Genoplasty in Rd1 Mice in vivo

Abstract

Abstract: : Purpose: Evaluate the genoplasty strategy on the rd1 mice and study the intraretinal penetration and action of specific ODNs designed to correct the rd1 mutation. In rd1 mice, the rapid retinal degeneration results from a C to A point mutation in the rod c-GMP-phosphodiesterase-ß subunit (PDE), creating a stop codon and subsequent truncated protein. Rod photoreceptor death begins at about postnatal day 13 and retinal degeneration is completed by four weeks. A genoplasty strategy, capable of site-specific correction of point mutations has been developed using RNA-DNA hybrid oligonucleotides (ODNs), or more recently single strand DNA ODNs, both in vitro and in vivo. Methods: Intravitreal injections of fluorescent labeled ODNs designed to correct the rd1 mutation in mice was performed and associated or not to a current application on the eye surface. The penetration of labeled ODNs in all nuclear layers of the retina was evaluated at one hour after injection +/- current, by fluorescent microscopy of eye sections. In the purpose to screen the potential of ODNs (specific chimeric, phosphodiester, phosphorothioate and polyU ODNs targeting coding or non coding DNA strand), photoreceptor survival was indirectly evaluated by opsin-immunohistochemistry on whole flat-mounted retinas at PN28 after 1 to 3 treatments. Control animals received injection of the vehicle, scrambled or sense ODNs, and retinas were processed in the same conditions (n=3). Results: Labeled ODNs could be localized in the ganglion cell layer and internal part of the inner nuclear layer, one hour after a single intravitreal injection. When current application was coupled to the injection, ODNs could be clearly localized in the retinal inner nuclear layer and the outer nuclear layer, where most of the nuclei were positively stained. Among all the ODNs tested, only the coding phosphorothioate single strand DNA ODN leads to a significant and reproductible dose-dependent survival of photoreceptors, three weeks after treatment. Control injected animals did not lead to significant survival. Conclusions: Intravitral injection of ODNs requires the addition of a current application to quickly and efficiently penetrate into photoreceptor nuclei. The specific coding phosphorothioate ODN, used in these conditions allows a dose-dependent photoreceptor survival, measured by opsin-immunohistochemistry. Correction at the DNA level remains now to be demonstrated under the defined treatment conditions.

Keywords: gene transfer/gene therapy • retinal degenerations: cell biology • immunohistochemistry