title: Significant inhibition of corneal scarring in vivo with tissue-selective, targeted AAV5 decorin gene therapy.
abstract: OBJECTIVE
  This study tested a hypothesis that tissue-selective targeted decorin gene therapy delivered to the stroma with adeno-associated virus serotype 5 (AAV5) inhibits corneal fibrosis in vivo without significant side effects.
  
  
  METHODS
  An in vivo rabbit model of corneal fibrosis was used. Targeted decorin gene therapy was delivered to the rabbit cornea by a single topical application of AAV5 (100 μL; 6.5 × 10(12) μg/mL) onto the bare stroma for 2 minutes. The levels of corneal fibrosis were determined with stereomicroscopy, slit lamp biomicroscopy, α-smooth muscle actin (αSMA), fibronectin, and F-actin immunocytochemistry, and/or immunoblotting. CD11b, F4/80 immunocytochemistry, and TUNEL assay were used to examine immunogenicity and cytotoxicity of AAV5 to the cornea. Transmission electron microscopy (TEM) was used to investigate ultrastructural features. Slot-blot-quantified the copy number of AAV5-delivered decorin genes.
  
  
  RESULTS
  Selective decorin delivery into the stroma showed a significant (P < 0.01) decrease in corneal haze (1.3 ± 0.3) compared with the no-decorin-delivered control rabbit corneas (3 ± 0.4) quantified using slit lamp biomicroscopy. Immunostaining and immunoblot analyses detected significantly reduced levels of αSMA, F-actin, and fibronectin proteins (59%-73%; P < 0.001 or <0.01) in decorin-delivered rabbit corneas compared with the no-decorin-delivered controls. The visual clinical eye examination, slit lamp clinical studies, TUNEL, CD11b, and F4/80 assays revealed that AAV5-mediated decorin gene therapy is nonimmunogenic and nontoxic for the cornea. TEM studies suggested that decorin gene delivery does not jeopardize collagen fibrillogenesis as no significant differences in collagen fibril diameter and arrangement were observed in decorin-delivered and no-decorin-delivered control corneas.
  
  
  CONCLUSIONS
  Tissue-targeted AAV5-mediated decorin gene therapy is effective and safe for treating corneal fibrosis in vivo.
authors.affiliation: Harry S. Truman Memorial Veterans' Hospital, Columbia, Missouri, USA. mohanr@health.missouri.edu
authors.forename: Rajiv R
authors.initials: RR
authors.lastname: Mohan
authors.forename: Ashish
authors.initials: A
authors.lastname: Tandon
authors.forename: Ajay
authors.initials: A
authors.lastname: Sharma
authors.forename: John W
authors.initials: JW
authors.lastname: Cowden
authors.forename: Jonathan C K
authors.initials: JC
authors.lastname: Tovey
mesh_terms: D000287:Administration, Topical; D000818:Animals; D017209:Apoptosis; D002921:Cicatrix / Q000209:etiology / Q000473:pathology / Q000628:therapy*; D003315:Cornea / Q000648:ultrastructure*; D003318:Corneal Opacity / Q000209:etiology / Q000473:pathology / Q000517:prevention & control*; D058575:Decorin / Q000008:administration & dosage*; D000229:Dependovirus; D004195:Disease Models, Animal; D018450:Disease Progression; D005260:Female; D015316:Genetic Therapy / Q000379:methods*; D015151:Immunoblotting; D020287:In Situ Nick-End Labeling; D017348:Microscopy, Electron, Scanning Transmission; D010957:Plasmids; D011817:Rabbits
chemical_list: D058575:Decorin