ORIGINAL ARTICLE Novel Hydrophobically Modified Asymmetric RNAi Compounds (sd-rxRNA) Demonstrate Robust Efficacy in the Eye Michael Byrne, 1, * Radouil Tzekov, 2, * ,{ Yi Wang, 2 Amanda Rodgers, 1 James Cardia, 1 Glenna Ford, 1 Katherine Holton, 1 Lakshmipathi Pandarinathan, 1 Jennifer Lapierre, 1 William Stanney, 1 Karen Bulock, 1 Sharon Shaw, 1 Lyn Libertine, 1 Kevin Fettes, 1 Anastasia Khvorova, 1,{ Shalesh Kaushal, 2 and Pamela Pavco 1 Abstract 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 Ò ), are extensively modified, have a small duplex region of < 15 base pairs, contain a fully phosphor- othioated 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. Introduction R NA interference (RNAi) is a highly conserved fun- damental cellular process for silencing genes that can be harnessed for the discovery and development of new drugs. 1,2 RNAi can be initiated by the introduction of small interfering RNAs (siRNAs) into cells, followed by entry of one of the strands of the duplex (the guide strand) into the RNA-induced silencing complex (RISC). Once loaded with the guide strand, RISC finds and cleaves a complementary target mRNA, resulting in a truncated mRNA molecule un- able to be translated into a fully functional protein product. 3 The potential for RNAi-based therapeutics is in the ability to generate RNA compounds that target and silence disease- causing genes. The advantages over small-molecule drugs include the ability to target genes previously considered ‘‘undruggable’’ as well as a promise of a long duration of effect based on a unique mechanism of action. While RNAi- based drugs represent a new and potentially significant therapeutic paradigm, demonstration of clinical utility has been hampered by poor tissue and cellular delivery in vivo. 4 Antisense oligonucleotides (ASOs), another class of oli- gonucleotide therapeutic, have demonstrated robust pre- clinical and clinical efficacy that has recently resulted in an approval by the FDA (ie, Mipomersen). 5–7 The favorable pharmacokinetic (PK) properties of ASOs are believed to result from the ability of a fully phosphorothioated single- stranded oligonucleotide to bind proteins, enter cells and tissues and, in combination with stabilizing chemical modi- fications, be protected from both endo- and exonucleases. 1 RXi Pharmaceuticals, Corp., Westborough, Massachusetts. 2 Department of Ophthalmology, University of Massachusetts Medical School, Worcester, Massachusetts. { Current affiliation: The Roskamp Institute, Sarasota, Florida. { Current affiliation: RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, Massachusetts. *These authors contributed equally to this work. JOURNAL OF OCULAR PHARMACOLOGY AND THERAPEUTICS Volume 00, Number 00, 2013 ª Mary Ann Liebert, Inc. DOI: 10.1089/jop.2013.0148 1