All siRNAs, including two that are currently in clinical development as treatments for wet age-related macular degeneration, suppress neovascularization regardless of their sequences or targets due to the activation of a cellular immune response, according to a paper appearing in this week’s Nature.
In the study, researchers from the University of Kentucky reported that any dsRNA at least 21 nucleotides in length activated toll-like receptor 3, which in turn suppressed angiogenesis, in the retinas of a mouse in which a laser injury triggered choroidal neovascularization.
Among those siRNAs tested by the investigators were ones targeting the same sequences as the AMD drug candidates bevasiranib, which targets vascular endothelial growth factor and is being developed by Opko Health, and AGN211745 (formerly Sirna-027), which targets VEGF receptor-1 and was licensed by Allergan from Sirna Therapeutics (see RNAi News, 6/28/2007 and 10/7/2005).
Though the paper raises questions about the mechanism by which these two RNAi drugs appear to be working, at least two industry insiders say it mostly reinforces what is already known: that RNA-based drugs have the potential to trigger an unintended immune responses.
But Sam Reich, executive vice president of ophthalmologics at Opko, charged that the paper’s findings, at least in regards to bevasiranib, are simply inaccurate and could be the result of the authors’ use of a mouse model known to yield “variable outcomes.”
“We respectfully disagree with the conclusions [the authors] have drawn,” he told RNAi News this week. “We stand by our data [that] shows that our molecule mediates VEGF silencing, which is anti-angiogenic, using the appropriate controls.”
‘Practiced in the Art’
“Anyone who is practiced in the art is not surprised that toll-like receptors are involved in nucleic acid recognition and downstream signaling leading to interferon production, et cetera,” Alan Sachs, vice president of RNA therapeutics at Merck Research Laboratories, told RNAi News this week. “In many ways, [the paper] confirms what we [at Merck] believe: that heavily chemically modified RNA is going to be a differentiation that will be needed to play in the marketplace.”
Another researcher, City of Hope’s John Rossi, said that In addition to “smart use” of chemical modifications, the use of delivery vehicles for siRNA drugs will likely be key to overcoming the kind of effect described in the Nature paper.
Neither Sachs or Rossi participated in the study. However, Rossi reviewed the paper for Nature prior to its publication.
Jayakrishna Ambati, a University of Kentucky professor and senior author of the Nature paper, agreed. “If one wants to realize the real sequence-specific, gene-silencing potential of these molecules, [the activation of toll-like receptors] has to be taken into account and somehow perturbed either through siRNA modification or some other mechanism,” he told RNAi News.
The paper “doesn’t detract from how powerful RNAi is. It just tells people that when you are doing these experiments, you’ve got to consider the innate immune response as something that is very critical for the interpretation of your data and results.”
Overall, the paper “doesn’t detract from how powerful RNAi is,” Rossi noted. “It just tells people that when you are doing these experiments, you’ve got to consider the innate immune response as something that is very critical for the interpretation of your data and results.
“We go marching along thinking that [the drugs in] all of these clinical trials that are taking place are working by the mechanism that you think they are,” he added. “Here’s a clear case where they’re not, at least in the mouse model that they used.”
It is, in fact, that mouse model that Opko’s Reich argued may be the source of what he believes are inaccuracies in the data.
Calling the laser injury-induced CNV model “technically difficult,” Reich said that “everyone who works with this model knows that you must take the results with great caution. It is a first model that is tested and followed up by many other models to look for confirmation.”
But Ambati and his colleagues did not provide any such confirmation, he noted. To fully understand the data in the Nature paper requires “confirmation of this model by other independent labs because it is very common for different labs to get different findings … using the laser-induced mouse model of CNV.”
Opko’s data showing the RNAi-mediated silencing of VEGF, meantime, has been validated by the company in multiple models, as well as by independent labs, Reich said.
“We are resting on a comprehensive body of data that goes well beyond the laser-induced model to show the safety and efficacy and VEGF silencing of bevasiranib including an entire [pharmacology and safety data] package that was submitted to [the US Food and Drug Administration], as well as clinical results from three clinical trials supporting the biological effect and showing a signal indicative of efficacy,” he said. “We’ve very confident bevasiranib mediates VEGF silencing.”
A representative from Allergan directed request for comment to Merck, which acquired Sirna about a year ago (see RNAi News, 1/4/2007), although a Merck spokesman told RNAi News that his company could not comment directly on AGN211745 since it had been licensed to another company.
Unraveling a Mechanism
According to Ambati, his discovery of siRNAs’ anti-angiogenic properties began a few years ago when his lab, which focuses on the vascular biology of the eye, began routinely using RNAi as a research tool.
“We found that no matter what siRNA we used, as far as blood vessel growth in the retina was concerned, the result was always the same: that is, angiogenesis was suppressed,” he said. “We were tremendously intrigued by this sequence-independent effect and proceeded to try to unravel the mechanism that were underlying it.”
After enlisting the aid of collaborators to reproduce this phenomenon in order to make sure that they weren’t “seeing some artifact of contamination or other types of non-biological events,” Ambati and his colleagues began scouring the literature for clues.
Their search led them to TLR3, a member of the innate immune system and a known sensor for dsRNA. “It had never been precisely delineated how short a double-stranded RNA could be and still activate TLR3, so that was a natural candidate for us to interrogate,” he said.
Initial tests with untargeted siRNAs revealed that these oligos were activating both TLR3 and downstream signaling cascades, Ambati explained. Importantly, all of the observed anti-angiogenic effects associated with the siRNAs “were lost in mice that didn’t have TLR3 or any of the downstream signaling genes.”
The investigators then began examining siRNAs that targeted the same sequences as bevasiranib and AGN211745 and found that they suppressed choroidal neovascularization only in mice with TLR3. “The so-called targeted siRNAs didn’t have any biological effect … when you looked at TLR3-knockout mice or mice that don’t have the TLR3 signaling cascade,” Ambati said.
To determine whether siRNA-induced anti-angiogenesis occurred in organs other than the eye, the research team experimented in human endothelial cells from the aorta, dermis, lung, and umbilical vein.
They found surface TLR3 on all of the cells and observed a suppression of dermal angiogenesis with 21 nucleotide-long, 2’ O-methyl-modified siRNAs on par with the kind of anti-angiogenic effect seen with a similar siRNA targeting VEGFA. A seven nucleotide-long version of the siRNAs did not have any effect.
“Our results demonstrate that 21-nucleotide or longer dsRNAs are anti-angiogenic … [and that] two investigational siRNAs in clinical trials owe their anti-angiogenic effect in mice not to target knockdown but to TLR3 activation,” the authors wrote in Nature.
Still, there is still potential for these two drugs, they added.
“Our findings support investigations of non-targeted dsRNAs as generic anti-CNV agents as effective as anti-VEGFA antibodies, the current standard of care, while avoiding the potential neurotoxicity resulting from chronic administration of the latter,” they wrote.