Abstract
We engineered a phage vector for targeted gene delivery to mammalian cells by inserting a mammalian reporter gene expression cassette (copGFP) into the vector backbone. Engineered phage vectors are a good strategy for gene delivery because they best rating are lack natural tropism to mammalian cells and can be genetically modified for specific applications. We have genetically modified filamentous M13 bacteriophage to deliver genes or (gene delivery) to mammalian cells. In previous studies have been demonstrated that 3VGR19 Nanobody specifically binds VEGFR2 on the surface of 293KDR and HUVECs cells. Here, we describe that phage vector can be engineered to infect eukaryotic cells resulting in expression of a reporter gene inserted into the M13 bacteriophage genome. To achieve reporter gene expression, pHEN4/3VGR19 phage were engineered to package the copGFP reporter gene driven by the CMV promoter. CopGFP is a green fluorescent protein cloned from copepod Pontellina plumata, has been genetically fused to many protein in various species to produce stable chimeras which apparently retain their original biological activity as well as retaining the fluorescent properties of native GFP. Here, we have engineered a phagemid vector that express the Nanobody against VEGFR2 for fusion with pIII phage gene (pHEN4/3VGR19) for targeted mammalian cell gene transfer by inserting a CMV-promoter-regulated CopGFP expression cassette. This phage when applied to cells undergo VEGFR2-mediated endocytosis leading to copGFP expression.
These data suggest the possibility of using phage-based vectors, with targeting molecules displayed on the surface include Nanobody, leading to specific targeted in cancer gene therapy.
Keywords: Targeted Gene Delivery, Bacteriophage, Receptor-Mediated Gene Transfer, Nanobody, VEGFR2
1- INTRODUCTION
The widespread application of gene therapy requires the ability to deliver therapeutic genes to the appropriate cells type with high efficiency while minimizing vector-associated toxicity [1-3]. Bacteriophage vectors have many of the favorable properties of both viral and non-viral vectors without the significant drawbacks. Filamentous bacteriophage lack intrinsic tropism for mammalian cells and can be conveniently produced at high titer in bacterial culture making production potentially simpler and more economical than either nonviral or viral vectors. Moreover phage particles are simply packaging. the most significant of filamentous phage advantage is genetic flexibility that allows a wide variety of peptides, proteins and antibodies can displayed on the phage surface, so allowing phage to be targeted genetically to cell surface receptors [3, 4]. Bacteriophage vectors can be modified and engineered to display foreign peptides, proteins and antibodies as fusions to their coat proteins such as pIII for monovalent or pVIII for polyvalent expression of the fusions [5, 6]. Gene transfer by targeted phage is dependent to ligand, time and dose transduction and is specific for the appropriate cell surface receptors[5] .
Two types of vectors including phage and phagemid vector have been used for display of exogenous genes on the surfaces of filamentous phage. In phage vector system, peptides or proteins can be displayed as fusion to gIII through cloning of gene of interest directly within the phage genome. Therefore, a highly display level of foreign peptides will be obtained of phage vector system, because all pIII molecules are originally fusions with exogenous genes. However, there is limitation on the use of phage vectors in size of exogenous proteins that fused with pIII. Because large foreign proteins fusion with N-terminal of pIII prevents the interaction of pIII with pili on bacterium that for infection step this interaction is required. Therefore for the display of larger molecules such as antibodies, a phagemid vector preferred. In a phagemid vector system, DNA of exogenous proteins are cloned with gIII in a phagemid vector, and packaging and releasing of recombinant phage are provided by a helper phage such as VCSM13. Thereby in this system, phage particles display pIII in both wild-type gIII of the helper phage (that is required for the successful reinfection of recombinant phage for amplification) and the fusion pIII from the resident phagemid [6-8].
Poul and Marks have shown that filamentous phage vectors displaying the anti-ErbB2 scFv F5 phage as a genetic fusion with the phage minor coat protein pIII can directly infected mammalian cells expressing ErbB2 leading to expression of a reporter gene contained in the phage genome [1].
In similar findings, Larocca et al demonstrated that when a phage is genetically engineered to display the growth factor ligand, FGF2, it obtains the ability to deliver a gene to mammalian cells via the FGF receptor resulting in transduced cells. They constructed FGF2 display phage by fusing the FGF2 gene to the pIII gene and phage were modified for transduction by mammalian cells by inserting a green fluorescent protein (GFP) reporter gene that obtained of transcription driven by the CMV promoter [9].
Initially phage-mediated transduction efficiency were 1-4% of transfected cells. However, later optimized the phage vector and transduction levels as high as 10% are obtained in human prostate carcinoma cells that transfected with multivalent phagemid vectors. [1, 5, 10].
Behdani initially demonstrated that engineered bacteriophage could be targeted to mammalian cell-surface receptors for gene delivery using phage particles that expressed 3VGR19 Nanobody against VEGFR2 reseptor on surface of cells [11]. In this study, we demonstrated mammalian cell tropism to an M13 phage vector by inserting a CMV regulated reporter gene (copGFP) and genetically targeting with VEGFR2.
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Here, we have an engineered phagemid vector for targeted mammalian cell gene transfer by inserting 3VGR19 Nanobody gene for fusion with pIII coat protein against VEGFR2 reseptor that have been constructed by Behdani [11]. We engineered phagemid vector for gene transfer to mammalian cells by inserting a CMV/copGFP gene for fiusion pIII gene into 3VGR19 phagemid that genetically targeting with VEGFR2 reseptor.
2- MATERIALS AND METHODS
1-2 Cell Lines and Cell Culture
HEK293T, 293KDR cells (obtained from the Pasteur Institute of Iran) used in this study were cultured in Dulbecco’s modified Eagle’smedium (DMEM) supplemented with 10% FBS (Fetal Bovin Serum), 100 units/ml penicillin, 100 µg/ml streptomycin, 2 mM l-glutamine and incubated at 37°C in 5% CO2 incubator. 293KDR is a stably transfected cell line for VEGFR2 [12].
2-2 Construction of phagemid for mammalian cell reporter gene
The final phagemid vector which named pHEN4/3VGR19/copGFP was constructed in two sequential sub-cloning as shown in figure 1. Briefly, the GFP expressing cassette was amplified by PCR from pCMV/myc/ER/GFP plasmid as template. The PCR was done with pCMV (EcoRI)5ï‚¢-GTACCGAATTCACATTGATTATTG-3ï‚¢ and BGH(NarI) 5ï‚¢-actgggcgccCGCCTCAGAAGCCATAGAG-3ï‚¢) primers. The resulted PCR product was cloned in pHENE4/3VGR19 phagemid (Fig.1a) [11].
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(b)
Figure 1. (a) Construction of pHEN4/3VGR19/GFP expression plasmid. A PCR reaction was performed with pCMV (EcoRI) and BGH (NarI) primers and cloned in the pHEN4/3VGR19 vector, restricted with EcoRI and NarI and ligated to each other. (b) Construction of pHEN4/3VGR19/copGFP. A PCR reaction was performed with copGFP (NheI) and copGFP (SacI) primers and cloned in pHEN4/3VGR19/GFP phagemid. In this cloning copGFP sequence was replacing with GFP sequence. The final construct was named pHEN4/3VGR19/copGFP.
pHEN4/3VGR19/copGFP was generated by replacing copGFP sequence. copGFP fragment from pCDH-CMV-MCS-EF1-cGFP-T2A-Puro plasmid was amplified using primers with NheI and SacI restriction endonuclease recognition site extensions (copGFP (NheI) 5ï‚¢-ACGGCTAGCGAGAGCGACGAGAGCG-3ï‚¢ and copGFP (SacI)5ï‚¢-ACGGAGCTCGCGAGATCCGGTGGAG-3ï‚¢). The resulting plasmid, pHEN4/3VGR19/copGFP was confirmed by colony-PCR, digestion and sequencing.
3-2 Analysis of copGFP Expression
copGFP expression in pHEN4/3VGR19/copGFP phagemid was confirmed in HEK293 and 293KDRcell line. The cells was transfected using standard calcium phosphate method [13, 14]. HEK293 and 293KDR cells were seeded in a 6-wells plates until 70-80% confluency.
Three microgram µg of pHEN4/3VGR19/copGFP and pCDH-CMV-MCS-EF1-cGFP-T2A-Puro(control) plasmids was added in a sterile 15 mL falcon with TE 1X, CaCl2 (2.5M), 2x HBSS and buffered water. The resulting transfection mixture was added to wells in a dropwise manner after mixing. The plate was incubated at 37°C.
Until 48 hr post-transfection, the transfection result of the reporter gene selected, copGFP, was observed under fluorescence microscopy.
4-2 Construction of VEGFR2-specific Nb display M13 phage
The pHEN4/3VGR19/copGFP was transformed in E. TG1 .An aliquot of bacterial cells, was grown in 300 ml 2xTY medium supplemented with 4% (w/vol) glucose and 100 µg ml-1 of ampicillin at 37°C and 250 rpm up to (or until) an OD600 of 0.5 (∼2–3 hr) and infected with 2 × 1012 VCSM13 helper phage for 30-60 min at 37°C. Infected cells were harvested by centrifugation,pellets was resuspend in 300 ml of 2xTY medium supplemented with 0.1% (wt/vol) glucose, 100 µg ml-1 of ampicillin and 50 µg ml-1 of kanamycin and incubated overnight at 30°C, 250 rpm. Phage particles were precipitated from culture supernatant with PEG/NaCl solution and centrifugation. Phage titers was estimated by measuring optical density (OD) at 260 nm. Titers was estimated according to the following empirical formula: phage ml-1 = OD260 × 100 × 22.14 × 1010.
phage ml-1 = OD260 × dilution 100 × 22.143 × 1010
5-2 Analysis of Coloning on Phage Particles.
The confirmation of coloning in phagemid particle was performed using Colony-PCR methods. To prove the presence of pHEN4/3VGR19/copGFP phagemid in recombinant phage as genome PCR reaction was used. For this purpose, E. TG1 cell were infected with recombinant phage and helper phage as control, and colonies grown on the selective medium for the presence of pHEN4/3VGR19/copGFP phagemid, were used in Colony-PCR methods. In this reaction, the primers pCMV (EcoRI) and BGH (NarI), copGFP (NheI) and copGFP (SacI) and pHEN4 phagemid Universal primers (Rp and GIII) were used.
6-2 In vitro phage particle transduction and detection of reporter gene
The ability of copGFP phage particles to specific target and transduce cells was evaluated using the 293KDR cell. Cells (50,000 cells/well) were seeded in 24-wells plate 24 hr before phage particle addition. The phage were added at doses indicated (from 107 to 1011 cfu/ml) and incubated for 72 hr at 37°C in complete medium prior to determination of copGFP-positive cells. Transduction efficiency was measured as the percentage of total cells that were GFP positive as detected by fluorescence microscopy analysis.
3- RESULTS
3-1 Construction of phagemid for mammalian cell reporter gene
Construction of Phagemid Vectors
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Generation of Phagemid and Phage Particle
We engineered phagemid vector for gene transfer to mammalian cells (Fig. 1 (a) and (b)). The vector pHEN4/3VGR19/GFP was constructed by inserting a CMV/GFP gene into the pHENE4/3VGR19 expression vector containing pIII gene and vector pHEN4/3VGR19/copGFP was constructed by replacing copGFP sequence with GFP sequence in pHEN4/3VGR19/GFP phagemid. Monovalent display of GFP (or Nb?) is created when either phagemid is rescued with helper phage VCSM13 (Fig. 3).
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Figure 3. GFP display phagemid vectors for mammalian cell gene delivery and ligand display strategy. The phagemid particles produced when either vector is rescued with wild-type VCSM13 helper phage are predicted to display one or fewer Nb molecules per particle (monovalent display).
We prepared monovalent phage from the vector described above with the helper phage.
3-2 Cloning CMV/GFP and copGFP fragments into the pHENE4/3VGR19 expression vector
According to previous research, 3VGR19 Nb molecule against receptor VEGFR2 was constructed and was cloned in pHENE4 vector [11].
After digestion of the vector and the PCR product, finally copGFP with NheI and SacI restriction endonuclease and ligation process was cloned in pHEN4/3VGR19/GFP and the resulting clones were analyzed by colony-PCR (Fig. 4), and then digestion assay for pHEN4/3VGR19/copGFP phagemid, with NheI – SacI, NheI – EcoRI and EcoRI enzymes was performed and cloning accuracy in positive clones was confirmed (Fig. 4).
Figure 4. Agarose gel electrophoresis 2 % or Results PCR reaction for confirmation of pHEN4/3VGR19/copGFP digestion. lanes 1, undigested pHEN4/3VGR19/copGFP plasmid; M, 1 kb marker lanes; lanes 2, digested plasmid with NheI & SacI enzymes, as a result the fragment was created during 755 bp. This fragment is related to copGFP gene; lanes 3, digested with NheI & EcoRI enzymes, as a result the fragment was created during 790 bp; lanes 4, digested with EcoRI enzyme.
3-3 Transfection of pHEN4/3VGR19/copGFP
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Targeted phagemid transfer into 293KDR cell line
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Phagemid transfection and detection of reporter gene
We used and 293KDR cell to demonstrate the internalization copGFP-targeted phagemid vector.
Also the ability of copGFP exprestion in phagemid particles was evaluated using the HEK293T and 293KDR cell line, that was grown in DMEM medium containing 10% FBS, 100 units/ml penicillin, 100 µg/ml streptomycin, 2 mM l-glutamine. Cells were transferred into six-well plates at densities of 500,000 (600,000) cells 24 h prior to phagemid particle addition and transfection was performed using a standard calcium phosphate method. Internalization and copGFP exprestion of the pHEN4/3VGR19/copGFP phagemid was about 30% (Fig. 5).
Figure 5. In vitro transfection of pHEN4/3VGR19/copGFP phagemid particles in 293KDR and HEK 293T cell lines. (a) HEK 293T cells; (b) Transfection of pCDH-CMV-MCS-EF1-GFP-T2A-Puro plasmid with calcium phosphate method as positive control; (c) Transfeced 293KDR cells visualized under a fluorescent microscope; (d) Transfeced HEK 293T cells.
Figure 5. In vitro transfection of pHEN4/3VGR19/copGFP phagemid particles in 293KDR cell line. (a) HEK 293T cells; (b) Transfeced 293KDR cells with calcium phosphate method visualized under a fluorescent microscope; (c) Transfection of pCDH-CMV-MCS-EF1-GFP-T2A-Puro plasmid as positive control.
3-4 Preparation of Phage Particles.
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Targeted phagemid and phage transfer into 293KDR cell line
We used 293KDR cell to demonstrate the internalization copGFP-targeted phage vector.
3-4 Preparation of VEGFR2-binding fluorescents phages for expression in eukaryotic cells.
To make monovalent phage containing a reporter gene, initially we cloned the gene for copGFP driven by the CMV promoter into the pHEN4/3VGR19/GFP phagemid vector generating the pHEN4/3VGR19/copGFP vector. Escherichia coli TG1 containing pHEN4/3VGR19/copGFP phagemid with ampicillin resistant were infected with VCSM13 helper phage (kanamycin resistant) and phage were purified by standard polyethylene glycol precipitation and high titers of monovalent pHEN4/3VGR19/copGFP phage were obtained (Fig.6). To prove the presence of pHEN4/3VGR19/copGFP phagemid in recombinant phage as genome Colony-PCR method was used. The results of these reactions shown in Fig.7 (primers pCMV (EcoRI) and BGH (NarI)), Fig.8 (copGFP (NheI) and copGFP (SacI)) and Fig.9 (pHEN4 phagemid Universal primers (Rp and GIII)).
Or Fig.7 (a) (primers pCMV (EcoRI) and BGH (NarI)), (b) (copGFP (NheI) and copGFP (SacI)) and (c) (pHEN4 phagemid Universal primers (Rp and GIII)).
Figure 6. Schematic of obtaining recombinant monovalent phage with releasing by helper phage.
Figure 7. Results Colony-PCR reaction for confirmation of obtained recombinant phage. (a) Colony-PCR reaction with specific primers pCMV (EcoRI) and BGH (NarI). M, 1 kb marker lanes; lanes 1, 4 and 5, are approved colonies; lanes (-), negative control and lanes (+), positive control. (b) Colony-PCR reaction with specific primers copGFP(NheI) and copGFP(SacI). M, 1 kb marker lanes; lanes 1-7, are approved colonies; lanes p12, PCR reaction with copGFP/3VGR19/pHEN4 phagemid; lanes (-), negative control and lanes (+), positive control. (c) Colony-PCR reaction with universal primers of pHEN4 phagemid (GIII and Rp). M, 1 kb marker lanes; lanes 1 and 2, are approved colonies; lanes p12, PCR reaction with copGFP/3VGR319/pHEN4 phagemid; lanes (-), negative control and lanes (+), PCR reaction with pHEN4 phagemid as positive control.
Figure 7.
Figure 8.
Figure 9.
3-5 Internalization of VEGFR2 binding monovalent phage particle by VEGFR2 expressing cell.
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Targeted phage-mediated gene transfer into 293KDR cell.
To test the functionality of the fusion protein in vitro, 5 × 104 KDR293 cells (VEGFR2-positive cell lines), were incubated with 5 ×1011 cfu/ml pHEN4/3VGR19/copGFP recombinant phage and helper phage as negative control at 37°C for 2, 24, 48 and 72 hr at the indicated doses and then analyzed by fluorescence microscopy to determine the percentage of copGFP expressing cells. Or Cells were analyzed for copGFP expression by fluorescence microscopy after 2, 24, 48 and 72 hr.
copGFP expression was not detected in KDR293 cells incubated with helper phage. These findings indicate that the VEGFR2 ligand on the surface of the phage particles results in VEGFR2-dependent binding and internalization of phage because expression of the phage DNA encoded copGFP requires nuclear transport, transcription, and translation in the target cell.
6- DISCUSSION
In this study, we fused a VEGFR2-specific Nanobody, with copGFP in phagemid to generate fluorescent, antigen-binding nanobodies (flubody) that can be expressed in living cells. We demonstrate that this structure can be expressed in 293KDR cells with optimized calcium phosphate transfection method. Construction of recombinant phage was performed using a standard PEG/NaCl method.
Phage-mediated gene transfer offers an alternative method of gene delivery into specific cell types because bacteriophages such as filamentous bacteriophages can be easily engineered to transfer genes to mammalian cells by binding a targeting ligand to the phage surface that can be occur either noncovalently [15] or genetically [1, 9, 10, 16]. Effective gene delivery and thereafter protein expression can be detected using a reporter gene such as GFP, inhibitors such as immunotoxins, neomycin phosphotransferase or β-galactosidase [17, 18]. in this strategy, any gene can be expressed in mammalian cells using appropriate mammalian transcriptional promoter and polyadenylation signal into a phage vector, therefore this combination confers to phage vector, tropism to mammalian cell.
We engineered a targeted phagemid gene delivery system by using VCSM13 helper phage to rescue a phagemid that displays an Nb-pIII fusion protein and carries a mammalian gene expression cassette. Indeed, we have demonstrated that vascular endothelial growth factor targeted phage can be used to deliver genes to cells bearing the appropriate receptors.
Or, We demonstrate that recombinant phage displaying specific Nb of VEGFR2 receptor as a genetic fusion with pIII can be targeted mammalian cell expression the VEGFR2 receptores.
We also examined whether the phagemid vector could be internalized by KDR293 and 293T cells. As shown in Figure 5, the phagemid vector was actively endocytosed by using a standard calcium phosphate method in the cells after a 24 h incubation (Figure 5, B and C), while the internalization of phagemid particles rescued by VCSM13 was barely detectable (Figure 10). Also confirmed the presence of recombinant phage using PCR reaction (Figure 7, 8 and 9) demonstrate that the Nb-pIII-GFP was displayed on the coat of the phagemid particles. However, substantial GFP expression was only observed in about 1% of the cells (Figure 10) transfected with racombinant phagemid particles packaged by VCSM13.
This suggested that there are other topic that need to be resolved, such as endosome release, translocation into nucleus, DNA release, and transformation from ssDNA into dsDNA and etc [19].
These data extend previous findings, that phage vector transduction efficiency was 1–4% with targeted phage [1, 10]. However, transduction levels as high as 10% are obtained in human prostate carcinoma cells transfected with multivalent phagemid vectors [5].