Hypoxia-specific gene expression for ischemic disease gene therapy

Advanced Drug Delivery Reviews 61 (2009) 614–622

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Advanced Drug Delivery Reviews j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / a d d r

Hypoxia-specific gene expression for ischemic disease gene therapy☆ Hyun Ah Kim a, Ram I. Mahato b, Minhyung Lee a,⁎ a b

Department of Bioengineering, College of Engineering, Hanyang University, Seoul 133-791, Republic of Korea Department of Pharmaceutical Sciences, University of Tennessee Health Science Center, Memphis, TN 38103-3308, USA

a r t i c l e

i n f o

Article history: Received 30 November 2008 Accepted 4 April 2009 Available online 23 April 2009 Keywords: Gene regulation Hypoxia response element Untranslated region Oxygen dependent degradation domain Ischemic disease Gene therapy

a b s t r a c t Gene therapy for ischemic diseases has been developed with various growth factors and anti-apoptotic genes. However, non-specific expression of therapeutic genes may induce deleterious side effects such as tumor formation. Hypoxia-specific regulatory systems can be used to regulate transgene expression in hypoxic tissues, in which gene expression is induced in ischemic tissues, but reduced in normal tissues by transcriptional, translational or post-translational regulation. Since hypoxia-inducible factor 1 (HIF-1) activates transcription of genes in hypoxic tissues, it can play an important role in the prevention of myocardial and cerebral ischemia. Hypoxia-specific promoters including HIF-1 binding sites have been used for transcriptional regulation of therapeutic genes. Also, hypoxia-specific untranslated regions (UTRs) and oxygen dependent degradation (ODD) domains have been investigated for translational and posttranslational regulations, respectively. Hypoxia-specific gene expression systems have been applied to various ischemic disease models, including ischemic myocardium, stroke, and injured spinal cord. This review examines the current status and future challenges of hypoxia-specific systems for safe and effective gene therapy of ischemic diseases. © 2009 Elsevier B.V. All rights reserved.

Contents 1. 2.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Hypoxia-regulated gene expression . . . . . . . . . . . . . . . . . . . 2.1. Transcriptional regulation . . . . . . . . . . . . . . . . . . . . 2.2. Post-transcriptional regulation . . . . . . . . . . . . . . . . . . 2.3. Post-translational regulation . . . . . . . . . . . . . . . . . . . 3. Ischemic disease-specific gene therapy with . . . . . . . . . . . . . . . 3.1. Transcriptional regulatory systems for ischemic disease gene therapy 3.2. Post-transcriptional regulation for ischemic disease gene therapy . . 3.3. Post-translational regulation for ischemic disease gene therapy . . . 3.4. Combination of regulatory strategies . . . . . . . . . . . . . . . 4. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction Hypoxia is a pathological condition that deprives adequate oxygen supply to organs or tissues and thus it regulates many cellular processes.

☆ This review is part of the Advanced Drug Delivery Reviews theme issue on “Gene Regulation for Effective Gene Therapy”. ⁎ Corresponding author. Department of Bioengineering, College of Engineering, Hanyang University, Haengdang-dong, Seongdong-gu, Seoul 133-791, Republic of Korea. Tel.: +82 2 2220 0484; fax: +82 2 2220 1998. E-mail address: [email protected] (M. Lee). 0169-409X/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.addr.2009.04.009

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Hypoxia has been implicated in many disease processes, including ischemic brain, ischemic myocardium, and injured spinal cord. In ischemic heart disease, the coronary arteries are narrowed and the blood supply to the myocardium is decreased. Due to low blood supply, oxygen and nutrients concentrations are not enough to maintain normal heart function. Decreased oxygen concentration activates hypoxia-inducible genes such as vascular endothelial growth factor (VEGF) [1–8]. Many gene products induced under hypoxia can protect cells from apoptosis and recover blood supply through neovascularization [9,10]. Hypoxia is a physiological signal for specific types of growth factors, anti-apoptotic and angiogenic genes. In stroke, hypoxia is an important

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hallmark of disease state. Blocking of cerebral artery causes low oxygen concentration in the brain. The ischemic brain undergoes physiological responses, which are similar to ischemic myocardium such as induction of protective factors and angiogenic factors [11–13]. In the case of stroke, blood supply must be recovered immediately. After reperfusion, blood supply is not fully recovered and brain cells are still under hypoxia, since microvessels are damaged during ischemia and reperfusion [14,15]. Due to reperfusion injury, the brain undergoes secondary injury by excitotoxic neurotransmitters and infarction area increases with time [16]. In spinal cord injury, the spinal cord also undergoes secondary injury from excitotoxicity and hypoxia [17]. Tumor hypoxia is a common feature to most solid tumors due to malformed vasculature and inadequate perfusion [18–20]. Tumor growth relies on the formation of new blood vessels, and in this process, several angiogenic factors including VEGF are induced [21,22]. In addition, severe hypoxia in the core of tumor often induces necrosis of the cells. Recently, it was suggested that high mobility group box-1 (HMGB-1) was released from the necrotic tissues [23]. Inside cells, HMGB-1 is a nuclear protein involved in gene regulation [24], while outside the cells, it is a cytokine, which binds to tall like receptors of infiltrating immune cells and induces nuclear factor-kappa B (NF-κB) [23,25–27]. This process increases VEGF gene expression in the tumor and promotes angiogenesis. Hypoxia is an important factor determining overall efficiency of tissue transplantation. Isolated islets from a donor are subjected to hypoxia due to the disruption of islet microvasculature during isolation from the pancreas. The hypoxic condition causes the islets to undergo apoptosis [28-30]. In addition, after transplantation, the blood supply to the islets is not sufficient for a significant period until new blood vessels are formed and oxygen supply is recovered. For this reason, islets after transplantation in diabetic patients suffer from high rates of cell death due to hypoxic damage [28-30]. Non-specific gene expression may induce deleterious effects and thus gene expression should be regulated. There are several reports on severe side effects when VEGF gene therapy is used for treating ischemic diseases [31]. VEGF is a potent angiogenic factor [4,32-39], and endogenous VEGF and its receptors are induced in the ischemic tissues [10]. However, this endogenous response to ischemic condition is usually not enough to recover normal state. Therefore, exogenous VEGF gene delivery may be beneficial for treating ischemic diseases. Since VEGF receptor is up-regulated in ischemic tissue, but not in normal tissue [10], non-specific VEGF expression may have minimal effect on normal tissue. Since unregulated VEGFmediated angiogenesis has the potential to promote tumor growth, accelerate diabetic proliferative retinopathy, and promote rupture of atherosclerotic plaque. [31,40], VEGF gene expression should be regulated. Targeted gene therapy can be achieved by two approaches. One approach is site-specific gene delivery by conjugating targeting ligands to gene carriers. The other approach to achieve targeted gene therapy is to regulate transcription with a cell-specific promoter. Several endogenous genes are regulated by hypoxia-specific transcription factors. Among them, hypoxia-inducible factor-1 (HIF-1) [7,41] is the key transcription factor, which binds to hypoxia response elements (HREs) and induces transcription in a hypoxic environment [1,42]. In addition to transcriptional regulation, posttranscriptional regulation or post-translational regulation strategies have been employed for hypoxia-inducible gene therapy. However, promoters specific to certain cell types or dependent on certain physiological condition usually have low promoter activity. Also, there are leaky expressions by hypoxia-specific promoters in normal tissue [43,44], possibly due to the basal promoter activity of these promoters. Various approaches have been taken to overcome these obstacles. In this review, basic regulatory mechanisms of hypoxia-specific gene expression are introduced and their applications to ischemic gene therapy are discussed.

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2. Hypoxia-regulated gene expression 2.1. Transcriptional regulation Several genes are induced in response to hypoxia at the transcriptional level. The most important transcription factor for hypoxia responsive gene expression is HIF-1 [1,6,7,12,45,46], which binds to specific cis-regulatory elements in promoters and facilitates transcription under hypoxic condition [1,7,47,48]. HIF-1 specific cis-regulatory elements are hypoxia response elements (HREs) and have a consensus sequence of (G/C/T)ACGTGC(G/C). Most hypoxia-inducible genes have multiple copies of HREs at their 5′-regulatory regions. HIF-1 is a heterodimeric protein composed of two subunits, HIF-1α, which is accumulated under hypoxic conditions, and HIF-1β, which is a constitutive subunit [45]. The level of HIF-1β is stable regardless of tissue oxygen concentration. However, HIF-1α levels change rapidly in response to oxygen concentration. The regulation of HIF-1α levels is an important regulatory step for hypoxia-specific gene expression. HIF-1α is specifically stabilized under hypoxic conditions and degraded rapidly under normoxia [49], a behavior that is mediated by the ubiquitin–proteasome pathway [40,49–52]. HIF-1α is hydroxylated by proline hydroxylases (PHDs), which are specifically activated under normoxia [49,50,53-56]. PHDs recognize specific proline residues in the oxygen dependent degradation (ODD) domain of HIF-1α. These hydroxylated prolines in the ODD domain are then recognized by the von Hippel Lindau tumor suppressor protein (pVHL). pVHL contains E3 ubiquitin ligase activity and polyubiquitnates the ODD domain. The proteasome-mediated pathway degrades the polyubiquitinated HIF-1α. However, PHDs are rapidly deactivated under hypoxia, which in turn stabilizes HIF-1α. Therefore, the accumulation of HIF-1α activates the transcription of specific genes under hypoxia. The HIF-1α level is also up-regulated by the stabilization of HIF-1α mRNA under hypoxia [57]. The half-life of the HIF-1α mRNA is higher under hypoxia than normoxia, which increases the translational level of the protein. As a result, the increased level of HIF-1α activates the transcription of hypoxia-inducible protein. Other transcription factors are also involved in hypoxia-inducible gene expression. For an example, stimulating protein-1 (SP-1) is a sequence-specific DNA-binding protein and is up-regulated under hypoxia condition [58–62]. Thus, Sp1 has been implicated in the regulation of various genes, since its binding sites are a recurrent motif in regulatory sequences of the hypoxia-inducible genes. Cyclooxygenease-2 (COX-2) or RTP801 was induced by Sp1 under hypoxia [58]. However, it is likely that Sp1 activates gene expression in cooperation with HIF-1. For example, the RTP801 and endoglin promoters have Sp1 and HIF-1 binding sites. It was suggested that Sp1 forms multiprotein complex with Smad3 and HIF-1, in which Smad3 is a coactivator and adaptor protein. Sp1–Smad3–HIF-1 complex on the promoter may cooperate to induce gene expression [59]. Hypoxic regulation of genes by transcription factors other than HIF-1 is not fully understood. However, advances in molecular biology for hypoxia-inducible gene expression may make it possible to develop more sophisticated regulatory systems for hypoxia-specific gene therapy. 2.2. Post-transcriptional regulation Post-transcriptional regulation can be achieved by controlling mRNA stability. For example, the erythropoietin (Epo) mRNA has prolonged half-life in hypoxic cells [8,63]. The up-regulation of Epo expression is mediated mainly by transcriptional regulation [64]. The Epo enhancer has HREs and the transcription level was induced by HIF1 under hypoxia. An Epo mRNA binding peptide (ERBP) binds to the AT-rich region of the Epo 3′-UTR under hypoxia, which increases the stability and steady-state level of the Epo mRNA [8]. As a result, the translation level of the mRNA increases, producing more protein. This hypoxia-specific stabilization is found in various genes. For example,

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VEGF mRNA is stabilized under hypoxia by protein binding to the VEGF UTR [10,65–67]. The stability of the VEGF mRNA was regulated by the cooperation of the 5′- and 3′-UTRs and coding region [67]. A previous report suggested that the VEGF 3′-UTR increased the stability of the target mRNA, when it was fused to a gene. However, the VEGF 3′-UTR also has an AT-rich region that destabilizes the mRNA and therefore, the 3′-UTR is not enough to increase the steady-state level of the mRNA [21]. Therefore, the combination of the 5′- and 3′-UTR of the VEGF mRNA is important for effective stabilization of a target mRNA. Tyrosine hydroxylase (TH) mRNA also has 3′-UTR and it was reported that the TH 3′-UTR increased the mRNA stability under hypoxia [65,68]. The same protein bound to the Epo, VEGF, and TH mRNA UTRs, suggesting that the mRNAs are stabilized by the same mechanism. The transferrin receptor and ferritin 3′-UTR also play a role in stabilizing mRNA under hypoxia [69]. The transferrin and ferritin 3′-UTRs have iron responsive elements (IREs), which may be responsible for hypoxic stabilization of these mRNAs [69]. As described above, the HIF-1α 3′-UTR contributes to the accumulation of the mRNA under hypoxia [57]. These UTRs seem to stabilize the linked mRNAs irrespective of their sequences. Epo 3′-UTR cDNA has previously been linked to the luciferase cDNA [70]. After transcription, Epo 3′-UTR specifically stabilized the luciferase mRNA, resulting in increased amount of luciferase protein. This suggests that the hypoxia-specific UTRs can be applied to various genes for gene therapy. Until now, the Epo 3′-UTR and VEGF 3′-UTR have been studied and only the Epo 3′-UTR showed successful results [70,71]. Studies with the various hypoxia-specific UTRs will provide more useful UTRs for gene therapy applications. 2.3. Post-translational regulation The HIF-1α level is regulated mainly by the control of protein stability [49]. The ODD domain in the middle of HIF-1α is responsible for this regulation. The ODD domain facilitates HIF-1α degradation under normoxia through proteasome-mediated pathways [49,51,53,72,73]. Protein stability regulation by the ODD domain had not been found in other hypoxia-inducible proteins. However, recently a novel ODD domain was identified in the activating transcription factor-4 (ATF-4) [74]. ATF-4 was induced by hypoxia and it seems that the novel ODD domain is involved in this process. However, this process is independent of VHL, suggesting the ATF-4 ODD domain follows the mechanism other than the HIF-1 ODD domain. The ODD domain can be applied to hypoxia-specific gene therapy. A fusion protein of the ODD domain and a therapeutic protein is stabilized under hypoxia and degraded rapidly under normoxia [75]. The rapid degradation under normoxia is useful to reduce protein level by leaky expression of hypoxia-specific promoters. In our study, the fusion protein of luciferase and the ODD domain was stabilized under hypoxia [75]. Luciferase levels were about 10 fold higher under hypoxia than normoxia. Therefore, a fusion protein of therapeutic protein with the ODD domain may be useful for ischemic regionspecific gene therapy. Finding of new ODD domains such as the ATF-4 ODD will increase the selection of ODD and eventually improve the efficiency of hypoxia-specific gene therapy. 3. Ischemic disease-specific gene therapy with hypoxia-inducible systems 3.1. Transcriptional regulatory systems for ischemic disease gene therapy Hypoxia-specific transcription systems have been developed using HREs, The simplest approach is to combine multiple copies of HREs with the viral basal promoters as hybrid systems [21,76–79]. The HREs from the Epo gene was combined with the SV40 promoter [79]. This Epo HREs-SV40 promoter was applied to VEGF gene therapy for ischemic heart disease. The Epo HREs-SV40 promoter regulated VEGF

gene was delivered to ischemic myocardium using adeno-associated viral (AAV) vectors or plasmid/lipopolymer complexes [79,80]. VEGF expression was induced in an ischemic myocardium by this system up to 20 folds. Since the physiological outcomes from the increased VEGF expression were not determined in these studies, further assessment of the hypoxia-inducible VEGF vector is required. Also, these studies showed that a considerable level of VEGF detected in normal myocardium, possibly due to its leaky expression. When various HREs were combined with basal promoters [77,81], the HRE from phosphoglycerate kinase-1 (PGK-1) gene showed the highest transcription activity under hypoxia. This hybrid promoter of PGK-1, HRE and SV40 promoter was referred to Oxford Biomedica HRE (OBHRE) [77,81]. The HREs from the Epo gene was also used for treating other ischemic diseases [82,83]. The Epo enhancer, which contained HREs, was combined with the SV40 promoter, and this promoter system was applied to gene therapy of spinal cord injuries [83]. In spinal cord injuries, the immediate damage is due to trauma, but delayed damage is due to excitotoxicity, altered ion balance, ischemia and immunological reaction [17,84]. Since it can protect neuronal cells from apoptosis and also promotes the formation of new blood vessels [84– 86], the Epo enhancer-SV40 promoter mediated VEGF expression vector was constructed and evaluated for treating injured spinal cord [82,83]. The hypoxia-specific VEGF vector reduced apoptotic cell death in the ischemic epicenter, compared with the VEGF vector without the Epo enhancer (Fig. 1). A behavioral examination using the Basso, Beattie, and Bresnahan (BBB) test showed that the Epo enhancer mediated VEGF expression vector facilitates recovery of the injured spinal cord animal model [83]. Apart from HREs and basal promoter hybrid systems, cellular hypoxia-inducible promoters have been used for gene therapy applications. These promoters have their own HREs and promoter function. For example, the RTP801 and the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoters have been used for hypoxia-specific gene expression [60,87-90]. The main advantage of these mammalian promoters is the reduction of promoter silencing effect. Although viral promoters induce much higher levels of transcription than mammalian promoters, they often fail to sustain transgene expression in vivo. Most viral promoters are prone to inactivation and silencing in vitro and in vivo due to hypermethylation in viral promoter region [72,91]. The promoter silencing effect is minimal in case of eukaryotic promoters [72]. Therefore, the use of strong eukaryotic promoters will allow us to achieve long-term expression in vivo. Moreover, the use of a cell-specific eukaryotic promoter adds an additional layer of specificity, and hence safety to gene transfer protocols by minimizing ectopic transgene expression. RTP801 gene is induced at the transcriptional level in response to various stresses such as hypoxia, glucocorticoids, and DNA damage. RTP801 promoter activity is greatly induced under hypoxia [92] and thus has been extensively investigated for hypoxia-specific gene therapy. We cloned the RTP801 promoter-based VEGF expression vector [60,87,89]. The RTP801 promoter showed stronger promoter activity and similar induction fold as Epo enhancer-SV40 promoter in 293 cells. Yockman et al. compared the RTP801 promoter to the SV40 promoter in terms of therapeutic efficacy in ischemic myocardium VEGF gene therapy, and found that VEGF expression was induced in ischemic myocardium injected with the RTP801-regulated VEGF gene [89]. In addition, fibrous and infracted tissue was reduced, suggesting that the RTP801 promoter is suitable for therapeutic application. We also tested the efficacy of the RTP801 promoter for erectile dysfunction gene therapy [88]. First, we showed that the HIF-1α protein was induced in the hypoxic corpus cavernosum using a high cholesterol diet erectile dysfunction animal model. Second, the VEGF gene may be useful to treat erectile dysfunction by promoting the formation of blood vessels to the corpus cavernosum. RTP801 promoter gene exhibited higher expression efficiency than the Epo

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Fig. 1. Anti-apoptotic effects of the hypoxia-inducible VEGF expression. PBS, pSV-VEGF, pRTP801-VEGF, and pEpo-SV-VEGF were injected directly to inured spinal cord. The spinal cord tissues were obtained at 2 days after plasmid injection. Tissue sections around the lesion area were examined for apoptotic cells by TUNEL staining. (A) The TUNEL positive cells were shown in green color and the nucleus was in red color. (B) The counting result was presented as mean values with standard errors from 3 independent experiments in the histogram. Reproduced with permission from Choi et al. J. Neurosurg. Spine, 7: 59, 2007.

enhancer-SV40 promoter in the corpus cavernosum. Kim et al. evaluated the role of the RTP801 promoter in ischemic region-specific expression of granulocyte-macrophage colony-stimulating factor (GM-CSF) by transfecting hypoxia-inducible GM-CSF plasmids (pEpo-SV-GM-CSF and pRTP801-GM-CSF) into SK-N-BE(2)C cells and demonstrated induced expression of GM-CSF under hypoxia and decrease in the hypoxia-induced cell death [93]. Since hypoxia-specific promoters usually have weak promoter activity, the two-step transcription amplification (TSTA) method has been employed to increase the promoter activity [94–98]. This approach includes two expression units (Fig. 2). The first expression unit expresses the fusion protein of the Gal4 DNA-binding domain (Gal4DBD) and the p65 transactivation domain (p65TAD) under the control of the hypoxiaspecific promoter system. The Gal4DBD-p65TAD fusion protein is a transcriptional activator, which is highly induced under hypoxia and binds to its target sequence, the Gal4 upstream activation sequence (UAS) in the second expression unit. Then, the therapeutic protein is over-expressed under the control of Gal4 UAS. Gal4 is a yeast transcription factor and its target sequence is not found in mammalian cells. Therefore, the binding specificity of Gal4DBD-p65TAD is extremely high and the TSTA system does not induce endogenous gene expression. Tang et al. showed that the TSTA system with the HRE-SV40 minimal promoter increased gene expression levels by more than 100 times in vitro, compared with a single expression vector [94]. In vivo evaluation of the hypoxia-specific TSTA system also showed much higher levels of

transgene expression in ischemic tissues [95–98]. However, the induction fold of the TSTA system under hypoxia compared with normoxia was hampered. Therefore, careful optimization of the TSTA system is required for high levels of hypoxia-specific gene expression [94]. The hypoxia-specific TSTA system was applied to ischemic myocardium gene therapy with the human heme-oxygenase-1 (hHO-1) gene [98]. hHO-1 is the key enzyme in heme degradation. hHO-1 expression is

Fig. 2. Amplification of hypoxia-specific promoter activity: Two-step transcription amplification system. The first unit produces an artificial transcription factor, which then binds to the control element of the second unit and induces expression.

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up-regulated in various ischemic tissues, including ischemic myocardium [2,99]. However, the over-expression of hHO-1 may induce tumor growth, necessitating careful regulation of hHO-1 expression [98]. hHO1 expression was restricted to the ischemic region when the hypoxiaspecific TSTA system of hHO-1 was injected into ischemic myocardium. Also, fibrous tissue in the infarct region was much reduced when the hypoxia-specific TSTA system of hHO-1 was used. The activity of each promoter system is different depending on the tissue type in which it is observed. For example, the effects of the Epo enhancer-SV40 promoter are stronger than those of the RTP801 promoter in ischemic neuronal tissue [82], but weaker in the ischemic corpus cavernosum [88]. In addition, transcription amplification of hypoxia-specific promoter had a tendency to reduce hypoxia-specific gene expression. Therefore, a hypoxia-specific promoter should be carefully optimized with the TSTA systems. Therefore, fine-tuning and careful selection of promoters may be required to increase therapeutic efficacy. 3.2. Post-transcriptional regulation for ischemic disease gene therapy Unlike transcriptional regulation, translational regulation of therapeutic genes in ischemic disease has not been thoroughly investigated. A limited number of studies, focusing on the UTRs of hypoxiaspecific genes, indicate its usefulness for ischemic disease gene therapy. The Epo 3′-UTR has been reported to increase the stability and half-life of Epo mRNA specifically under hypoxic conditions [70,71]. The Epo 3′-UTR could stabilize the mRNA regardless of its type, when it was linked to an mRNA [8,63]. This suggests that Epo 3′UTR can be used for translational regulation of various types of therapeutic genes. To test this possibility, we constructed a pSV-LucEpoUTR, in which Epo 3′-UTR sequence was located downstream of luciferase gene. The construct expressed a fusion mRNA of luciferase gene and Epo 3′-UTR [70]. An in vitro transfection assay showed that Epo 3′-UTR stabilized the mRNA in a hypoxia-specific manner. Luciferase activity assay showed luciferase level to be about 10 times higher in pSV-Luc-EpoUTR transfected cells than pSV-Luc transfected cells. Epo 3′-UTR was also integrated into a VEGF expression vector. An in vitro transfection assay showed that the level of VEGF mRNA was induced under hypoxia by Epo 3′-UTR [70]. Also, VEGF protein level was also increased by Epo 3′-UTR, compared with the VEGF vector without Epo 3′-UTR. Luciferase expression vector with Epo 3′-UTR was evaluated in injured spinal cord in vivo [71]. In spinal cord injury animal model, luciferase expression was upregulated by Epo 3′-UTR. Luciferase mRNA level was also increased by Epo 3′-UTR, suggesting that the increase in luciferase expression is due to the enhanced mRNA stability. Previous reports indicate that the same protein binds to Epo 3′UTR and VEGF 3′-UTR, suggesting that VEGF 3′-UTR may be useful for ischemic disease gene therapy [65]. While VEGF mRNA is stabilized by the cooperation of the 5′- and 3′-UTRs [67], its 3′-UTR has a

destabilizing AT-rich region [21]. Therefore, further optimization of VEGF 3′-UTR is required before it can be widely applied for ischemic disease gene therapy. The tyrosine hydroxylase (TH) 3′-UTR and HIF-1α 3′-UTR are other examples of hypoxia-specific UTRs [57,65]. Transferrin 3′-UTR and ferritin 3′-UTR both have iron-response elements (IRE) [69], which were reported to be involved in the stabilization of mRNAs under hypoxia. The evaluation of UTRs for ischemic disease gene therapy is ongoing. 3.3. Post-translational regulation for ischemic disease gene therapy The stability of therapeutic proteins can be regulated using the ODD domain. We previously constructed a hypoxia-specific luciferase expression vector using the ODD domain [75], in which the ODD domain was located downstream of the luciferase gene. For the expression of the luciferase and ODD domain fusion protein, the termination codon of luciferase cDNA was eliminated and a new termination codon was located at the 3′-end of the ODD domain. Luciferase-ODD construct showed 10-fold higher expression under hypoxia than normoxia. A reoxygenation study showed that gene expression rapidly decreased when oxygen levels recovered. The ODD fused therapeutic gene may have a lower protein activity. The ODD domain is relatively long, composed of about 200 amino acids, and may interfere with the normal folding of the therapeutic protein, impairing its activity. The ODD may also reduce the secretion of proteins such as VEGF, when VEGF is fused with the ODD domain. A shorter ODD domain may be useful to facilitate effective folding and secretion of therapeutic proteins. The previous report showed that 18 core amino acids in the HIF-1α ODD have oxygen dependent protein regulation effect [100]. Therefore, the creation of short ODD domains may increase their therapeutic effects in ischemic disease gene therapy. Tang et al. [96] combined the ODD domain with the TSTA system by inserting it between the Gal4DBD and the p65TAD promoters (Fig. 3). The resulting transcription factor was active under hypoxia, but rapidly degraded under normoxia. Under hypoxia, Gal4DBD-ODDp65TAD was stabilized and bound to the Gal4 UAS, increasing gene expression. This system was applied to stroke gene therapy, in which a neuronal specific promoter controlled the expression of Gal4DBDODD-p65TAD in neuronal cells. 3.4. Combination of regulatory strategies Transcriptional, translational, and post-translational regulation of gene expression is independent of each other. Therefore, a simple combination of regulation strategies may increase the specificity of gene therapy (Fig. 4). One example is the combination of transcriptional regulation with the Epo enhancer-SV40 promoter and posttranslational regulation with the ODD domain [75] (Fig. 5A). We showed that the combination of these two strategies increased specificity. When the construct, pEpo-SV-Luc-ODD, was transfected

Fig. 3. Hypoxia-specific amplification of promoter activity. The ODD domain was combined with the TSTA system by inserting it between the Gal4DBD and the p65TAD promoters. The resulting transcription factor was specifically active under hypoxia, but rapidly degraded under normoxia. Under hypoxia, Gal4DBD-ODD-p65TAD was stabilized and bound to the Gal4 UAS, increasing gene expression.

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Fig. 4. A combination of transcriptional, translational and post-translational regulation.

into neuro2A cells, luciferase expression was induced by more than 1,000-fold under hypoxia compared with normoxia (Fig. 5B). This upregulation was highly specific in ischemic tissue, specific enough for ischemic tissue imaging [101]. The combination of transcriptional regulation via the Epo enhancerSV40 promoter and translation regulation with the Epo 3′-UTR has also been investigated [70]. Constructs combining luciferase with the Epo enhancer-SV40 promoter or the Epo 3′-UTR demonstrated luciferase induction (Fig. 6A). When the two regulatory elements were combined

Fig. 5. pEpo-SV-Luc-ODD, a combination plasmid of transcriptional and posttranslational regulations. (A) Map of pEpo-SV-Luc-ODD, (B) pSV-Luc, pEpo-SV-Luc, and pEpo-SV-Luc-ODD were transfected into Neuro2A cells. The cells were incubated under normoxia or hypoxia for 20 h and assessed for luciferase activity. The data expressed as mean values (± standard deviation) of four experiments. Reproduced with permission from Kim et al., J. Control. Release, 121: 222, 2007.

into one expression plasmid, the combination of the Epo enahncer-SV40 promoter and the Epo 3′-UTR increased specificity of gene expression in 293 cells (Fig. 6B). However, in neuronal cells, transcriptional regulation with the Epo enhancer-SV40 promoter was dominant and the effect of the Epo 3′-UTR was negligible [71]. This may be due to the high transcription activity of the Epo enhancer in neuronal cells. Therefore, a combination of regulatory strategies should be optimized depending on the target disease and tissue type.

Fig. 6. pEpo-SV-Luc-Epo 3′-UTR, a combination plasmid of transcriptional and posttranscriptional regulations. (A) Map of pEpo-SV-Luc-Epo 3′-UTR, (B) pSV-Luc, pEpo-SVLuc, pSV-Luc-Epo 3′-UTR, pEpo-SV-Luc-Epo 3′-UTR were transfected into A7R5 cells. The cells were incubated under normoxia or hypoxia for 44 h and assessed for luciferase activity. The luciferase activity of each plasmid under normoxia was set as 100% and the relative luciferase activity under hypoxia was presented. The data expressed as mean values (± standard deviation) of three experiments. Reproduced with permission from Lee et al., J. Control. Release, 115: 116, 2006.

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4. Conclusion Hypoxia is a serious impediment to normal tissue function. To maintain cell function, endogenous genes have their own expression regulatory systems for hypoxic conditions. In ischemic diseases, these regulatory systems are turned on and may actively increase or decrease specific gene expression. For ischemic gene therapy, these regulatory systems can be converted for therapeutic gene expression. Using such systems, clinicians may avoid unwanted side effects and maximize therapeutic efficacy. Many researchers have been trying to develop more specific and safer hypoxia-specific gene expression systems. While the efficacy of such systems has been improved, there are still challenges to be addressed. First, there is basal level expression in the hypoxia-specific gene expression system. Although the basal promoter has low promoter activity, a basal level of exogenous therapeutic gene expression in unintended regions may induce a serious deleterious effect. For this reason, implementation of normoxia-reducible systems, such as the ODD domain, may be as important as application of hypoxia-inducible gene expression systems. Previously, the diphtheria toxin was fused to the ODD domain [102], resulting in significantly reduced expression of DTAODD in normal tissue and restricting any possible toxic effects. Second, the hypoxia-specific promoter often has low promoter activity. Therefore, the TSTA system was applied to a hypoxia-specific gene expression system. The TSTA system increased basal gene expression levels, but induction folding was decreased. Another possible strategy is to combine a hypoxia-specific promoter with strong viral enhancers, which may increase overall expression levels. To avoid the compromise of hypoxia specificity, careful optimization of the combination system is required. Third, modification of the fused therapeutic protein with the ODD domain may reduce the biological and therapeutic effect of the protein. Therefore, a more compact and shorter ODD domain should be developed. The applications of hypoxia-specific gene expression systems are broad, with several possible target diseases including ischemic heart disease, cancer, stroke, spinal cord injury, and erectile dysfunction. Hypoxia is also an important factor for the successful outcome of islet transplantation. Anti-apoptotic or growth factor genes have been investigated to protect these islets from ischemic damage. In the previous study, hemangioma was induced in VEGF transfected islets, even though the VEGF gene was delivered to the islets with a liposomal carrier [103]. Since liposomal carriers have far less transfection efficiency in islets than viral vectors, it is important to regulate VEGF expression in islets. Hypoxia-specific gene expression systems are important for safe and effective gene therapy. Recent advances in molecular biology will help develop effective hypoxia responsive gene regulation systems. These regulatory systems will be a combination of transcriptional, translational, and post-translational regulations. With various efforts to develop highly hypoxia-specific gene expression systems, gene regulatory systems will be useful for developing safe and effective ischemic disease gene therapy.

[3]

[4]

[5]

[6]

[7]

[8]

[9]

[10]

[11]

[12]

[13]

[14] [15]

[16] [17] [18]

[19] [20]

[21] [22]

[23]

[24] [25]

[26]

Acknowledgments [27]

This work was supported by Korea Ministry of Knowledge Economy under the KORUS Tech Program (Grant No. KT-2008-NT-APFS0-0001). [28]

References [1] N.V. Iyer, L.E. Kotch, F. Agani, S.W. Leung, E. Laughner, R.H. Wenger, M. Gassmann, J.D. Gearhart, A.M. Lawler, A.Y. Yu, G.L. Semenza, Cellular and developmental control of O2 homeostasis by hypoxia-inducible factor 1 alpha, Genes Dev. 12 (2) (1998) 149–162. [2] S.F. Yet, R. Tian, M.D. Layne, Z.Y. Wang, K. Maemura, M. Solovyeva, B. Ith, L.G. Melo, L. Zhang, J.S. Ingwall, V.J. Dzau, M.E. Lee, M.A. Perrella, Cardiac-specific expression

[29]

[30] [31]

of heme oxygenase-1 protects against ischemia and reperfusion injury in transgenic mice, Circ. Res. 89 (2) (2001) 168–173. A. Dembinska-Kiec, J. Dulak, L. Partyka, R. Krzesz, D. Dudek, S. Bartus, M. Polus, I. Guevara, I. Wybranska, T. Krzeminski, Induction of nitric oxide synthase (NOS) and vascular endothelial growth factor (VEGF) in experimental model of angioplasty and heart ischemia, Adv. Exp. Med. Biol. 433 (1997) 163–167. P. Carmeliet, Basic Concepts of (Myocardial) Angiogenesis: Role of Vascular Endothelial Growth Factor and Angiopoietin, Curr. Interv. Cardiol. Rep. 1 (4) (1999) 322–335. L. Jingjing, B. Srinivasan, X. Bian, H.F. Downey, R.S. Roque, Vascular endothelial growth factor is increased following coronary artery occlusion in the dog heart, Mol. Cell. Biochem. 214 (1–2) (2000) 23–30. G.L. Semenza, P.H. Roth, H.M. Fang, G.L. Wang, Transcriptional regulation of genes encoding glycolytic enzymes by hypoxia-inducible factor 1, J. Biol. Chem. 269 (38) (1994) 23757–23763. G.L. Wang, G.L. Semenza, General involvement of hypoxia-inducible factor 1 in transcriptional response to hypoxia, Proc. Natl. Acad. Sci. USA 90 (9) (1993) 4304–4308. I.J. Rondon, L.A. MacMillan, B.S. Beckman, M.A. Goldberg, T. Schneider, H.F. Bunn, J.S. Malter, Hypoxia up-regulates the activity of a novel erythropoietin mRNA binding protein, J. Biol. Chem. 266 (25) (1991) 16594–16598. R. Kacimi, J. Chentoufi, N. Honbo, C.S. Long, J.S. Karliner, Hypoxia differentially regulates stress proteins in cultured cardiomyocytes: role of the p38 stressactivated kinase signaling cascade, and relation to cytoprotection, Cardiovasc. Res. 46 (1) (2000) 139–150. E. Brogi, G. Schatteman, T. Wu, E.A. Kim, L. Varticovski, B. Keyt, J.M. Isner, Hypoxiainduced paracrine regulation of vascular endothelial growth factor receptor expression, J. Clin. Invest. 97 (2) (1996) 469–476. H. Beck, T. Acker, C. Wiessner, P.R. Allegrini, K.H. Plate, Expression of angiopoietin1, angiopoietin-2, and tie receptors after middle cerebral artery occlusion in the rat, Am. J. Pathol. 157 (5) (2000) 1473–1483. G.L. Semenza, F. Agani, D. Feldser, N. Iyer, L. Kotch, E. Laughner, A. Yu, Hypoxia, HIF-1, and the pathophysiology of common human diseases, Adv. Exp. Med. Biol. 475 (2000) 123–130. M.D. Ginsberg, Adventures in the pathophysiology of brain ischemia: penumbra, gene expression, neuroprotection: the 2002 Thomas Willis Lecture, Stroke 34 (1) (2003) 214–223. J.M. McCord, Oxygen-derived free radicals in postischemic tissue injury, N. Engl. J. Med. 312 (3) (1985) 159–163. K. Blomgren, C. Zhu, U. Hallin, H. Hagberg, Mitochondria and ischemic reperfusion damage in the adult and in the developing brain, Biochem. Biophys. Res. Commun. 304 (3) (2003) 551–559. S.J. Won, D.Y. Kim, B.J. Gwag, Cellular and molecular pathways of ischemic neuronal death, J. Biochem. Mol. Biol. 35 (1) (2002) 67–86. C.H. Tator, M.G. Fehlings, Review of the secondary injury theory of acute spinal cord trauma with emphasis on vascular mechanisms, J. Neurosurg. 75 (1) (1991) 15–26. G.U. Dachs, A.V. Patterson, J.D. Firth, P.J. Ratcliffe, K.M. Townsend, I.J. Stratford, A.L. Harris, Targeting gene expression to hypoxic tumor cells, Nat. Med. 3 (5) (1997) 515–520. G.U. Dachs, G.J. Dougherty, I.J. Stratford, D.J. Chaplin, Targeting gene therapy to cancer: a review, Oncol. Res. 9 (6–7) (1997) 313–325. S. Kress, A. Stein, P. Maurer, B. Weber, J. Reichert, A. Buchmann, P. Huppert, M. Schwarz, Expression of hypoxia-inducible genes in tumor cells, J. Cancer Res. Clin. Oncol. 124 (6) (1998) 315–320. T. Shibata, A.J. Giaccia, J.M. Brown, Development of a hypoxia-responsive vector for tumor-specific gene therapy, Gene Ther. 7 (6) (2000) 493–498. D.G. Bae, Y.S. Gho, W.H. Yoon, C.B. Chae, Arginine-rich anti-vascular endothelial growth factor peptides inhibit tumor growth and metastasis by blocking angiogenesis, J. Biol. Chem. 275 (18) (2000) 13588–13596. T. Sasahira, T. Kirita, U.K. Bhawal, M. Ikeda, A. Nagasawa, K. Yamamoto, H. Kuniyasu, The expression of receptor for advanced glycation end products is associated with angiogenesis in human oral squamous cell carcinoma, Virchows Arch. 450 (3) (2007) 287–295. S. Banerjee, T.K. Kundu, The acidic C-terminal domain and A-box of HMGB-1 regulates p53-mediated transcription, Nucleic Acids Res. 31 (12) (2003) 3236–3247. J. Qiu, M. Nishimura, Y. Wang, J.R. Sims, S. Qiu, S.I. Savitz, S. Salomone, M.A. Moskowitz, Early release of HMGB-1 from neurons after the onset of brain ischemia, J. Cereb. Blood Flow Metab. 28 (5) (2008) 927–938. M.A. van Zoelen, H. Yang, S. Florquin, J.C. Meijers, S. Akira, B. Arnold, P.P. Nawroth, A. Bierhaus, K.J. Tracey, T. van der Poll, Role of Toll-Like Receptors 2 and 4, and the Receptor for Advanced Glycation End Products (Rage) in Hmgb1 Induced Inflammation in Vivo, Shock 31 (3) (2009) 280–284. M.A. van Zoelen, H. Yang, S. Florquin, J.C. Meijers, S. Akira, B. Arnold, P.P. Nawroth, A. Bierhaus, K.J. Tracey, T. Poll, Role of toll-like receptors 2 and 4, and the receptor for advanced glycation end products in high-mobility group box 1-induced inflammation in vivo, Shock 31 (3) (2009) 280–284. K.E. Dionne, C.K. Colton, M.L. Yarmush, Effect of oxygen on isolated pancreatic tissue, ASAIO Trans. 35 (3) (1989) 739–741. J. Schrezenmeir, L. Gero, C. Laue, J. Kirchgessner, A. Muller, A. Huls, R. Passmann, H.J. Hahn, L. Kunz, W. Mueller-Klieser, The role of oxygen supply in islet transplantation, Transplant. Proc. 24 (6) (1992) 2925–2929. H.A. Kim, B.W. Lee, D. Kang, J.H. Kim, S.H. Ihm, M. Lee, Delivery of hypoxia-inducible VEGF gene to rat islets using polyethylenimine, J. Drug Target. 17 (1) (2009) 1–9. R.J. Lee, M.L. Springer, W.E. Blanco-Bose, R. Shaw, P.C. Ursell, H.M. Blau, VEGF gene delivery to myocardium: deleterious effects of unregulated expression, Circulation 102 (8) (2000) 898–901.

H.A. Kim et al. / Advanced Drug Delivery Reviews 61 (2009) 614–622 [32] S. Takeshita, L.P. Zheng, E. Brogi, M. Kearney, L.Q. Pu, S. Bunting, N. Ferrara, J.F. Symes, J.M. Isner, Therapeutic angiogenesis. A single intraarterial bolus of vascular endothelial growth factor augments revascularization in a rabbit ischemic hind limb model, J. Clin. Invest. 93 (2) (1994) 662–670. [33] Z.G. Zhang, L. Zhang, Q. Jiang, R. Zhang, K. Davies, C. Powers, N. Bruggen, M. Chopp, VEGF enhances angiogenesis and promotes blood-brain barrier leakage in the ischemic brain, J. Clin. Invest. 106 (7) (2000) 829–838. [34] K.A. Vincent, K.G. Shyu, Y. Luo, M. Magner, R.A. Tio, C. Jiang, M.A. Goldberg, G.Y. Akita, R.J. Gregory, J.M. Isner, Angiogenesis is induced in a rabbit model of hindlimb ischemia by naked DNA encoding an HIF-1alpha/VP16 hybrid transcription factor, Circulation 102 (18) (2000) 2255–2261. [35] P. Carmeliet, E. Storkebaum, Vascular and neuronal effects of VEGF in the nervous system: implications for neurological disorders, Semin. Cell Dev. Biol. 13 (1) (2002) 39–53. [36] M.J. Kutryk, D.J. Stewart, Angiogenesis of the heart, Microsc. Res. Tech. 60 (2) (2003) 138–158. [37] A.J. Siddiqui, P. Blomberg, E. Wardell, I. Hellgren, M. Eskandarpour, K.B. Islam, C. Sylven, Combination of angiopoietin-1 and vascular endothelial growth factor gene therapy enhances arteriogenesis in the ischemic myocardium, Biochem. Biophys. Res. Commun. 310 (3) (2003) 1002–1009. [38] A.V. Benest, A.H. Salmon, W. Wang, C.P. Glover, J. Uney, S.J. Harper, D.O. Bates, VEGF and angiopoietin-1 stimulate different angiogenic phenotypes that combine to enhance functional neovascularization in adult tissue, Microcirculation 13 (6) (2006) 423–437. [39] J.K. Ryu, C.H. Cho, H.Y. Shin, S.U. Song, S.M. Oh, M. Lee, S. Piao, J.Y. Han, I.H. Kim, G.Y. Koh, J.K. Suh, Combined angiopoietin-1 and vascular endothelial growth factor gene transfer restores cavernous angiogenesis and erectile function in a rat model of hypercholesterolemia, Mol. Ther. 13 (4) (2006) 705–715. [40] M.L. Springer, A.S. Chen, P.E. Kraft, M. Bednarski, H.M. Blau, VEGF gene delivery to muscle: potential role for vasculogenesis in adults, Mol. Cell 2 (5) (1998) 549–558. [41] G.L. Semenza, HIF-1 and mechanisms of hypoxia sensing, Curr. Opin. Cell Biol. 13 (2) (2001) 167–171. [42] G. Ambrosini, A.K. Nath, M.R. Sierra-Honigmann, J. Flores-Riveros, Transcriptional activation of the human leptin gene in response to hypoxia. Involvement of hypoxia-inducible factor 1, J. Biol. Chem. 277 (37) (2002) 34601–34609. [43] S.A. Nicklin, P.N. Reynolds, M.J. Brosnan, S.J. White, D.T. Curiel, A.F. Dominiczak, A.H. Baker, Analysis of cell-specific promoters for viral gene therapy targeted at the vascular endothelium, Hypertension 38 (1) (2001) 65–70. [44] D.Y. Hwang, W.A. Carlezon Jr., O. Isacson, K.S. Kim, A high-efficiency synthetic promoter that drives transgene expression selectively in noradrenergic neurons, Hum. Gene Ther. 12 (14) (2001) 1731–1740. [45] B.H. Jiang, E. Rue, G.L. Wang, R. Roe, G.L. Semenza, Dimerization, DNA binding, and transactivation properties of hypoxia-inducible factor 1, J. Biol. Chem. 271 (30) (1996) 17771–17778. [46] R.H. Wenger, Mammalian oxygen sensing, signalling and gene regulation, J. Exp. Biol. 203 (Pt 8) (2000) 1253–1263. [47] B.H. Jiang, J.Z. Zheng, S.W. Leung, R. Roe, G.L. Semenza, Transactivation and inhibitory domains of hypoxia-inducible factor 1alpha. Modulation of transcriptional activity by oxygen tension, J. Biol. Chem. 272 (31) (1997) 19253–19260. [48] K. Miyazaki, T. Kawamoto, K. Tanimoto, M. Nishiyama, H. Honda, Y. Kato, Identification of functional hypoxia response elements in the promoter region of the DEC1 and DEC2 genes, J. Biol. Chem. 277 (49) (2002) 47014–47021. [49] L.E. Huang, J. Gu, M. Schau, H.F. Bunn, Regulation of hypoxia-inducible factor 1alpha is mediated by an O2-dependent degradation domain via the ubiquitin– proteasome pathway, Proc. Natl. Acad. Sci. USA 95 (14) (1998) 7987–7992. [50] J.W. Lee, S.H. Bae, J.W. Jeong, S.H. Kim, K.W. Kim, Hypoxia-inducible factor (HIF-1) alpha: its protein stability and biological functions, Exp. Mol. Med. 36 (1) (2004) 1–12. [51] N. Masson, C. Willam, P.H. Maxwell, C.W. Pugh, P.J. Ratcliffe, Independent function of two destruction domains in hypoxia-inducible factor-alpha chains activated by prolyl hydroxylation, Embo J. 20 (18) (2001) 5197–5206. [52] A.J. Soitamo, C.M. Rabergh, M. Gassmann, L. Sistonen, M. Nikinmaa, Characterization of a hypoxia-inducible factor (HIF-1alpha ) from rainbow trout. Accumulation of protein occurs at normal venous oxygen tension, J. Biol. Chem. 276 (23) (2001) 19699–19705. [53] L.E. Huang, E.A. Pete, M. Schau, J. Milligan, J. Gu, Leu-574 of HIF-1alpha is essential for the von Hippel-Lindau (VHL)-mediated degradation pathway, J. Biol. Chem. 277 (44) (2002) 41750–41755. [54] G. D'Angelo, E. Duplan, N. Boyer, P. Vigne, C. Frelin, Hypoxia up-regulates prolyl hydroxylase activity: a feedback mechanism that limits HIF-1 responses during reoxygenation, J. Biol. Chem. 278 (40) (2003) 38183–38187. [55] S. Khanna, S. Roy, M. Maurer, R.R. Ratan, C.K. Sen, Oxygen-sensitive reset of hypoxia-inducible factor transactivation response: prolyl hydroxylases tune the biological normoxic set point, Free Radic. Biol. Med. 40 (12) (2006) 2147–2154. [56] Y. Pan, K.D. Mansfield, C.C. Bertozzi, V. Rudenko, D.A. Chan, A.J. Giaccia, M.C. Simon, Multiple factors affecting cellular redox status and energy metabolism modulate hypoxia-inducible factor prolyl hydroxylase activity in vivo and in vitro, Mol. Cell. Biol. 27 (3) (2007) 912–925. [57] A. Gorlach, G. Camenisch, I. Kvietikova, L. Vogt, R.H. Wenger, M. Gassmann, Efficient translation of mouse hypoxia-inducible factor-1alpha under normoxic and hypoxic conditions, Biochim. Biophys. Acta 1493 (1–2) (2000) 125–134. [58] Q. Xu, Y.S. Ji, J.F. Schmedtje Jr., Sp1 increases expression of cyclooxygenase-2 in hypoxic vascular endothelium. Implications for the mechanisms of aortic aneurysm and heart failure, J. Biol. Chem. 275 (32) (2000) 24583–24589. [59] T. Sanchez-Elsner, L.M. Botella, B. Velasco, C. Langa, C. Bernabeu, Endoglin expression is regulated by transcriptional cooperation between the hypoxia and transforming growth factor-beta pathways, J. Biol. Chem. 277 (46) (2002) 43799–43808.

621

[60] M. Lee, M. Bikram, S. Oh, D.A. Bull, S.W. Kim, Sp1-dependent regulation of the RTP801 promoter and its application to hypoxia-inducible VEGF plasmid for ischemic disease, Pharm. Res. 21 (5) (2004) 736–741. [61] M. Lee, S.U. Song, J.K. Ryu, J.K. Suh, Sp1-dependent regulation of the tissue inhibitor of metalloproteinases-1 promoter, J. Cell. Biochem. 91 (6) (2004) 1260–1268. [62] E.P. Cummins, C.T. Taylor, Hypoxia-responsive transcription factors, Pflugers Arch. 450 (6) (2005) 363–371. [63] E.C. McGary, I.J. Rondon, B.S. Beckman, Post-transcriptional regulation of erythropoietin mRNA stability by erythropoietin mRNA-binding protein, J. Biol. Chem. 272 (13) (1997) 8628–8634. [64] P.H. Maxwell, C.W. Pugh, P.J. Ratcliffe, Inducible operation of the erythropoietin 3′ enhancer in multiple cell lines: evidence for a widespread oxygen-sensing mechanism, Proc. Natl. Acad. Sci. USA 90 (6) (1993) 2423–2427. [65] A.B. Scandurro, B.S. Beckman, Common proteins bind mRNAs encoding erythropoietin, tyrosine hydroxylase, and vascular endothelial growth factor, Biochem. Biophys. Res. Commun. 246 (2) (1998) 436–440. [66] A.P. Levy, N.S. Levy, M.A. Goldberg, Post-transcriptional regulation of vascular endothelial growth factor by hypoxia, J. Biol. Chem. 271 (5) (1996) 2746–2753. [67] J.A. Dibbens, D.L. Miller, A. Damert, W. Risau, M.A. Vadas, G.J. Goodall, Hypoxic regulation of vascular endothelial growth factor mRNA stability requires the cooperation of multiple RNA elements, Mol. Biol. Cell 10 (4) (1999) 907–919. [68] W.R. Paulding, M.F. Czyzyk-Krzeska, Regulation of tyrosine hydroxylase mRNA stability by protein-binding, pyrimidine-rich sequence in the 3′-untranslated region, J. Biol. Chem. 274 (4) (1999) 2532–2538. [69] B.D. Schneider, E.A. Leibold, Effects of iron regulatory protein regulation on iron homeostasis during hypoxia, Blood 102 (9) (2003) 3404–3411. [70] M. Lee, D. Choi, M.J. Choi, J.H. Jeong, W.J. Kim, S. Oh, Y.H. Kim, D.A. Bull, S.W. Kim, Hypoxia-inducible gene expression system using the erythropoietin enhancer and 3′-untranslated region for the VEGF gene therapy, J. Control. Release 115 (1) (2006) 113–119. [71] B.H. Choi, Y. Ha, C.H. Ahn, X. Huang, J.M. Kim, S.R. Park, H. Park, H.C. Park, S.W. Kim, M. Lee, A hypoxia-inducible gene expression system using erythropoietin 3′ untranslated region for the gene therapy of rat spinal cord injury, Neurosci. Lett. 412 (2) (2007) 118–122. [72] D.R. Gill, S.E. Smyth, C.A. Goddard, I.A. Pringle, C.F. Higgins, W.H. Colledge, S.C. Hyde, Increased persistence of lung gene expression using plasmids containing the ubiquitin C or elongation factor 1alpha promoter, Gene Ther. 8 (20) (2001) 1539–1546. [73] F. Wang, H. Sekine, Y. Kikuchi, C. Takasaki, C. Miura, O. Heiwa, T. Shuin, Y. FujiiKuriyama, K. Sogawa, HIF-1alpha-prolyl hydroxylase: molecular target of nitric oxide in the hypoxic signal transduction pathway, Biochem. Biophys. Res. Commun. 295 (3) (2002) 657–662. [74] J. Koditz, J. Nesper, M. Wottawa, D.P. Stiehl, G. Camenisch, C. Franke, J. Myllyharju, R.H. Wenger, D.M. Katschinski, Oxygen-dependent ATF-4 stability is mediated by the PHD3 oxygen sensor, Blood 110 (10) (2007) 3610–3617. [75] H.A. Kim, K. Kim, S.W. Kim, M. Lee, Transcriptional and post-translational regulatory system for hypoxia specific gene expression using the erythropoietin enhancer and the oxygen-dependent degradation domain, J. Control. Release 121 (3) (2007) 218–224. [76] T. Shibata, N. Akiyama, M. Noda, K. Sasai, M. Hiraoka, Enhancement of gene expression under hypoxic conditions using fragments of the human vascular endothelial growth factor and the erythropoietin genes, Int. J. Radiat. Oncol. Biol. Phys. 42 (4) (1998) 913–916. [77] K. Binley, S. Iqball, A. Kingsman, S. Kingsman, S. Naylor, An adenoviral vector regulated by hypoxia for the treatment of ischaemic disease and cancer, Gene Ther. 6 (10) (1999) 1721–1727. [78] N. Koshikawa, K. Takenaga, M. Tagawa, S. Sakiyama, Therapeutic efficacy of the suicide gene driven by the promoter of vascular endothelial growth factor gene against hypoxic tumor cells, Cancer Res. 60 (11) (2000) 2936–2941. [79] H. Su, J. Arakawa-Hoyt, Y.W. Kan, Adeno-associated viral vector-mediated hypoxia response element-regulated gene expression in mouse ischemic heart model, Proc. Natl. Acad. Sci. USA 99 (14) (2002) 9480–9485. [80] M. Lee, J. Rentz, M. Bikram, S. Han, D.A. Bull, S.W. Kim, Hypoxia-inducible VEGF gene delivery to ischemic myocardium using water-soluble lipopolymer, Gene Ther. 10 (18) (2003) 1535–1542. [81] K. Boast, K. Binley, S. Iqball, T. Price, H. Spearman, S. Kingsman, A. Kingsman, S. Naylor, Characterization of physiologically regulated vectors for the treatment of ischemic disease, Hum. Gene Ther. 10 (13) (1999) 2197–2208. [82] M. Lee, E.S. Lee, Y.S. Kim, B.H. Choi, S.R. Park, H.S. Park, H.C. Park, S.W. Kim, Y. Ha, Ischemic injury-specific gene expression in the rat spinal cord injury model using hypoxia-inducible system, Spine 30 (24) (2005) 2729–2734. [83] B.H. Choi, Y. Ha, X. Huang, S.R. Park, J. Chung, D.K. Hyun, H. Park, H.C. Park, S.W. Kim, M. Lee, Hypoxia-inducible expression of vascular endothelial growth factor for the treatment of spinal cord injury in a rat model, J. Neurosurg. Spine 7 (1) (2007) 54–60. [84] H. Xiaowei, Z. Ninghui, X. Wei, T. Yiping, X. Linfeng, The experimental study of hypoxia-inducible factor-1alpha and its target genes in spinal cord injury, Spinal Cord 44 (1) (2006) 35–43. [85] F. Facchiano, E. Fernandez, S. Mancarella, G. Maira, M. Miscusi, D. D'Arcangelo, G. Cimino-Reale, M.L. Falchetti, M.C. Capogrossi, R. Pallini, Promotion of regeneration of corticospinal tract axons in rats with recombinant vascular endothelial growth factor alone and combined with adenovirus coding for this factor, J. Neurosurg. 97 (1) (2002) 161–168. [86] J. Widenfalk, A. Lipson, M. Jubran, C. Hofstetter, T. Ebendal, Y. Cao, L. Olson, Vascular endothelial growth factor improves functional outcome and decreases secondary degeneration in experimental spinal cord contusion injury, Neuroscience 120 (4) (2003) 951–960.

622

H.A. Kim et al. / Advanced Drug Delivery Reviews 61 (2009) 614–622

[87] D. Choi, M. Lee, D.A. Bull, R. Reiss, C.W. Chang, L. Christensen, S.W. Kim, Hypoxiainducible VEGF gene therapy using the RTP801 promoter, Mol. Ther. 9 (2004) S74–S75. [88] M. Lee, J.K. Ryu, S. Piao, M.J. Choi, H.A. Kim, L.W. Zhang, H.Y. Shin, H.I. Jung, I.H. Kim, S.W. Kim, J.K. Suh, Efficient gene expression system using the RTP801 promoter in the corpus cavernosum of high-cholesterol diet-induced erectile dysfunction rats for gene therapy, J. Sex. Med. 5 (6) (2008) 1355–1364. [89] J.W. Yockman, D. Choi, M.G. Whitten, C.W. Chang, A. Kastenmeier, H. Erickson, A. Albanil, M. Lee, S.W. Kim, D.A. Bull, Polymeric gene delivery of ischemia-inducible VEGF significantly attenuates infarct size and apoptosis following myocardial infarct, Gene Ther. 16 (1) (2009) 127–135. [90] H. Lu, Y. Zhang, D.D. Roberts, C.K. Osborne, N.S. Templeton, Enhanced gene expression in breast cancer cells in vitro and tumors in vivo, Mol. Ther. 6 (6) (2002) 783–792. [91] Z.Y. Chen, C.Y. He, L. Meuse, M.A. Kay, Silencing of episomal transgene expression by plasmid bacterial DNA elements in vivo, Gene Ther. 11 (10) (2004) 856–864. [92] T. Shoshani, A. Faerman, I. Mett, E. Zelin, T. Tenne, S. Gorodin, Y. Moshel, S. Elbaz, A. Budanov, A. Chajut, H. Kalinski, I. Kamer, A. Rozen, O. Mor, E. Keshet, D. Leshkowitz, P. Einat, R. Skaliter, E. Feinstein, Identification of a novel hypoxiainducible factor 1-responsive gene, RTP801, involved in apoptosis, Mol. Cell. Biol. 22 (7) (2002) 2283–2293. [93] J.M. Kim, M. Lee, K.H. Kim, Y. Ha, J.K. Choi, S.R. Park, H. Park, H.C. Park, C.H. Ahn, S.W. Kim, B.H. Choi, Gene therapy of neural cell injuries in vitro using the hypoxiainducible GM-CSF expression plasmids and water-soluble lipopolymer (WSLP), J. Control. Release 133 (1) (2009) 60–67. [94] Y. Tang, M. Jackson, K. Qian, M.I. Phillips, Hypoxia inducible double plasmid system for myocardial ischemia gene therapy, Hypertension 39 (2 Pt 2) (2002) 695–698. [95] M.I. Phillips, Y. Tang, K. Schmidt-Ott, K. Qian, S. Kagiyama, Vigilant vector: heartspecific promoter in an adeno-associated virus vector for cardioprotection, Hypertension 39 (2 Pt 2) (2002) 651–655.

[96] Y. Tang, K. Schmitt-Ott, K. Qian, S. Kagiyama, M.I. Phillips, Vigilant vectors: adenoassociated virus with a biosensor to switch on amplified therapeutic genes in specific tissues in life-threatening diseases, Methods 28 (2) (2002) 259–266. [97] Y.L. Tang, Y. Tang, Y.C. Zhang, K. Qian, L. Shen, M.I. Phillips, Protection from ischemic heart injury by a vigilant heme oxygenase-1 plasmid system, Hypertension 43 (4) (2004) 746–751. [98] Y.L. Tang, K. Qian, Y.C. Zhang, L. Shen, M.I. Phillips, A vigilant, hypoxia-regulated heme oxygenase-1 gene vector in the heart limits cardiac injury after ischemia– reperfusion in vivo, J. Cardiovas. Pharmacol. Ther. 10 (4) (2005) 251–263. [99] R.S. Foo, R.C. Siow, M.J. Brown, M.R. Bennett, Heme oxygenase-1 gene transfer inhibits angiotensin II-mediated rat cardiac myocyte apoptosis but not hypertrophy, J. Cell. Physiol. 209 (1) (2006) 1–7. [100] H. Harada, M. Hiraoka, S. Kizaka-Kondoh, Antitumor effect of TAT-oxygendependent degradation-caspase-3 fusion protein specifically stabilized and activated in hypoxic tumor cells, Cancer Res. 62 (7) (2002) 2013–2018. [101] H. Harada, S. Kizaka-Kondoh, S. Itasaka, K. Shibuya, A. Morinibu, K. Shinomiya, M. Hiraoka, The combination of hypoxia-response enhancers and an oxygendependent proteolytic motif enables real-time imaging of absolute HIF-1 activity in tumor xenografts, Biochem. Biophys. Res. Commun. 360 (4) (2007) 791–796. [102] N. Koshikawa, K. Takenaga, Hypoxia-regulated expression of attenuated diphtheria toxin A fused with hypoxia-inducible factor-1alpha oxygen-dependent degradation domain preferentially induces apoptosis of hypoxic cells in solid tumor, Cancer Res. 65 (24) (2005) 11622–11630. [103] H.Y. Chae, B.W. Lee, S.H. Oh, Y.R. Ahn, J.H. Chung, Y.K. Min, M.S. Lee, M.K. Lee, K.W. Kim, Effective glycemic control achieved by transplanting non-viral cationic liposome-mediated VEGF-transfected islets in streptozotocin-induced diabetic mice, Exp. Mol. Med. 37 (6) (2005) 513–523.