Hypoxia- and radiation-activated Cre/loxP 'molecular switch' vectors for gene therapy of cancer

Although a significant negative prognostic factor, tumor hypoxia can be exploited for gene therapy. To maximize targeting within the tumor mass, we have developed synthetic gene promoters containing hypoxia-responsive elements (HREs) from the erythropoietin (Epo) gene as well as radiation-responsive CArG elements from the early growth response (Egr) 1 gene. Furthermore, to achieve high and sustained expression of the suicide gene herpes simplex virus thymidine kinase (HSVtk), our gene therapy vectors contain an expression amplification system, or 'molecular switch', based on Cre/loxP recombination. In human glioma and breast adenocarcinoma cells exposed to hypoxia and/or radiation, the HRE/CArG promoter rapidly activated Cre recombinase expression leading to selective and sustained HSVtk synthesis. Killing of transfected tumor cells was measured after incubation with the prodrug ganciclovir (GCV; converted by HSVtk into a cytotoxin). In vitro, higher and more selective GCV-mediated toxicity was achieved with the switch vectors, when compared with the same inducible promoters driving HSVtk expression directly. In tumor xenografts implanted in nude mice, the HRE/CArG-switch induced significant growth delay and tumor eradication. In conclusion, hypoxia- and radiation-activated 'molecular switch' vectors represent a promising strategy for both targeted and effective gene therapy of solid tumors.     

Optimizing radiation-responsive gene promoters for radiogenetic cancer therapy

We have been developing synthetic gene promoters responsive to clinical doses of ionizing radiation (IR) for use in suicide gene therapy vectors. The crucial DNA sequences utilized are units with the consensus motif CC(A/T)(6)GG, known as CArG elements, derived from the IR-responsive Egr1 gene. In this study we have investigated the parameters needed to enhance promoter activation to radiation. A series of plasmid vectors containing different enhancer/promoters were constructed, transiently transfected into tumor cells (MCF-7 breast adenocarcinoma and U-373MG glioblastoma) and expression of a downstream reporter assayed. Results revealed that increasing the number of CArG elements, up to a certain level, increased promoter radiation-response; from a fold-induction of 1.95 +/- 0.17 for four elements to 2.74 +/- 0.17 for nine CArGs of the same sequence (for MCF-7 cells). Specific alteration of the core A/T sequences caused an even greater positive response, with fold-inductions of 1.71 +/- 0.23 for six elements of prototype sequence compared with 2.96 +/- 0.52 for one of the new sequences following irradiation. Alteration of spacing (from six to 18 nucleotides) between elements had little effect, as did the addition of an adjacent Sp1 binding site. Combining the optimum number and sequence of CArG elements in an additional enhancer was found to produce the best IR induction levels. Furthermore, the improved enhancers also performed better than the previously reported prototype when used in in vitro and in vivo experimental GDEPT. We envisage such enhancers will be used to drive suicide gene expression from vectors delivered to a tumor within an irradiated field. The modest, but tight expression described in the present study could be amplified using a molecular 'switch' system as previously described using Cre/LoxP. In combination with targeted delivery, this strategy has great potential for significantly improving the efficacy of cancer treatment in the large number of cases where radiotherapy is currently employed.     

Increasing endothelial cell specific expression by the use of heterologous hypoxic and cytokine-inducible enhancers

One of the current challenges in gene therapy is to construct a vector that will target specific tissues. Targeting expression to endothelium is of particular interest in the treatment of several pathologies. We have shown previously that defined regions of the E-selectin and KDR promoters confer endothelial cell specific expression following retroviral delivery. However, the levels of expression were low. In an attempt to increase expression but to preserve the tissue specificity we have examined hypoxic and cytokine-inducible enhancer elements in combination with the KDR and E-selectin promoters. Both enhancers should be active in the tumour environment, boosting expression and giving additional specificity of gene expression in the tumour endothelium. The hypoxia response element (HRE) of the murine phosphoglycerate kinase-1 (PGK-1) promoter was used as a hypoxic enhancer and the tandem-binding site for NFKB from the murine vascular cell adhesion molecule-1 (VCAM-1) promoter as a cytokine-inducible enhancer. The HRE conferred hypoxia inducibility to the KDR and E-selectin promoters. Endothelial specificity of expression was retained with the KDR but not the E-selectin promoter. The NFKB-binding site conferred responsiveness to TNF-alpha to the KDR promoter, however the level of induction was less than that achieved with the HRE. Retrovirus combining both enhancer elements transferred inducibility by hypoxia and TNF-alpha, and reached the highest expression levels upon stimulation. These results confirm that heterologous enhancer elements may operate on a single endothelial cell specific promoter. These findings make the use of inducible enhancers a promising strategy for increasing tissue specific gene expression.     

A novel adenoviral vector which mediates hypoxia-inducible gene expression selectively in neurons

Selective gene expression in neurons is still a challenge. We have developed several expression vectors using a combination of neuron restrictive silencer elements (NRSEs), hypoxia responsive elements (HREs) and CMV minimal promoter (CMVmp). These elements were packaged into replication defective adenovirus to target gene expression selectively in neurons in a hypoxia-regulated manner. Neuronal selectivity and responsiveness to hypoxia of these novel constructs were determined empirically in both neural cell lines and primary cerebellar granule neurons (CGNs). The construct p5HRE-3NRSE exhibited not only the highest level of reporter gene expression in neuronal cells but also in an oxygen concentration-dependent manner when compared with all other constructs. As expected, this construct did not elicit reporter gene expression in non-neuronal cells including human HEK293A and HT29 cells, rat NRK cells, mouse 3T6 cells and 3T3 L1 cells. This construct was packaged into a replication defective adenoviral vector (Ad/5HRE-3NRSE) to determine neuron-selective and hypoxia-inducible gene expression in cultured mouse postmitotic primary CGNs and differentiated human NT2 neurons (NT2/Ns). Remarkably, in response to hypoxia, Ad/5HRE-3NRSE showed strong hypoxia-inducible gene expression selectively in neurons (12-fold induction in CGNs and 22-fold in NT2/Ns), but not in glial cells. Taken together, this vector with restricted gene expression to neurons under the regulation of hypoxia will be a useful tool for investigations of mechanisms of neuronal damage caused by ischemic insult.     

The radiation-inducible pE9 promoter driving inducible nitric oxide synthase radiosensitizes hypoxic tumour cells to radiation

Driving high-level transgene expression in a tumour-specific manner remains a key requirement in the development of cancer gene therapy. We have previously demonstrated the strong anticancer effects of generating abnormally high levels of intracellular NO(*) following the overexpression of the inducible nitric oxide synthase (iNOS) gene. Much of this work has focused on utilizing exogenously activated promoters, which have been primarily induced using X-ray radiation. Here we further examine the potential of the pE9 promoter, comprising a combination of nine CArG radio-responsive elements, to drive the iNOS transgene. Effects of X-ray irradiation on promoter activity were compared in vitro under normoxic conditions and various degrees of hypoxia. The pE9 promoter generated high-level transgene expression, comparable with that achieved using the constitutively driven cytomegalovirus promoter. Furthermore, the radio-resistance of radiation-induced fibrosarcoma-1 (RIF-1) mouse sarcoma cells exposed to 0.1 and 0.01% O(2) was effectively eliminated following transfection with the pE9/iNOS construct. Significant inhibition of tumour growth was also observed in vivo following direct intratumoural injection of the pE9/iNOS construct compared to empty vector alone (P<0.001) or to a single radiation dose of 10 Gy (P<0.01). The combination of both therapies resulted in a significant 4.25 day growth delay compared to the gene therapy treatment alone (P<0.001). In summary, we have demonstrated the potential of the pE9/iNOS construct for reducing radio-resistance conferred by tumour cell hypoxia in vitro and in vivo, with greater tumour growth delay observed following the treatment with the gene therapy construct as compared with radiotherapy alone.     

Tumor-specific gene expression using the survivin promoter is further increased by hypoxia

Increasing evidence indicates that survivin, an inhibitor of apoptosis protein (IAP), is expressed in human cancer cells but is absent from most normal adult tissues. Here, we examined the feasibility of using a survivin promoter (Sur-P) to direct therapeutic expression of a proapoptotic gene specifically in human tumor cells. First, we demonstrated that this promoter was highly active in human tumor cells but not in normal cells. Second, we found that Sur-P activity was upregulated by hypoxia in tumor cells. Third, to further enhance this promoter's activity under hypoxia, we added a hypoxia-responsive element (HRE) from the vascular endothelial growth factor gene promoter in its 5' region, and showed that this combination resulted in a further increase in the level of gene expression in hypoxic tumor cells. Finally, we demonstrated that expression of an autocatalytic reverse caspase-3 gene by this promoter specifically induced apoptotic cell death in human tumor cells but not in normal cells. These findings support the use of promoters Sur-P or chimeric HRE-Sur-P for generating novel vectors for cancer gene therapy.     

5' CArG degeneracy in smooth muscle alpha-actin is required for injury-induced gene suppression in vivo

CC(A/T)6GG-dependent (CArG-dependent) and serum response factor-dependent (SRF-dependent) mechanisms are required for gene expression in smooth muscle cells (SMCs). However, an unusual feature of many SMC-selective promoter CArG elements is that they contain a conserved single G or C substitution in their central A/T-rich region, which reduces binding affinity for ubiquitously expressed SRF. We hypothesized that this CArG degeneracy contributes to cell-specific expression of smooth muscle alpha-actin in vivo, since substitution of c-fos consensus CArGs for the degenerate CArGs resulted in relaxed specificity in cultured cells. Surprisingly, our present results show that these substitutions have no effect on smooth muscle-specific transgene expression during normal development and maturation in transgenic mice. However, these substitutions significantly attenuated injury-induced downregulation of the mutant transgene under conditions where SRF expression was increased but expression of myocardin, a smooth muscle-selective SRF coactivator, was decreased. Finally, chromatin immunoprecipitation analyses, together with cell culture studies, suggested that myocardin selectively enhanced SRF binding to degenerate versus consensus CArG elements. Our results indicate that reductions in myocardin expression and the degeneracy of CArG elements within smooth muscle promoters play a key role in phenotypic switching of smooth muscle cells in vivo, as well as in mediating responses of CArG-dependent smooth muscle genes and growth regulatory genes under conditions in which these 2 classes of genes are differentially expressed.     

Hypoxia-inducible gene expression system using the erythropoietin enhancer and 3'-untranslated region for the VEGF gene therapy.

Gene therapy with the vascular endothelial growth factor (VEGF) gene is a potential treatment for many disorders or injuries with ischemia. However, unregulated expression of VEGF may induce pathological angiogenesis, promoting tumor growth, diabetic proliferative retinopathy and rupture of atherosclerotic plaque. Therefore, the effective regulation of the gene expression is one of the requirements for the VEGF gene therapy. In this research, we evaluated the hypoxia-inducible gene expression system with the erythropoietin (Epo) enhancer and the Epo 3'-untranslated region (UTR). The luciferase plasmids were constructed with the Epo enhancer (pEpo-SV-Luc), the Epo 3'-UTR (pSV-Luc-EpoUTR) or both (pEpo-SV-Luc-EpoUTR). The polyethylenimine/plasmid complexes were transfected to 293 or A7R5 cells and the cells were incubated under normoxia or hypoxia. The results showed that the Epo enhancer or Epo 3'-UTR increased the target gene expression under hypoxia. pEpo-SV-Luc-EpoUTR showed the highest luciferase expression. The VEGF expression plasmid with the Epo enhancer and 3'-UTR was also constructed. The VEGF expression by pEpo-SV-VEGF-EpoUTR showed the highest specificity of the gene expression in the hypoxic cells. The results suggest that the VEGF plasmid with the Epo enhancer and the Epo 3'-UTR may be useful for gene therapy for ischemic diseases.     

Adeno-associated viral vector-mediated hypoxia-regulated VEGF gene transfer promotes angiogenesis following focal cerebral ischemia in mice

Uncontrolled expression of vascular endothelial growth factor (VEGF) in vivo may cause unexpected side effects, such as brain hemangioma or tumor growth. Because hypoxia-inducible factor-1 (HIF-1) is upregulated during cerebral ischemia and regulates gene expression by binding to a cis-acting hypoxia-responsive element (HRE), we therefore used a novel HRE, originating in the 3'-end of the erythropoietin (Epo) gene, to control gene expression in the ischemic brain. A concatemer of nine copies (H9) of the consensus sequence of HRE was used to mediate hypoxia induction. Three groups of adult CD-1 mice received AAVH9-VEGF, AAVH9-lacZ or saline injection, and then underwent 45 min of transient middle cerebral artery occlusion (tMCAO). Results show that HIF-1 was persistently expressed in the ischemic brain. VEGF was overexpressed in the ischemic perifocal region in AAVH9-VEGF-transduced mice. Double-labeled immunostaining showed that VEGF expressed in neurons and astrocytes but not endothelial cells, suggesting that adeno-associated virus (AAV) vectors transduced neurons and astrocytes predominantly. The total number of microvessels/enlarged microvessels was greatly increased in the AAVH9-VEGF-transduced mice (180+/-29/27+/-4) compared to the AAVH9-lacZ (118+/-19/14+/-3) or saline-treated (119+/-20/14+/-2) mice after tMCAO (P<0.05). Cell proliferation examination demonstrated that these microvessels were newly formed. Regional cerebral blood flow recovery in the AAVH9-VEGF-transduced mice was also better than in AAVH9-lacZ or saline-treated mice (P<0.05). Our data indicated that HRE is a novel trigger for the control of VEGF expression in the ischemic brain. VEGF overexpression through AAVH9-VEGF gene transfer showed stable focal angiogenic effects in post-ischemic repair process, providing an opportunity to rebuild injured brain tissue.     

Transcriptional control of viral gene therapy by cisplatin

Ionizing radiation (IR) and radical oxygen intermediates (ROIs) activate the early growth response-1 (Egr1) promoter through specific cis-acting sequences termed CArG elements. Ad.Egr.TNF.11D, a replication-deficient adenoviral vector containing CArG elements cloned upstream of the cDNA for human recombinant TNF-alpha was used to treat human esophageal adenocarcinoma and rat colon adenocarcinoma cells in culture and as xenografts in athymic nude mice. Cisplatin, a commonly used chemotherapeutic agent, causes tumor cell death by producing DNA damage and generating ROIs. The present studies demonstrate induction of TNF-alpha production in tumor cells and xenografts treated with the combination of Ad.Egr.TNF.11D and cisplatin. The results show that the Egr1 promoter is induced by cisplatin and that this induction is mediated in part through the CArG elements. These studies also demonstrate an enhanced antitumor response without an increase in toxicity following treatment with Ad.Egr.TNF.11D and cisplatin, compared with either agent alone. Chemo-inducible cancer gene therapy thus provides a means to control transgene expression while enhancing the effectiveness of commonly used chemotherapeutic agents.     

Evaluation of a synthetic CArG promoter for nitric oxide synthase gene therapy of cancer

Nitric oxide synthase gene therapy has been shown to be effective at inducing apoptosis in experimental tumours and sensitizing them to radiotherapy. We have also shown that expression of inducible nitric oxide synthase (iNOS) can be effectively restricted to the tumour volume by the use of the radiation inducible promoter (WAF1) to drive the transgene in clinically relevant protocols. A synthetic construct (pE9), incorporating nine radiosensitive CArG elements from the Egr1 promoter, has recently been developed for cancer gene therapy. We have now investigated basal gene expression of transgenes driven by this promoter to assess its suitability for use in iNOS gene therapy protocols in vivo. Transfection of human microvascular endothelial cells (HMEC-1) with pE9iNOS, using a cationic lipid vector, resulted in progressively increasing (<5-fold) levels of iNOS protein expression up to 8 h after transfection. Transfection of an ex vivo rat artery preparation with pE9iNOS caused 83% inhibition of response to the vasoconstrictor phenylephrine (PE). CMViNOS transfection also reduced response to PE, but by only 52%. A single injection of 25 microg of pE9iNOS DNA in a lipid vector into the centre of a murine sarcoma (RIF1) induced iNOS protein expression by four-fold and increased nitrite concentration eight-fold. This caused a 7-day delay in tumour growth and was more effective than the constitutive CMV-driven construct. Our data suggest that generation of NO*, as a result of iNOS overexpression, is capable of further activating the E9 promoter, through a positive feedback loop, yielding stronger and sustained levels of NO*. This pE9iNOS combination may, therefore, be particularly useful in an anticancer gene therapy strategy as its antitumour effect in vivo was clearly superior to that of the strong constitutive promoter, CMV.