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• W2043915150 abstract "The endothelium imposes a structural barrier to the extravasation of systemically delivered oncolytic adenovirus (Ad). Here, we introduced a transendothelial route of delivery in order to increase tumor accumulation of virus particles (vp) beyond that resulting from convection-dependent extravasation alone. This was achieved by engineering an Ad encoding a syncytium-forming protein, gibbon ape leukemia virus (GALV) fusogenic membrane glycoprotein (FMG). The expression of GALV was regulated by a hybrid viral enhancer-human promoter construct comprising the human cytomegalovirus (CMV) immediate-early enhancer and the minimal human endothelial receptor tyrosine kinase promoter (“eTie1”). Endothelial cell-selectivity of the resulting Ad-eTie1-GALV vector was demonstrated by measuring GALV mRNA transcript levels. Furthermore, Ad-eTie1-GALV selectively induced fusion between infected endothelial cells and uninfected epithelial cells in vitro and in vivo, allowing transendothelial virus penetration. Heterofusion of infected endothelium to human embryonic kidney 293 (HEK 293) cells, in mixed in vitro cultures or in murine xenograft models, permitted fusion-dependent transactivation of the replication-deficient Ad-eTie1-GALV, due to enabled access to viral E1 proteins derived from the HEK 293 cytoplasm. These data provide evidence to support our proposed use of GALV to promote Ad penetration through tumor-associated vasculature, an approach that may substantially improve the efficiency of systemic delivery of oncolytic viruses to disseminated tumors. The endothelium imposes a structural barrier to the extravasation of systemically delivered oncolytic adenovirus (Ad). Here, we introduced a transendothelial route of delivery in order to increase tumor accumulation of virus particles (vp) beyond that resulting from convection-dependent extravasation alone. This was achieved by engineering an Ad encoding a syncytium-forming protein, gibbon ape leukemia virus (GALV) fusogenic membrane glycoprotein (FMG). The expression of GALV was regulated by a hybrid viral enhancer-human promoter construct comprising the human cytomegalovirus (CMV) immediate-early enhancer and the minimal human endothelial receptor tyrosine kinase promoter (“eTie1”). Endothelial cell-selectivity of the resulting Ad-eTie1-GALV vector was demonstrated by measuring GALV mRNA transcript levels. Furthermore, Ad-eTie1-GALV selectively induced fusion between infected endothelial cells and uninfected epithelial cells in vitro and in vivo, allowing transendothelial virus penetration. Heterofusion of infected endothelium to human embryonic kidney 293 (HEK 293) cells, in mixed in vitro cultures or in murine xenograft models, permitted fusion-dependent transactivation of the replication-deficient Ad-eTie1-GALV, due to enabled access to viral E1 proteins derived from the HEK 293 cytoplasm. These data provide evidence to support our proposed use of GALV to promote Ad penetration through tumor-associated vasculature, an approach that may substantially improve the efficiency of systemic delivery of oncolytic viruses to disseminated tumors. The ability of intravenously (i.v) administered adenoviruses (Ads) to infect tumor cells within cancer deposits is confounded by many obstacles, notably the vascular endothelium separating the blood circulation from the tumor parenchyma. Only a small fraction of the input dose of virus particles (vp) achieves extravasation within solid tumors and is able to infect cancer cells.1Lyons M Onion D Green NK Aslan K Rajaratnam R Bazan-Peregrino M et al.Adenovirus type 5 interactions with human blood cells may compromise systemic delivery.Mol Ther. 2006; 14: 118-128Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar In contrast, circulating vp interact readily with the tumor-associated endothelium, which is directly accessible from the blood stream. Because tumor-associated endothelial cells express a range of receptors that allow natural or retargeted infection by various viruses, including Ad and vaccinia,2Shinozaki K Suominen E Carrick F Sauter B Kähäri VM Lieber A et al.Efficient infection of tumor endothelial cells by a capsid-modified adenovirus.Gene Ther. 2006; 13: 52-59Crossref PubMed Scopus (32) Google Scholar,3Kirn DH Wang Y Le Boeuf F Bell J Thorne SH Targeting of interferon-beta to produce a specific, multi-mechanistic oncolytic vaccinia virus.PLoS Med. 2007; 4: e353Crossref PubMed Scopus (158) Google Scholar targeted infection of tumor-associated endothelial cells may allow efficient delivery of vp to disseminated tumors. One of the most promising cancer gene therapy approaches makes use of oncolytic viruses, however oncolytic Ads are generally designed so that their replication is triggered by tumor-associated molecular changes.4Alemany R Cancer selective adenoviruses.Mol Aspects Med. 2007; 28: 42-58Crossref PubMed Scopus (93) Google Scholar Accordingly they cannot achieve lytic self-amplification within noncancerous, and therefore nonpermissive, endothelial cells. This turns the tumor endothelium from a potentially useful target into a significant structural barrier to oncolytic agents, which must gain infection of target tumor cells in order to fulfill their therapeutic potential.5Jain RK Vascular and interstitial barriers to delivery of therapeutic agents in tumors.Cancer Metastasis Rev. 1990; 9: 253-266Crossref PubMed Scopus (423) Google Scholar This impediment could be partially overcome by conditioning the tumor endothelium with VEGF165 to transiently support the replication of oncolytic agents such as reovirus and vesicular stomatitis virus,6Kottke T Hall G Pulido J Diaz RM Thompson J Chong H et al.Antiangiogenic cancer therapy combined with oncolytic virotherapy leads to regression of established tumors in mice.J Clin Invest. 2010; 120: 1551-1560Crossref PubMed Scopus (64) Google Scholar or by the use of an oncolytic agent such as vaccinia virus which naturally targets tumor-associated endothelium.3Kirn DH Wang Y Le Boeuf F Bell J Thorne SH Targeting of interferon-beta to produce a specific, multi-mechanistic oncolytic vaccinia virus.PLoS Med. 2007; 4: e353Crossref PubMed Scopus (158) Google Scholar However, the activity of most oncolytic agents remain limited by their inability to penetrate through the endothelium. In the present study, we investigate a new approach to achieving transendothelial delivery by exploiting endothelial-specific production of a viral fusogenic membrane glycoprotein (FMG), thereby utilizing endothelial–epithelial fusion as a means of creating an access route for therapeutic viruses from the blood circulation into the tumor parenchyma. The use of FMGs to enhance cell-to-cell spread of oncolytic agents has been demonstrated in previous studies.7Ahmed A Jevremovic D Suzuki K Kottke T Thompson J Emery S et al.Intratumoral expression of a fusogenic membrane glycoprotein enhances the efficacy of replicating adenovirus therapy.Gene Ther. 2003; 10: 1663-1671Crossref PubMed Scopus (33) Google Scholar,8Allen C McDonald C Giannini C Peng KW Rosales G Russell SJ et al.Adenoviral vectors expressing fusogenic membrane glycoproteins activated via matrix metalloproteinase cleavable linkers have significant antitumor potential in the gene therapy of gliomas.J Gene Med. 2004; 6: 1216-1227Crossref PubMed Scopus (18) Google Scholar,9Bateman A Bullough F Murphy S Emiliusen L Lavillette D Cosset FL et al.Fusogenic membrane glycoproteins as a novel class of genes for the local and immune-mediated control of tumor growth.Cancer Res. 2000; 60: 1492-1497PubMed Google Scholar,10Diaz RM Bateman A Emiliusen L Fielding A Trono D Russell SJ et al.A lentiviral vector expressing a fusogenic glycoprotein for cancer gene therapy.Gene Ther. 2000; 7: 1656-1663Crossref PubMed Scopus (60) Google Scholar FMGs such as gibbon ape leukemia virus (GALV) have been explored as therapeutic transgenes to arm oncolytic viruses, inducing the formation of multinucleated syncytia which may facilitate viral spread from infected to adjacent uninfected cells.7Ahmed A Jevremovic D Suzuki K Kottke T Thompson J Emery S et al.Intratumoral expression of a fusogenic membrane glycoprotein enhances the efficacy of replicating adenovirus therapy.Gene Ther. 2003; 10: 1663-1671Crossref PubMed Scopus (33) Google Scholar,8Allen C McDonald C Giannini C Peng KW Rosales G Russell SJ et al.Adenoviral vectors expressing fusogenic membrane glycoproteins activated via matrix metalloproteinase cleavable linkers have significant antitumor potential in the gene therapy of gliomas.J Gene Med. 2004; 6: 1216-1227Crossref PubMed Scopus (18) Google Scholar,9Bateman A Bullough F Murphy S Emiliusen L Lavillette D Cosset FL et al.Fusogenic membrane glycoproteins as a novel class of genes for the local and immune-mediated control of tumor growth.Cancer Res. 2000; 60: 1492-1497PubMed Google Scholar,10Diaz RM Bateman A Emiliusen L Fielding A Trono D Russell SJ et al.A lentiviral vector expressing a fusogenic glycoprotein for cancer gene therapy.Gene Ther. 2000; 7: 1656-1663Crossref PubMed Scopus (60) Google Scholar The ability of FMGs to synergize with virotherapy and enhance their antitumor efficacy has been demonstrated in previous studies,7Ahmed A Jevremovic D Suzuki K Kottke T Thompson J Emery S et al.Intratumoral expression of a fusogenic membrane glycoprotein enhances the efficacy of replicating adenovirus therapy.Gene Ther. 2003; 10: 1663-1671Crossref PubMed Scopus (33) Google Scholar,11Hoffmann D Bangen JM Bayer W Wildner O Synergy between expression of fusogenic membrane proteins, chemotherapy and facultative virotherapy in colorectal cancer.Gene Ther. 2006; 13: 1534-1544Crossref PubMed Scopus (36) Google Scholar,12Li H Haviv YS Derdeyn CA Lam J Coolidge C Hunter E et al.Human immunodeficiency virus type 1-mediated syncytium formation is compatible with adenovirus replication and facilitates efficient dispersion of viral gene products and de novo-synthesized virus particles.Hum Gene Ther. 2001; 12: 2155-2165Crossref PubMed Scopus (44) Google Scholar,13Simpson GR Han Z Liu B Wang Y Campbell G Coffin RS Combination of a fusogenic glycoprotein, prodrug activation, and oncolytic herpes simplex virus for enhanced local tumor control.Cancer Res. 2006; 66: 4835-4842Crossref PubMed Scopus (55) Google Scholar,14Fu X Tao L Jin A Vile R Brenner MK Zhang X Expression of a fusogenic membrane glycoprotein by an oncolytic herpes simplex virus potentiates the viral antitumor effect.Mol Ther. 2003; 7: 748-754Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar raising the possibility that FMGs may function as effective cytotoxics as well as transduction agents to complement virotherapy. Here, we report the GALV-driven formation of heterocellular syncytia between tumor-associated endothelial cells and tumor cells as a means of promoting Ad penetration from the bloodstream into the tumor. This was accomplished through transcriptionally directing GALV expression to activated, proliferating endothelium using a construct containing a 460 base pair enhancer from the human cytomegalovirus (CMV) and a 880 base pair promoter of human endothelial receptor tyrosine kinase (Tie1).15Korhonen J Lahtinen I Halmekytö M Alhonen L Jänne J Dumont D et al.Endothelial-specific gene expression directed by the tie gene promoter in vivo.Blood. 1995; 86: 1828-1835PubMed Google Scholar Tie1 is expressed almost exclusively on endothelial cells,16Partanen J Armstrong E Mäkelä TP Korhonen J Sandberg M Renkonen R et al.A novel endothelial cell surface receptor tyrosine kinase with extracellular epidermal growth factor homology domains.Mol Cell Biol. 1992; 12: 1698-1707Crossref PubMed Scopus (294) Google Scholar mainly during embryonic vasculogenesis but also in adults during both physiological and tumor angiogenesis.17Dumont DJ Fong GH Puri MC Gradwohl G Alitalo K Breitman ML Vascularization of the mouse embryo: a study of flk-1, tek, tie, and vascular endothelial growth factor expression during development.Dev Dyn. 1995; 203: 80-92Crossref PubMed Scopus (449) Google Scholar,18Korhonen J Partanen J Armstrong E Vaahtokari A Elenius K Jalkanen M et al.Enhanced expression of the tie receptor tyrosine kinase in endothelial cells during neovascularization.Blood. 1992; 80: 2548-2555PubMed Google Scholar,19Korhonen J Polvi A Partanen J Alitalo K The mouse tie receptor tyrosine kinase gene: expression during embryonic angiogenesis.Oncogene. 1994; 9: 395-403PubMed Google Scholar,20Kaipainen A Vlaykova T Hatva E Böhling T Jekunen A Pyrhönen S et al.Enhanced expression of the tie receptor tyrosine kinase mesenger RNA in the vascular endothelium of metastatic melanomas.Cancer Res. 1994; 54: 6571-6577PubMed Google Scholar,21Puri MC Bernstein A Requirement for the TIE family of receptor tyrosine kinases in adult but not fetal hematopoiesis.Proc Natl Acad Sci USA. 2003; 100: 12753-12758Crossref PubMed Scopus (104) Google Scholar,22Tang Y Borgstrom P Maynard J Koziol J Hu Z Garen A et al.Mapping of angiogenic markers for targeting of vectors to tumor vascular endothelial cells.Cancer Gene Ther. 2007; 14: 346-353Crossref PubMed Scopus (29) Google Scholar We engineered an Ad vector encompassing this fusogenic system which expressed high levels of the transgene exclusively in endothelial cells, and mediated endothelial-tumor cell fusion in vitro and in vivo. Formation of heterocellular syncytia following infection of endothelial cells resulted in fusion-dependent infection of human embryonic kidney 293 (HEK 293) cells in vitro and HEK 293 xenografts in vivo, resulting in dramatically increased viral titers. We have therefore provided the first demonstration of using heterocellular syncytium formation to assist viral penetration through the tumor vasculature, ultimately allowing target cell transduction. This strategy may be applicable to oncolytic Ads, e.g., AdEHE2F, vKH6, and CV706,4Alemany R Cancer selective adenoviruses.Mol Aspects Med. 2007; 28: 42-58Crossref PubMed Scopus (93) Google Scholar which are reliant on gain of function mutations in tumor cells, whereby cancer-specific factors could then become imported into endothelial nuclei-containing virus genomes to transactivate replication. The use of Ad to achieve the fusion of tumor-associated endothelium to tumor cells requires tight transcriptional regulation to minimize the risk of off-target toxicity. One means of achieving such regulation is through the use of tissue-specific promoters. To select the optimum endothelial-selective promoter to drive GALV expression, the minimal promoter sequences of Tie115Korhonen J Lahtinen I Halmekytö M Alhonen L Jänne J Dumont D et al.Endothelial-specific gene expression directed by the tie gene promoter in vivo.Blood. 1995; 86: 1828-1835PubMed Google Scholar and the kinase insert domain receptor (KDR) of vascular endothelial growth factor23Modlich U Pugh CW Bicknell R Increasing endothelial cell specific expression by the use of heterologous hypoxic and cytokine-inducible enhancers.Gene Ther. 2000; 7: 896-902Crossref PubMed Scopus (55) Google Scholar,24Jaggar RT Chan HY Harris AL Bicknell R Endothelial cell-specific expression of tumor necrosis factor-alpha from the KDR or E-selectin promoters following retroviral delivery.Hum Gene Ther. 1997; 8: 2239-2247Crossref PubMed Scopus (52) Google Scholar were isolated from primary human umbilical vein endothelial cells (HUVECs), and tested in the context of luciferase-expressing reporter Ads based on the AdEasy system (Qbiogene, MP Biomedicals, Montreal, Quebec, Canada). Replication-deficient vectors were constructed with each promoter cloned upstream of luciferase or GALV (Figure 1a), allowing quantitative comparisons of promoter strength and cell-type selectivity (Figure 1b). Tie1 or KDR-driven luciferase-expressing reporter viruses, Ad-KDR-Luc and Ad-Tie1-Luc, were used to infect HUVECs, human microvascular endothelial cells or SW480 (human colorectal cancer) cells. Activity of Ad-KDR-Luc was close to background levels in all cells tested; in contrast, Ad-Tie1-Luc exhibited high endothelial specificity, with a 100-fold greater luciferase expression per genome copy in HUVECs (P < 0.01) and human microvascular endothelial cells (P < 0.001) than in SW480 cells. In contrast the CMV promotor driven Ad-Luc control virus showed no selectivity for endothelial cells (P > 0.05) (Figure 1b). However, the corresponding GALV-expressing viruses (Ad-Tie1-GALV and Ad-KDR-GALV) did not drive adequate levels of GALV expression in HUVECs to permit fusion (data not shown). We therefore sought to increase the functional output of the tightly endothelial-selective Tie1 promoter by introducing the CMV immediate-early enhancer element, producing transcriptional control by a hybrid CMV enhancer-Tie1 promoter (Ad-eTie1-GALV). This virus induced extensive syncytium formation in cultured HUVECs, leading to loss of cell morphology and clustering of nuclei as shown by DAPI staining; tubulin organization in syncytia was shown by staining with an anti-α tubulin-fluorescein isothiocyanate antibody (Figure 1c). These effects were not observed for the control Ad-Luc vector (Figure 1d). The cell-type specificity of the CMV enhancer-Tie1 promoter transcriptional activation system of Ad-eTie1-GALV was tested in a range of human cell lines including those of epithelial (PC-3, prostate cancer), mesenchymal (MRC5v2, immortalized fibroblast and HT1080, fibrosarcoma) or endothelial (HUVEC) origin. As GALV-mediated fusion is dependent on the recognition of the cellular transmembrane receptor Pit-1,25O'Hara B Johann SV Klinger HP Blair DG Rubinson H Dunn KJ et al.Characterization of a human gene conferring sensitivity to infection by gibbon ape leukemia virus.Cell Growth Differ. 1990; 1: 119-127PubMed Google Scholar the ability of each of the nonendothelial cell lines to undergo GALV-induced homocellular fusion was tested by transfection with a control CMV-driven GALV-expressing plasmid (CMV-GALV) (left panel images, Figure 2a). In each of these cell lines, the CMV-GALV plasmid induced extensive syncytium formation with the exception of HUVECs (where the absence of syncytia reflects low transfection efficiency, as confirmed using a CMV-GFP plasmid, data not shown). In contrast, following infection with Ad-eTie1-GALV syncytium formation was observed exclusively in HUVECs with no observable syncytia in any of the nonendothelial cell lines tested (right panel images, Figure 2a). In order to provide quantitative analysis of fusion activity in different cell lines, a fusion index was calculated for each experimental condition as described in the methods section. The average fusion indexes for MRC5v2, HT1080, and PC-3 cells following transfection with the CMV-driven, GALV-expressing plasmid were 0.22, 0.09, and 0.13, respectively (Figure 2a). The average fusion index for HUVECs following infection with Ad-eTie1-GALV was 0.10 (Figure 2a), whereas in MRC5v2, HT1080, and PC-3 cells it was 0. To quantify differential expression of GALV, mRNA levels were determined by reverse-transcription real-time quantitative PCR (RT-QPCR) (Figure 2b). At 24 hours postinfection ~8.2 × 107 GALV transcripts were obtained from infection of 10,000 HUVECs with Ad-eTie1-GALV at 5,000 vp/cell, whereas GALV mRNA was undetectable in all other cell lines. This equates to ~19 GALV mRNA molecules from each virus genome, and ~8,200 transcripts per HUVEC. Statistical analysis confirms significant differences between transcript levels found in HUVECs versus all epithelial cell lines (P < 0.001 for all groups). Ad-Luc infection of each cell line served as negative controls for the design of GALV-specific primers and probe; no GALV mRNA was detected in these controls. To account for possible differences in virus infectivity in different cell lines, viral genome copy numbers were determined at 24 hours postinfection by real-time QPCR. No significant difference in the levels of Ad-eTie1-GALV associated with each cell line was detected (Figure 2c). These results demonstrate the strict endothelial specificity of the CMV enhancer-Tie1 promoter system. Coculturing experiments were performed to test whether Ad-eTie1-GALV could induce fusion between two cell types. Coculturing of Ad-Luc infected HUVECs with each nonendothelial cell line resulted in the maintenance of morphologically distinct mixed cell populations (left panel images, Figure 3a). In contrast, widespread heterocellular syncytium formation was observed within 12 hours of the addition of uninfected PC-3, HCT116, or HEK 293 cells to HUVECs that had been preinfected with Ad-eTie1-GALV (right panel images in Figure 3a). The average fusion indexes (F) for PC-3, HCT116, and HEK 293 cells cocultured with HUVECs preinfected with Ad-eTie1-GALV were 0.59, 0.38, and 0.21, respectively. For Ad-Luc the F value was 0. In order to characterize the composition of the syncytia formed under such mixed culture conditions PC-3 cells were visualized using an antibody against epithelial cell adhesion molecule (EpCAM, in green), and HUVECs preinfected with Ad-eTie1-GALV were visualized using an antibody against CD31 (in red). When the Ad-Luc control was used only single cells stained either red or green were observed (Figure 3b). However, abundant syncytial structures were apparent when HUVECs were preinfected with Ad-eTie1-GALV (Figure 3c). An image gallery of these structures is shown in Figure 3d, they share the features of multinucleation (as shown by DAPI) and are comprised of both HUVEC endothelial (red) and PC-3 epithelial cells (green). Single stained PC-3 and HUVEC which have yet to be incorporated into the syncytia are also apparent at the margins of these images. These data confirmed the ability of GALV expressing HUVECs to form heterocellular syncytia with a range of tumor cell types. The activation of an oncolytic Ad within the endothelial nucleus of a heterocellular syncytium would generally require the conversion of the nucleus into a site for virus production by the nuclear import of tumor-associated cytoplasmic factors. To assess the feasibility of this approach, HUVECs were infected with the E1/E3-deleted Ad-eTie1-GALV or Ad-Luc virus before coculture with HEK 293 cells, which stably express adenoviral E1 gene products and should be capable of transcomplementing missing viral functions in these replication-deficient vectors. Mixed cultures containing HUVECs preinfected with the control Ad-Luc virus did not give observable cytopathic effect (Figure 4a) and distinct HUVEC and HEK 293 populations were still observable after 72 hours. In contrast complete cytopathic effect was achieved 72 hours after mixing Ad-eTie1-GALV-infected HUVECs with uninfected HEK 293 cells (Figure 4a), where the disappearance of syncytia at 48 and 72 hours is linked to the raised cytotoxic replication (Figure 4b). QPCR analysis of the number of Ad genome copies in the culture supernatant revealed an ~10,000-fold increase in Ad genomes between 24 and 72 hours when using Ad-eTie1-GALV, whereas Ad-Luc gave only a 100-fold rise over the same period (Figure 4b). This statistically significant difference (P < 0.001) in genome copy numbers obtained from cocultures with or without GALV demonstrate the successful transactivation of the replication-deficient virus in heterocellular syncytia. The feasibility of testing the Ad-eTie1-GALV vector in a murine model was demonstrated by the successful induction of murine endothelial (bEnd.3) fusion to human epithelial (HEK 293) cells in vitro. When Ad-Luc-infected bEnd.3 cells were mixed with uninfected HEK 293 cells no multinucleated syncytia were obtained (Figure 5a). However, when uninfected HEK 293 cells were mixed with bEnd.3 cells which had been preinfected with Ad-eTie1-GALV syncytia were obtained (Figure 5b), demonstrating murine (i.e., Pit-1 negative) endothelial cells producing GALV could undergo fusion with human HEK 293 (Pit-1 positive) cells. The demonstration of fusion-mediated viral transduction of noninfected cells in vitro encouraged the testing of this strategy in vivo to provide a more relevant, three-dimensional model. Ad-Luc or Ad-eTie1-GALV was given i.v. at 3 × 1010 vp/animal to mice-bearing subcutaneous HEK 293 xenografts, or control PC-3 tumors which do not express E1 gene products and therefore cannot provide transcomplementation. Xenografts and tumors were harvested after 48 hours. Measurement of Ad genomes copies in the HEK 293 xenografts revealed a threefold higher level of Ad-eTie1-GALV (1.2 × 108/xenograft) than Ad-Luc (4.0 × 107vp/xenograft) (P = 0.008), providing evidence for enhanced access of the replication-deficient virus to HEK 293 cells in the presence of GALV expression (Figure 5c). This likely to be a conservative measure of the extent to which endothelial GALV expression has increased virus access to xenografted cells, as plaque assays revealed that the titer of the control Ad-Luc used here was approximately tenfold higher than that of Ad-eTie1-GALV. In contrast to the significantly higher levels of Ad-eTiel-GALV compared to Ad-Luc found in HEK 293 xenografts, there was no statistically significant difference between the quantities of the viruses in the control E1-deficient PC-3 tumors (P = 0.38) (Figure 5d), demonstrating that the higher levels of Ad-eTie1-GALV was a result of increased virus access to and replication in HEK 293 xenografts. Since GALV expression is endothelial-selective, our data suggest that virus infection of tumor-associated endothelial cells has enabled GALV-mediated spread of the virus to adjacent xenografted epithelial cells. In order to confirm the in vivo formation of heterocellular syncytia between murine endothelium and HEK 293 cells, immunohistochemistry of tissue cross-sections was performed. Distinct endothelial (red) and epithelial (green) cells were observed in xenograft cross-sections from Ad-Luc treated animals (Figure 6a) with no evidence of colocalized staining. However, dual-stained, multinucleated structures were observed in sections obtained from animals treated with Ad-eTie1-GALV (Figure 6b). Heterocellular structures were rare; examination of 50 nonconsecutive tissue sections afforded observations of four dual-stained syncytia. The areas of intense yellow staining (see arrows) are indicative EpCAM and CD31 colocalization made possible by the GALV-mediated fusion of epithelial cells to endothelial cells. These data confirm the GALV-mediated formation of mouse endothelial-human epithelial heterocellular syncytia. The tumor-associated vasculature presents a highly accessible target for vp circulating in the bloodstream,3Kirn DH Wang Y Le Boeuf F Bell J Thorne SH Targeting of interferon-beta to produce a specific, multi-mechanistic oncolytic vaccinia virus.PLoS Med. 2007; 4: e353Crossref PubMed Scopus (158) Google Scholar,26Herz J Gerard RD Adenovirus-mediated transfer of low density lipoprotein receptor gene acutely accelerates cholesterol clearance in normal mice.Proc Natl Acad Sci USA. 1993; 90: 2812-2816Crossref PubMed Scopus (504) Google Scholar enabling active targeting approaches.27Bazan-Peregrino M Seymour LW Harris AL Gene therapy targeting to tumor endothelium.Cancer Gene Ther. 2007; 14: 117-127Crossref PubMed Scopus (27) Google Scholar In the present study, we explored a strategy to enhance viral vascular penetration in order to increase the therapeutic index of a systemically delivered Ad. We tested the hypothesis that vascular transcriptional targeting and subsequent induction of site-specific syncytium formation can be combined to turn a major structural obstacle imposed by the tumor endothelium into an entry route toward tumor cells. A hybrid viral enhancer/human promoter system encompassing the CMV enhancer and the Tie1 promoter was used to mediate conditional activation of GALV expression upon Ad infection of proliferating endothelium. Using this vector (Ad-eTie1-GALV) we achieved tight endothelial-selective transcriptional activity and targeted induction of endothelial–epithelial fusion in vitro and in vivo, presenting the first demonstration of exploiting a viral FMG to induce heterocellular syncytium formation in order to create access to target tumor cells. Upon endothelial infection and the induction of heterofusion, the ability of a GALV-expressing Ad to successfully replicate in heterocellular syncytia is crucial for subsequent vp dissemination. As there is no internuclear spread of Ad DNA upon GALV-induced syncytium formation,28Guedan S Gros A Cascallo M Vile R Mercade E Alemany R Syncytia formation affects the yield and cytotoxicity of an adenovirus expressing a fusogenic glycoprotein at a late stage of replication.Gene Ther. 2008; 15: 1240-1245Crossref PubMed Scopus (17) Google Scholar the ability of transactivating factors to be imported into nuclei-containing Ad genomes would be essential for triggering replication. Tumor-associated endothelial cells are noncancerous and should therefore be unable to support the replication of conditionally replicating Ad; therefore, the capacity of heterocellular syncytia to support virus transactivation following fusion of a virus-infected, nonpermissive cell type with a permissive cell type was investigated. The initiation of Ad replication upon fusion between endothelial cells infected with the E1, E3-deleted vector Ad-eTie1-GALV and cocultured permissive HEK 293 cells demonstrated the successful transactivation of the replication-defective virus in vitro. Dramatically increased (~2-log) levels of virus were recovered from these cocultures compared to those infected with a luciferase-expressing control vector, correlating with more rapid development of cytopathic effect. This is a novel demonstration that the cytoplasmic factors of an epithelial cell can be shared by the nuclei of an Ad-infected endothelial cell in a heterocellular syncytium, thereby drivin" @default.
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• W2043915150 title "Active Adenoviral Vascular Penetration by Targeted Formation of Heterocellular Endothelial–epithelial Syncytia" @default.
• W2043915150 cites W1605004657 @default.
• W2043915150 cites W1972320565 @default.
• W2043915150 cites W1975185686 @default.
• W2043915150 cites W1987723433 @default.
• W2043915150 cites W1990130844 @default.
• W2043915150 cites W1990545247 @default.
• W2043915150 cites W1993546257 @default.
• W2043915150 cites W2003516776 @default.
• W2043915150 cites W2007430864 @default.
• W2043915150 cites W2014583909 @default.
• W2043915150 cites W2018893865 @default.
• W2043915150 cites W2023743453 @default.
• W2043915150 cites W2030555272 @default.
• W2043915150 cites W2050934472 @default.
• W2043915150 cites W2055183613 @default.
• W2043915150 cites W2057913071 @default.
• W2043915150 cites W2058025467 @default.
• W2043915150 cites W2061581324 @default.
• W2043915150 cites W2082032640 @default.
• W2043915150 cites W2105754648 @default.
• W2043915150 cites W2120489698 @default.
• W2043915150 cites W2121627838 @default.
• W2043915150 cites W2125733229 @default.
• W2043915150 cites W2128054910 @default.
• W2043915150 cites W2129818141 @default.
• W2043915150 cites W2142040409 @default.
• W2043915150 cites W2161700314 @default.
• W2043915150 cites W2315349161 @default.
• W2043915150 cites W2345949512 @default.
• W2043915150 cites W29168110 @default.
• W2043915150 doi "https://doi.org/10.1038/mt.2010.209" @default.
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