Protein-based nanoparticles for non-viral retinal gene therapy.

Purpose: Protein-based nanoparticles for DNA compactation have been widely studied for different applications. In this work two different peptidic constructions (R9-GFP-His and HNRK) have been tested on in-vitro and in-vivo models for its application in non-viral retinal gene therapy.

Methods: R9-GFP-His and HNRK nanoparticles have been produced in bacteria and purified. Plasmidic DNA compactation efficacy has been studied by agarose gel electrophoresis assay. Transfection efficiency of both nanoparticles using a plasmid encoding tdTomato fluorescent protein has been first evaluated in-vitro in two cell lines, and therefore the best formulation has been used for in-vivo transfection assays by sub-retinal injections in Long-Evans rats. In vivo transfection efficiencies have been evaluated by direct fluorescence observation in cryosections and flat-mounts.

Results: R9-GFP-His and HNRK nanoparticles induce DNA condensation, producing a retardation in agarose gel electrophoresis. The in-vitro transfection with ptdTomato-NP complexes at different ratios showed lower values compared with the commercial reactive used as a positive control, being the highest NP-DNA ratios the most efficient. In rat retinas, sub-retinal administration transfected cells mainly in the RPE layer.

Conclusions: This study establishes the base for the application of these non-viral transfection systems for future treatments of retinal disorders.