title: Canine corneal fibroblast and myofibroblast transduction with AAV5.
abstract: OBJECTIVE
  The aims of this study were (1) to determine the efficacy of adeno-associated vector serotype 5 (AAV5) for delivering gene therapy to canine corneal fibroblasts (CCFs) and myofibroblasts (CCMs) using enhanced green fluorescent protein (GFP) marker gene and (2) to evaluate the cytotoxicity of AAV5 to CCFs and CCMs using an in vitro model.
  
  
  METHODS
  Healthy donor canine corneas were used to generate primary CCFs by growing cultures in minimal essential medium supplemented with 10% fetal bovine serum. Canine corneal myofibroblasts were produced by growing cultures in serum-free medium containing transforming growth factor β1 (1 ng/mL). An AAV5 titer (6.5 × 10(12) μg/mL) expressing GFP under control of hybrid cytomegalovirus + chicken β-actin promoters (AAV5-gfp) was used to transduce CCF and CCM cultures. Delivered gene expression in CCFs and CCMs was quantified using immunocytochemistry, fluorescent microscopy, and real-time PCR. Transduction efficacy of the AAV5 vector was determined by counting DAPI-stained nuclei and EGFP-positive cells in culture. Phase-contrast microscopy, trypan blue, and dUTP nick end labeling (TUNEL) assays were used to determine the toxicity and safety of AAV5 in this canine corneal model.
  
  
  RESULTS
  Topical AAV5 application successfully transduced a significant population of CCFs (42.8%; P < 0.01) and CCMs (28%; P < 0.01). Tested AAV5 did not affect CCF or CCM phenotype or cellular viability and did not cause significant cell death.
  
  
  CONCLUSIONS
  The tested AAV5 is an effective and safe vector for canine corneal gene therapy in this in vitro model. In vivo studies are warranted.
authors.affiliation: Harry S. Truman Veterans Memorial Hospital, Columbia, MO, USA.
authors.forename: Ann P
authors.initials: AP
authors.lastname: Bosiack
authors.forename: Elizabeth A
authors.initials: EA
authors.lastname: Giuliano
authors.forename: Rangan
authors.initials: R
authors.lastname: Gupta
authors.forename: Rajiv R
authors.initials: RR
authors.lastname: Mohan
mesh_terms: D000256:Adenoviridae; D000818:Animals; D002478:Cells, Cultured; D003315:Cornea / Q000166:cytology*; D004285:Dogs / Q000502:physiology*; D005347:Fibroblasts / Q000378:metabolism*; D005786:Gene Expression Regulation; D005822:Genetic Vectors / Q000592:standards; D049452:Green Fluorescent Proteins / Q000235:genetics; D007150:Immunohistochemistry; D058628:Myofibroblasts / Q000378:metabolism*; D060888:Real-Time Polymerase Chain Reaction; D014161:Transduction, Genetic / Q000379:methods / Q000662:veterinary*
chemical_list: C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins