Abstract
Purpose:
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.
Methods:
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).
Results:
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.
Conclusions:
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.