Transplantation of a donor cornea to restore vision is the most frequently performed transplantation\nin the world. Corneal endothelial cells (CEC) are crucial for the outcome of a graft\nas they maintain corneal transparency and avoid graft failure due to corneal opaqueness.\nGiven the characteristic of being a monolayer and in direct contact with culture medium during\ncultivation in eye banks, CEC are specifically suitable for gene therapeutic approaches\nprior to transplantation. Recombinant adeno-associated virus 2 (rAAV2) vectors represent\na promising tool for gene therapy of CEC. However, high vector titers are needed to achieve\nsufficient gene expression. One of the rate-limiting steps for transgene expression is the\nconversion of single-stranded (ss-) DNA vector genome into double-stranded (ds-) DNA.\nThis step can be bypassed by using self-complementary (sc-) AAV2 vectors. Aim of this\nstudy was to compare for the first time transduction efficiencies of ss- and scAAV2 vectors\nin CEC. For this purpose AAV2 vectors containing enhanced green fluorescent protein\n(GFP) as transgene were used. Both in CEC and in donor corneas, transduction with\nscAAV2 resulted in significantly higher transgene expression compared to ssAAV2. The difference\nin transduction efficiency decreased with increasing vector titer. In most cases, only\nhalf the vector titer of scAAV2 was required for equal or higher gene expression rates than\nthose of ssAAV2. In human donor corneas, GFP expression was 64.7�±11.3%(scAAV) and\n38.0�±8.6% (ssAAV) (p<0.001), respectively. Furthermore, transduced cells maintained\ntheir viability and showed regular morphology. Working together with regulatory authorities,\na translation of AAV2 vector-mediated gene therapy to achieve a temporary protection of\ncorneal allografts during cultivation and transplantation could therefore become more\nrealistic.
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