Epidermal Growth Factor Receptor-Targeted Phage Gene Delivery System

Tumorigenesis is a very complex process requiring the accumulated alteration ofmultiple growth-promoting or growth-controlling genes. So far, a majority ofevidences in molecular biology have documented the role of genetic alterations intumorigenesis and have led to the development of potential new therapeuticapproaches. Among these therapeutic approaches, gene therapy seems the mostpotential. However, limitations still exist to the widespread application of genetherapy. One of the main issues remaining to be resolved is lack of an efficienttargeting gene transfer system. Therefore, it is very urgent to develop an efficienttargeting gene delivery agent. Modified filamentous bacteriophage is a new type ofgene therapy vector. Compared with other animal virus vector systems, it offersmany advantages. It is the easiest and most stable viruses to grow and purify. Aftermodified to display some peptide ligand, It can efficiently target to the mammaliancells which expressing the receptors of the peptide ligand. Moreover, it can easilysimultaneously display different peptides on the surface. So it should be a verypotential gene delivery system. Epidermal growth factor receptor (EGFR) plays anextremely important role in tumorigenesis. During the last two decades, manystudies have shown that the overexpression and mutation of EGFR have beenfrequently found in a variety of epithelial tumors, including non-small cell lung cancer,head and neck, breast, bladder, colorectal and ovarian cancer. Several significantobservations have established EGFR as a potential therapeutic target for thereceptor-mediated gene delivery system in cancer treatment. Here, we have attemptedto construct efficient EGFR-mediated phage gene delivery system. It will provide newtheoretic and practical guidance for targeting cancer gene therapy.SectionⅠApplication of EGFP-EGF Fusions to Explore Mechanism ofEndocytosis of Epidermal Growth Factor 【Objective】To explore the endocytic pathway of EGFP-EGF fusion protein inVitro.【Methods】Enhanced green fluorescent protein (EGFP) was used as anautofluorescent tag to label EGF ligand, pET28a-EGFP-EGF plasmid was constructedand expressed in prokaryotic expression systems. Protein expression and purificationwere performed by using Ni-NTA beads and Amicon Ultra-4 centrifugal filter devices.SDS-PAGE and Western blot analysis were used to detect the expression of theEGFP-EGF protein. The cell-binding and internalization activity of EGFP-EGF wereanalyzed by fluorescence-activated cell sorting (FACS) and confocal microscopy.【Results】EGFP-tagged EGF (EGFP-EGF) protein was expressed in Escherichiacoli and purified. A cell-binding assay demonstrated that the EGFP-EGF proteincould bind efficiently to the cells expressing EGFR. The binding and internalizationof EGFP-EGF can be visualized even at a very low concentration under confocalmicroscopy. The FACS-based assay for internalization activity indicated theaccumulation of internalized EGFP-EGF over time. Furthermore, the results of thecompetition assay indicated its EGFR binding specificity. Using such a method, itdoes not need to label EGF with chemicals and avoid light in the experimental process.【Conclusion】The fusion protein EGFP-EGF can be internalized by normalendocytic pathway. It has several characters including high sensitivity, stability andspecificity, and is a powerful tool for the study of EGF endocytosis.SectionⅡExploring Mechanism of Endocytosis of Epidermal GrowthFactor Receptor-Targeted Phagemid Particles【Objective】To investigate the endocytic pathway and distribution ofEGFR-targeted phage gene delivery vector in vitro.【Methods】EGF encodingsequence was inserted into the geneⅢof M13KO7 helper phage. EGF-modifiedhelper phage genome (plasmid) was transformed into F~+ bacterial cells and theresulted cells were designated as LMP cells. The phagemid encoding genes of interestwas then transformed into the LMP cells to make phagemid particles. LMP cells wasused to package phagemid such as pEGFP-Amp. ELISA was used to quantify the titerof the progeny virus particles, ssDNA was extracted and analyzed by agarose gelelectrophoresis to evaluate the percentage of the phagemid particles. The level of EGF protein displayed in the phagemid particles was examined by western blot. Theinternalization of the phagemid particles was further determined by incubating themwith PC-3 and NCI-H1299 cells. The endocytic pathway and distribution in cells ofEGF-displaying phage gene delivery vector was investigated by internalization assayand confocal microscopy.【Results】LMP cells was successfully used to packagephagemid such as pEGFP-Amp. The phagemid particles occupied most of theprogeny particles. The protein EGF was displayed efficiently in the phagemidparticles. Phagemid particles prepared by M13KO7EGFCT can be efficientlyinternalized into PC-3 cells while those by M13KO7 can not. In in vitro phagemidparticle transduction, reporter gene (EGFP) was found to be expressed in 10% ofPC-3 cells. The colocolization assay showed that low doses of phagemid particles (theconcentration of displayed EGF is about 0.5 ng/ml) were predominantly internalizedthrough clathrin-dependent pathway and high doses of phagemid particles (theconcentration of displayed EGF is about 7.5 ng/ml) were internalized throughclathrin-dependent and caveolin-mediated pathway. Furthermore, the overlap wasobserved between the internalized EGF-diplaying phagemid particles and earlyendosome marker EEA1 and lysosome marker LAMP1.【Conclusion】We havedeveloped a novel efficient technique to prepare targeted phagemid particles. TheEGF-diplaying phagemid particles can be internalized into EGFR expressing cellsthrough normal endocytic pathway.SectionⅢModification of Epidermal Growth Factor Receptor-TargetedPhage Gene Delivery vector【Objective】To improve the gene transduction efficiency of the epidermal growthfactor receptor-targeted phage gene delivery vector.【Methods】Peptides NLS andRGD-4C encoding sequence were inserted into the carboxyl terminal of geneⅥandthe amino terminal of geneⅦof M13KO7EGFCT helper phage. Modified helperphage genome (plasmid) and phagemid such as pEGFP-Amp were cotransformed intoF~+ bacterial cells and obtained the phagemid particles simultaneously displaying different peptides on the surface. ELISA was used to quantify the titer of the progenyvirus particles, ssDNA was extracted and analyzed by agarose gel electrophoresis toevaluate the percentage of the phagemid particles. The level of NLS and RGD-4Cpeptides displayed in the phagemid particles was examined by western blot. Theinternalization of the phagemid particles was further determined by incubating themwith HeLa and 7721 cells.【Results】Plasmids of displying peptides NLS andRGD-4C, M13KO7EGFCT-pⅥ-NLS, M13KOTEGFCT-pⅥ-RGD, M13KO7RGDCT,M13KO7EGFCT-pⅦ-NLS and M13KO7EGFCT-pⅦ-RGD were successfullyconstructed. Among of these, M13KO7EGFCT-pⅦ-NLS, M13KO7EGFCT-pⅦ-RGD and M13KO7RGDCT were used to package phagemid such as pEGFP-Ampwhile M13KO7EGFCT-pⅥ-NLS and M13KO7EGFCT-pⅥ-RGD can not. Thephagemid particles occupied most of the progeny particles. The peptides NLS andRGD-4C were displayed efficiently in the phagemid particles. Phagemid particlesprepared by M13KO7EGFCT-pⅦ-NLS, M13KO7EGFCT-pⅦ-RGD andM13KOTRGDCT can be efficiently internalized into HeLa and SMMC-7721 cells.However, the internalization assay showed that the effiency of transferring intonuclear of modified EGFR-targeted phagemid particle M13KOTEGFCT-pⅦ-NLSwas not clearly enhanced. In in vitro phagemid particle transduction, the expressionefficiency of reporter gene (EGFP) was also not increased evidently in cells.【Conclusion】The nuclear-transferring effiency of modified EGFR-targetedphagemid particle and the transduction efficiency in vitro were not significantlyimproved.SectionⅣDevelopment of efficient RNA interference system usingphagemid particles displaying EGF ligand【Objective】To develop an efficient RNA interference system using phagemidparticles displaying EGF ligand.【Methods】pSilencer1.0-siEGFP andpSilencer4.1-siAkt plasmids were constructed by gene clone technology. Modifiedhelper phage genome (plasmid) helper phage M13KO7EGFCT was used to package phagemid such as pSilencer1.0-siEGFP and pSilencer4.1-siAkt. ELISA was used toquantify the titer of the progeny virus particles, ssDNA was extracted and analyzed byagarose gel electrophoresis to evaluate the percentage of the phagemid particles. Theexpression level of report gene (EGFP) was determined by transducing phagemidparticles packaging pSilencer1.0- siEGFP into cells. The level of Akt gene expressionin cells transduced phagemid particles packaging pSilencer4.1-siAkt was examined bywestern blot. Hydroxycamptothecin was used to enhance the gene transductionefficiency.【Results】RNAi vectors pSilencer1.0-siEGFP and pSilencer4.1-siAktwere successfully constructed. M13KO7EGFCT helper phage was used in thepackageing of phagemid such as pSilencer1.0-siEGFP and pSilencer4.1-siAkt.Thephagemid particles occupied most of the progeny particles. Phagemid particlespackaging with siRNA can be efficiently internalized into NCI-H1299 cells. In vitrophagemid particle transduction, the expression of reporter gene (EGFP) was found tobe down-regulated. Western-blot assay showed that the down-regulated level of Aktgene expression can be increased when NCI-H1299 cells were treated with 2.5μMhydroxycamptothecin and the efficacy of suppression was dose-dependent.【Conclusion】Applying EGFR-targeted phage gene delivery system to deliverseveral siRNA into mammalian cells treated with hydroxycamptothecin, it candown-regulate the expression of exogenous and endogenous target gene to extent invitro.