title: AAV serotype influences gene transfer in corneal stroma in vivo.
abstract: This study evaluated the cellular tropism and relative transduction efficiency of three AAV serotypes, AAV6, AAV8 and AAV9, for corneal gene delivery using mouse cornea in vivo and donor human cornea ex vivo. The AAV6, AAV8 and AAV9 serotypes having AAV2 plasmid encoding for alkaline phosphatase (AP) gene were generated by transfecting HEK 293 cell line with pHelper, pARAP4 and pRep/Cap plasmids. Viral vectors (10(9) vg/microl) were topically applied onto mouse cornea in vivo and human cornea ex vivo after removing the epithelium. Human corneas were processed for transgene delivery at day 5 after viral vector application. Mouse corneas were harvested at 4, 14 and 30 days after vector application for AP staining. Transduction efficiency was calculated by quantifying pixels of AP-stained area using Image J software and also confirmed by functional AP enzyme activity in the corneal lysates. Cellular toxicity of the three AAV serotypes was tested with TUNEL assay. Inflammatory response was detected by immunostaining for CD11b and F4/80. All three AAV serotypes successfully transduced mouse and human corneas. The order of transduction efficiency was AAV9 > AAV8 > AAV6. The transduction efficiency of AAV9 was 1.1-1.4 fold higher (p > 0.05) as compared to AAV8 and 3.5-5.5 fold higher (p < 0.01) as compared to AAV6. The level of transgene expression for all the three serotypes was greater at 14 days compared to 4 days and this high level of transgene expression was maintained up to the tested time point of 30 days. Corneas exposed to any of the three AAV serotypes did not show significant TUNEL positive cells or any inflammatory response as tested by CD11b or F4/80 staining suggesting that tested AAV serotypes do not induce cell death or inflammation and are safe for corneal gene therapy.
authors.affiliation: Harry S Truman Memorial Veterans' Hospital, Columbia, MO 65201, USA.
authors.forename: Ajay
authors.initials: A
authors.lastname: Sharma
authors.forename: Jonathan C K
authors.initials: JC
authors.lastname: Tovey
authors.forename: Arkasubhra
authors.initials: A
authors.lastname: Ghosh
authors.forename: Rajiv R
authors.initials: RR
authors.lastname: Mohan
mesh_terms: D000469:Alkaline Phosphatase / Q000235:genetics*; D000818:Animals; D000943:Antigens, Differentiation / Q000378:metabolism; D039481:CD11b Antigen / Q000378:metabolism; D003319:Corneal Stroma / Q000201:enzymology*; D000229:Dependovirus / Q000145:classification / Q000235:genetics*; D005260:Female; D019084:Fluorescent Antibody Technique, Indirect; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology*; D005822:Genetic Vectors; D006801:Humans; D020287:In Situ Nick-End Labeling; D051379:Mice; D008810:Mice, Inbred C57BL; D010957:Plasmids; D012703:Serotyping; D014161:Transduction, Genetic
chemical_list: D000943:Antigens, Differentiation; D039481:CD11b Antigen; C036763:monocyte-macrophage differentiation antigen; D000469:Alkaline Phosphatase