In situtransduction of target cells on solid surfaces by immobilized viral vectors
© Hobson et al; licensee BioMed Central Ltd. 2003
Received: 15 November 2002
Accepted: 9 May 2003
Published: 9 May 2003
For both in vitro and in vivo gene transfer applications, recombinant viral vectors have almost always been used free in solution. Some site-specificity of the delivery of viral vectors can be achieved by applying a solution containing viral particles specifically to the site of interest. However, such site-specificity is seriously limited since viral vectors can diffuse freely in solution after application.
We have developed a novel strategy for in situ transduction of target cells on solid surfaces by viral vectors. In this strategy, adenoviral vectors are attached stably to solid surfaces by using the extremely tight interaction between (strept)avidin and biotin, while maintaining the infectivity of the viral vectors. Target cells are cultured directly on such virus-coated solid surfaces, resulting in the transduction of the cells, in situ, on the solid surface. When compared using an equal number of viral particles present in each well (either immobilized or free), the efficiencies of such in situ transduction on solid surfaces were equivalent to those seen with the adenoviral vectors used free in solution. Since viral particles can be attached at desired locations on solid surfaces in any sizes, shapes, and patterns, the ultimate spatial arrangements of transduced cells on solid surfaces can be predetermined at the time of the preparation of the virus-coated solid surfaces.
We have devised a method of immobilizing adenoviral vectors, tightly and stably, on solid surfaces, while maintaining their ability to infect cells. Such immobilized viral vectors can infect target cells, in situ, on solid surfaces. This strategy should be very useful for the development of a variety of both in vitro and in vivo applications, including the creation of cell-based expression arrays for proteomics and drug discovery and highly site-specific delivery of transgenes for gene therapy and tissue engineering.
Recombinant viral vectors have become the primary agents for both in vitro and in vivo gene transfer applications [1–4]. Viruses are powerful gene transfer agents, since they have evolved specific, efficient machinery to deliver nucleic acids into cells. To our knowledge, viral vectors have almost always been used free in solution. For in vitro systems, a solution containing free viral vectors is applied to target cells. Similarly, free viral vectors in solution are administered locally or systemically for in vivo gene transfer applications. Some site-specificity of the delivery of viral vectors can be achieved by applying a solution containing viral particles specifically to the site of interest for both in vitro and in vivo settings. However, such site-specificity is seriously limited since viral vectors can diffuse freely in solution after application.
In this work, we have developed a novel strategy for virus-mediated in situ transduction of target cells on solid surfaces using adenoviral vectors as gene transfer agents. In this strategy, adenoviral particles are immobilized, tightly and stably, on solid surfaces, while maintaining the natural properties of the viral vectors. We reasoned that such immobilized viral vectors could infect cells only at the contact site between the solid surface and cells. Thus, the ultimate spatial arrangements of transduction sites on the solid surface could be controlled and determined, at will, by the strategic placement of viral vectors on the solid surface.
Virus-mediated in situtransduction of cells on solid surfaces
We have developed a novel strategy for virus-mediated in situ transduction of target cells on solid surfaces using adenoviral vectors as gene transfer agents. In this strategy, adenoviral particles are immobilized, tightly and stably, on solid surfaces (virus-coated solid surfaces), while maintaining their natural properties, including infectivity. We hypothesized that such viral vectors, immobilized on a solid surface, could infect cells only at the contact site between the solid surface and cells. This could allow the spatial control and arrangements of transduction sites on a solid surface, which can be determined by the strategic placement of viral particles on the solid surface.
Infectivity of biotinylated adenoviral vectors immobilized on solid surfaces
The feasibility of this strategy was tested by using recombinant adenoviral vectors carrying a transducable lacZ (β-galactosidase) gene (Ad5.CMV-LacZ) (Qbiogene, Montreal, Canada). The extremely tight interaction between the protein (strept)avidin and its ligand biotin (Kd ~ 10-14 M) [5–9] was chosen as a means of tethering viral particles to the solid surface. We previously showed that biotin moieties can be attached covalently to the outer surface of adenoviral vectors without appreciable effect on their infectivity under carefully controlled biotinylation conditions [10, 11]. Such biotinylated viral particles could be attached, specifically and stably, to a solid surface on which (strept)avidin is covalently immobilized (avidin- or streptavidin-coated solid surface).
Biotinylation of Ad5.CMV-LacZ was performed by using sulfo-NHS-LC-biotin (Pierce Chemical, Rockford, IL), a biotin derivative containing an N-hydroxysuccinimidyl ester (NHS) that reacts with primary amino groups. Ad5.CMV-LacZ was treated at varying concentrations (0 – 100 μg/ml) of sulfo-NHS-LC-biotin on ice for 2 h in PBS (pH 7.4), followed by the removal of non-virion-associated biotinylation reagent by ultrafiltration. The infectivities of the resulting adenoviral vectors were analyzed by using D-17 cells [canine osteosarcoma cell line; American Type Culture Collection (ATCC), Manassas, VA] (highly permissive to adenoviral infection)  as targets. Little effect on viral infectivity was seen when the adenoviral vectors were treated with sulfo-NHS-LC-biotin at concentrations up to approximately 40 μg/ml (data not shown). This is in agreement with our earlier study .
Efficiency of immobilization of biotinylated adenoviral vectors
Infectivity of immobilized adenoviral vectors
Adenoviral vector spots on solid surfaces
We previously reported a strategy similar to the one described in this study. In this strategy, biotinylated adenoviral vectors are immobilized on the surfaces of streptavidin-coated microbeads with diameters up to a few micrometers . For such adenovirus-microbead conjugates, great enhancement in transduction efficiency was seen with several different cell lines, including COLO205, the infection efficiency of which was not notably enhanced by adenoviral vectors immobilized on streptavidin-coated wells in the present study. The primary difference between the two strategies is derived from the size of solid surfaces (streptavidin-coated wells and streptavidin-coated microbeads) relative to target cells. The sizes of adenovirus-microbead conjugates are smaller than those of target cells. Thus, the entire adenovirus-microbead conjugates can be taken up by cells. Such internalization events appear to contribute substantially to the infectivity enhancement by virus-microbead conjugates. In contrast, the dissociation of viral particles from the solid surface must occur for adenoviral vectors, which are initially immobilized on streptavidin-coated wells, to be taken up by target cells. This may be one possible explanation of why infectivity enhancement was seen with adenovirus-microbead conjugates and was not with adenoviral vectors immobilized on streptavidin-coated wells, except for one cell line (C6).
Virus-coated solid surfaces should offer a variety of both in vitro and in vivo applications. One such in vitro application would be the creation of virus arrays for expression and functional analysis of genes [13, 14]. A recombinant viral vector library, each clone of which carries a different gene, could be immobilized in an ordered manner as discrete spots on a solid surface. Onto the resulting virus array, target cells are applied and cultured. This should result in the expression of encoded genes in target cells in an array format, in which each encoded gene should be expressed in a discrete cell spot while maintaining the original location of the viral vector spot in the virus array. Thus, expression and functional analysis of many different genes can potentially be performed with a single virus array for a given target cell line. The use of viral vectors should offer enhanced efficiencies and versatility for the expression of cloned genes in various cell types than approaches using expression plasmids that are printed on solid surfaces [15, 16]. This might be particularly true to primary cells, which often show poor expression of encoded genes when plasmid-based expression vectors are used. The availability of such viral vector libraries, particularly those based on adenoviral vectors [17, 18], should facilitate the realization of such applications. Virus-coated solid surfaces could also be used for in vitro tissue engineering applications, for which the shapes and sizes of areas/regions of genetically engineered cells can be predetermined two-dimensionally by strategic placement and patterning of viral vectors on solid surfaces, as exemplified in Fig. 5. Such precise control of the shapes and sizes of areas/regions of genetically engineered cells would be very difficult to achieve with free viral vectors, which can diffuse freely in solution.
Virus-coated solid surfaces could also be used in vivo for highly site-specific delivery of viral particles to target tissues or cells through the direct application of virus-coated solid surfaces to target sites. The delivery of viral vectors in a solid-surface-associated form should eliminate the migration of viral particles from the administration site, minimizing uncontrolled transduction of non-target tissues. For example, a virus-coated solid surface, the size of which has been adjusted to the intended transduction site, could be transplanted into a target tissue, allowing the specific transduction of the target site by immobilized viral vectors with minimal infection of surrounding tissues. As proposed for in vitro applications described above, the primary advantage for such in vivo gene transfer applications would be that the shapes and sizes of areas/regions of transduction sites can be predetermined by strategic placement and patterning of viral vectors on solid surfaces.
We have devised a method of immobilizing adenoviral vectors, tightly and stably, on solid surfaces using the (strept)avidin-biotin interaction, while maintaining their ability to infect cells. Such immobilized viral vectors can infect target cells, in situ, on solid surfaces with efficiencies equivalent to the same viral vectors used free in solution for the cell lines tested. For one cell line (C6), the infection efficiency by immobilized viral vectors was greater than the same viral vectors used free in solution, which showed a limited ability to infect the target cells. This strategy should be very useful for the development of a variety of both in vitro and in vivo applications, including the creation of cell-based expression arrays for proteomics and drug discovery and highly site-specific delivery of transgenes for gene therapy and tissue engineering.
The recombinant adenoviral vectors used are Ad5.CMV-LacZ (Qbiogene), which is derived from adenovirus serotype 5 with the deletion of the viral E1A, E1B, and E3 genes. Ad5.CMV-LacZ carries the bacterial lacZ (β-galactosidase) gene under the control of the human cytomegalovirus (CMV) immediate-early promoter with the human β-globin polyadenylation signal sequence. This viral vector had been purified by two rounds of CsCl gradient centrifugation.
The following two cell lines were used as targets for adenoviral infection: D-17 (canine osteosarcoma cells) and C6 (rat glioma cells), both of which were obtained from ATCC. These cell lines were grown at 37°C in a humidified atmosphere containing 5% CO2. D-17 cells were maintained in Dulbecco's modified Eagles medium (DMEM; BioWhittaker) supplemented with 6% fetal bovine serum (FBS; BioWhittaker), and C6 cells were in DMEM supplemented with 10% FBS.
Biotinylation of adenoviral vectors with sulfo-NHS-LC-biotin
Sulfo-NHS-LC-biotin (Pierce Chemical) was dissolved in dimethylformamide at a concentration of 3 mg/ml and added to Ad5.CMV-LacZ [1.0 × 109 viral particles in 100 μl PBS (pH 7.4)] to various final concentrations (0 – 100 μg/ml). The reaction mixtures were incubated on ice in the dark for 2 h, and the biotinylation reaction was terminated by the addition of 100 μl of 9 mM glycine in PBS (pH 7.4). The viral particles were subjected to three rounds of ultrafiltration with ZM-500 centrifugal filtration units (molecular mass cut-off, 500 kDa; Millipore) using PBS (pH 7.4) containing 0.05% Tween 20 (PBST) as a diluent to remove non-virion-associated biotinylation reagent. Finally, the viral particles were suspended in 100 μl of fresh PBST at a concentration of 1 × 1010 viral particles/ml.
For the infectivity analysis of biotinylated Ad5.CMV-LacZ, dilutions of biotinylated viral vectors were placed over monolayers of D-17 target cells on a 96-well cell culture plate (8 × 103 cells per well), followed by the incubation of cells at 37°C for 48 h. Then, cells were fixed with 0.5% glutaraldehyde and stained for β-galactosidase (LacZ) activity using 5-bromo-4-chloro-3-indoyl-β-D-galactopyronoside (X-Gal) as the substrate. The numbers of infected, lacZ-expressing cells for 10–15 randomly chosen microscopic fields (3.75 mm2) in each well were counted and used to assess the total number of infected cells in each well.
Preparation of wells coated with adenoviral vectors
Biotinylated Ad5.CMV-LacZ, prepared by treatment with 15 μg/ml sulfo-NHS-LC-biotin as above, was diluted in PBST and incubated for immobilization in streptavidin-coated wells (well diameter, 0.64 cm; Reacti-Bind Streptavidin Coated Polystyrene Wells, Pierce Chemical) (50 μl per well) for 2 h at 25°C with shaking on a rotary shaker at 150 rpm. The solution of each well, which contained unbound Ad5.CMV-LacZ, was collected and analyzed for infectivity on D-17 cells as above to estimate the number of unbound viral particles. The wells were washed with PBST and then with PBS (pH 7.4) without Tween 20. Onto the wells coated with biotinylated Ad5.CMV-LacZ, target cells were placed and cultured at 37°C for 48 h. Cells were fixed with glutaraldehyde and stained for the expression of the lacZ gene as described above. For control experiments, a solution containing free, unmodified Ad5.CMV-LacZ (50 μl per well) was applied to target cells grown on streptavidin-coated wells.
Repeated application of biotinylated adenoviral vectors to wells
Biotinylated Ad5.CMV-LacZ, prepared by treatment with 15 μg/ml sulfo-NHS-LC-biotin as above, was diluted in PBST and added to streptavidin-coated wells (2.5 × 106 viral particles in 50 μl PBST per well) and incubated with shaking on a rotary shaker at 150 nm at 25°C for 30 min. The solution in each well, which contained unbound viral particles, was collected, and the wells were washed three times with PBST. Then, the same number of fresh biotinylated Ad5.CMV-LacZ was added to the well and incubated in the same manner, followed by the collection of unbound viral particles and washing of the wells. These steps were repeated several more times. The unbound viral particle fraction, collected after each addition cycle, was subjected to infectivity analysis on D-17 cells to estimate the number of unbound Ad5.CMV-LacZ.
Preparation of adenoviral vector spots on solid surfaces
Six-well cell culture plates, coated with poly-D-lysine (well diameter, 3.5 cm; Biocoat Poly-D-Lysine Cellware 6-Well Plates, Becton Dickinson Labware), were used as the solid surface. First, spots of biotin moieties on the wells were created by using sulfo-NHS-LC-biotin. Plastic rings (inner diameter, 1.4 cm) were used to specify the biotinylation areas, and the remaining area of the wells was covered with a layer of 2% agarose in PBS. These spot areas within plastic rings were treated with 1 mg/ml sulfo-NHS-LC-biotin in PBS (pH 7.4) (50 μl per spot) for 2 h at 4°C and then washed with PBST to remove unreacted biotinylation reagent. Neutralite avidin (Southern Biotechnology Associates) (25 μg in 50 μl PBST per spot) was added to the spot areas within plastic rings, which had been treated with sulfo-NHS-LC-biotin, and allowed to bind to biotin moieties on the well surface. Unbound Neutralite avidin was removed by washing the wells with PBST. Biotinylated Ad5.CMV-LacZ (1 × 107 viral particles per spot), prepared by treatment with 15 μg/ml sulfo-NHS-LC-biotin as above, were incubated in the spot areas within plastic rings at 25°C for 2 h, followed by washing the wells with PBST to remove unbound viral particles. The plastic rings and agarose were removed from the wells, and the entire wells were washed with PBS. D-17 cells (2.5 × 105 per well) were applied to the entire wells and cultured at 37°C for 48 h. Cells were fixed with glutaraldehyde and stained for the expression of the lacZ gene as described above.
We thank Peter Thomas for providing us with cell lines. M.W.P. was supported by a postdoctoral fellowship (PC990029) from the U.S. Department of Army Prostate Cancer Research Program. This work was supported, in part, by Grant CA46109 from the National Institutes of Health.
- Verma IM, Somia N: Gene therapy – promises, problems and prospects. Nature. 1997, 389: 239-242. 10.1038/38410.View ArticleGoogle Scholar
- Kay MA, Glorioso JC, Naldini L: Viral vectors for gene therapy: the art of turning infectious agents into vehicles of therapeutics. Nature Med. 2001, 7: 33-40. 10.1038/83324.View ArticleGoogle Scholar
- Walther W, Stein U: Viral vectors for gene transfer: a review of their use in the treatment of human diseases. Drugs. 2000, 60: 249-271.View ArticleGoogle Scholar
- Pfeifer A, Verma IM: Gene therapy: Promises and problems. Annu Rev Genomics Hum Genet. 2001, 2: 177-211. 10.1146/annurev.genom.2.1.177.View ArticleGoogle Scholar
- Green NM: Avidin. Adv Protein Chem. 1970, 29: 85-133.View ArticleGoogle Scholar
- Wilchek M, Bayer EA: Introduction to avidin-biotin technology. Methods Enzymol. 1990, 184: 5-13.View ArticleGoogle Scholar
- Green NM: Avidin and streptavidin. Methods Enzymol. 1990, 184: 51-60.View ArticleGoogle Scholar
- Sano T, Vajda S, Reznik GO, Smith CL, Cantor CR: Molecular engineering of streptavidin. Annals NY Acad Sci. 1996, 799: 383-390.View ArticleGoogle Scholar
- Wilchek M, Bayer EA: Foreword and introduction to the book (strept)avidin-biotin system. Biomol Eng. 1999, 16: 1-4. 10.1016/S1050-3862(99)00032-7.View ArticleGoogle Scholar
- Pandori MW, Hobson DA, Sano T: Adenovirus-microbead conjugates possess enhanced infectivity: A new strategy for localized gene delivery. Virology. 2002, 299: 204-212. 10.1006/viro.2002.1510.View ArticleGoogle Scholar
- Pandori MW, Hobson DA, Olejnik J, Krzymanska-Olejnik E, Rothschild KJ, Palmer AA, Phillips TJ, Sano T: Photochemical control of the infectivity of adenoviral vectors using a novel photocleavable biotinylation reagent. Chem Biol. 2002, 9: 567-573. 10.1016/S1074-5521(02)00135-7.View ArticleGoogle Scholar
- Fechner H, Wang X, Wang H, Jansen A, Pauschinger M, Scherubl H, Bergelson JM, Schultheiss H-P, Poller W: Trans-complementation of vector replication versus Coxsackie-adenovirus-receptor overexpression to improve transgene expression in poorly permissive cancer cells. Gene Ther. 2000, 7: 1954-1968. 10.1038/sj.gt.3301321.View ArticleGoogle Scholar
- Wade-Martins R, Smith ER, Tyminski E, Chiocca EA, Saeki Y: An infectious transfer and expression system for genomic DNA loci in human and mouse cells. Nature Biotechnol. 2001, 19: 1067-1070. 10.1038/nbt1101-1067.View ArticleGoogle Scholar
- Lotze MT, Kost TA: Viruses as gene delivery vectors: Application to gene function, target validation, and assay development. Cancer Gene Ther. 2002, 9: 692-699. 10.1038/sj.cgt.7700493.View ArticleGoogle Scholar
- Ziauddin J, Sabatini DM: Microarrays of cells expressing defined cDNA. Nature. 2001, 411: 107-110. 10.1038/35075114.View ArticleGoogle Scholar
- Mitchell P: A perspective on protein microarrays. Nature Biotechnol. 2002, 20: 225-229. 10.1038/nbt0302-225.View ArticleGoogle Scholar
- Elahi SM, Qualikene W, Naghdi L, O'Connor-McCourt M, Massie B: Adenovirus-based libraries: efficient generation of recombinant adenoviruses by positive selection with the adenovirus protease. Gene Ther. 2002, 9: 1238-1246. 10.1038/sj.gt.3301793.View ArticleGoogle Scholar
- Michiels F, van Es H, van Rompaey L, Merchiers P, Francken B, Pittois K, van der Schueren J, Brys R, Vandermissen J, Beirinckx F, Herman S, Dokic K, Klaassen H, Narinx E, Hagers A, Laenen W, Piest I, Pavliska H, Rombout Y, Langemeijer E, Ma L, Schipper C, De Raeymaeker M, Schweicher S, Jans M, van Beeck K, Tsang I-R, van de Stolpe O, Tomme P: Arrayed adenoviral expression libraries for functional screening. Nature Biotechnol. 2002, 20: 1154-1157. 10.1038/nbt746.View ArticleGoogle Scholar
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