496. AAV-Mediated, Somatic Gene Transfer to RPE Cells in Order To Study the Pathogenesis of Macular Degeneration: Proteomic and Genomic Analysis

Top of pageAbstract The use of AAV-mediated gene therapy appears to be an exciting approach for the treatment of inherited retinal diseases. However, there is at present, minimal information regarding the effect on the genome and proteome of the target cell following rAAV infection. Additional studies are critically needed to determine which genes and proteins are differentially expressed as the result of rAAV infection and as a result of the inclusion of a transgene. Using ARPE-19, a retinal pigment epithelial (RPE) cell line and recombinant AAV2/5 viruses, AAV2/5.EGFP, AAV2/5.EGFP-RO (reverse orientation), AAV2/5.EFEMP1wt and EFEMP1mut, as well as uninfected cells, we have examined the proteomes by 2D differential in-gel electrophoresis (DIGE) and the genomes by RNA transcript profiling. EGFP encodes the enhanced green flourescent protein and infection of RPE cells with this virus allowed the determination of the optimal multiplicity of infection (MOI) which was 1 |[times]| 106. The AAV2/5.EFEMP1wt and AAV2/5 EFEMP1mut encode epidermal growth factor containing fibrillin-like extracellular matrix protein wild-type and mutant R345W, where an arginine codon is substituted for a tryptophan codon. In patients with Malattia Leventinese (ML), an autosomal dominant macular degenerative disease, the EFEMP1 protein has been shown to accumulate within RPE cells and between the RPE and drusen. In vitro studies have shown that the EFEMP1wt protein is secreted while the EFEMP1mut protein is misfolded, poorly secreted and retained within cells. These results suggest that a misfolding and aberrant accumulation of EFEMP protein may underlie drusen formation and other abnormalities in the retinas of patients with ML and AMD. Initial analysis of the DIGE data shows the presence of both downregulated and upregulated proteins. Analysis of the microarray data shows the downregulation of a number of cell cycle regulator, DNA replication, kinase, kinesin-like (motor), and survivin [baculoviral IAP (apoptosis inhibitor protein) repeat-containing 5 and survivin-beta] genes for rAAV infected cells compared with uninfected RPE cells. Interestingly, there appears to be very little or no change in immune response, inflammatory response or signal transduction genes. Additional proteomic studies using DIGE and SILAC (stable isotope labeling with amino acids in cell culture) as well as continuing microarray studies are in progress to provide further understanding of the effect on the proteome and genome of these important gene therapy tools for the treatment of inherited retinal diseases.