To elevate expression level of endogenous GRP78/BiP protein in human RPE primary culture via recombinant AAV virus vectors. Manipulation of UPR markers, e.g. upregulation of GRP78/BiP protein, could alter the rate of retinal degeneration and provide for the survival of retinal cells. The authors expect these approaches which has been made possible by viral vector-mediated augmentation of gene expression, considered as useful research tools for gene therapy in retinal degenerative disorders.

Human RPE cells were cultured in DMEM/F12 supplemented with 10% FBS. The recombinant AAV (rAAV) virus particles were prepared and purified using HiTrap heparin columns. Suspended cells were infected with rAAV/GRP78 at a multiplicity of infection (MOI) of 1500, according to quantitative real time PCR genomic titration, and plated in 60mm or 100mm culture dishes. Culture medium was changed 24 hours later and every two days. Transduction was assayed 4 or 10 days post-infection, through real time PCR (gene expression transcriptional level) and western blotting (gene expression translational level), respectively.

Real time PCR results showed that GRP78 expression has been elevated 3-5 fold at transcriptional level, and western blotting experiments resulted in 4.8 fold increase at translational level, calculating by Image J analysis software.

More than 80 rhodopsin mutations have been identified that account for 30% of Autosomal Dominant Retinitis Pigmentosa (ADRP) in humans. Since protein folding problems are believed to contribute to the pathogenesis of the disease, gene transfer of UPR markers is an attractive approach for ADRP therapy. Studies have demonstrated that AAV-mediated overexpression of GRP78/BiP makes significant improvement in electroretinogram amplitudes and survival of retinal cells through suppression of the UPR signaling pathway in ADRP animal models. At present study we have shown that expression level of endogenous GRP78/BiP protein in human RPE primary culture has been successfully increased. This would be an initial step toward investigating its effect on cell survival in our cellular model of UPR stress.