Purchase this article with an account.
L. Libertine, S. Noorwez, R. Tzekov, M. Byrne, Y. Wang, J. Cardia, P. Pavco, S. Kaushal; Novel Self-Delivering RNAi Compounds (sd-rxRNATM) Demonstrate Robust Ocular Cell Uptake in vitro and in vivo. Invest. Ophthalmol. Vis. Sci. 2010;51(13):3097.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
To evaluate retinal cell uptake of sd-rxRNATM in vivo after direct intravitreal or subretinal administration. It is well known that the introduction of small interfering RNAs (siRNAs) into cells results in potent and specific gene silencing by RNA interference (RNAi). While siRNA-based drugs represent a new, potentially significant therapeutic paradigm, effective application to ocular disease has been impeded by lack of non-toxic delivery systems. RXi has recently developed a new class of chemically modified RNAi compound (sd-rxRNATM) that spontaneously enter cells without requiring a delivery vehicle.
Cultured human retinal pigment epithelial cells (ARPE-19) were treated with fluorescently-labeled sd-rxRNATM (Fl-sd-rxRNATM) in the absence of any transfection reagent and target mRNA silencing was quantified using the QuantiGene b-DNA assay (Panomics) at 24 and 48 hours. For in vivo studies, Fl-sd-rxRNATM was administered using intravitreal or subretinal injection to 8-week old C57BL/6 mice. PBS was injected in the contralateral eye as a control. Fluorescence in cells and fixed ocular tissue was detected using laser scanning confocal microscopy.
Treatment of ARPE cells in vitro with Fl-sd-rxRNATM results in significant reduction of the targeted mRNA compared to untreated cells or treatment with a non-targeting Fl-sd-rxRNATMcontrol (>65%, P<0.0001). Robust cellular internalization of Fl-sd-rxRNATM is observed in direct contrast to the minimal uptake of a more standard siRNA lacking some chemical modifications present on sd-rxRNATM. When administered to mouse eyes via either injection route, retinal uptake of Fl-sd-rxRNATM was observed in treated eyes and absent in contralateral eyes. For example, 3 hours after intravitreal dosing of Fl-sd-rxRNATM in mice, fluorescence was present within the ganglion cell layer with full retina and RPE penetration observed at 24 hours.
These data demonstrate effective uptake of sd-rxRNA in retinal cells in vitro and in vivo and establish the potential value of sd-rxRNAsTM for the treatment of ocular disease.
This PDF is available to Subscribers Only