Inhibition of gene expression using RNA interference (RNAi) pathway in general uses double stranded RNA (siRNA) and requires cationic lipid formulation for delivery to the desired compartment of the cell and animals. In contrast, RNase H based single stranded antisense oligonucleotides have shown activity in multiple species (including humans) without formulation. Moreover, it has been demonstrated that a single stranded RNA (ssRNA) delivered to cells using cationic lipids has been shown to activate the RNAi pathway.  This suggests that a double stranded structure is not necessary to elicit RNAi. This observation made us think that the dsRNA structure could be simplified to a single stranded oligonucleotide that would activate RNAi in cells and in animals. Our initial mechanistic studies revealed that 5'-phosphate is necessary for ssRNA activity in cell culture. We have done an extensive SAR study of ssRNAs, and coupled this to biochemical studies on the mechanism of activation of the RNA induced silencing complex (RISC). These efforts provided highly potent compounds in cell culture which were shown to function via an argonaute-2 dependent mechanism.  These ssRNAs are active in cell culture without cationic lipids, and this activity translated to activity in animals at pharmacologically relevant doses with subcutaneous administration in saline formulations. These studies provide an outline for further optimization of the ssRNA structure for the potential development of human therapeutic agents.

Chemical approaches for achieving activation of RNA interference with single stranded oligonucleotides in animals

243rd National Meeting (2012)

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