Gene Expression Profiles of Retinal Degeneration Induced by Ribozyme–mediated Knockdown of Rod cGMP Phosphodiesterase –Subunit

Abstract

Abstract: : Purpose: Ribozymes can be used down–regulate both dominant pathogenic genes to affect therapy and normal genes to mimic disease. Here, we employ an AAV vector with a ribozyme to mediate in vivo somatic down–regulation of the γ–subunit of the rod cGMP phosphodiesterase (PDEγ) gene in wild type mice to create retinal degeneration and to profile the gene expression by microarray. Methods: Hammerhead ribozymes were designed to target the PDEγ gene in C57BL/6 mice and packaged into serotype 2 AAV vectors containing a murine rod opsin promoter. Right eyes were subretinally injected with approximately 10exp10 vector genomes and partner left eyes injected with AAV–GFP or PBS. ERGs were recorded at 3, 6 and 9 weeks post–injection. To identify gene expression changes in response to ribozyme treatment, retinal RNA from treated and control eyes were analyzed by Murine Genome U74A Oligonucletoide Genechip® arrays and by the Customized cDNA Arrays (mouse I–GENE array) from the University of Michigan (UM). Results: ERG amplitudes of treated eyes were 30∼90% lower than of partner control eyes at 3∼9 weeks post– vector injection. Histological analysis confirmed this observation. Using oligonucletoide microarrays with approximately 12,000 genes scored, 14 known retinal genes were significantly up–regulated and 7 were significantly down–regulated. Hierarchical cluster analysis showed that this pattern of gene expression in ribozyme–treated eyes clusters with RNA from rd mouse retinas but not with partner control or with wild–type mouse retinas. Using UM cDNA arrays, out of 6500 genes probed, significant changes (>2–fold) relative the untreated eyes occurred in 723 genes at 3 weeks post–injection, 185 genes at 6 weeks post–injection, and 337 genes at 9 weeks post injection. Ribozyme–treated eyes again clustered with rd mouse retinas and not with partner control eyes or with untreated wild type eyes. Conclusions: Two independent types of gene array analyses confirm that Rz–treated eyes have a pattern of gene expression similar to the rd mouse. Microarray analysis of such ribozyme–treated retinas may be a useful tool for identifying co–regulated gene expression changes related to retinal degenerations.

Keywords: gene microarray • gene/expression • retinal degenerations: hereditary