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Thomas Armin Fuchsluger, Marco Schmeer, Martin Schleef, Maria Schnoedt, Hildegard Buening, Friedrich E Kruse, Anja Katharina Gruenert; A new tool for transfection of corneal endothelial cells: minicircle-based AAV-vectors. Invest. Ophthalmol. Vis. Sci. 2017;58(8):1445. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Cell and gene therapy of corneal endothelial cells (EC) is a proven concept to alter EC’s functions. Thus, EC resistance against apoptosis could be increased by transfection with anti-apoptotic DNA. To achieve this, both non-viral and viral vectors can be used. If non-viral vectors are used plasmid backbone sequences are delivered, as they are an inherent part of the system. However, also in viral vectors plasmid backbone sequences can be detected. As such contamination may induce innate immune responses, we established usage of minicircles instead of plasmids for AAV vector production aiming to avoid unintended packaging of backbone sequences. We therefore propose a new tool for EC transfection by studying these minicircle-based adeno-associated viral vectors.
Four different constructs of AAV2 vectors containing a green fluorescent protein were manufactured (plasmid helper-minicircle transgene): plasmid-plasmid (p-p), plasmid-minicircle (p-m), minicircle-plasmid (mc-p) and minicircle-minicircle (mc-mc). Human corneal endothelial cells (SV40 transduced) were treated with each construct. Transfection efficacy, induced apoptosis (Po-Pro1, 7-AAD) and metabolic activity (Cell Counting Kit - 8 (CCK-8)) were evaluated by flow cytometry / confocal microscopy.
Transfection efficacy varied significantly amongst the specific vector constructs: p-mc constructs showed significantly higher GFP-positivity (50,8±7,5%) compared to mc-mc (43,3± 4,5%), p-p (40,2± 3,8%) and mc-p constructs (32,1± 3,1%) (p=0,03). Apoptosis induced by these vectors was low and insignificant between all groups (3,5-4,3%). Metabolic activity in the p-mc group (104,4± 6,4%) was significantly higher than in the p-p (95,9± 5,8%) and in the mc-mc groups (97,6± 4,7%) (mc-p: 97,2± 5,4%) (p<0,05).
Interestingly, we could demonstrate that plasmid-minicircle AAV2 vectors perform significantly better than p-p, mc-mc and mc-p constructs. This is independent of the vector prep solution. Notably, cells treated with this vector group also repeatedly showed higher metabolic activity. Plasmid-minicircle AAV2 seem to be a suitable tool for optimized transduction of corneal endothelial cells.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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