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Andrei A Kramerov, Mehrnoosh Saghizadeh, Ezra Maguen, Yaron S Rabinowitz, Alexander V Ljubimov; Optimization of Adenovirus Transduction of Cultured Human Limbal Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):3639.
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© ARVO (1962-2015); The Authors (2016-present)
Gene therapy of limbal epithelial stem cells may be a promising approach to treat corneal diseases caused by diabetes, alkali burns and limbal stem cell deficiency (LSCD). The purpose was to determine optimal conditions for adenovirus type 5 (AdV) transduction of cultured human limbal epithelial cells (LEC) that would allow for high level of expression of transduced genes without affecting cell viability.
Primary LEC were isolated from discarded normal and diabetic corneoscleral rims and cultured in EpiLife medium (Thermo Fisher). Cultured LEC were transduced with GFP-AdV (KeraFAST) or scrambled shRNA-GFP-AdV (Capital Biosciences) in a wide range of multiplicity of infection (MOI: 1-300 PFU/cell) for 4 h in a minimal volume (0.2 mL) followed by 20 h in 0.5 mL of the medium. After replacing medium for the one without AdV, cultured cells were incubated for 4 days, and GFP expression level was evaluated using Evos FL inverted fluorescent microscope. To enhance AdV transduction efficiency the following reagents were used: polycations (poly-L-lysine and polybrene; Sigma-Aldrich), ViraDuctin (Cell Biolabs), and ibiBoost (ibidi).
LEC transduction by AdV at high MOI (>100 PFU/cell) resulted in fairly high level of transduction (GFP expression in >50% cells) but was also toxic, causing cell death after 3-4 days of transduction, or severely impaired cell migratory ability. At lower MOI (10-30 PFU/cell), AdV transduction produced less or no cytotoxic effect, and resulted in a decreased number of GFP-expressing cells (5-15%). Transduction-enhancing reagents that facilitate AdV binding to cell surface showed different efficiency improvements in LEC transduction by AdV at MOI 10-30. Polycations were the most effective (3-4 fold increase) and non-toxic, ibiBoost was effective but slightly toxic, and ViraDuctin was non-effective and rather toxic. Normal and diabetic corneas yielded similar results.
Transduction of LEC with lower AdV titer (MOI 10-30) that did not cause cytotoxicity, in the presence of polycations allowed for transduction of >50% of LEC which may be sufficient for effective gene therapy.
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