Effect of immunomodulation with anti-CD40L antibody on adenoviral-mediated transgene expression in mouse anterior segment

Abstract

Purpose: Gene transduction using adenoviral vectors is an important research tool. To assess and optimize this technique for glaucoma research, we characterized green fluorescent protein (GFP) expression in the mouse eye after intraocular injection of adenoviral vector encoding GFP (Ad5.CMV-GFP) and evaluated the effect of anti-CD40L antibody administration on GFP expression.

Methods: Mice were injected with Ad5.CMV-GFP intracamerally (IC) or intravitreally (IVT) with or without anti-CD40L antibody treatment. GFP expression was assessed by in vivo fluorescence intensity with a standardized grading scale. Location of expression was analyzed histologically by fluorescence microscopy.

Results: Intraocular injection of Ad5.CMV-GFP induced titer-dependent expression of GFP in the anterior segment. In vivo fluorescence was detectable but low after IC injection. After IVT injection, fluorescence in the eye peaked at days 4-7 with a fluorescence grade of 3.0 +/-0.0 (mean +/-SEM, n=6; injection with 1x10(8) pfu vector). After day 7, GFP expression declined significantly. Treatment with anti-CD40L antibody increased fluorescence intensity after IC injection, and prolonged GFP expression in the IVT group. At day 43, fluorescence grades of the IVT group were 2.8 +/-0.7 (with anti-CD40L) and 1.2 +/-0.6 (without antibody). Three-Way ANOVA confirmed that GFP expression was significantly higher in the anti-CD40L than the no antibody group (p=0.013), significantly higher in the IVT than the IC group (p=0.003), and significantly higher in the high viral titer (1x10(8) pfu) than the low titer (1x10(7) pfu) group (p=0.010). Fluorescence microscopic examination of cross-sections of eyes indicated GFP expression in the trabecular meshwork (TM), corneal endothelium, and sporadically iris, ciliary body and lens epithelium.

Conclusions: Intraocular injection of Ad5.CMV-GFP induced GFP expression in the mouse anterior segment, including the TM. Expression was more prominent after IVT injection than IC injection. Anti-CD40L antibody treatment increased both intensity and duration of GFP expression. These findings provide important and practical means to improve duration and efficiency of adenovirus-mediated transgene expression in the eye.

Figure 1

Representative examples of mouse eyes with various fluorescence grades. The number under each image indicates the assigned grade. Definitions of the grades are listed in Table 1.

Figure 2

Effects of routes of intraocular injections, titers of viral vector, and use of anti-CD40L antibody on the intensity and duration of GFP expression evaluated by in vivo fluorescence grades. At day 0, Ad5.CMV-GFP, at a titer of 1x10⁷ pfu (A and C) or 1x0⁸ pfu (B and D), was injected intracamerally (A and B) or intravitreally (C and D) with (open symbols) or without (closed symbols) immunomodulation by intraperitoneal treatment of anti-CD40L antibody. In vivo fluorescence in the eyes was evaluated and fluorescence grades assigned at the indicated days by a researcher who was masked of the treatments. The fluorescence grades were further analyzed as the integrated fluorescence (E) by summation of the area under the curve of each animal. All data were presented as mean and SEM.

Figure 3

GFP fluorescence in mouse eyes six days following intravitreal injection. Images of mouse eyes six days after intravitreal injection of Ad5.CMV-GFP at 1x10⁷ pfu (left panels) or 10⁸ pfu (right panels) with (upper panels) or without (lower panels) anti-CD40L treatment. All eyes were expressing a substantial level of GFP with fluorescence grades of 2 or 3.

Figure 4

GFP fluorescence in mouse eyes 35 days following intravitreal injection. Images of mouse eyes 35 days after intravitreal injection of Ad5.CMV-GFP at 1x10⁷ pfu (left panels) or 10⁸ pfu (right panels) with (upper panels) or without (lower panels) anti-CD40L treatment. The eyes from mice treated with anti-CD40L antibody still expressed high level of GFP; their fluorescence grades were between 2 and 3. In contrast, eyes from mice without anti-CD40L treatment had minimal fluorescence with grades of 1 or below.

Figure 5

Representative fluorescence photomicrographs of cross-sections of mouse anterior segments treated with intracameral injection of Ad.CMV-GFP (1x10⁷ pfu). The animals were euthanized at either 5 or 43 days after vector injection with or without anti-CD40L antibody treatment. Green fluorescence=GFP, blue fluorescence=DAPI staining of cell nuclei. The occasional yellowish-green fluorescence represents auto-fluorescence of certain tissues, such as those in the outer nuclear layer and retinal pigmented epithelium, which was also observable in non-injected eyes (Data not shown). Abbreviations: C=cornea; CB=ciliary body; I=iris; L=lens; R=retina; TM=trabecular meshwork.

Figure 6

Representative fluorescence photomicrographs of cross-sections of mouse anterior segments treated with intracameral injection of Ad.CMV-GFP (1x10⁸ pfu). Representative fluorescence photomicrographs of cross-sections of mouse anterior segments treated with intracameral injection of Ad5.CMV-GFP (1x10⁸ pfu). See legend of Figure 5 for additional information.

Figure 7

Representative fluorescence photomicrographs of cross-sections of mouse anterior segments treated with intravitreal injection of Ad.CMV-GFP (1x10⁷ pfu). Representative fluorescence photomicrographs of cross-sections of mouse anterior segments treated with intravitreal injection of Ad5.CMV-GFP (1x10⁷ pfu). See legend of Figure 5 for additional information.

Figure 8

Representative fluorescence photomicrographs of cross-sections of mouse anterior segments treated with intravitreal injection of Ad.CMV-GFP (1x10⁸ pfu). Representative fluorescence photomicrographs of cross-sections of mouse anterior segments treated with intravitreal injection of Ad5.CMV-GFP (1x10⁸ pfu). See legend of Figure 5 for additional information.