Thursday, March 27, 2008
Journal Club: Study Shows TLR3 Induction by siRNAs with Anti-angiogenic Effects, Questioning Ongoing RNAi Clinical Trials for Wet AMD
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.
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