Down Regulation of TGFbeta–2 Expression in Rat Retinal Müller Glial Cells by Polyplexes of Polyethyleneimine / Phosphorothioate Antisense Oligodeoxynucleotides

Abstract

Abstract: : Purpose: To identify an optimal formulation of phosphorothioate antisense oligonucleotides (PS–AsODN) combined with polyethyleneimine (PEI) to down regulate TGFbeta–2 expression in rat retinal Müller glial cells in vitro. Methods: Branched PEI was combined to ODN to generate nanoparticular polyplexes. Rat RMG cells were transfected with PEI/PSODN polyplexes using antisense (As) and scramble (Sc) ODN sequences, PEI and naked PS–ODN (As and Sc). Fluorescent ODN transfection was observed by confocal microscopy. Levels of mRNA expression were measured by real time quantitative RT–PCR. Whether the down–regulation of TGFbeta–2 influence RMG cells proliferation was assayed by MTT method. Results: PEI/PS–ODN polyplexes form homogenous nanoparticles that are efficiently internalized in RMG cells. At a concentration below 100nM, the polyplexes did not affect RMG viability. At 72 hours after a stable transfection of RMG cells with PEI/PS–ODN polyplexes, the specific AsODN induces a dose dependant down–regulation of TGFbeta–2 mRNA. 100 nM PEI/PS–AsODN reduced by 61% the level of TGFbeta–2 mRNA when compared to the control cells (P= 0.0026). The down–regulation of TGFbeta–2 was associated to a significant 59 % reduction of RMG cells proliferation at 72 hours (P < 0.001). Conclusions:The present study has demonstrated that PEI/PS–AsODN were able to deliver PS–AsODN into rat RMG cells to induce a specific down–regulation of TGFbeta–2 mRNA expression. Moreover, our results show that this down regulation is correlated with a decrease in RMG cell number.

Keywords: retinal glia • gene transfer/gene therapy • gene/expression