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

In the second part of my review of mdRNA’s and RXi Pharmaceutical’s new trigger designs (part I: specificity of mdRNA's usiRNAs), I will try and somewhat de-mystify RXi’s ‘self-delivering rxRNAs’, the industry’s best-kept secret. First insights into the structure and chemistry of sd-rxRNAs were recently provided at the ARVO meeting in Florida and the publication of a related patent application (International Publication number WO 2010/033247 A2).

Shown above is a typical example of an sd-rxRNA. Sd-rxRNAs are based on a so-called ‘asymmetric siRNA’ structure in which the double-stranded region of the RNAi trigger is relatively small (less than 15bp) whereas the length of the guide strand is maintained around 19-23 nucleotides to maintain silencing efficacy. Although on average these RNAi trigger designs are significantly less potent than traditional Tuschl-type siRNAs, extensive screening in some cases allows for quite potent asymmetric siRNAs to be found with picomolar activities.

The double-stranded region is kept short because it is thought that structurally rigid double-stranded nucleic acids don’t wiggle well through the plasma membrane. Single-strand nucleic acids, however, as practiced in the antisense field can enter the cells more readily possibly because of the increased flexibility and an exposed nucleobase that is relatively less charged.Another benefit of keeping the dsRNA region below 15bp is that it avoids conflicting with competing IP estate such as Alnylam's Kreutzer-Limmer series, although in this case the scientific rationale, as just pointed out, should be strong enough to stand on its own feet. Having said that, I can remember conference presentations by a Korean group that reported on similar asymmetric siRNAs, and there was also a publication on 'asymmetric siRNAs' in Nature Biotech2 years ago, although in that case the dsRNA region was more centrally placed, whereas in the case of RXi’s sd-rxRNAs the shorter passenger strand base pairs with the 5’ end of the guide strand.

The guide strand carries a synthetic 5’ phosphate group. This is probably intended to address the less efficient phosphorylation of highly modified, conjugated siRNAs of unusual structure compared to traditional RNAi triggers that undergo rapid phosphorylation following introduction into the cell, a step that is necessary for activating the silencing potential of an siRNA.

In addition to asymmetric structure, there are a number of chemical bells and whistles that render the RNAi trigger more lipophilic (‘fat-loving’) and therefore membrane permeable. One important strategy here is to replace a number of the phophodiester bonds in the RNA backbone with phosphorothioate linkages, especially in the long 3’ overhang of the guide strand. Phosphorothioate backbones are well known in the oligonucleotide therapeutics field and e.g. widely applied to RNAseH-type antisense molecules and have the property of being 'sticky' and contributing to favorable pharmacologies. It is possible that these phosphorothioylated 3' overhangs may not only enhance the membrane permeability of the molecules, but harness specific oligonucleotide uptake receptors thought to play a role in RNaseH antisense delivery.

Based on my understanding of the effect of phosphorothioylation on RNAi performance, and also evident from a number of datasets in the patent application, such modifications should not be used too extensively as this often will compromise knockdown efficacy (best limited to the 3' overhang). Phosphorothioylation, like the extensive 2’-O-methyl and 2’-F modifications of the nucleotides, has the added benefit of stabilizing sd-rxRNAs which in many applications will be directly exposed to body fluids (note: such modifications may also be used to prevent innate immune stimulation and to enhance guide-strand specificity).

In addition to phosphorothioylation, the conjugation of a lipophilic group such as a cholesterol to the 3’ end of the passenger strand is intended to also enhance membrane permeability and cellular uptake. This, of course, is very similar to the cholesterol-conjugated siRNA delivery approach taken by Alnylam and first published 5 years ago (Soutschek et al., 2005). For the same reason, lipophilic groups may also be added to within the RNAi trigger structure itself.

The real question, of course, is how all this translates to silencing in vivo. Unfortunately, the presentations contained only very little in vivo efficacy data. The abundance of tissue culture experiments, however, indicate that compared to lipid-mediated transfection, much higher amounts of sd-rxRNAs are required to achieve similar silencing (50-1000 fold higher). There is, however, one dataset that shows that ~50mg/kg sd-rxRNAs can knock down a gene expressed in a mouse liver (compare this to the 1000-fold lower dosages required to the latest SNALP liposomes), but further improvements in potencies are required before sd-rxRNAs, without further formulation become therapeutically relevant.

Compared to the 2005 Nature study by Alnylam, I agree that sd-rxRNAs may be a little bit more potent on average. Consequently, the patent application is littered with references about the superiority of sd-rxRNA over Alnylam’s siRNA-cholesterol conjugate technology. This, however, is a little bit unfair in my opinion given that it appears to be Alnylam that was really the innovator in this field and that its conjugate technology should have progressed since the initial publication.

Maybe a little bit more surprising, or then again maybe not, were also regular references about the technical superiority compared to Dharmacon’s Accell self-delivering siRNAs. To be clear, references of technical superiority per se are nothing unusual in the patent literature. It becomes noticeable, however, when such references are quite frequent and apparently politically motivated. As discussed on this blog before, I have speculated that Accell is likely to use lipophilic chemistries, too, not least because there are patent applications by Dharmacon covering lipophilic siRNA conjugates. (e.g. WO/2008/036825). I don’t want to speculate any further here, but RXi Pharmaceutical would probably do well to take potential conflicts of interests serious and take the necessary steps to address them before yet another IP drama erupts.

In summary, sd-rxRNAs are an example of how one would aim to render siRNAs more membrane-permeable without sacrificing too much silencing efficacy. The concept and the means employed may not be that revolutionary, but credit has to be given for extensively testing which structures and chemistries could work. For now, sd-rxRNAs are probably most promising in direct RNAi applications such as for the skin (see recently announced TransDerm collaboration), eye, and lung (by inhalation) where the ability to administer large amounts of unformulated siRNAs to the target organ may outweigh the potency disadvantages compared to traditional siRNAs formulated in polyconjugate or nanoparticle formulations.

Dharmacon’s Accell Delivery Technology Signals Increasing Focus on non-Endosomal siRNA Uptake Strategies

[Update May 8, 2008: Based on a recent patent application by Dharmacon, WO 2008/036825, it appears that Accell siRNAs are in fact very similar in principle to Alnylam's 2004 cholesterol-conjugated siRNA paper in Nature and for which the Australian patent office has now accepted a related patent application (Australian Patent Application No. 2004206255)].

Dharmacon (now part of Thermo Fisher Scientific), the company that rose to prominence as one of the first and trusted siRNA suppliers that understood to maintain their leading position by staying at the cutting-edge of RNAi research, just announced the introduction of Accell siRNAs.

Accell siRNAs may not be just yet another type of modified siRNA, but appear to be inherently cell membrane permeable allowing for straight-forward in vitro gene knockdown in all cell types tested, including the notoriously difficult-to-transfect primary cells, without the need for prior formulation with special transfection reagents. This is in contrast to conventional siRNAs that, due to charge and size, are not cell permeable and are therefore require formulation with delivery reagents such that they may gain access to the cytoplasm (the site of Ago2-mediated RNAi) following endosomal uptake and lysis of that compartment. This can be toxic, either because of the chemistry of the specific formulation or the triggering of cytokine responses by engaging TLR receptors in the endosomes.

It is also clear that although potent gene silencing may be achieved following endosomal uptake, typically only a fraction of the siRNA that had been delivered to the cell is actually active in gene silencing. In fact, the rate-limiting step in vivo in many instances may not be getting the siRNA to the target cell, but getting it into the target cells once in close proximity. In addition to better understanding the endosomal uptake-release mechanism, research into alternative technologies may therefore prove highly rewarding.

Although technical details were not disclosed, it is reasonable to assume that Accell siRNAs have been rendered more cell permeable either by the addition of a small lipophile to the siRNA duplex or by modifying the highly charged siRNA backbone without compromising siRNA silencing activity. Stanley Crooke, the CEO of ISIS, has e.g. noted before that it is the amphiphilic nature of single-stranded antisense nucleic acids facilitates their cellular uptake, so that it is conceivable that Accell siRNAs may have been engineered to be similarly amphiphilic.

The current Accell siRNA technology, however, has some drawbacks in that a special tissue culture media is required and therefore does not allow them to be directly translated to in vivo applications, although formulating them in ways that get them to their target cells should not represent an insurmountable challenge, with small conjugates such as cholesterol being likely candidates. The recommended concentration of 1 micromolar siRNA (about 50-fold higher of what is typically used in vitro) further indicates that the process needs to be optimized before Accell siRNAs will be widely used for large-scale and routine tissue culture experimentation. The immediate market potential should therefore largely come from work with primary cells.

Accell siRNAs reminds us that systemic RNAi delivery is a multi-step process, starting with extravasation of the siRNAs from the blood stream into the tissues to cell attachment and cellular entry, and the potential for improving each step is significant. It is fascinating to see how intensely each of these tasks are being addressed and the many creative solutions coming out of these efforts.