mdRNA and RXi Coming Out with their RNAi Triggers (Part I)

In conference presentations this week, both mdRNA and RXi Pharmaceuticals finally lifted the veil of mystery shrouding the nature and performance of their claimed proprietary RNAi trigger designs. Independent of what they may mean in terms of IP, they are certainly worthy contributions to the science of RNAi triggers: the use of unlocked nucleic acids to increase the specificity of RNAi gene silencing (mdRNA), and the convergence of delivery and RNAi triggers through nucleic acid chemistry (RXi).

At the ‘RNAi and miRNA World Congress’, mdRNA presented data illustrating how selectively spiking siRNAs of the traditional Tuschl structure with a number of unlocked nucleic acid residues (usiRNAs) can reduce the level of off-targeting commonly provoked by ‘naked’ siRNAs. While similar strategies have been reported before and are broadly attempted in the industry, what differentiates mdRNA’s approach is that this particular nucleic acid analogue does not involve the addition of modifying chemical groups. Instead, in an unlocked nucleic acid the ribose ring is simply broken without making the residue any bulkier, but this is enough to significantly affect the interactions of the siRNA with its target mRNA and the Argonaute protein, the effector protein of the RNAi silencing pathway.

The simplest, and therefore widely practiced first step in reducing off-targeting is to modify the 5’ end of the passenger strand with a variety of chemistries. Because the 5’ end is important for the Argonaute protein to recognize and bind an RNA as the guide strand, half the off-targeting can be eliminated based on the sensitivity of this interaction to chemical alterations. It so happens that a UNA at the 5’ end of the passenger strand is similarly not tolerated…as has in effect recently also been shown in a publication by Sirna Therapeutics/Merck (Kenski et al., 2010). In the example provided by mdRNA at the conference, this meant a reduction in the number of genes that were changed in their expression by a factor of more than 2-fold (generally 2-3 fold) from ~390 to ~ 180, while on-target gene silencing activity was maintained at around 95% (data from RNA microarray experiments).

I should add, however, that additional usiRNA residues in the 3’ overhangs of both passenger and guide strands somewhat complicate determining the exact contribution of the passenger strand 5’ modification in reducing off-targeting. This is because it is known from structural studies that the position of the 3’ end of the guide strand relative to the Argonaute protein changes upon the transition from the microRNA-off-target (in the so called ‘PAZ pocket’) to the on-target cleavage (dislodged from the ‘PAZ pocket’) confirmations. 3’ ends that do not fit into the PAZ pocket may therefore change the ease with which the enzyme adopts either conformation and therefore affect the propensity of microRNA-like off-targeting. Structural studies with UNA residues at the 3’ end may be instructive here.

More interesting is mdRNA’s observation that when, on top of the above modifications, one normal base is replaced with its corresponding UNA analogue in the seed (= the first 2-8 nucleotides), the off-target activity is further reduced to about 35 genes that are changed by at least 2-fold, while again maintaining on-target efficacy. This was theorized to be due to the changes in guide RNA-target mRNA hybridization energies within the seed: the usiRNA lowered this energy sufficiently such that a microRNA-like interaction that is reliant on the seed becomes prohibitively weakened. However, the extended length of the guide RNA-target mRNA base-pairing compensates for the lost energy thus still allowing for Argonaute conformational change and on-target cleavage to ensue.

Previously described modifications have similarly been thought to function in reducing microRNA-like off-targeting by modulating seed energies. usiRNAs, however, could be a particularly attractive approach, because its small size should make it less likely to be detrimental to on-target activity. This, of course, would have to be shown with more examples, preferably in a peer-reviewed publication, and the Sirna Therapeutics paper has shown at least some limitations in where the UNA can be placed in the seed. Similarly, it remains to be demonstrated how much of the reduction in the off-target activity was due to eliminating microRNA-like interactions instead of a reduction in the innate immunostimulatory potential of these siRNAs as UNAs were also shown by mdRNA to mitigate such responses.

I believe this story shows how RNAi trigger design has matured to a point that RNAi Therapeutics truly lives up to its original promise of specificity which had temporarily (2003-6 period) been tarnished by the discovery of wide-spread microRNA-like off-targeting and the innate immunostimulatory potential of exogenously delivered synthetic siRNAs. It also demonstrates that at least on the level of RNAi trigger design mdRNA could certainly be a valuable partner to most in Big Pharma.

In my next entry (link here), I will provide my thoughts about RXi Pharmaceutical’s ‘self-delivering rxRNAs’ for which, until now to the best of my knowledge, the company has created so much expectations without actually disclosing the chemical nature thereof.