Abstract
Purpose :
Gene therapy approaches are explored to prevent EC apoptosis extending storage time of donor corneas and achieve post-operative grafts with higher EC quality. Retroviruses lead to an integration of the DNA of choice into the nucleus, a suboptimal feature in EC modification. We investigated the efficacy of integrase-deficient vector types as potential alternatives for EC transduction.
Methods :
GFP-transducing vectors of EIAV and of HIV origin and their integrase-defective variants were used to transfer genes into human corneal endothelium. Corneal grafts were gained from donors of different age and gender (age 45 ± 24) and stored hypothermic (SightLife, Seattle). Each quarter of a cut cornea was transduced with the respective vector variants for 24h at 37°C. Fluorescence microscopy was done at 37°C with a Zeiss Spinning Disc confocal microscope endothelium-down. Amount of green fluorescence was quantified via ImageJ. Additionally, life imaging via intravital microscopy with non-linear optics and ary scan technology was used to establish fluorescence imaging in full thickness human corneas.
Results :
Our studies showed that human corneas can be transduced with all used variations of lentiviral vectors namely EIAV (28 ±9%), EIAV integrase-defective (38 ±10%), HIV (17 ±7.0%) and HIV integrase deficient (24 ±7.5%). We noticed that integrase-deficient vectors transduced EC more effective than standard integrating vectors (p<0.05). Furthermore, we transduced a complete uncut cornea from the endothelial side and established life cell imaging throughout all layers of the cornea. We could visualize healthy and apoptotic as well as transduced corneal endothelial cells. The transduction efficiency for the corneal endothelium was 75% (± 9%) representing the higher vector to cell proportion in the "cornea-in-a-cup" transduction and we can state that only the endothelium was transduced showing the high specificity of our method.
Conclusions :
With integrase-deficient vectors performing better in the corneal endothelium, a crucial step towards patient treatment is achieved considering regulatory questions. To our knowledge, this study shows for the first time in the corneal endothelium that integrase-deficient vectors work as effective as or better than their integrating counterparts. Given confirmation of these data in further experiments in human corneal tissue, we will transfer this concept into an eye setting.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.