Non-viral siRNA delivery into the mouse retina in vivo

Gene silencing in the retina using RNA interference could open broad possibilities for functional studies of genes in vivo and for therapeutic interventions in eye disorders. Therefore, there is a considerable demand for protocols to deliver siRNA into the vertebrate retina. In this work we explored a possibility to deliver synthetic 21 bp siRNA into the mouse retina after intravitreal application using a non-viral carrier.

Fluorescently labelled synthetic 21 bp siRNA duplex was combined with Transit-TKO transfection reagent and injected intravitreally into adult mice eyes. Eyes cryostat sections and whole mount retinas were prepared 24-48 h post-injection, stained with either Hoechst 33342 (cell nuclei) or immunostained with anti-GFAP antibody (astroglia cells marker). Distribution of fluorescent siRNA signal in the retina was investigated.

Single intravitreal injection of as little as 5 ng of siRNA combined with Transit-TKO transfection reagent by a modified protocol provided robust and non-toxic delivery of the siRNA into the retina. However, siRNA accumulation was predominantly confined to ganglion cells layer as analysed 24 h post-injection. Furthermore, siRNA containing particles were localized along GFAP cytoskeleton of retinal astroglial cells hinting on intracellular localization of the siRNA CONCLUSIONS: In this work we demonstrated that siRNA can be efficiently delivered into the vertebrate retina in vivo with low-toxicity using a non-viral carrier, specifically Transit-TKO transfection reagent. However, the capacity of siRNA delivered by our protocol to induce gene silencing in the retina has to be further evaluated. Our report could raise a closer look on Transit-TKO transfection reagent as a promising siRNA carrier in vivo and be of interest for the researchers and companies who work on development of ocular RNAi techniques.