Novel hydrophobically modified asymmetric RNAi compounds (sd-rxRNA) demonstrate robust efficacy in the eye.

PURPOSE: The major challenges of developing an RNAi therapeutic include efficient delivery to and entry into the cell type of interest. Conventional ("naked" and chemically stabilized) small interfering RNAs (siRNAs) have been used in the eye in the past but they demonstrated limited clinical efficacy. Here we investigated a recently developed class of small, hydrophobic, asymmetric RNAi compounds. These compounds, termed "self-delivering rxRNAs" (sd-rxRNA(®)), are extensively modified, have a small duplex region of <15 base pairs, contain a fully phosphorothioated single-stranded tail, and readily enter cells and tissues without the requirement for a delivery vehicle. METHODS: We compared sd-rxRNA compounds with stabilized siRNAs in vitro (in ARPE-19 cells) and in vivo (intravitreal injection in mouse and rabbit eyes). Specifically, we investigated the retinal uptake, distribution, efficacy, and preliminary safety of sd-rxRNAs. RESULTS: Treatment with sd-rxRNAs resulted in uniform cellular uptake and full retina penetration in both animal models while no detectable cellular uptake was observed with stabilized siRNAs either in vitro or in vivo. Further, both in vitro and in vivo delivery (without any transfection reagent or formulation) resulted in a significant reduction of the targeted mRNA levels, which lasted 14-21 days in vivo. Retinal morphology and function were unaltered following a single administration of sd-rxRNAs. CONCLUSION: These data support the potential of developing sd-rxRNAs as a therapeutic for ocular disease.

Potent and systematic RNAi mediated silencing with single oligonucleotide compounds.

RNA interference (RNAi) has been established as an important tool for functional genomics studies and has great promise as a therapeutic intervention for human diseases. In mammalian cells, RNAi is conventionally induced by 19-27-bp RNA duplexes generated by hybridization of two complementary oligonucleotide strands (oligos). Here we describe a novel class of RNAi molecules composed of a single 25-28-nucleotide (nt) oligo. The oligo has a 16-nt mRNA targeting region, followed by an additional 8-10 nt to enable self-dimerization into a partially complementary duplex. Analysis of numerous diverse structures demonstrates that molecules composed of two short helices separated by a loop can efficiently enter and activate the RNA-induced silencing complex (RISC). This finding enables the design of highly effective single-oligo compounds for any mRNA target.