Self‐complementary vectors for optimization of AAV2‐mediated gene‐therapy of corneal endothelial cells

Abstract

PurposeRecombinant adeno‐associated viruses 2 (rAAV2) represent a nonpathogenic and safe alternative to other viral delivery systems. However, their transduction efficiency in corneal endothelial cells (CEC) is limited. As the level of transgene expression is dependent on the conversion of single‐stranded (ss)‐ into double‐stranded (ds)‐DNA, self‐complementary (sc)‐AAV vectors have been developed to circumvent this problem. The aim of this study was to evaluate the use of scAAV2 in terms of transduction efficiency in CEC. Additionally, the impact of transduction on cell viability was investigated.MethodsA human corneal endothelial cell line (HCEC‐12) as well as organ‐cultured human donor corneas were transduced with different titers of ss‐ or sc‐AAV2. Transduction efficiencies were compared by means of GFP‐transgene expression. GFP‐expression in HCEC‐12 cells was evaluated by flow cytometry over a period of 28 days. GFP‐expression in human donor corneas was analyzed by confocal microscopy on day 6. 7‐AAD staining and flow cytometry as well as MTT‐assay were performed to determine cell viability after transduction.ResultsGFP‐expression was significantly higher in cells transduced with scAAV2 than in cells transduced with ssAAV2. The difference in transduction efficiency decreased with increasing vector titer. The highest transgene expression rate using scAAV2 was 86.9% compared to 80.5 % using ssAAV2. In human donor corneas GFP‐expression was observed in 72.2% (scAAV) and 44.1% (ssAAV) of CEC respectively. There was no significant difference between viability of transduced and untreated cells.ConclusionsScAAV2 vectors are an effective tool to enhance transduction efficiency in CEC. Allowing higher transduction rates with lower vector titers, this could improve AAV2‐mediated gene therapy to protect CEC in corneal allografts.