Over the course of the last few years, we have developed a novel gene transfer technology, in which adenoviral vectors are attached stably to the surfaces of microbeads (nanoparticles) using the extremely strong (strept)avidin-biotin interaction and delivered to target cells in the form of adenovirus-microbead conjugates (Pandori et al, 2002; Pandori and Sano, 2005). When analyzed in vitro, such adenovirus-microbead conjugates showed infectivities equivalent to or even greater than adenoviral vectors used free in solution. In particular, the infectivity for target cells, which are poorly permissive to infection by free adenoviral vectors, can be enhanced considerably. In addition, the use of microbeads as virus carriers allows the co-attachment of other materials to the microbead surface to enhance or control the functionality of the adenovirus-microbead conjugates. In the present study, we investigated if this gene transfer technology with adenovirus-microbead conjugates could be used for efficient transduction of the inflamed colon by adenoviral vectors toward its application to gene therapy of inflammatory bowel disease (IBD), such as Crohn’s disease and ulcerative colitis (for reviews, Podolsky, 2002; Strober et al, 2002; Bouma and Strober, 2003; Dignass et al, 2004; Korzenik and Podolsky, 2006).

The colorectal system is potentially an attractive target for in vivo somatic gene therapy since it is readily accessible externally. However, the presence of the mucous coat on the epithelium and the dynamic fluidic properties of the colorectal system act as barriers for the access to the colonic tissue by viral vectors that are administered intra-colonically. In IBD, chronic intestinal inflammation occurs, which causes severe destruction of the mucosal layer. This exposes the colonic tissue, making it directly accessible by viral vectors that are administered intra-colonically. However, the dynamic fluidic properties of the colorectal system limit the direct, stable contact of administered viral vectors with the colonic tissue. This considerably reduces the overall transduction efficiency of the colonic tissue by viral vectors. Thus, previous attempts for intra-colonical delivery of viral vectors to the inflamed colon involved the use of large amounts of viral vectors to achieve sufficient levels of transgene expression (Lindsay et al, 2003; Wirtz et al, 1999, 2002). This suggests that, if viral vectors could be made capable of associating stably with colonic cells, the transduction of the colonic tissue by viral vectors could be enhanced considerably. In the present study, we tested if intra-colonical administration of adenoviral vectors in the form of virus-microbead conjugates could enhance the transduction of the inflamed colon. In particular, we investigated if the co-attachment of an anchoring agent to adenovirus-microbead conjugates could provide the conjugates with the abilities to associate stably with the colonic tissue and to transduce the inflamed colon efficiently. We chose a lectin, concanavalin A (Con A), as a potential anchoring agent. Con A, isolated from Canavalia ensiformis (Jack bean) seeds, binds to α-D-glucopyranosyl and α-D-mannopyranosyl moieties, which exist abundantly in carbohydrate chains on the cell surfaces (Lis and Sharon, 1986, 1998; Sharon and Lis, 1989, 1995). We previously showed that the co-attachment of Con A can restore the ability of adenovirus-microbead conjugates containing chemically inactivated adenoviral vectors to associate stably with target cells (Pandori and Sano, 2005). We hypothesized that the co-attachment of Con A allows adenovirus-microbead conjugates to associate stably with the colonic tissue upon intra-colonical administration, resulting in efficient transduction of the inflamed colon.

II. Materials and Methods

A. Adenoviral vectors

Two adenoviral vector constructs, both of which are derived from adenovirus serotype 5 with the deletion of the viral E1 and E3 genes, were used in this study. One adenoviral vector construct, Ad5.CMV-LacZ (Qbiogene, Montreal, Canada), carries the bacterial lacZ (β-galactosidase) gene under the control of the human cytomegalovirus (CMV) immediate/early promoter. The other adenoviral vector construct, Ad5.CMV-IL10, carries the mouse interleukin-10 (IL-10) gene containing the coding sequence for the signal peptide under the control of the CMV immediate/early promoter (a generous gift from Dr. Andrea Gambotto, University of Pittsburgh School of Medicine).

B. Cell lines

The following four cell lines were used as targets: HeLa (human cervical adenocarcinoma), COLO 205 (human colorectal adenocarcinoma), MIP-101 (human colonic carcinoma), and SW620 (human colorectal adenocarcinoma). These cell lines were obtained from the American Type Culture Collection (Manassas, VA, USA), except for MIP-101 that is a generous gift from Dr. Peter Thomas, Boston University School of Medicine. HeLa and SW620 cells were maintained in Dulbecco’s modified Eagle’s medium (BioWhittaker) supplemented with 10% fetal bovine serum (BioWhittaker). COLO 205 and MIP-101 cells were maintained in RPMI 1640 (BioWhittaker) supplemented with 10% fetal bovine serum, 4.5 mg/ml glucose, 1.5 mg/ml sodium bicarbonate, and 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid.

C. Preparation of adenovirus-microbead conjugates with and without the co-attachment of Con A

Adenovirus-microbead conjugates were prepared by the method described previously (Pandori et al, 2002). Briefly, purified adenoviral vectors (Ad5.CMV-LacZ or Ad5.CMV-IL10) were biotinylated using sulfo-NHS-LC-biotin (Pierce) at 20 μg/ml, at which concentration the viral infectivity can be maintained (Pandori et al, 2002; Hobson et al, 2003). After non-virion-associated biotinylation reagent was removed by repeated ultrafiltration, the resulting biotinylated adenoviral particles were attached to avidin-coated polystyrene microbeads (diameter, 0.48 μm; specific gravity, 1.06 g/cm³; Spherotech) at appropriate ratios. The co-attachment of Con A to the microbead surfaces was done by the addition of excess biotinylated Con A (Vector Laboratories) to adenovirus-microbead conjugates (2.5 μg biotinylated Con A per 1.67 x 10⁷ microbeads), followed by the removal of unbound Con A. The addition of excess biotinylated Con A is essential for the prevention of the formation of aggregates, which have considerably reduced infectivity. Under these conditions, the surfaces of the microbeads, to which adenoviral vectors had been attached, should be saturated with biotinylated Con A.

D. Infectivity analysis of adenovirus-microbead conjugates containing Ad5.CMV-LacZ

The infectivity of adenovirus-microbead conjugates with and without the c-attachment of Con A, prepared using Ad5.CMV-LacZ as above, was analyzed on HeLa and COLO 205 cell lines. Cells were cultured in wells (5 x 10⁴ cells per well) at 37 °C for 24 hr. An appropriate amount of adenovirus-microbead conjugates or free Ad5.CMV-LacZ was applied to each well (the actual amount of adenoviral particles added to each well is given in the legends to Figures 1 and 3). Cells were incubated for 37 °C for 48 hr, fixed with 0.5% glutaraldehyde, and stained for β-galactosidase (LacZ) activity using X-gal (5-bromo-4-chloro-3-indoyl-β-D-galactopyronoside) as the substrate. The numbers of infected cells, which were stained blue, were counted under a light microscope.

E. Cell-binding analysis of adenovirus-microbead conjugates

Adenovirus-microbead conjugates with and without the co-attachment of Con A were prepared using Ad5.CMV-LacZ at 50 adenoviral particles per microbead, as described above. These conjugates were applied to HeLa and COLO 205 cells grown at 37 °C on glass cover slips. At 4 hr and 24 hr after the application of adenovirus-microbead conjugates, cells were washed with PBS (phosphate-buffered saline) and fixed with 4% paraformaldehyde. Cell nuclei were stained with DAPI (4'-6-diamidino-2-phenylindole; blue fluorescence), and stained cells were examined under a fluorescence microscope with appropriate filters (Axioscop 2, Carl Zeiss). Association of adenovirus-microbead conjugates with target cells can be detected by cell-associated red fluorescence, derived from the microbeads used that contain a rhodamine derivative.

F. Analysis of in vitro production of mouse IL-10 upon transduction by adenovirus-microbead conjugates containing Ad5.CMV-IL10

Adenovirus-microbead conjugates with and without the co-attachment of Con A were prepared using Ad5.CMV-IL10 at 50 adenoviral particles per microbead, as above. The ability of these conjugates to produce mouse IL-10 upon transduction was analyzed by using three colonic cell lines (COLO 205, MIP-101, and SW620), along with HeLa cells, as targets. Cells were cultured in wells (5 x 10⁴ cells per well) at 37 °C for 24 hr. Appropriate amounts of adenovirus-microbead conjugates with and without the co-attachment of Con A, along with free Ad5.CMV-IL10, were applied to target cells (the actual amount of adenoviral particles added to each well is given in the legend to Figure 5). Cells were incubated at 37 °C for 24 hr, and the amounts of mouse IL-10, which had been produced and secreted into the culture media, were determined quantitatively by enzyme-linked immunosorbent assays (ELISA) (OptEIA Mouse IL-10 ELISA kit; BD Pharmingen). Purified recombinant mouse IL-10 (BD Pharmingen) was used as the standard for quantitation.

G. Transduction of the inflamed colon in mice upon intra-colonical administration of adenovirus-microbead conjugates containing Ad5.CMV-LacZ

All animal procedures were carried out in accordance with NIH guidelines following approval by the Harvard Medical Area Standing Committee on Animals. Adenovirus-microbead conjugates with and without the co-attachment of Con A were prepared using Ad5.CMV-LacZ at 50 adenoviral particles per microbead, as above. A mouse acute colitis model was prepared by intra-colonical administration of 0.75 mg TNBS (2,4,6-trinitrobenzenesulfonic acid), dissolved in 100 μl of 50% ethanol (TNBS-induced colitis) (Jurjus et al, 2004), into Balb/c mice (6 - 8 weeks old; Taconic) by enema. At 48 hr after the administration of TNBS, adenovirus-microbead conjugates with and without the co-attachment of Con A, along with free Ad5.CMV-LacZ, were administered intra-colonically into mice by enema (5 x 10⁸ adenoviral particles in 100 μl PBS per mouse). At 48-hr post-administration, mice were euthanized, and their colons were collected. The colon samples were frozen in tissue freezing media (Tissue-Tek O.C.T. compound, Miles), followed by the preparation of cryosections (thickness, 5-7 μm). These colon sections were subjected to the analysis of transduction by Ad5.CMV-LacZ or the detection of microbeads, used as adenovirus carriers. For transduction analysis, colon sections were stained for β-galactosidase activity using X-gal as the substrate, with counter-staining with neutral red. Stained colon sections were examined under a light microscope. For the detection of microbeads (red fluorescence), colon sections were counter-stained with DAPI and examined under a fluorescence microscope.

H. Production of mouse IL-10 in the inflamed colon upon intra-colonical administration of adenovirus-microbead conjugates containing Ad5.CMV-IL10

Adenovirus-microbead conjugates with and without the co-attachment of Con A were prepared using Ad5.CMV-IL10 at 50 adenoviral particles per microbead, as described above. These adenovirus-microbead conjugates, along with free Ad5.CMV-IL10, were administered intra-colonically into mice with TNBS-induced colitis by enema (1 x 10⁹ adenoviral particles in 100 μl PBS per mouse) (3 mice per sample). At 24-hr post-administration, mice were euthanized, and their colons were collected. Each colon sample was homogenized in 2 ml of 0.25 mM Tris-Cl (pH 7.8) using a glass Potter homogenizer. The resulting homogenates were centrifuged at 4 °C at 1,600 x g for 20 min, and the supernatants were subjected to the quantitation of mouse IL-10 by ELISA in triplicate (OptEIA Mouse IL-10 ELISA kit), as described in F above. The total protein contents of the supernatants were also determined by the protein assay method of Bradford (Bradford, 1976) using bovine serum albumin as the standard.

III. Results

A. Effect of the number of viral particles per microbead on the infectivity of adenovirus-microbead conjugates

First, we analyzed the effect of the number of adenoviral particles per microbead on the infectivity of adenovirus-microbead conjugates using cultured cells. An adenoviral vector construct carrying the lacZ (β-galactosidase) gene (Ad5.CMV-LacZ) was used. Adenovirus-microbead conjugates were prepared by the method, described in the Materials and Methods section, at varying numbers of adenoviral particles per microbead. The infectivity of the resulting adenovirus-microbead conjugates, along with free Ad5.CMV-LacZ as a control, was analyzed in vitro using two cell lines, HeLa (moderately permissive to infection by free adenoviral vectors) and COLO 205 (very poorly permissive to infection by free adenoviral vectors) (Fechner et al, 2000) (Figure 1). On HeLa cells, the infectivity of adenovirus-microbead conjugates was approximately 60 - 70% of that of free Ad5.CMV-LacZ. However, the infectivity of the conjugates was hardly affected by the number of adenoviral particles per microbead tested (up to 50 adenoviral particles per microbead). In contrast, when COLO 205 cells were used as targets, the infectivity of adenovirus-microbead conjugates slightly increased with increasing the number of adenoviral particles per microbead. At 50 or 100 adenoviral particles per microbead, the infectivity of the conjugates became even higher than that of free Ad5.CMV-LacZ. However, the overall effect of the number of adenoviral particles per microbead on the infectivity of adenovirus-microbead conjugates was found to be relatively small in the range tested (up to 100 adenoviral particles per microbead), in agreement with a previous study (Pandori et al, 2002). From these results, we decided to use adenovirus-microbead conjugates containing 50 adenoviral particles per microbead in subsequent experiments.

B. Effect of the co-attachment of Con A on the cell-binding ability and the infectivity of adenovirus-microbead conjugates

The effect of the co-attachment of Con A on the ability of adenovirus-microbead conjugates to associate with target cells was analyzed using HeLa and COLO 205 cells as targets. Adenovirus-microbead conjugates with and without the co-attachment of Con A were prepared using Ad5.CMV-LacZ at 50 adenoviral particles per microbead. These conjugates were applied to target cells, and cell-associated red fluorescence, derived from the microbeads that contain a rhodamine derivative (red fluorescence), was visualized under a fluorescence microscope (Figure 2). When Con A was attached to adenovirus-microbead conjugates, the amount of cell-associated red fluorescence became greater then that seen with adenovirus-microbead conjugates without Con A for both HeLa and COLO 205 cells. In particular, the co-attachment of Con A allowed adenovirus-microbead conjugates to associate efficiently with COLO 205 cells, for which no appreciable association of the conjugates was seen in the absence of the co-attachment of Con A. This result demonstrates that the co-attachment of Con A can considerably enhance the ability of adenovirus-microbead conjugates to associate with target cells, in agreement with a previous study with adenovirus-microbead conjugates containing chemically inactivated adenoviral vectors (Pandori and Sano, 2005).

The infectivity of adenovirus-microbead conjugates was also investigated in the absence and presence of the co-attachment of Con A using HeLa and COLO 205 cells as targets. Adenovirus-microbead conjugates with and without Con A were applied to target cells. At 48-hr post-application, cells were analyzed for lacZ expression (Figure 3). Adenovirus-microbead conjugates showed higher infectivities than free Ad5.CMV-LacZ on both HeLa and COLO 205 cells. The co-attachment of Con A to adenovirus-microbead conjugates further enhanced the infectivity of the conjugates. These results reveal that the co-attachment of Con A makes adenovirus-microbead conjugates capable of associating more efficiently with target cells, resulting in enhanced transduction of the cells.

C. In vivo transduction of the inflamed colon in mice by adenovirus-microbead conjugates containing Ad5.CMV-LacZ

The ability of adenovirus-microbead conjugates to transduce the inflamed colon was investigated in vivo using a mouse TNBS-induced colitis model. Adenovirus-microbead conjugates with and without the co-attachment of Con A were prepared using Ad5.CMV-LacZ at 50 adenoviral particles per microbead. These conjugates, along with free Ad5.CMV-LacZ, were administered intra-colonically by enema into mice with TNBS-induced colitis (a total of 5 x 10⁸ adenoviral particles per mouse). No appreciable effect on the health and behavior of mice was seen upon intra-colonical administration of free Ad5.CMV-LacZ and its microbead conjugates with and without the co-attachment of Con A until they were euthanized. When free Ad5.CMV-LacZ was used, no appreciable transduction was detected in colon sections (Figure 4A). Similarly, little transduction of the colon was seen when adenovirus-microbead conjugates without the co-attachment of Con A was administered intra-colonically (Figure 4B). In contrast, the use of adenovirus-microbead conjugates containing Con A resulted in efficient transduction of colonic cells (Figures 4C and 4D). Transduction was seen primarily near the surfaces of mucosal layers, to which administered adenovirus-microbead conjugates should have easy access due to their destruction caused by colonic inflammation.

Colon sections were also analyzed under a fluorescence microscope for the presence of adenovirus-microbead conjugates. When adenovirus-microbead conjugates were used without the co-attachment of Con A, few red fluorescence spots, derived from the microbeads used that contain a rhodamine derivative, were seen in colon sections (Figure 4E). In contrast, red fluorescent spots were seen in many colon sections when adenovirus-microbead conjugates with Con A were administered intra-colonically (Figure 4F). This result reveals that adenovirus-microbead conjugates can associate stably with

E. Local production of IL-10 in the inflamed colon upon intra-colonical administration of adenovirus-microbead conjugates containing Ad5.CMV-IL10

Adenovirus-microbead conjugates containing Con A were used to test if local IL-10 levels in the colons of mice with TNBS-induced colitis could be raised upon intra-colonical administration of the conjugates. Adenovirus-microbead conjugates with and without Con A (50 adenoviral particles per microbead), along with free Ad5.CMV-IL10, were administered intra-colonically into mice with TNBS-induced colitis (a total of 1 x 10⁹ adenoviral particles per mouse). No appreciable changes of the health and behavior were seen with mice upon intra-colonical administration of free Ad5.CMV-IL10 and its microbead conjugates with and without the co-attachment of Con A. At 24-hr post-administration, the local level of mouse IL-10 in the colon was determined quantitatively by ELISA (Figure 6). When free Ad5.CMV-IL10 was used, the level of mouse IL-10 in the colon became slightly higher than that of control mice, which received no adenoviral vectors. The use of adenovirus-microbead conjugates without Con A slightly reduced the local IL-10 level in the colon, as compared to that of control mice. In contrast, when Ad5.CMV-IL10 was administered intra-colonically in the form of adenovirus-microbead conjugates containing Con A, the amount of IL-10 in the colon was raised considerably to a level that is almost an order of magnitude higher than that of control mice.

We also tested if either avidin-coated microbeads, used as virus carriers, or Con A, used as an anchoring agent for adenovirus-microbead conjugates, contributed to the elevated IL-10 level in the colon, seen with intra-colonical administration of adenovirus-microbead conjugates containing Con A above. In particular, Con A might have contributed to the elevated IL-10 level in the colon since repeated, intravenous administration of Con A can induce IL-10 production (Louis et al, 2000). Biotinylated Con A was attached to avidin-coated microbeads to saturate the microbead surface, followed by the removal of unbound Con A. The resulting avidin-coated microbeads containing Con A were administered intra-colonically into mice with TNBS-induced colitis by enema (a total of 2 x 10⁷ microbeads in 100 μl PBS per mouse; the same amount of microbeads as that used for adenovirus-microbead conjugates containing Ad5.CMV-IL10 above). At 24-hr post-administration, mice were euthanized, and the local IL-10 levels in the colons were determined quantitatively by ELISA. No appreciable changes in the local IL-10 levels were seen, as compared to control mice that received PBS alone (P > 0.4), suggesting that neither avidin-coated microbeads nor conjugated Con A induced the production of IL-10 in the colon (data not shown). These results indicate that the elevated IL-10 level in the colon upon intra-colonical administration of adenovirus-microbead conjugates containing Con A (Figure 6) was indeed derived from the transduction of the colon by Ad5.CMV-IL10. These results reveal that intra-colonical administration of Ad5.CMV-IL10 in the form of adenovirus-microbead conjugates containing Con A allows for efficient transduction of the colon with TNBS-induced colitis, raising the local IL-10 level considerably.

IV. Discussion

We have demonstrated that the use of adenovirus-microbead conjugates containing Con A allows for efficient transduction of the inflamed colon by adenoviral vectors upon intra-colonical administration by enema. The co-attachment of Con A as an anchoring agent has shown to be essential for enhanced transduction of the inflamed colon by adenovirus-microbead conjugates. Without the co-attachment of Con A, adenovirus-microbead conjugates showed a limited ability to transduce the inflamed colon, and their transduction efficiency was similar to that of free adenoviral vectors. These results suggest the potential for the gene transfer technology with adenovirus-microbead conjugates containing Con A to serve as an effective means for the delivery of therapeutic transgenes to the inflamed colon for the therapy of IBD. In addition, the size of adenovirus-microbead conjugates and the use of Con A as an anchoring agent could effectively inhibit systemic absorption of the conjugates. This could reduce uncontrolled migration of adenoviral vectors to and subsequent transduction of non-target organs. Furthermore, since adenovirus-microbead conjugates containing Con A have higher infectivity and broader tropism than free adenoviral vectors, a smaller amount of adenoviral vectors should be needed to achieve a given level of transgene expression. Hence, the use of adenovirus-microbead conjugates containing Con A for the delivery of therapeutic transgenes to the inflamed colon could also offer safety enhancement by minimizing both undesirable transduction of non-target organs and the number of adenovirus vectors required.

With an efficient transduction system for the inflamed colon now in hand, it should be possible to investigate, rigorously, the effect of local expression of the IL-10 and other therapeutic genes in the colon on the amelioration of established colitis. Studies are currently in progress by using a few different mouse colitis models, including the one with TNBS-induced colitis used in this study, to ask if the intra-colonical delivery of Ad5.CMV-IL10 to the inflamed colon in the form of adenovirus-microbead conjugates containing Con A could offer enhanced amelioration of colitis. These studies address several key questions, including the relationship between the local levels of IL-10 in the colon and the therapeutic effect on established colitis and whether the use of adenoviral vectors in the form of adenovirus-microbead conjugates containing Con A could minimize uncontrolled migration of viral particles to non-target organs. In addition, what cell types in the colon can be transduced by adenoviral vectors upon intra-colonical administration of adenovirus-microbead conjugates containing Con A is being determined, since this serves as a critical factor that determines the persistency of the expression of the IL-10 and other therapeutic genes.

Acknowledgments

We would like to thank Andrea Gambotto for providing Ad5.CMV-IL10, Andrew Keates for the instructions on the preparation of a mouse colitis model, and Peter Thomas for providing the MIP-101 cell line. We also thank Khashayarsha Khazaie, William Faubion, Cox Terhorst, and Mark Pandori for useful suggestions. AJ was supported by a training grant from the National Cancer Institute (CA59367; awarded to Dr. Melvin E. Clouse). This work was supported, in part, by the Broad Medical Research Program of The Eli and Edythe L. Broad Foundation (IBD-0078).

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Research Article

Received: 18 July 2006; Revised: 21 August 2006

Accepted: 23 August 2006; electronically published: August 2006

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