Abstract
Purpose :
Gene therapy is a promising way for the treatment of inherited retinal degenerations. However, there are still certain problems and limitations with gene therapy, such as the biological safety of viral vectors and the limited gene delivery efficiency of non-viral vectors. In order to achieve highly efficient transfection of retinal tissue without compromising biological safety, we have established Sleeping Beauty (SB) transposon-based non-viral gene transfer via electroporation. The experiments were performed on isolated retinas from rd10 mice, a well-established model of retinal degeneration.
Methods :
Using the NEPA21 type II electroporator, we introduced the SB100X transposase plasmid and the pT2/CAGGS-Venus transposon plasmid (1:16 ratio) into the retinal tissues of rd10 mice at different stages of degeneration (P25, P61, P90, and P180) as well as age-matched wild-type (WT) mice (C57BL/6J). Transfection efficiency of whole mount retinas was assessed by fluorescence microscopy one day after incubation. Hematoxylin and eosin (HE) staining and immunofluorescence staining were performed to evaluate the degree of retinal integrity and to identify the transfected cell types, respectively.
Results :
With regard to transfection efficiency and retinal integrity, the following electroporation parameters were identified: 10 V (poring pulse voltage), 5 ms (pulse length), 50 ms (pulse interval), 2 (pulse number), 10% (pulse decay rate), +/- (pulse polarity); 5 V (transfer pulse voltage), 50 ms (pulse length), 50 ms (pulse interval), 5 (pulse number), 40% (pulse decay rate), +/- (pulse polarity). Optimal transfection efficiency was observed when a concentration of 0.1 µg/µl purified plasmid mixture was used for transfection together with Opti-MEM electroporation buffer. Retinas of rd10 mice older than P61 were easily transfected. The transfected cells were Müller cells, which could be identified by co-labelling with anti-glutamine synthetase. However, it was not possible to transfect WT mice retinas.
Conclusions :
The non-viral, electroporation-based gene transfer method shown here allows for distinct transfection in degenerated rd10 retinas. The cell type that could be transfected in this way is the Müller cell. Further experiments will be conducted to determine why WT retinas and younger rd10 retinas did not show successful transfection and why only Müller cells can be transfected.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.