title: Transduction efficiency of AAV 2/6, 2/8 and 2/9 vectors for delivering genes in human corneal fibroblasts.
abstract: In the present study, cellular tropism and relative transduction efficiency of AAV2/6, AAV2/8 and AAV2/9 vectors have been tested for the cornea using primary cultures of human corneal fibroblasts. The AAV6, AAV8 and AAV9 serotypes having AAV2 ITR plasmid encoding for alkaline phosphatase (AP) gene were generated by transfecting HEK293 cell line with pHelper, pARAP4 and pRep/Cap plasmids. Primary cultures of human corneal fibroblasts were exposed to AAV infectious particles at two different doses (1 x 10(5) and 2 x 10(5) MOI). Cytochemistry and enzyme assays were used to measure delivered transgene expression in samples collected at 4 and 30 h after AAV infection by counting AP-stained cells or quantifying AP enzyme activity. Cellular toxicity of AAVs was evaluated with TUNEL and trypan blue assays. All three AAV serotypes transduced human corneal fibroblasts. The order of transduction efficiency was AAV2/6>>>AAV2/9>AAV2/8. The transduction efficiency of AAV2/6 was 30-50-fold higher (p < 0.001) for the human corneal fibroblasts compared to the AAV2/8 or AAV2/9 at two tested doses. The level of transgene expression at 4h was considerably low compared to 30 h suggesting that the transgene delivery did not reach its peak at 4h. Cultures exposed to any of the three AAV serotypes showed more than 97% cellular viability and less than 5 TUNEL positive cells suggesting that tested AAV serotypes do not induce significant cell death and are safe for corneal gene therapy.
authors.affiliation: Mason Eye Institute, School of Medicine, University of Missouri-Columbia, 1 Hospital Dr. Columbia, MO 65212, USA.
authors.forename: Ajay
authors.initials: A
authors.lastname: Sharma
authors.forename: Arkasubhra
authors.initials: A
authors.lastname: Ghosh
authors.forename: Eric T
authors.initials: ET
authors.lastname: Hansen
authors.forename: Jason M
authors.initials: JM
authors.lastname: Newman
authors.forename: Rajiv R
authors.initials: RR
authors.lastname: Mohan
mesh_terms: D000469:Alkaline Phosphatase / Q000235:genetics / Q000378:metabolism*; D000704:Analysis of Variance; D017209:Apoptosis / Q000235:genetics; D002470:Cell Survival / Q000502:physiology; D002478:Cells, Cultured; D003315:Cornea / Q000166:cytology*; D000229:Dependovirus / Q000145:classification / Q000235:genetics*; D005347:Fibroblasts / Q000201:enzymology*; D005822:Genetic Vectors / Q000145:classification; D006801:Humans; D020287:In Situ Nick-End Labeling / Q000379:methods; D007211:Indoles; D013053:Spectrophotometry / Q000379:methods; D014161:Transduction, Genetic / Q000379:methods*
chemical_list: D007211:Indoles; C007293:DAPI; D000469:Alkaline Phosphatase