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

Millar JC, Pang IH, Wang WH, Wang Y, Clark AF - Mol. Vis. (2008)

Bottom Line: After day 7, GFP expression declined significantly.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).These findings provide important and practical means to improve duration and efficiency of adenovirus-mediated transgene expression in the eye.

View Article: PubMed Central - PubMed

Affiliation: Alcon Research, Ltd., Fort Worth, TX 76134, USA.

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.

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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.
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f8: 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.

Mentions: As indicated by the in vivo fluorescence grades, IVT injection produced more efficient expression of the injected GFP vector than IC injection. Fluorescence microscopic observation substantiated this conclusion. At day 5 after IVT injection of 1x107 pfu Ad5.CMV-GFP, with or without anti-CD40L antibody treatment, significant fluorescence was noted in the TM, corneal endothelium, and occasionally the iris (Figure 7). With the anti-CD40L treatment, a similar distribution of fluorescence was still present at day 43, whereas in animals without anti-CD40L antibody, minimal fluorescence was detected at 43 days after injection (Figure 7). Higher titer of the vector produced more prominent GFP expression. Thus, five days after IVT injection of 1x108 pfu, the TM, corneal endothelium, and sometimes the iris, lens epithelium and ciliary body demonstrated fluorescence (Figure 8). Treatment with anti-CD40L antibody prolonged this expression profile till at least 43 days after vector injection, while without the antibody, fluorescence faded and became faint at day 43 (Figure 8).


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

Millar JC, Pang IH, Wang WH, Wang Y, Clark AF - Mol. Vis. (2008)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2267727&req=5

f8: 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.
Mentions: As indicated by the in vivo fluorescence grades, IVT injection produced more efficient expression of the injected GFP vector than IC injection. Fluorescence microscopic observation substantiated this conclusion. At day 5 after IVT injection of 1x107 pfu Ad5.CMV-GFP, with or without anti-CD40L antibody treatment, significant fluorescence was noted in the TM, corneal endothelium, and occasionally the iris (Figure 7). With the anti-CD40L treatment, a similar distribution of fluorescence was still present at day 43, whereas in animals without anti-CD40L antibody, minimal fluorescence was detected at 43 days after injection (Figure 7). Higher titer of the vector produced more prominent GFP expression. Thus, five days after IVT injection of 1x108 pfu, the TM, corneal endothelium, and sometimes the iris, lens epithelium and ciliary body demonstrated fluorescence (Figure 8). Treatment with anti-CD40L antibody prolonged this expression profile till at least 43 days after vector injection, while without the antibody, fluorescence faded and became faint at day 43 (Figure 8).

Bottom Line: After day 7, GFP expression declined significantly.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).These findings provide important and practical means to improve duration and efficiency of adenovirus-mediated transgene expression in the eye.

View Article: PubMed Central - PubMed

Affiliation: Alcon Research, Ltd., Fort Worth, TX 76134, USA.

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.

Show MeSH
Related in: MedlinePlus