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• W2037993233 abstract "Developing nanomaterials that are effective, safe, and selective for gene transfer applications is challenging. Bacteriophages (phage), viruses that infect bacteria only, have shown promise for targeted gene transfer applications. Unfortunately, limited progress has been achieved in improving their potential to overcome mammalian cellular barriers. We hypothesized that chemical modification of the bacteriophage capsid could be applied to improve targeted gene delivery by phage vectors into mammalian cells. Here, we introduce a novel hybrid system consisting of two classes of nanomaterial systems, cationic polymers and M13 bacteriophage virus particles genetically engineered to display a tumor-targeting ligand and carry a transgene cassette. We demonstrate that the phage complex with cationic polymers generates positively charged phage and large aggregates that show enhanced cell surface attachment, buffering capacity, and improved transgene expression while retaining cell type specificity. Moreover, phage/polymer complexes carrying a therapeutic gene achieve greater cancer cell killing than phage alone. This new class of hybrid nanomaterial platform can advance targeted gene delivery applications by bacteriophage. Developing nanomaterials that are effective, safe, and selective for gene transfer applications is challenging. Bacteriophages (phage), viruses that infect bacteria only, have shown promise for targeted gene transfer applications. Unfortunately, limited progress has been achieved in improving their potential to overcome mammalian cellular barriers. We hypothesized that chemical modification of the bacteriophage capsid could be applied to improve targeted gene delivery by phage vectors into mammalian cells. Here, we introduce a novel hybrid system consisting of two classes of nanomaterial systems, cationic polymers and M13 bacteriophage virus particles genetically engineered to display a tumor-targeting ligand and carry a transgene cassette. We demonstrate that the phage complex with cationic polymers generates positively charged phage and large aggregates that show enhanced cell surface attachment, buffering capacity, and improved transgene expression while retaining cell type specificity. Moreover, phage/polymer complexes carrying a therapeutic gene achieve greater cancer cell killing than phage alone. This new class of hybrid nanomaterial platform can advance targeted gene delivery applications by bacteriophage. Successful delivery of gene expression to desired sites in vivo following systemic administration will have a major impact on the practice of medicine,1Liu G Swierczewska M Lee S Chen X Functional nanoparticles for molecular imaging guided gene delivery.Nano Today. 2010; 5: 524-539Crossref PubMed Scopus (125) Google Scholar in particular on the advance of gene therapy, genetic imaging, and DNA vaccine applications. Moreover, targeting systemic gene delivery to diseased tissue presents an efficient and safer approach to “theragnostics,” i.e., both gene therapy and genetic imaging combined into one vector system. Most progress in gene delivery has been made with eukaryotic viruses such as adenovirus, adeno-associated virus (AAV), and lentivirus, which, unquestionably, provide superior gene delivery vectors.2Verma IM Weitzman MD Gene therapy: twenty-first century medicine.Annu Rev BioChem. 2005; 74: 711-738Crossref PubMed Scopus (484) Google Scholar However, systemic administration using these eukaryotic viruses has had limited success due to undesired uptake by the liver and the reticulo-endothelial system, insertional mutagenesis, immunogenicity, pre-existing antibodies, and broad tropism for mammalian tissues.3Waehler R Russell SJ Curiel DT Engineering targeted viral vectors for gene therapy.Nat Rev Genet. 2007; 8: 573-587Crossref PubMed Scopus (533) Google Scholar Numerous materials are being developed to increase gene transfer function. The widely used approaches involved the complexation of naked DNA with cationic polymers or cationic lipids.4Wang B Yi WJ Zhang J Zhang QF Xun MM Yu XQ TACN-based cationic lipids with amino acid backbone and double tails: materials for non-viral gene delivery.Bioorg Med Chem Lett. 2014; 24: 1771-1775Crossref PubMed Scopus (21) Google Scholar Recently, bacteriophages, which are among the most promising new type of biological nanomaterials, have attracted attention as safe and new class of vectors for targeted systemic delivery of transgenes. They have no intrinsic tropism for mammalian cell receptors but can be modified to display tissue-specific ligands on the coat proteins without disruption of their virus structure.5Di Giovine M Salone B Martina Y Amati V Zambruno G Cundari E et al.Binding properties, cell delivery, and gene transfer of adenoviral penton base displaying bacteriophage.Virology. 2001; 282: 102-112Crossref PubMed Scopus (44) Google Scholar,6Hart SL Knight AM Harbottle RP Mistry A Hunger HD Cutler DF et al.Cell binding and internalization by filamentous phage displaying a cyclic Arg-Gly-Asp-containing peptide.J Biol Chem. 1994; 269: 12468-12474Abstract Full Text PDF PubMed Google Scholar,7Ivanenkov V Felici F Menon AG Uptake and intracellular fate of phage display vectors in mammalian cells.Biochim Biophys Acta. 1999; 1448: 450-462Crossref PubMed Scopus (79) Google Scholar,8Larocca D Witte A Johnson W Pierce GF Baird A Targeting bacteriophage to mammalian cell surface receptors for gene delivery.Hum Gene Ther. 1998; 9: 2393-2399Crossref PubMed Scopus (98) Google Scholar,9Piersanti S Cherubini G Martina Y Salone B Avitabile D Grosso F et al.Mammalian cell transduction and internalization properties of lambda phages displaying the full-length adenoviral penton base or its central domain.J Mol Med (Berl). 2004; 82: 467-476Crossref PubMed Scopus (31) Google Scholar Such vectors have many advantages over animal viral vectors and nonviral gene delivery systems due to a number of promising characteristics. Firstly, bacteriophage is a nano-sized natural system capable of efficiently condensing and packaging DNA. The high tolerance for phage coat protein mutations allows insertions of foreign peptides to achieve ligand-directed targeting to the desired cell types and unlike eukaryotic viral vectors, targeting bacteriophage vectors does not require elimination of native tropism.10Monaci P Urbanelli L Fontana L Phage as gene delivery vectors.Curr Opin Mol Ther. 2001; 3: 159-169PubMed Google Scholar They are safe, having long been used for both prophylaxis and treatment of bacterial infections, both in adults and children, with no safety concerns being identified.11Monk AB Rees CD Barrow P Hagens S Harper DR Bacteriophage applications: where are we now?.Lett Appl Microbiol. 2010; 51: 363-369Crossref PubMed Scopus (146) Google Scholar They have also been approved by the US Food and Drug Administration for use as safe antibacterial food additives.12Lang LH FDA approves use of bacteriophages to be added to meat and poultry products.Gastroenterology. 2006; 131: 1370PubMed Scopus (72) Google Scholar Large-scale production and purification of phage vectors are simple and economical. Finally, they have a large cloning capacity for insertion of foreign DNA.13Greenstein D Brent R Introduction to vectors derived from filamentous phages.Curr Protoc Mol Biol. 2001; Chapter 1: Unit1.14PubMed Google Scholar We previously introduced an M13 phage-based vector displaying the double cyclic RGD (CDCRGDCFC, RGD4C) ligand to target overexpressed αv integrin receptors in tumors, and incorporating a mammalian transgene cassette flanked by inverted terminal repeats from AAV2. This vector can selectively deliver transgenes to tumors in rodents and pet dogs after intravenous administration, while sparing normal organs.14Hajitou A Lev DC Hannay JA Korchin B Staquicini FI Soghomonyan S et al.A preclinical model for predicting drug response in soft-tissue sarcoma with targeted AAVP molecular imaging.Proc Natl Acad Sci USA. 2008; 105: 4471-4476Crossref PubMed Scopus (61) Google Scholar,15Hajitou A Rangel R Trepel M Soghomonyan S Gelovani JG Alauddin MM et al.Design and construction of targeted AAVP vectors for mammalian cell transduction.Nat Protoc. 2007; 2: 523-531Crossref PubMed Scopus (85) Google Scholar,16Hajitou A Trepel M Lilley CE Soghomonyan S Alauddin MM Marini 3rd FC et al.A hybrid vector for ligand-directed tumor targeting and molecular imaging.Cell. 2006; 125: 385-398Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar,17Tandle A Hanna E Lorang D Hajitou A Moya CA Pasqualini R et al.Tumor vasculature-targeted delivery of tumor necrosis factor-alpha.Cancer. 2009; 115: 128-139Crossref PubMed Scopus (71) Google Scholar,18Trepel M Stoneham CA Eleftherohorinou H Mazarakis ND Pasqualini R Arap W et al.A heterotypic bystander effect for tumor cell killing after adeno-associated virus/phage-mediated, vascular-targeted suicide gene transfer.Mol Cancer Ther. 2009; 8: 2383-2391Crossref PubMed Scopus (44) Google Scholar Bacteriophage is thus the first system that has been experimentally shown to offer safe and efficient delivery of transgenes to target tissues after systemic administration in vivo. Unfortunately, phage particles are comparatively poor biomaterial vectors, as they have evolved to infect bacteria only. Unlike eukaryotic viruses, they have no intrinsic strategies for delivering genes to mammalian cells.19Przystal JM Umukoro E Stoneham CA Yata T O'Neill K Syed N et al.Proteasome inhibition in cancer is associated with enhanced tumor targeting by the adeno-associated virus/phage.Mol Oncol. 2013; 7: 55-66Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar,20Stoneham CA Hollinshead M Hajitou A Clathrin-mediated endocytosis and subsequent endo-lysosomal trafficking of adeno-associated virus/phage.J Biol Chem. 2012; 287: 35849-35859Crossref PubMed Scopus (44) Google Scholar As a result, although phage-derived vectors undoubtedly have great promise, due to this inherent limitation, they need to be improved if they are going to find wide clinical applications. As a proof-of-concept study, we report a hybrid platform of phage and synthetic materials. They are self-assembled complexes consisting of a recombinant M13 bacteriophage displaying the RGD4C-targeting ligand and containing a eukaryotic transgene cassette, coupled with a synthetic cationic polymer material (Figure 1). We have investigated their physical and chemical properties (surface chemistry, electrokinetic behavior, size, and morphology), and their biological activity (transgene expression, cytotoxicity, cell–vector interaction, and delivery mechanisms). Integration of phage with polymers successfully generated positively charged, large-sized particles with increased potential for binding to the surface of eukaryotic cells, better buffering capacity suggesting enhanced endosomal escape and subsequently improved gene transfer efficiency. Importantly, gene delivery by the hybrid vector remained targeted and specific, inducing cancer cell killing. We believe this innovation represents a major advance in phage-mediated gene transfer with potential for clinical applications. We sought to assess whether the efficiency of gene delivery by the RGD4C-phage to eukaryotic cells can be improved if phage viral particles are integrated with cationic polymers. We therefore studied the efficacy with which RGD4C-phage/polymer complexes transduce human M21 melanoma cells, which are known to express high levels of αv integrin receptors for the RGD4C ligand.17Tandle A Hanna E Lorang D Hajitou A Moya CA Pasqualini R et al.Tumor vasculature-targeted delivery of tumor necrosis factor-alpha.Cancer. 2009; 115: 128-139Crossref PubMed Scopus (71) Google Scholar,21Hood JD Bednarski M Frausto R Guccione S Reisfeld RA Xiang R et al.Tumor regression by targeted gene delivery to the neovasculature.Science. 2002; 296: 2404-2407Crossref PubMed Scopus (782) Google Scholar To rule out the possibility that the observed effects are not cell or species specific, we also assessed the efficacy on the rat 9L glioblastoma cells, which have previously been shown to be transduced by the RGD4C-phage.19Przystal JM Umukoro E Stoneham CA Yata T O'Neill K Syed N et al.Proteasome inhibition in cancer is associated with enhanced tumor targeting by the adeno-associated virus/phage.Mol Oncol. 2013; 7: 55-66Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar,22Kia A Przystal JM Nianiaris N Mazarakis ND Mintz PJ Hajitou A Dual systemic tumor targeting with ligand-directed phage and Grp78 promoter induces tumor regression.Mol Cancer Ther. 2012; 11: 2566-2577Crossref PubMed Scopus (30) Google Scholar We first sought to determine the optimal ratio of two cationic polymers (poly-d-lysine (PDL) and DEAE-DEX) and RGD4C-phage using RGD4C-phage vector carrying the firefly luciferase (Luc) reporter gene. Quantification of luciferase activity in 9L and M21 cells at 72-hour post-cell transduction showed that Luc gene expression by the RGD4C-phage dramatically improved with increased concentrations of PDL and DEAE.DEX polymers (Figure 2a), as compared with RGD4C-phage alone (0 μg/ml of polymer). Maximum gene transfer levels were achieved in both M21 and 9L cells at polymer/phage ratios of 30 ng/μg for PDL and 60 ng/μg for DEAE.DEX, respectively, after which a gradual decrease in Luc gene expression occurred (Figure 2a). To determine whether the decreased transgene expression at high amounts of cationic polymers was associated with PDL and DEAE.DEX cytotoxicity, we performed cell viability assays and showed that this range of polymer concentrations was not associated with any toxic effects (Figure 2b). Next, we used the previously established optimal ratios of polymer and phage to assess the efficacy of gene transfer by the hybrid RGD4C-phage/polymer complexes over a period of 5 days following transduction of M21 and 9L cells (Figure 2c). Four different vector systems were investigated: non-targeted phage (NT), targeted RGD4C-phage (RGD4C) displaying the tumor-targeting ligand on pIII minor coat protein, RGD4C-phage complexed with PDL (termed RGD4C-PDL), and RGD4C-phage complexed with DEAE.DEX (termed RGD4C-DEAE.DEX). Considerable increase in expression of the Luc transgene was detected in both M21 and 9L cells transduced with the hybrid RGD4C-PDL and RGD4C-DEAE.DEX phage/polymer complexes at day 5 post-transduction (Figure 2c). This Luc gene expression increased rapidly over time, whereas gene expression remained low in cells transduced by the RGD4C-phage, and none was detected in cells incubated with a control NT phage. For instance, at day 5 post-transduction, treatment with RGD4C-PDL and RGD4C-DEAE.DEX phage/polymer resulted in ~10.3- and ~6.6-fold increase in Luc gene expression in 9L cells and ~10.0- and ~15.0-fold in M21 cells, respectively, compared with RGD4C-phage alone (Figure 2c). Next, to further explore the superiority of the RGD4C-phage vector when combined with cationic polymers, we assembled a panel of cancer and normal cell lines. The human LN229 and SNB19 glioma cells were incubated with vectors bearing the Luc reporter transgene. Marked and dose-dependent increase in gene delivery was detected with RGD4C-PDL or RGD4C-DEAE.DEX compared with uncomplexed RGD4C-phage (Supplementary Figure S1a). Similar effects of the DEAE.DEX polymers were also observed in the rat C6 brain tumor cells (Supplementary Figure S1a). These data were confirmed by using vectors carrying the green fluorescent protein (GFP) reporter gene (Supplementary Figure S1b). Finally, we also evaluated efficacy in a nontumor cellular model using the human embryonic kidney (HEK293) cell line. These cells have extensively been used as a standard in vitro model to characterize cell transduction by RGD4C-phage vectors since they express high levels of αvβ3 and αvβ5 integrins.15Hajitou A Rangel R Trepel M Soghomonyan S Gelovani JG Alauddin MM et al.Design and construction of targeted AAVP vectors for mammalian cell transduction.Nat Protoc. 2007; 2: 523-531Crossref PubMed Scopus (85) Google Scholar,16Hajitou A Trepel M Lilley CE Soghomonyan S Alauddin MM Marini 3rd FC et al.A hybrid vector for ligand-directed tumor targeting and molecular imaging.Cell. 2006; 125: 385-398Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar Thus, HEK293 cells were treated with vectors bearing the Luc or GFP reporter transgenes. Quantitative analysis of Luc activity at day 3 post-vector transduction showed that Luc gene expression by the RGD4C-DEAE.DEX or RGD4C-PDL was significantly enhanced with increased concentrations of DEAE.DEX and PDL polymers (Supplementary Figure S2a), as compared with RGD4C-phage alone (0 μg/ml of polymer). Maximum transduction efficiency was achieved at optimal polymer/phage ratios of 250 ng/μg for DEAE.DEX and 125 ng/μg for PDL, followed by a gradual decrease in Luc gene expression (Supplementary Figure S2a). Importantly, no effect on HEK293 cell viability was induced by this range of polymer concentrations (Supplementary Figure S2b). These data were confirmed with microscopic imaging of GFP expression in HEK293 cells, revealing increase in GFP expression in those treated with RGD4C-phage/polymer complexes (Supplementary Figure S3). No GFP expression was detected in cells treated with the control NT phage. Altogether, these data validate that the integration of cationic polymers with bacteriophage boosts gene transfer efficiency. To gain insight into the characterization of the hybrid phage/polymer complexes, we sought ways to explore the electrostatic charge on the phage capsid. We first investigated the charge characteristics of the bacteriophage viral particles by measuring their ζ-potential using electrophoresis (Figure 3a). As expected, we found that bacteriophage is negatively charged at physiological pH (Figure 3a). The data also indicate that RGD4C-phage possesses an acidic surface, with an isoelectric point of pH = 3 as determined from ζ-potential = f(pH) (Figure 3b). Next, we analyzed the ζ-potential of bacteriophage vectors following hybridization with cationic polymers PDL and DEAE.DEX (Figure 3c). We found that addition of increasing concentrations of both cationic polymers PDL and DEAE.DEX resulted in gradual shift of the ζ-potential from a negative value for unmodified bacteriophage to a positive value for the phage complexed with polymers to reach maximum positive values after which the ζ-potential started to drop significantly. For instance, incorporation of 25-ng PDL with 1 μg of phage resulted in shifting the ζ-potential from −5 mV to +15 mV, after which the ζ-potential dropped sharply toward a zero value. This increase in ζ-potential can be attributed to surface adsorption of the polymer counteracting the inherent negative charge of the bacteriophage particles. Next, we conducted a second set of experiments to assess whether addition of cationic polymers leads to aggregation of the phage viruses resulting in the formation of large size particles. Size measurements of phage/polymer complexes revealed that increasing the polymer concentrations resulted in gradual increase in the average size of hybrid complexes (Figure 3d). These results were confirmed by confocal microscopy and anti-phage staining showing the morphology of uncomplexed phage as single filamentous particles, whereas the addition of cationic polymers resulted in aggregation of the phage particles (Figure 3e). Moreover, size distribution of the RGD4C-phage and RGD4C-phage/polymer complexes revealed marked heterogeneity in size and populations of the RGD4C-PDL and RGD4C-DEAE.DEX complexes (Figure 3f). Finally, we investigated whether complex formation was required prior to treatment of cells. Thus, 9L cells were first treated with cationic polymers alone followed by addition of the RGD4C-phage. We found that separate treatments of cells starting with cationic polymers and then with RGD4C-phage had no significant effect on gene delivery efficacy (Supplementary Figure S4). These data suggest that formation of the phage/polymer complex, prior to incubation with cells, is required to improve transduction efficiency. Altogether, these results indicate that negatively charged phage particles are physically incorporated with cationic polymers to form large complex aggregates. We also investigated the phage/polymer cell surface accessibility to determine whether gene delivery efficiency by the RGD4C-phage is limited by inefficient access to the negatively charged cell surface. We therefore carried out a supernatant-depletion assay, where the free cell-unbound phage in the external fluid phase above the adherent cell layer was quantified by infection of host bacteria followed by colony counting (Figure 4a). A large amount of free phage particles (90% of input phage particles) were recovered from the supernatant of cells treated with the RGD4C-phage vector (Figure 4b), showing that only a small fraction (10% of input phage) was bound to the cell surface. By contrast, very little phage (6%) was recovered from the supernatant of cells incubated with RGD4C-PDL and RGD4C-DEAE.DEX phage/polymer complexes, indicating that most of the phage (94%) was bound to the surface of cells (Figure 4b). No phage depletion was observed in the supernatant of cells treated with the control NT phage (Figure 4b). Confocal microscopic imaging following immunofluorescence with an anti-phage antibody revealed greater cell surface localization of the RGD4C-phage/polymer aggregates than the uncomplexed RGD4C-phage alone (Figure 4c). No phage was observed on cells incubated with the control NT phage (Figure 4c). These data strongly suggest that incorporation of phage into a cationic complex increases phage accessibility to the cell surface. Finally, internalization assays revealed that incorporation of phage with the cationic polymers PDL or DEAE.DEX does not increase cellular entry of the RGD4C-phage (Supplementary Figure S5). Endosomal escape is an important factor to be considered for the design of gene delivery vectors. This mechanism is associated with the buffering capacity of gene vectors within the pH range, in which vectors traffic from the extracellular environment into acidified endosomal compartments. Polycations with high buffering capacity can mediate efficient escape from the endosome to the cytosol triggered by the acidic endosome via a mechanism termed the “proton sponge effect.”23Nel AE Mädler L Velegol D Xia T Hoek EM Somasundaran P et al.Understanding biophysicochemical interactions at the nano-bio interface.Nat Mater. 2009; 8: 543-557Crossref PubMed Scopus (5226) Google Scholar A buffering capacity should allow the complexes to absorb protons pumped into the endosomes resulting in an influx of Cl− ions to prevent the build-up of a charge gradient. This influx of both protons and Cl− ions increases the osmolarity of the endosomes leading to osmotic swelling, subsequent endosomal destabilization and release of their contents into the cytoplasm. We previously identified the endosomal-lysosomal degradative pathway as an intracellular barrier to RGD4C-phage-derived particles, which are sequestered and degraded within the lysosomes, reducing their ability to deliver genes to mammalian cells.20Stoneham CA Hollinshead M Hajitou A Clathrin-mediated endocytosis and subsequent endo-lysosomal trafficking of adeno-associated virus/phage.J Biol Chem. 2012; 287: 35849-35859Crossref PubMed Scopus (44) Google Scholar It was reported that cationic polymers increase the gene transfer efficiency of nonviral vectors due to their ability to induce endosomal escape.23Nel AE Mädler L Velegol D Xia T Hoek EM Somasundaran P et al.Understanding biophysicochemical interactions at the nano-bio interface.Nat Mater. 2009; 8: 543-557Crossref PubMed Scopus (5226) Google Scholar Therefore to assess the buffering capacity of the phage/polymer complexes, we performed acid–base titrations to compare the polymer, RGD4C-phage alone and the hybrid complexes with a sodium chloride (NaCl) control solution (Figure 5a). Moreover, we extended the list of cationic polymers to include the poly(ethyleneimine) (PEI), since this polymer has been reported for its buffering capacity and potential to enhance endosomal escape of delivery vectors.24Akinc A Thomas M Klibanov AM Langer R Exploring polyethylenimine-mediated DNA transfection and the proton sponge hypothesis.J Gene Med. 2005; 7: 657-663Crossref PubMed Scopus (1106) Google Scholar We found that both complexes RGD4C-DEAE.DEX and RGD4C-PEI have high buffering capacity similar to DEAE.DEX and PEI polymers, respectively (Figure 5a), and required additional HCl, compared to control NaCl, to lower the pH from 7.0 to 4.0 (Figure 5a). The uncomplexed RGD4C-phage vector displayed a titration curve similar to NaCl and therefore has no buffering capacity (Figure 5a). An important finding was that RGD4C-PDL complex had no buffering capacity compared with RGD4C-DEAE.DEX and RGD4C-PEI (Figure 5a). This result is consistent with the Luc gene expression analyses demonstrating higher transduction efficacies of RGD4C-DEAE.DEX and RGD4C-PEI than RGD4C-PDL (Figure 5b). These data provide strong evidence that the buffering capacity of the RGD4C-DEAE.DEX and RGD4C-PEI hybrids contributes to their gene delivery efficiency. To confirm these findings, we performed transduction experiments in the presence of bafilomycin A1, a specific inhibitor of the vacuolar ATPase proton pump that prevents endosomal escape.20Stoneham CA Hollinshead M Hajitou A Clathrin-mediated endocytosis and subsequent endo-lysosomal trafficking of adeno-associated virus/phage.J Biol Chem. 2012; 287: 35849-35859Crossref PubMed Scopus (44) Google Scholar The results showed a significant decrease in transduction efficiencies of RGD4C-DEAE.DEX and RGD4C-PEI in the presence of bafilomycin A1; in contrast, there was no significant effect on RGD4C-PDL (Figure 5b). These data give further evidence to an additional mechanism of improved gene delivery by the RGD4C-DEAE.DEX and RGD4C-PEI phage/polymer complexes by facilitating escape from the endosome-lysosome degradative pathway. We want to confirm that the targeting properties of the RGD4C-phage vector remain intact in the phage/polymer complex and that transduction of cells is specific and mediated by binding of the RGD4C ligand to the αv integrin receptors. Therefore, cell transduction efficiency of the RGD4C-phage/polymer complex was compared with that of complexes between polymer and either NT phage or phage displaying a mutant version of the RGD4C sequence (RGE4C, D–E), previously reported as NT controls for the RGD4C-phage.14Hajitou A Lev DC Hannay JA Korchin B Staquicini FI Soghomonyan S et al.A preclinical model for predicting drug response in soft-tissue sarcoma with targeted AAVP molecular imaging.Proc Natl Acad Sci USA. 2008; 105: 4471-4476Crossref PubMed Scopus (61) Google Scholar,16Hajitou A Trepel M Lilley CE Soghomonyan S Alauddin MM Marini 3rd FC et al.A hybrid vector for ligand-directed tumor targeting and molecular imaging.Cell. 2006; 125: 385-398Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar As shown in Figure 6a, Luc gene expression in cells treated with the RGE4C-phage/polymer was nonsignificant and comparable to that of NT phage/polymer complex. Next, to confirm specificity, we carried competition experiments by incubation with a phage lacking a gene expression cassette, but expressing the RGD4C peptide on the capsid, because use of significant excess of the soluble RGD ligand causes cell toxicity. The data showed that the RGD4C-expressing phage inhibits gene delivery by the targeted RGD4C-PDL and RGD4C-DEXAE.DEX phage/polymer complexes in a dose-dependent manner (Figure 6b). Finally, to prove the ability of the RGD4C-phage/polymer complexes to target gene delivery to αv integrin-expressing cells specifically, but not cells lacking the αv integrin receptors, we compared transduction efficiencies between the 9L tumor cells and the normal C2C12 myoblast mouse cell line using RGD4C-PDL and RGD4C-DEAE.DEX complexes containing increasing concentrations of either PDL or DEAE.DEX cationic polymers (Figure 6c). While the RGD4C-phage/polymer complexes reproduced the expected pattern of Luc transgene expression in 9L cells, no transgene expression was induced in C2C12 cells by any concentration of either PDL or DEAE.DEX polymers (Figure 6c). We also confirmed the expression of the αv integrin receptors on the 9L tumor cells, in contrast to the C2C12 myoblast cells lacking this integrin (Figure 6d). These findings confirm that gene delivery by the RGD4C-phage/polymer complex is targeted, specific, and dependent on αv integrin receptors. After establishing the integration of cationic polymers with bacteriophage vectors substantially increases their gene transfer efficacy, we sought to assess the effect of polymer on a specific clinical application of phage, namely gene therapy. To test the efficacy of tumor cell killing of the RGD4C-phage/polymer complex, we constructed RGD4C-phage vector carrying the HSVtk gene. When combined with the GCV prodrug, this gene can serve as a suicide gene. We chose to conduct our experiments in the 9L glioblastoma model as this tumor type is highly aggressive and remains a major clinical challenge. We compared the RGD4C-phage vector alone with the complexes RGD4C-PDL and RGD4C-DEAE.DEX. HSVtk suicide gene therapy was induced at day 3 post-transduction by daily treatment with GCV for 5 days. Morphological characteristics of 9L cells 3 days post-GCV treatment were visualized by brightfield microscopy. Figure 7a shows normal morphology of confluent cells in all treatments in the absence of GCV and in cells treated with a combination of NT phage and GCV. There was a significant increase in cells detaching from the substrate by the RGD4" @default.
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• W2037993233 date "2014-01-01" @default.
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• W2037993233 title "Hybrid Nanomaterial Complexes for Advanced Phage-guided Gene Delivery" @default.
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• W2037993233 doi "https://doi.org/10.1038/mtna.2014.37" @default.
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