Next-Generation RNAi

Listening to a webcast by Rosetta Genomics last weekend, I noticed their concerted effort to brand themselves as the Next-Generation RNAi company. Although this is a misnomer as their efforts are really centred around microRNAs, and is likely driven by a desire to get the attention of Wall Street, it made me reflect on what I expect from the next generation of RNAi drugs, specifically the design of RNAi triggers.

Ideally, the next development cycle will yield siRNAs with higher specificity and potency. This should allow for the use of lower amounts of drugs in the clinic for obvious reasons of safety, but also cost. At the moment, algorithms can pretty well predict siRNA sequences that will give a decent knockdown in tissue culture experiments in the low nanomolar range. However, once in a while, we stumble across those “super-silencers” that have IC50s in the mid-to-low picomolar range, yet we do not understand what makes them so good.

I expect that the intense study of the RNAi-related pathways in both model organisms and human cells will ultimately explain their behaviour and reveal rules for designing better and better siRNAs. Exemplary are recent studies by the Zamore group in the fruit fly system that showed that small RNAs are partitioned into separate RNAi effector complexes based on their structure as double-stranded precursors prior to loading into the activated RNAi effector complex. Similar to flies and most other multicellular eukaryotes, there are also a number of related RNAi effector complexes in human cells. It is, however, still unclear how much they differ from each other or what their functional overlap is. It is therefore intriguing to speculate that it were possible, similar to what has just been demonstrated for flies, to introduce small RNAs that would specifically harness the RNAi-cleavage pathway, while remaining invisible to the complexes responsible for the non-cleavage silencing pathways. This is because the microRNA-like non-cleavage pathways are responsible for most RNAi off-target effects, and it would further minimise competition with the endogenous microRNA pathway.

The use of different RNAi triggers (PolII::sh-miR; PolIII::shRNA; Dicer-substrate; Tuschl siRNA; 3-stranded siRNA) or, possibly even more exciting from a drug development perspective, chemical modifications and structural variations to the siRNAs may allow us to introduce the desired bias into which effector complex the small RNA will be incorporated. Along these lines, Dharmacon reported not long ago the use of chemical modification at the 2nd nucleotide position of the guide RNA that would still allow for on-target cleavage activity, but almost eliminated microRNA-like off-target silencing by the siRNA in tissue culture. Although a recent abstract by Alnylam scientists for the Annual Meeting of the Society for Neuroscience suggests that this particular modification may not always be neutral to on-target activity, a combination of chemical modification guided by a deepening understanding of RNAi pathways in humans should yield next-generation RNAi molecules with higher clinical success rates.