Adenoviral Context Research Articles

Increased glia‐specific transgene expression with glial fibrillary acidic protein promoters containing multiple enhancer elements

The ability to direct transgene expression to astrocytes has become increasingly important as the roles for these cells continue to expand. Promoters consisting of the 5'-flanking region of the human or mouse glial fibrillary acidic protein (GFAP) gene have generally proved satisfactory. However, a more powerful promoter would be advantageous for several applications, such as expression of dominant negative RNAs or proteins, or for gene therapy. We investigated the possibility of increasing the transcriptional activity of the human GFAP promoter by inserting into it one or three additional copies of putative GFAP enhancer regions. The promoters enhanced with three additional copies gave 75-fold higher LacZ expression levels upon plasmid transfection into GFAP-expressing U251 cells than the parental gfa2 promoter. Surprisingly, in a transgenic mouse model, the enhanced promoters resulted in no or only very low expression of marker genes, probably caused by toxicity. When various cell lines were infected with replication-deficient adenoviral vectors, the enhanced promoters gave LacZ expression levels that were approximately 10-fold higher than those with the parental gfa2 promoter, while retaining specificity for GFAP-expressing cells. Injection of the adenoviral vectors carrying the enhanced promoters into nude mouse brain showed that LacZ expression was limited to GFAP-positive cells. We conclude that gfa2 enhanced promoters are useful for production of short-term, glia-specific, high expression levels of genes in an adenoviral context. Adenoviral vectors containing these enhanced promoters may be useful in glioma gene therapy.

The carcinoma-specific epithelial glycoprotein-2 promoter controls efficient and selective gene expression in an adenoviral context

Adenoviral vectors are widely used in cancer gene therapy. After systemic administration however, the majority of the virus homes to the liver and the expressed transgene may cause hepatotoxicity. To restrict transgene expression to tumor cells, tumor- or tissue-specific promoters are utilized. The tumor antigen epithelial glycoprotein-2 (EGP-2), also known as Ep-CAM, is expressed in many cancers from different epithelial origins. In this study, the EGP-2 promoter was shown to restrict the expression of luciferase and thymidine kinase in an adenoviral context in different cell lines. In vivo, the EGP-2 promoter mediated efficient expression of luciferase in tumors but showed a 3-log lower activity in liver tissue when compared with the cytomegalovirus (CMV) promoter. Similarly, the EGP-2 promoter mediated specific cell killing after ganciclovir treatment in EGP-2-positive cells. Moreover, in vivo, this treatment regiment did not cause any rise in the liver enzymes aspartate aminotransferase (ASAT) and alanine aminotransferase (ALAT), demonstrating absence of liver toxicity. In contrast, CMV-mediated expression of thymidine kinase in combination with ganciclovir treatment resulted in high ASAT and ALAT values. This study demonstrates the value of the EGP-2 promoter to restrict transgene expression to a broad range of tumor types, thereby preventing liver toxicity.

543. Enhanced Glia-Specific Expression Using GFAP-'Super'Promoters in an Adenoviral Context In Vitro and In Vivo

Background: New promoters for high level short-term glia-specific expression have been created. These 'super' promoters are based on the promoter of the gene for glial fibrillary acidic protein (GFAP). Addition of three extra copies of the glia-specific enhancer elements A, B and D to the glia-specific 2.2 kb GFAP-promoter fragment (gfa2) resulted in 2 log higher expression levels after plasmid transfections. We describe the characteristics of these promoters in the context of the Ad5 E1 region in vitro and in vivo.

314. The Epithelial Glycoprotein-2 Promoter for Highly Specific and Efficient Cancer Gene Therapy for a Broad Range of Tumors

Top of pageAbstract Adenoviral cancer gene therapy is hampered by the lack of specific and efficient transgene expression in tumor cells. Employing tissue specific promoters to restrict transgene expression will increase specificity towards the tumor tissue. In addition, specific promoters can be used to increase the efficiency of adenoviral gene therapy by controlling adenoviral replication. However, many specific promoters are restricted to specific tumor types. The tumor specific promoter epithelial glycoprotein-2 (EGP-2) is known to be active in various epithelial-derived cancers including ovarian, colon, and breast cancer and might therefore be a good candidate to target a broad range of tumor types. In this study, the EGP-2 promoter was employed to improve the therapeutic potential of adenoviral cancer gene therapy. The EGP-2 promoter demonstrated an outstanding specificity and activity profile in vivo. In mouse liver tissue, 3 log lower activity compared to the strong constitutively active CMV promoter was shown compared with only 1 log lower activity in EGP-2 positive tumor tissue. Subsequently, the EGP-2 promoter was employed to control the expression of the suicide gene thymidine kinase (AdEGP-2 SR39TK). In vitro selective cell killing after infection with AdEGP-2 SR39TK and GCV administration was seen in EGP-2 positive cell lines, but not in EGP-2 negative cells. After iv injection of AdEGP-2 SR39TK in mice an absence of liver toxicity was demonstrated after administration of GCV, in contrast to AdCMV SR39TK which showed clear liver damage. These promising findings are currently being explored in tumor bearing mice. To successfully treat cancer, all tumor cells have to be eradicated. However, even after local administration adenoviral vectors will infect only a limited amount of tumor cells. Therefore, to increase the efficiency of the adenovirus the EGP-2 promoter was utilized to control the E1A gene, which is essential for adenoviral replication. This conditionally replicating adenovirus (CRAd) should replicate selectively in EGP-2 positive tumor cells, causing cell lysis and spread into the tumor mass. This cycle of selective replication and cell lysis is continued until no further tumor cells can be infected. The EGP-2 based CRAd was as efficient in cell killing as wild-type adenovirus in EGP-2 positive cell lines. In contrast, in EGP-2 negative cell lines replication of the EGP-2 based CRAd was attenuated up to 2 log when compared with the wild-type adenovirus. Currently, the EGP-2 based CRAd is further investigated in tumor bearing mice. Overall, it can be concluded that the EGP-2 promoter demonstrates high specificity and efficiency in adenoviral context and can therefore be a powerful tool in cancer gene therapy for the treatment of a broad range of tumor types.

Effects of the Ad5 upstream E1 region and gene products on heterologous promoters

All recombinant adenovirus vectors contain the upstream region of the E1A gene comprising the viral origin of replication, encapsidation signal, and cis-acting regulatory elements for transcription of the E1A and other early genes. Using different reporter genes, some previous studies demonstrated the maintenance of heterologous promoter specificity in the adenoviral context, while others reported that adenoviral sequences interfere with promoter activity. Plasmid DNA-based luciferase reporter gene assays and adenovirus type 5 (Ad5) infection were combined to examine the effect of the Ad5 (nt 1-353) element and/or adenoviral gene products on tissue-specific (Midkine (MK) and COX-2), cell cycle associated (Ki-67 and E2F1) and viral promoters (Ad5 E1, Ad5 E4 and SV40). As a proof of concept, data were verified in the setting of recombinant replication-defective and replication-competent adenoviral vectors. Viral and E2F1 promoter activities were enhanced by the Ad5 (nt 1-353) segment by approximately 100% and 145%, respectively, regardless of its position. A polyadenylation sequence (polyA) upstream of the promoter had no effect, confirming an enhancer element within the Ad5 (nt 1-353) segment. Ad5 (nt 1-353) increased COX-2 promoter activity by 146% but was blocked by an upstream polyA, indicating a cryptic transcription start site. When placing the reporter gene cassette in a replication-defective adenovirus, similar data were obtained. In the plasmid vector-based system, adenoviral gene products transactivated the E2F1 and viral promoters by 194%, 19%, 67%, and 16%, respectively. Tissue-specific promoter activities were not significantly affected by the Ad5 (nt 1-353) segment, nor adenoviral gene products. In concert with these data, we were able to target replication-competent adenoviral vectors with the COX-2 promoter, but not with the cell cycle associated promotor. The adenovirus E1A upstream regulatory region and gene products interact with some but not all heterologous promoters. Often, the basal promoter activity can be reduced with an upstream polyA. Since the data obtained in our plasmid vector-based assay with internal control and infection with adenovirus could be confirmed in the adenoviral setting, our system might be suitable to speed up the identification of promoters which maintain their specificity in the adenoviral context and circumvent the problems associated with determining infectious adenovirus titers.

Transcriptional targeting in renal cancer cell lines via the human CXCR4 promoter

Metastatic renal cell carcinoma (RCC) is often resistant to standard treatment, thereby requiring new therapeutic strategies. In this regard, tumor cell migration and metastasis have recently been shown to be regulated by chemokines and their respective receptors (e.g., SDF-1alpha/CXCR4). In the context of RCC, up-regulation of CXCR4 expression is closely related to the development of invasive cancer. Thus, we hypothesized that the CXCR4 pathway could be exploited for RCC targeting with gene therapy vectors. In this regard, targeting adenoviral vectors to tumor cells is critically dependent on tumor-specific gene expression. Toward the end of RCC tumor targeting, we evaluated the utility of the CXCR4 promoter in an adenoviral context. First, overexpression of CXCR4 was confirmed in several RCC cell lines. Next, an adenoviral vector was constructed, whereby the human CXCR4 promoter drives the expression of a reporter gene. We tested the activity of the CXCR4 promoter in vitro and in vivo in relevant models. Our data indicate that the human CXCR4 promoter is highly active in RCC cells but not in normal human cells. Finally, biodistribution studies in mice demonstrated dramatic repression of the CXCR4 promoter in the liver but not in the kidney. In conclusion, the unique activity of the CXCR4 promoter in RCC lines and its repression in normal human cells and in the murine liver underscore its potential utility as a novel candidate for transcriptional targeting of RCC.

292. Modification of the p53 Transgene of a Replication-Competent Adenovirus Improves p53 Stability and Cancer Cell Killing

Adenoviruses are widely used as vectors to deliver genetic material into cells. Although these vectors are currently the most efficient vectors used, they are not specific and not efficient enough to result in a good clinical outcome in the treatment against cancer. To increase the specificity, a transgene can be placed under the control of a tumor specific promoter, such as the epithelial glycoprotein-2 (EGP2) promoter. This promoter is active in a broad range of epithelial derived cancers. The length of the EGP2 promoter fragment used in initial studies is 3.4 kb. This promoter fragment showed in adenoviral context high activity and selectivity when tested both in vitro and in vivo. However due to limited space in the adenoviral genome, this promoter fragment is too large for the incorporation of several expression cassettes.

Inclusion of Moloney murine leukemia virus elements upstream of the transgene cassette in an E1-deleted adenovirus leads to an unusual genomic integration in epithelial cells

Classically, the 5′ and 3′ long terminal repeats (LTRs) are considered necessary but not sufficient for retroviral integration. Recently, we reported that inclusion of these and additional elements from Moloney murine leukemia virus (MoMLV) facilitated transgene integration, without retroviral integrase, when placed in an adenoviral context (AdLTR-luc vector) (Nat. Biotech. 18 (2000), 176; Biochem. Biophys. Res. Commun. 300 (2003), 115). To help understand this nonhomologous DNA recombination event, we constructed another vector, AdELP-luc, with 2.7 kb of MoMLV elements identically placed into an E1-deleted adenovirus type 5 backbone upstream of a luciferase cDNA reporter gene. Unlike AdLTR-luc, no MoMLV elements were placed downstream of the expression cassette. AdELP-luc readily infected epithelial cells in vitro. Southern hybridizations with DNA from cloned cells showed that disruption of the MoMLV sequences occurred. One cell clone, grown in vitro without any special selection medium for 9 months, exhibited stable vector integration and luciferase activity. Importantly, both Southern hybridization and FISH analyses showed that in addition to the MoMLV elements and expression cassette, substantial adenoviral sequence downstream of the luciferase cDNA was genomically integrated. These results suggest that the 2.7 kb of MoMLV sequence included in AdELP-luc have cis-acting functions and mediates an unusual integration event.

931. Specificity of gfa2 Derived Promoters in Adenoviral Vectors for Conditional Replication in Glioblastoma

4602 The limited spread of non-replicating adenoviral vectors after injection into glial tumors may contribute to the lack of significant tumor reduction in many human gene therapy trials. To enhance viral spread in the tumor while keeping it restricted to the the brain, we designed conditionally replicating viruses in which the E1A gene is under the control of the promoter of the glia specific intermediate filament Glial Fibrillary Acidic Protein (GFAP). Because of the moderate activity of the normal GFAP promoter (gfa2) compared to the CMV promoter, we also evaluated constructs containing additional glial specific enhancers: gfa2(B)3 and gfa2(ABD)3 promoters. The gfa2 derived promoters followed by a beta galactosidase reporter gene were cloned into the pShuttle plasmid, in an orientation inverse to wildtype E1 expression . Viral vectors were produced according to the Ad-Easy protocol and viruses giving rise to blue plaques upon infection of U251 but not A549 cells were selected and grown to high titers. To check the specificity and effectivity of the promoter constructs, a panel of human cell lines (glial and non-glial) was infected with the gfa2 (triple enhancer) promoter-beta galactosidase vectors. An adenoviral vector containing a CMV-beta galactosidase cassette was included to estimate cell entry. Expression of beta galactosidase was evaluated both in situ using X-gal staining and quantitatively using ONPG staining. Results: Of 11 glial cell lines tested, four were positive for GFAP on a Western blot incubated with anti-GFAP (U251, SNB19, U373 and GOS3). Furthermore, of 12 primary cultures of glial tumors one was positive for GFAP using immunohistochemistry. This positive primary culture was also GFAP positive on Western and was added to the panel of cell lines, as were fourteen non glial cell lines (all GFAP negative on Western). Of the three gfa2 derived promoters, the gfa2 (ABD)3-LacZ construct gives rise to high beta galactosidase expression levels in the GFAP positive cell lines. Beta galactosidase expression levels in some GFAP positive lines are at a level comparable to the CMV promoter construct. The ONPG ratio of GFAP positive cell lines to GFAP negative cell lines (both glial and non-glial) is best for the gfa2-(B)3 promoter containing virus. Preliminary results indicate that conditionally replicating adenovirus in which E1A is regulated by the gfa2-B3 promoter replicate better on the GFAP positive U251 cell line than on the GFAP negative Hep2 cells. Conclusion: The gfa2 promoter constructs are highly active in an adenoviral context and have retained their specificity. Constructs for conditionally replicating viruses using these promoters have been prepared and conditionally replicating virus is being produced.

Reannotation of the CELO genome characterizes a set of previously unassigned open reading frames and points to novel modes of host interaction in avian adenoviruses

BackgroundThe genome of the avian adenovirus Chicken Embryo Lethal Orphan (CELO) has two terminal regions without detectable homology in mammalian adenoviruses that are left without annotation in the initial analysis. Since adenoviruses have been a rich source of new insights into molecular cell biology and practical applications of CELO as gene a delivery vector are being considered, this genome appeared worth revisiting. We conducted a systematic reannotation and in-depth sequence analysis of the CELO genome.ResultsWe describe a strongly diverged paralogous cluster including ORF-2, ORF-12, ORF-13, and ORF-14 with an ATPase/helicase domain most likely acquired from adeno-associated parvoviruses. None of these ORFs appear to have retained ATPase/helicase function and alternative functions (e.g. modulation of gene expression during the early life-cycle) must be considered in an adenoviral context. Further, we identified a cluster of three putative type-1-transmembrane glycoproteins with IG-like domains (ORF-9, ORF-10, ORF-11) which are good candidates to substitute for the missing immunomodulatory functions of mammalian adenoviruses. ORF-16 (located directly adjacent) displays distant homology to vertebrate mono-ADP-ribosyltransferases. Members of this family are known to be involved in immuno-regulation and similiar functions during CELO life cycle can be considered for this ORF. Finally, we describe a putative triglyceride lipase (merged ORF-18/19) with additional domains, which can be expected to have specific roles during the infection of birds, since they are unique to avian adenoviruses and Marek's disease-like viruses, a group of pathogenic avian herpesviruses.ConclusionsWe could characterize most of the previously unassigned ORFs pointing to functions in host-virus interaction. The results provide new directives for rationally designed experiments.

The secretory leukoprotease inhibitor (SLPI) promoter for ovarian cancer gene therapy.

Adenoviruses allow efficient transduction of dividing and non-dividing cells and their safety for the treatment of cancer has been established in clinical trials. However, one disadvantage is their promiscuous tropism. In this regard, tissue-specific promoters (TSPs) could be useful for directing transgene expression to target tissues and for reducing adverse effects in non-target tissues. We hypothesize that selective adenovirus-mediated transgene expression could be achieved through the use of the secretory leukoprotease inhibitor (SLPI) promoter in the context of ovarian cancer. Adenoviruses containing the SLPI promoter driving reporter and suicide gene expression were created and tested in ovarian cancer cell lines and primary tumor cells isolated from patients. To evaluate the in vivo activation of the SLPI promoter in comparison to a ubiquitous promoter, intraperitoneal delivery was performed in tumor-bearing mice, followed by analysis of survival or gene expression in normal organs and tumor. The SLPI promoter retained its fidelity in an adenoviral context and was activated in both cell lines and primary cancer cells. The SLPI promoter was induced to a high degree in ovarian cancer cells while showing significantly reduced activity in normal tissues. The therapeutic efficacy of SLPI promoter-controlled gene expression was similar to the ubiquitous promoter in vitro and in an orthotopic murine model of peritoneally disseminated ovarian cancer, with higher activity than controls. The SLPI promoter is a potentially useful TSP for ovarian cancer and facilitates further development of targeting strategies for improved gene therapy of ovarian carcinomas.

Stimulation of angiogenesis by Cyr61 gene: a new therapeutic candidate.

Cyr61 is a secreted, cysteine-rich heparin-binding protein that is associated with extracellular matrix and cell surface, and has been demonstrated to be proangiogenic in vitro. In the present study we evaluated the angiogenic effect of human Cyr61 in an adenoviral context in the rabbit ischemic hindlimb model. For this purpose, three randomized groups of New Zealand White rabbits received intramuscular injections of 5 x 10(8) infectious units of an adenovirus carrying either the Cyr61 gene (Ad-Cyr61), the vascular endothelial growth factor gene (Ad-VEGF(165)) used as the angiogenic gene of reference, or no transgene (Ad-Null), 10 days after femoral artery excision in one limb. Perfusion of the ischemic limb was evaluated before adenoviral treatment (day 10) and 30 days postinjection (day 40). Angiographic, hemodynamic, and histologic parameters indicated that animals in the Ad-Cyr61 group had significantly better perfusion than in the Ad-Null group. Interestingly, this improvement exceeded that achieved with Ad-VEGF(165). In conclusion, Cyr61 gene transfer appears potent in stimulating limb revascularization, thereby promoting great improvement in tissue perfusion in the ischemic limb. These findings indicate that Cyr61 could be a promising therapeutic candidate for treating severe peripheral ischemic diseases.

Midkine promoter-based adenoviral suicide gene therapy to midkine-positive pediatric tumor

Background/Purpose: Suicide gene therapy based on the delivery of the herpes simplex virus thymidine kinase gene combined with ganciclovir (HSV-tk/GCV) is a promising approach for cancer treatment. Adenoviral (Ad) vectors are useful gene delivery vehicles for this approach; however, because these agents possess a high natural tropism for the liver, systems must be designed to avoid potential hepatotoxicity induced by expression of the therapeutic gene in this organ. It has been reported that Wilms' tumors or neuroblastomas express a high level of midkine (MK), a heparin-binding growth factor. In addition, no MK expression is observed in mouse or human liver. The authors investigated the application of MK promoter-based adenoviral gene therapy for MK-positive tumors, especially Wilms' tumors or neuroblastomas, and have shown that the MK promoter retains its fidelity in the adenoviral context, having low activity in liver and high activity in MK-positive tumor cells. We present herein the efficacy of in vivo tumor regression as well as prevention of lethal hepatic toxicity by using the MK promoter in an Ad vector–based HSV-tk/GCV treatment approach. Methods: Ad vectors, AdMKTK or AdCMVTK, encoding HSV-tk under the control of the MK or cytomegalovirus promoters, respectively, were injected systemically into mice, then hepatotoxicity and survival was monitored after GCV administration. In concurrent studies, the therapeutic impact of AdMKTK/GCV versus AdCMVTK/GCV on subcutaneous G-401 Wilms' tumors in nude mice was assessed. Results: By day 8 of systemic viral treatment, 4 of 5 mice treated with AdCMVTK/GCV had died, whereas all mice treated with AdMKTK/GCV survived at least 10 days. In the subcutaneous tumor study, equivalent regression of tumor was seen in the group that received AdMKTK as the group that received AdCMVTK intratumoral injection. Conclusion: These data indicate that the MK promoter–based adenoviral suicide gene therapy is a unique therapeutic candidate for MK-positive tumors, Wilms' tumors, or neuroblastomas, by virtue of its mild hepatotoxicity and high efficacy against MK-positive tumors. J Pediatr Surg 37:588-592. Copyright 2002, Elsevier Science (USA). All rights reserved.