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Thomas Fuchsluger, Christian Mueller, Reza Dana; Comparison of Adeno-Associated Viral Vector Serotypes for Gene Transfer To Corneal Endothelial Cells. Invest. Ophthalmol. Vis. Sci. 2013;54(15):2718.
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Due to the anatomical location and its monolayer layout the corneal endothelium (EC) is an ideal target for gene therapy. The aim of this study was to determine the efficacy and efficiency of the non-pathogenic recombinant adeno-associated viral vectors (rAAV) for gene transfer to EC. Therefore, different rAAV pseudotypes were compared to assess the expression and kinetic patterns of a reporter gene (GFP).
A comparison of GFP expression and kinetics after EC transduction using a AAV 2/1, 2/2, 2/5, 2/8, 2/9, 2/10 was performed on both murine EC (Balb/C) and human EC (corneas, cell line and primary cells). In addition, the effects of different vector concentrations (3x10^3/10^4, 10^5, 10^6, 10^7, 10^8IU/µl) were investigated. Analyses were performed using laser scanning microscopy and flow cytometry.
We detected significant differences between human and murine EC as well as primary and immortalized EC. Whereas in murine corneas transduction of EC with AAV2/2, 2/5 and 2/9 resulted in considerable protein expression, AAV 2/5 did not show considerable expression in human corneas. We also detected a slow onset of GFP expression both in immortalized and primary EC, leading to peak and stable expression around 2-3 weeks. However, peak expression was reached earlier in primary EC and immortalized murine EC compared to immortalized human EC. In addition, the overall plateau of expression was highest in primary EC (~80%).
Recombinant AAV pseudotypes differ in their tropism and transduction efficiency between murine and human corneas. In general high levels of gene expression can be obtained with several of these pseudotypes. Characteristic slow-onset kinetics of protein expression have to be taken into account when applying AAVs in translational applications, e.g. in corneal storage in eye banks.
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