title: Effect of immunomodulation with anti-CD40L antibody on adenoviral-mediated transgene expression in mouse anterior segment.
abstract: OBJECTIVE
  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.
authors.affiliation: Alcon Research, Ltd., Fort Worth, TX 76134, USA.
authors.forename: J Cameron
authors.initials: JC
authors.lastname: Millar
authors.forename: Iok-Hou
authors.initials: IH
authors.lastname: Pang
authors.forename: Wan-Heng
authors.initials: WH
authors.lastname: Wang
authors.forename: Yu
authors.initials: Y
authors.lastname: Wang
authors.forename: Abbot F
authors.initials: AF
authors.lastname: Clark
mesh_terms: D000256:Adenoviridae; D000818:Animals; D000869:Anterior Eye Segment / Q000378:metabolism*; D000906:Antibodies / Q000494:pharmacology*; D023201:CD40 Ligand / Q000276:immunology*; D005123:Eye / Q000378:metabolism; D005453:Fluorescence; D015870:Gene Expression / Q000187:drug effects*; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D007155:Immunologic Factors / Q000494:pharmacology*; D007267:Injections; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D008856:Microscopy, Fluorescence; D014018:Tissue Distribution; D019076:Transgenes; D014822:Vitreous Body
chemical_list: D000906:Antibodies; D007155:Immunologic Factors; C485192:green fluorescent protein, Aequorea victoria; D023201:CD40 Ligand; D049452:Green Fluorescent Proteins