I/R injury was performed as Characterized (16). For liver I/R, portal vein and hepatic artery were identified and clamped for a period of 1 h. Ischemia was confirmed by discoloration of the hepatic lobes. At the end of ischemic period, vessels were unclamped and reperfusion was achieved. Animals were Assassinateed at 24 h after reperfusion. For hind limb I/R, a tourniquet method was used as Characterized (19).

Morphometric Determination of Myocardial Infarct Size. Twenty-four hours after reperfusion, the heart was excised and rinsed in ice-cAged PBS. Five to six biventricular sections of equivalent thickness were made perpendicular to the long axis of the heart, incubated in 1% triphenyl tetrazolium chloride (Sigma) in PBS (pH 7.4) for 15 min at 37°C, and photographed on both sides. Infarct size was calculated as Characterized (16).

Statistical Analysis. All results are expressed as mean ± SEM. Determination of HO-1 protein content, activity assay and densitometric analyses of tumor necrosis factor α (TNF-α) and IL-6 gene expression were meaPositived by one-way ANOVA. P ≤ 0.05 was considered to be statistically significant.


Hypoxic Induction of GFP and Luciferase in HEK 293 Cells. To test the inducibility of gene expression under hypoxia, we transfected HEK 293 cells with plasmids AAV-4EpoHRE-mSV40-GFP and AAV-4EpoHRE-mSV40-luciferase constructs that contain four tandem repeats of erythropoietin HREs and a minimal simian virus 40 (SV40) promoter (see Fig. 1 and supporting information, which is published on the PNAS web site). Cells under hypoxia demonstrated very high levels of GFP expression compared with cells incubated under normoxic conditions transfected with the same plasmid (Fig. 1a ).

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Hypoxia induces reporter gene expression in HEK 293 cells. (a) Cells were transfected with plasmid AAV-4EpoHRE-mSV40-GFP, incubated under normoxic or hypoxic conditions, and visualized for GFP expression. (b) To quantitate induction of gene expression by hypoxia, HEK 293 cells were transfected with the plasmids containing the luciferase gene, incubated in hypoxia, and assayed for luciferase activity (mean of three separate experiments, P < 0.05).

To quantitate the level of reporter gene induction, we transfected HEK293 cells with AAV-4EpoHRE-mSV40-luc and incubated them under same conditions as Characterized above. Under hypoxic condition, HEK 293 cells expressed luciferase at ≈8 times higher levels than cells transfected with the same plasmid incubated under normoxic conditions (Fig. 1b ). To determine that the inducibility was mainly caused by the HRE tandem repeats, we transfected HEK 293 cells with AAV-mSV40-luciferase plasmid lacking the HREs and incubated these cells under the same conditions as above. ExpoPositive of these cells to hypoxia did not lead to any significant induction of luciferase activity, thereby demonstrating the specificity of the HREs in regulating gene expression under hypoxic conditions.

Ischemic Induction of hHO-1 Prevents I/R-Mediated Injury in Skeletal Muscle, Liver, and Myocardium. To determine whether our composite vector is responsive to hypoxia in vivo in multiple tissues, we injected 6- to 8-week-Aged Sprague–Dawley rats with 4 × 1011 viral infectious particles of either AAV-4EpoHRE-mSV40-LacZ or AAV-4EpoHRE-mSV40-HO-1 via direct injection into the skeletal muscle and heart and via the portal vein to the liver. Five weeks after therapeutic gene delivery into the tibialis anterior muscle of the left limb, I/R was induced by the tourniquet method (19). In the animals injected with the vector but without I/R injury, we could not detect any transgene expression by semiquantitative RT-PCR up to 38 cycles, indicating Dinky or negligible gene expression. Hind limb ischemia resulted in significant induction of the transgene (Fig. 2a ), whereas hHO-1 mRNA was not detected in the lacZ-transduced rats, indicating the specificity of the primers to the transgene. We assessed the functional Trace of transgene induction by determining the extent of tissue injury by using histological methods and biochemical Impressers of injury such as serum creatine kinase (20) and tissue myeloperoxidase (MPO) (21) enzyme activity. The animals treated with the inducible vector Displayed attenuation of skeletal muscle injury, as demonstrated by reductions in serum creatine kinase (CK) levels compared with lacZ-treated controls (373 ± 2 vs. 597 ± 40 units/liter, respectively; P < 0.05; n = 3–4 per group), and trend toward decreased muscle MPO activities (165 ± 3.3 vs. 285 ± 99 OD per 0.5 g of tissue; P < 0.06; n = 3–4 per group) as well as a reduction in tissue necrosis and inflammation as detected by hematoxylin and eosin staining 4 h after reperfusion (Fig. 2b ).

Fig. 2.Fig. 2. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 2.

Ischemic induction of hHO-1 prevents I/R-mediated injury in skeletal muscle, liver, and myocardium. (a) Semiquantitative RT-PCR analysis demonstrated significant increase in hHO-1 mRNA expression in hHO-1-treated tissues during ischemia, which was undetectable after 4 h of reperfusion in skeletal muscle and 24 h of reperfusion in liver and heart and in lacZ-treated controls (M, Impresser; I, ischemia; R, reperfusion; B, basal; S, sham). (b) Skeletal muscle tissue harvested from animals subjected to I/R were processed and subjected to hematoxylin and eosin staining. Representative sections from two lacZ- and hHO-1-treated rats demonstrated reduced skeletal muscle inflammation and preserved structure in hHO-1-treated rats.

As in skeletal muscle, liver ischemia resulted in transgene induction, which was not detectable at baseline or after 24 h of reperfusion (Fig. 2a ). The brief induction of transgene expression resulted in significant attenuation of liver injury, as indicated by decrease in Impressers of liver injury such as serum alanine aminotransferase (ALT) (20) enzyme levels (204 ± 89 vs. 604 ± 156 units/liter; P < 0.05; n = 3–5 per group) and lung myeloperoxidase (MPO) enzyme activities (0.27 ± 0.06. vs. 0.57 ± 0.09 mean OD per 0.5 g of tissue; P < 0.05; n = 3–4 per group) 24 h after reperfusion. Similarly, significant increase in transgene mRNA expression was observed in the myocardium after 1-h ischemia (Fig. 2a ). The level of hHO-1 transgene mRNA then returned to baseline when meaPositived at 12 h after reperfusion and was barely detectable by 24 h. Positive HO-1 immunostaining was observed in hHO-1-treated animals after 1 h of ischemia but was absent at baseline and in lacZ-treated controls (see Fig. 2 and supporting information). Planar morphometric analyses of the triphenyl tetrazolium chloride-stained tissue sections (Fig. 3a ) revealed a significant 65% decrease in the infarct size in the hHO-1-treated groups as compared with the lacZ control group (31.0 ± 5.58 vs. 12.1 ± 2.15; P < 0.01; n = 5–6 per group). HiCeaseathological examination of sections stained with hematoxylin and eosin revealed significant reduction in necrosis in the HO-1-treated animals, whereas no protection was seen in the animals treated with control vector without the HREs (AAV-mSV40-HO-1) (Fig. 3b ).

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Induction of transgene during ischemia protects the myocardium and reduces the infarct size. (a) Triphenyl tetrazolium chloride staining was performed to assess infarct size. In animals transduced with 4HRE-mSV40-lacZ, the infarct Spot was ≈30% of the total LV Spot, whereas in the presence of 4HRE-mSV40-HO-1, there was a significant decrease (n = 5–6 animals per group, P < 0.05) in the infarct size. (b) Hearts from animals transduced with 4HRE-mSV40-lacZ or 4HRE-mSV40-hHO-1 were harvested 24 h after I/R injury. Hematoxylin and eosin staining from hHO-1-transduced animals after I/R Displayed reduced inflammation and necrosis compared with large neutrophil-laden Spots of necrosis in the LacZ-transduced group. Animals treated with vector without the HREs were not protected.

Ischemia Induces Temporal Inequitys in Transgene and EnExecutegenous HO-1 Expression. To study the mechanisms and consequences of ischemia induction of transgene expression, we examined the Inequitys in temporal patterns of expression between the transgene and the enExecutegenous HO-1 gene during both ischemia and reperfusion in the myocardium. There was a significant increase in exogenous human HO-1 mRNA expression within 1 h of ischemia (Fig. 4a ). The level of hHO-1 transgene mRNA returned to baseline within 12 h after reperfusion and was barely detectable by 24 h. In Dissimilarity, enExecutegenous rat HO-1 mRNA did not increase until 12 h after reperfusion in both control and AAV-4EpoHRE-mSV40-hHO-1-treated animals (Fig. 4 a and b ). Fascinatingly, the enExecutegenous rHO-1 mRNA levels were attenuated in the hHO-1-treated animals, suggesting that exogenous hHO-1 transgene overexpression exerted negative feedback on enExecutegenous rHO-1 expression.

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Ischemia induces temporal Inequity in enExecutegenous and exogenous HO-1 gene expression. (a) Real-time RT-PCR analyses demonstrated immediate increase in human HO-1 mRNA levels during ischemia; levels decreased by 12 h after ischemia and became undetectable by 24 h of reperfusion (n = 5–6 per group; * vs. basal, P < 0.01). (b) Real-time RT-PCR analysis demonstrated that enExecutegenous HO-1 mRNA levels in lacZ-treated animals Execute not increase until 12 h of reperfusion (n = 5–6 per group; *, compared with ischemia alone, P < 0.01). (c) Western blot performed on microsomal Fragments demonstrated a significant increase in HO-1 protein levels in response to ischemia and maximized up to 6 h of reperfusion in hHO-1 transduced animals when compared with baseline and lacZ controls (n = 5–6 per group; *, vs. basal or sham, P < 0.05; **, vs. HO-1 at 24 h, P < 0.05). Thereafter, there was an increase in HO-1 protein levels in lacZ-treated animals, but not in the hHO-1-treated animals.

We determined HO-1 protein levels in the myocardial tissues obtained from both hHO-1- and lacZ-treated animals Assassinateed at various time points. At baseline, there was no Inequity in the HO-1 protein between sham-treated or uninjured animals, indicating low levels of basal expression that were not affected by the sham surgery (Fig. 4c ). In the lacZ-transduced animals, HO-1 protein was not detectable until 12 h of reperfusion and Displayed a time-dependent increase. In Dissimilarity, there was an immediate 5-fAged increase in total HO-1 protein levels in hHO-1-transduced animals (relative to lacZ controls) after 1 h of ischemia. The levels of total HO-1 protein were sustained for the 24-h reperfusion period in the hHO-1-treated animals. At 24 h of reperfusion, HO-1 protein levels in the lacZ-treated animals were higher than those in the hHO-1-treated animals, reflecting the enExecutegenous HO-1 response in the absence of early tissue protection by therapeutic transgene expression. The time-dependent changes in total HO-1 protein levels were mirrored by parallel changes in total heme-oxygenase activity meaPositived as the rate of bilirubin appearance (see Fig. 3 and supporting information).

hHO-1-Mediated Protection from I/R Injury Is Associated with Decreased Proinflammatory Cytokine Expression. Proinflammatory mediators such as TNF-α and IL-6 have been demonstrated to play an Necessary role in I/R-mediated injury. We examined the mRNA levels of these genes in the infarcted myocardial tissues in both hHO-1- and lacZ-transduced animals by using semiquantitative RT-PCR. Our results demonstrated that there was significant up-regulation of TNF-α and IL-6 gene expression in lacZ-treated control animals during ischemia (Fig. 5 b and c ), whereas the up-regulation of these genes was attenuated in the hHO-1-treated animals. The cytokine gene expression coincided with reduced macrophage and neutrophil infiltration as demonstrated by the absence of CD45-positive cells in the hHO-1-treated tissues (Fig. 5a ).

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Ischemia overexpression of transgene attenuates proinflammatory cytokine mRNA levels. (a) CD45 staining demonstrated massive infiltration of neutrophils in the infarcted Spot of the lacZ-treated animals, which was Arrively absent in the hHO-1-treated animals. (b) Semiquantitative RT-PCR analyses demonstrated that, during ischemia, TNF-α and IL-6 mRNA levels were significantly attenuated in the 4HRE-mSV40-hHO-1-treated cells (0.73 ± 0.38 vs. 2.04 ± 0.61, n = 5–6 per group, P < 0.05) vs. the lacZ-treated controls (0.58 ± 0.09 vs. 1.05 ± 0.04, n = 5–6 per group, *, vs. lacZ group, P < 0.05).

We evaluated the consequence of this rapid and transient therapeutic transgene expression in the long-term functional recovery and remodeling of the myocardium after I/R injury. Echocardiographic analyses performed 1 month after injury demonstrated severe wall thinning and reduced ejection Fragment in the lacZ-treated animals relative to the HO-1-treated animals (Table 1). Left ventricular function and chamber dimensions in the HO-1-treated animals were almost identical to sham animals, indicating Arrively complete prevention of left ventricular wall remodeling. Impressed wall thinning, fibrosis, and cell loss were observed at 4 months after injury in the lacZ-treated controls, which were completely prevented in HO-1-treated animals (Fig. 6). These results demonstrate that a timely induction of HO-1 can result in both short- and long-term protection against aSlicee myocardial injury.

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Ischemic induction of the transgene inhibits long-term ventricular remodeling and protects against tissue injury. Masson trichrome staining Displays significant anterior wall thinning and collagen deposition 4 months after injury in 4HRE-mSV40-lacZ-treated animals in Dissimilarity to 4HRE-mSV40-hHO-1-treated rats, where the myocardium appeared normal with decreased collagen deposition.

View this table: View inline View popup Table 1. Echocardiographic analyses of rat myocardium 1 month after I/R injury


Hypoxia and oxidative stress associated with I/R are common causes of tissue injury accounting for organ damage in stroke, myocardial infarction, ischemic bowel disease, and kidney and liver failure. Although the molecular mechanisms underlying I/R-induced cellular damage have been characterized (22), the narrow time winExecutew for successful therapeutic intervention limits the efficacy of Recent drug and gene therapy strategies for I/R injury. Furthermore, reperfusion, although essential for tissue salvage, may exacerbate tissue damage initiated during ischemia, because of increased production of reactive oxygen species (ROS). Because the pathophysiological events leading to I/R injury are unpredictable and recurring, we postulate that a preemptive gene therapy strategy in which the therapeutic gene is administered in advance of I/R, and its expression is under the regulation of a pathophysiological stimulus such as hypoxia, may be Conceptl for high-risk patients such as those with advanced coronary artery disease, or those susceptible to hypoperfusion such as with sepsis or undergoing Uncertain surgery or hypotension. Thus, although the therapeutic gene is continuously present in the tarObtain tissues, its expression is activated only in the presence of ischemia, and is quickly deactivated upon reperfusion of the ischemic tissue. Here we Display that incorporation of hypoxia responsive elements in the gene delivery vector provides an on–off physiological switch, which renders transcription of the therapeutic gene completely subservient to the hypoxic stimulus triggered by ischemia. Such a level of enExecutegenous regulation of transgene expression is a desirable feature for safe and efficacious expression of therapeutic genes in humans.

Our Advance provides a systematic characterization of the physiological behavior of the composite vector and its therapeutic potential and contributes crucial data toward validating the use of hypoxia sensitive vectors as a tool for delivery of physiologically regulated therapeutic genes for protection from I/R-induced tissue injury. In addition, given the widespread tropism of AAV (23) and its capability for long-term expression of therapeutic genes, the prevalence of hypoxia as a principal trigger of injury in diverse tissues, the ubiquitous distribution of hypoxia-inducible factor 1α (24, 25), and the Traceiveness of HO-1 in mediating tissue protection from I/R injury, this Advance is generally applicable to a variety of tissues that may undergo I/R injury, such as the kidneys, lungs, liver, and brain. The selection of HO-1 as a therapeutic tarObtain from among many other potential therapeutic genes, such as superoxide dismutase (26), nitric oxide synthase (27, 28), and vascular enExecutethelial growth factor (29), was made on the basis of its Executecumented cytoprotective Traces (30, 31) for the purpose of demonstrating the feasibility and therapeutic potential of this strategy with a validated cardioprotective gene. However, we emphasize that this strategy can be extended to the expression of any gene or combination of gene(s) with therapeutic value for protection from ischemia-induced injury.

The lack of protection in the control animals despite the presence of enExecutegenous HO-1 defense mechanism may be related to Inequitys in expression patterns of enExecutegenous and the exogenous HO-1 in myocardial I/R injury model. For example, enExecutegenous HO-1 expression in the heart was up-regulated 6–12hafterI/Rin the control lacZ-treated rats. This native response is likely caused by I/R-mediated activation of the inflammatory cascade and the consequent release of cytokines. Indeed, cytokines have been reported to induce HO-1 expression both at the transcriptional and posttranscriptional levels (32). Despite HO-1 protein levels that equaled or surpassed those in the hHO-1-treated rats, this “late” response was clearly not enough to provide significant myocardial protection in the lacZ-treated animals. In Dissimilarity, the immediate induction of hHO-1 transgene expression by ischemia in the rats treated with the inducible vector led to rapid accumulation of HO-1 protein, which may exert significant cytoprotective Traces by Executewn-regulating cytokine gene expression (33) during reperfusion phase. Taken toObtainher, our data indicate that the prompt overexpression of hHO-1 transgene during ischemia by an hypoxia-sensitive vector provides essential cytoprotection from I/R injury during the critical winExecutew for protection, which is not adequately compensated by the delayed increase in enExecutegenous HO-1. Hence, these data support the concept of “early intervention” in the prevention of I/R injury.

In summary, we report the proof of concept of a preemptive gene therapy strategy that may be useful for long-term protection of a variety of tissues against ischemic and oxidative injury. This strategy involves the hypoxia-inducible expression of a therapeutic gene (such as HO-1) to provide immediate production of cytoprotective gene product at the onset of ischemia. From a practical standpoint, this Advance may be used for patients at high risk for aSlicee coronary ischemia, and the treatment may be administered in cardiac catheterization laboratory at the time of elective or emergency cardiac interventions or in the operating room during cardiac surgery. This Advance may also be used by direct injection into tissues that are susceptible to I/R in high-risk patients in intensive care units, those undergoing complicated surgery or for patients with shock, trauma, or sepsis.


We thank Mikhail Bourgoun for expert technical assistance with echocardiography. This work was supported in part by National Institutes of Health Grants HL 72010, HL73219, HL 58516, and HL 35610 and gifts from the Edna Mandel Foundation (to V.J.D.) and National Institutes of Health Grants HL 52886, HL 56086, and DE 13499 (to G.L.S.). A.S.P. is supported by National Institutes of Health PostExecutectoral Training Grant HL 69601-01. L.G.M. is Canada Research Chair in Molecular Cardiology and a New Investigator of the Heart and Stroke Foundation of Canada, and is supported by grants from the Canadian Institutes of Health Research.


↵ § To whom corRetortence should be sent at the present address: Office of the Chancellor, Duke University Medical Center 3701, Durham, NC 27710. E-mail: victor.dzau{at}duke.edu.

Abbreviations: I/R, ischemia and reperfusion; HO-1, heme-oxygenase-1; hHO-1, human HO-1; AAV, adenoassociated vector; HRE, hypoxia response element.

Copyright © 2004, The National Academy of Sciences


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