Abstract
Purpose: :
The aim of the study is to evaluate the use of minicircle technology for the expression of two anti-angiogenic factors, pigment epithelium-derived factor (PEDF) and somatostatin (SST), for non-viral gene therapy of diabetic retinopathy
Methods: :
Three plasmid vectors carrying either PEDF, SST, or green fluorescent protein (GFP) cDNA were produced by insertion into the expression cassette of the pMC.BESPX vector, between the attP and attB sites. The vectors were transformed into the E.coli strain ZYCY10P3S2T and the growth media was supplemented with L-arabinose to induce formation of minicircles. The products, consisting of DNA vectors devoid of the bacterial backbone, were analyzed by restriction digest and were tested for in vitro transfection efficiency using the retinal pigment epithelium cell line ARPE-19 and lipofectamine. GFP expression was evaluated by direct visualization by fluorescence microscopy, and SST and PEDF expression was analyzed by Western blot of cell lysates and conditioned culture media. GFP minicircles were also tested in vivo by subretinal injection with lipofectamine in a rat model. GFP expression was evaluated by direct observation of whole retina under a confocal microscope.
Results: :
Restriction analysis of minicircle induction products showed bands corresponding to the minicircle vectors for each gene, demonstrating the elimination of the plasmid backbone in all the cases compared with the precursor vectors. Transfection studies in the ARPE-19 cell line with the minicircles were positive in all three cases: transfection with GFP minicircles produced green cells, with the same transfection efficiency as that of the conventional plasmid; the cell lysates and the culture supernatants from the ARPE-19 cells transfected with PEDF and SST minicircles, analyzed by Western blot, showed a higher expression of the peptides in both cases compared with the control non-transfected cells. The in vivo transfection study of GFP minicircles in the rat model, analyzed at 72h post-transfection, showed significant expression of green fluorescent protein in the transfected retinas compared with the control eye, where there was no fluorescence.
Conclusions: :
Our data suggest that minicircle vectors are capable of expressing high and persistent levels of therapeutic product in vitro and in vivo in the retina and have a great potential to serve as episomal vectors for the treatment of diabetic retinopathy.
Keywords: diabetic retinopathy • gene transfer/gene therapy • retinal neovascularization