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Anja Katharina Gruenert, Hildegard Buening, Maria Schnoedt, Marco Schmeer, Martin Schleef, Friedrich E Kruse, Thomas Armin Fuchsluger; Self-complementary AAV2 vectors improve transduction efficiency of corneal endothelial cells. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):3647.
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© ARVO (1962-2015); The Authors (2016-present)
Previous studies have shown that corneal endothelial cells (CEC) can be successfully transduced by recombinant adeno-associated virus (AAV)-2 vectors, which represent a promising, non-pathogenic alternative to other viral delivery systems. However, especially in the early period after transduction, gene expression remains low. One of the rate-limiting steps for transduction is the conversion of single-stranded (ss) DNA vector genome into double-stranded (ds) DNA prior to gene expression. This step can be bypassed by using self-complementary (sc) AAV2 vectors, which contain a dimeric inverted repeat genome that can fold into dsDNA, thus enhancing transduction efficiency. To evaluate this effect in CEC, we compared transduction efficiencies of ss- and sc AAV2 vectors.
A human corneal endothelial cell line (HCEC-12) was incubated with different concentrations of AAV2 vectors containing ss- or sc-green fluorescent protein (GFP) as transgene for 48 hours. In addition, human and murine corneal tissue was transduced accordingly. Culture medium was changed every other day. GFP-expression in HCEC-12 cells was analyzed by fluorescence-activated cell sorting (FACS) directly after the transduction period (day 2) as well as on day 5. GFP-expression in human and murine endothelial cells was evaluated using confocal microscopy.
After 48 hours, GFP-expression was observed in up to 11.5 % of the cells transduced with ssAAV2. Using the scAAV2 vector resulted in significantly higher expression levels of GFP (up to 27.3 %). The difference in transduction efficiency decreased with increasing vector titer. While lower titers of scAAV2 achieved 5-fold higher amounts of transfected cells relative to ssAAV2, for higher vector titers transfection rates were only 2-3 times higher. For both vectors, GFP-expression increased over time. On day 5, it was up to 3 times higher compared to day 2.
Transduction efficiency of AAV2-vectors in CEC can be increased by using a sc- instead of a ss-transgene. The use of scAAV2 yielded not only a faster onset but also higher levels of gene expression. This could have an impact on AAV2-mediated gene therapy to protect CEC in corneal allografts prior to transplantation.
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