RNAi is a relatively young therapeutic platform and we are rapidly learning about the gene-specific and non-specific effects of RNAi triggers in vivo. Ultimately, the value of RNAi Therapeutics lies in the gene-specific knockdown of therapeutic target genes, but it is clear that non-specific class effects may confound the analysis of pre-clinical and clinical results. This is especially true for a number of the early RNAi Therapeutics candidates that entered the clinic at a time when less was known about issues such as off-targeting, cytokine induction via TLR-7, and now also TLR3.
The study by Kleinman et al. from the University of Kentucky that appeared yesterday in the high-profile journal Nature (Nature doi 1038/Nature 06765), investigated siRNA therapy for wet age-related macular degeneration (AMD). Of note, there are at least 3 RNAi clinical trials ongoing for AMD: a phase III candidate by Opko Health, bevasiranib, involving intravitreal injection of an unmodified siRNA against VEGF; a phase II candidate by Allergan/Merck(Sirna Therapeutics) involving a chemically modified siRNA against VEGF-R1, also intravitreally injected; and last but not least a phase I, likely intravitreally injected “AtuRNAi” compound targeting a novel, non-VEGF pathway gene by Pfizer/Quark Biotech. Kleinman and colleagues showed that pretty much all of the siRNAs they injected, 2’O-methyl modified (Allergan drug) or not (Opko), suppressed laser-induced choroidal neovascularisation (CNV) in mice, a commonly used model for wet AMD, independent of whether their sequence was directed against an angiogenesis gene or not. Furthermore, such siRNAs were not taken up by the cells in the back of the eye, consistent with a lack of target gene knockdown. Through a series of elegant experiments, they showed that this non-specific antiangiogenic effect was mediated by binding of the dsRNA to the TLR3 receptor on the cell-surface of endothelial cells and the subsequent induction of IL-12 and interferon gamma, both of which alone could account for the observed CNV suppression.
At the moment, I cannot explain the discrepancy between these data and studies by the Opko (formerly Acuity) and Sirna Therapeutics groups that showed sequence-specific down-regulation of target-genes and cellular uptake of siRNAs using the same methods employed by the Kentucky group. Be that as it may, since TLR3 receptors are known to bind dsRNA and upregulate the IL-12 cytokine and interferon-gamma and it always amazed me how unformulated siRNAs may so efficiently be taken up in the eye, the conclusions of the studies appear credible. This receptor is different from the cytokine induction potential via TLR-7 which can be abrogated by chemical modifications, particularly 2’O-methyl.
Well, that’s the bad news. But there is also reason to be optimistic. Importantly, the authors showed that by conjugating a VEGF siRNA to cholesterol, the siRNAs were taken up into the cells and were able to knockdown its target gene and reduce CNV, even in mice lacking TLR3. All of this was achieved at the remarkably low amount of only 1ug administered siRNA per eye. This shows that RNAi could still be used very efficiently in a gene-specific manner to address AMD. Interestingly, a cholesterol-conjugated siRNA for VEGF-R1, the target for the Allergan/Merck drug, failed to ameliorate CNV, casting a doubt on the viability of this gene target that’s been relatively little characterized in the context of wet AMD. However, one should add that only one siRNA was tested for this and at the low 1ug dosage.
Cholesterol conjugation for siRNA delivery was first pioneered by Alnylam, and from recent presentations it appears that this is becoming an increasingly important technology, particularly with the elucidation of its uptake pathway in a recent Nature Biotech paper.
The authors further found that TLR induction was dependent on the length of the siRNA. DsRNAs of 21bp or longer induced this response, smaller ones did not. Also, it is very likely that this response to 21bp dsRNAs and longer could be abrogated by chemical modifications. Moreover, it would be interesting to speculate that since TLR3 is a dsRNA-specific binding protein on the cell surface, one may even harness this binding property for siRNA delivery if binding could be separated from TLR3 activation and demonstrated in the article for small dsRNAs.
Finally, as we learn more about these potential non-specific class effects of RNAi triggers (in this case synthetic dsRNAs, not DNA-directed RNAi), strategies can be designed to either avoid them by the judicious design of siRNAs (chemical modifications, length and structure of RNAi trigger) or even harnessed for a synergistic therapeutic effect. Although the number of supplemental figures of this paper (!) would indicate that there had been considerable resistance to the publication of this study, studies like this are extremely valuable in informing future RNAi development programs. This information can also be used to better monitor the safety of RNAi drug candidates already in clinical trials that may very well depend on such non-specific effects for therapeutic efficacy. Even for these drugs, not all hope is lost as firstly it remains to be seen whether and how these mouse studies would translate into the human setting. It is also true that many approved drugs work, but not through their anticipated mechanism of action, and some of the future RNAi drugs may be no exception to this.
4 comments:
If this can be generalized, how to understand Alnylam's RSV program which is using naked sRNA to inhibit virus? Are something still not discovered there for specific viral gene inhibition?
Excellent question. It appears that some mucosa, such as those of the lung and cervix, for some reason take up unformulated siRNA quite well. Alnylam has also shown that fluorescently labeled siRNAs went into the same cells in the mouse lung where RSV replicates. But in any case, there is a reasonable chance that cytokine activation may play a role in the antiviral activity of ALN-RSV01. While the true value of RNAi Therapeutics will be specific gene inhibition, any individual drug will get approved or not based on its efficacy and safety profile, while its mechanism of action may be secondary.
It is good point that it is hard to exclude the role of interferon response played in the inhibition of viral replication in respiratory epithelial cells. As you know, the publication of RNAi on influenza virus originated for Galenea and later licensed to Nastech showed viral inhibition in mice with delivery vehicle (I assume that naked siRNA wasn't working because they used PEI as delivery vehicle). I heard Alnylam started influenza program and later dropped or at least the program is not in company's pipeline. This makes me wonder if siRNA w/o delivery vehicle inhibit RSV, why not influenza virus. Of course, each virus may have different growth and infection characterization which may response to the inhibition differently. But in principle, it should work on other reparatory viruses too, if it works on RSV. The infected epithelial cells may change plasma membrane permeability for siRNA or other substance to enter into cells which may explain the results.
I don't want to comment on the Galenea studies, but note that there is a widespread feeling that the flu mouse models may be tricky to work with.
With regards to ALN-RSV01 it was reported in the latest study that efficacy wasn't correlated with innate immune activation, although one cannot rule out that the specific cytokines looked at, the timing of the analysis, and taking samples from the lung vs blood may have missed something.
This doesn't mean that such a finding would necessarily be a showstopper, but adds to our knowledge of RNAi Therapeutics in man. Unfortunately, it is a sad truth that often it is a mistake to be overly self-critical as the FDA may latch onto it.
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