The RNAi field continued to advance during 2008 with a growing number of drugs based on the gene-silencing technology heading to the clinic and the commercialization of microRNA-based diagnostics.
But to a handful of key industry insiders, it is a growing appreciation of therapeutic RNAi’s potential for immune stimulation and the need for rigorous experimentation standards, highlighted by two key publications, that is likely to define the past year.
At the same time, worries persist about how the troubled economy will impact RNAi research in coming years, especially work being conducted at academic institutions, darkening some people’s views of the road ahead.
In late March, a research team led by the University of Kentucky’s Jayakrishna Ambati reported data in Nature showing that all siRNAs suppress neovascularization regardless of their sequences or targets due to the activation of a cellular immune response.
The paper noted that this effect, which was a result of toll-like receptor 3 activation, was observed with two siRNAs currently in the clinic as treatments for wet age-related macular degeneration, namely Opko Health’s phase III candidate bevasiranib and Allergan’s AGN211745, which is in phase II testing (see RNAi News, 3/27/2008).
Although this paper was initially viewed from some corners as an attack on therapeutic RNAi, “in reality, I don’t think [the paper was] a big blow” to the field, Stanford University’s Mark Kay told RNAi News earlier this month. “I just think it teaches us that we have to be careful in how we interpret results and [raises awareness about] the kinds of things we need to really accomplish moving forward.”
In light of the ongoing work with chemical modifications and an increased understanding of innate immune responses, “we can get past these problems,” Kay said. “It’s just an important part of the learning curve.”
That learning curve was shown to be slightly steeper than expected about five months later when investigators from Protiva Biotherapeutics — now Tekmira Pharmaceuticals — reported how previously demonstrated antiviral effects of siRNAs targeting influenza in a mouse model were actually due to immune responses triggered by the duplexes, rather than by the RNAi mechanism (see RNAi News, 9/4/2008).
“The issue we see over and over again in evaluating external opportunities, as well as in reviewing papers, in the area is the hesitancy to report directly on RNA knockdown as a [pharmacodynamic] readout for whatever it is being measured.”
“The potential influence of siRNA-mediated immune responses on key readouts of therapeutic efficacy is a critical consideration when designing and interpreting in vivo RNAi studies,” the team wrote in Human Gene Therapy at the time. “However, surprisingly few of the reported studies have adequately tested or controlled for the potential effects of siRNA-mediated immune stimulation, making the many published claims of therapeutic efficacy a collective liability for the RNAi field that remains to be addressed.”
While these two papers garnered a lot of attention, to University of Massachusetts Medical School investigator Phillip Zamore “they were much ado about nothing … because the problem is with one or two siRNAs,” rather than RNAi itself.
“I’m not sure everyone shares my view of that, but I think we know how to design around innate immune problems,” he added. “I don’t think that there is some terrible hidden problem. Most people are testing their small RNAs in assays that should pretty easily find innate immunity activation.”
John Rossi, a researcher at the City of Hope, agreed.
The two papers don’t “say anything negative about … RNAi as a therapeutic modality, they just point to the fact that you need to carefully look at your data and use the appropriate controls,” he said.
They also show that naked siRNAs aren’t likely to be effective therapeutics, he added. “In my mind, that’s the important message: that [siRNAs] have to be wrapped in something that is going to [keep] them from these toll-like receptors,” he said, noting that the Protiva team has also shown that 2’-O-methyl modifications can be used to evade certain TLR responses.
In the end, the Nature and Human Gene Therapy papers may prove to be positives for the RNAi drugs space, reminding researchers of the need for stricter standards when conducting experiments and interpreting data — something that is often lacking when it comes to RNAi, according to Alan Sachs, vice president of RNA therapeutics at Merck Research Laboratories.
“The issue we see over and over again in evaluating external opportunities, as well as in reviewing papers, in the area is the hesitancy to report directly on RNA knockdown as a [pharmacodynamic] readout for whatever it is being measured,” he told RNAi News. “Instead, what we see is a lot of surrogate endpoints like tumor volume … [or] viral titer.”
While the “simplest way” to demonstrate an RNAi effect is to look directly at gene knockdown, followed by 5’ RACE analysis, “for reasons that I don’t understand, that does not seem to be the required, gold-standard beginning step for any claim on, for example, potency in vivo,” Sachs said.
“I think everyone is coming to terms with the fact that if you don’t have these types of data, you really shouldn’t be getting your papers accepted for publication and you really shouldn’t be considered as a business opportunity,” he added.
Despite the scientific challenges facing those developing RNAi therapeutics, it may be something far more mundane that proves to be the field’s biggest hurdle: money.
To Stanford’s Kay, the current financial crisis has made already limited government funding even harder to obtain, threatening a major source of scientific innovation.
“Basic scientific research, especially government-funded research, is the key to new discoveries that are eventually going to [turn into something] with clinical relevance,” he said. “If this slows down, which it has, there will be long-term repercussions.”
Zamore also expressed concerns about the effect of the economic slowdown on RNAi research, noting that while certain companies may have the financial wherewithal to survive, “there are others that don’t, and it’s worrying.
“There are lots of good people doing RNAi research in lots of companies, and not all of them have the kind of capital that’s required to survive in a climate where people aren’t making investments,” he said. “That’s true of academics, as well. The NIH is our venture capitalist … and they are not making the investments required in bright young people’s labs.
“Young people are having a really hard time getting money, and those are the people who will do most of the really exciting work in the next 15 years,” Zamore cautioned. “If we don’t give them what it takes to flourish, to really thrive, many of them will come away with having learned the lesson that they should do really safe things. That will be a disaster.”
And Still, Delivery
Although it may not have been first in people’s minds, as in past years delivery remains one of the key challenges for the RNAi drugs field.
“I wish I could say it were otherwise … [but] we are without a doubt making incremental progress” when it comes to addressing the delivery issue, Zamore said.
“It’s very much like the situation with laptop computers: every year the processor gets twice as good and the hard disk space gets twice as big … [but] the batteries get 10 percent better,” he said. “What we know about RNAi, the kinds of molecules we can design, our ability to improve the efficacy and control off-target effects, and screen innate immune effects — those things are far outstripping our advances in delivery. This is not to say we’re not making advances in delivery, it’s just at a much more incremental pace.”
Indeed, “there hasn’t been a huge amount happening in the [delivery] area,” Bill Marshall, president and CEO of Miragen Therapeutics and former Dharmacon executive, noted. “The recognition and promise of RNAi therapeutics has helped to ramp up the number of things being evaluated as delivery reagents … [but] I wouldn’t say we’ve seen any home runs out there.”
Nonetheless, “any time you see more and more stuff clicking out is a good sign …that you’re going to find something meaningful in terms of delivery to tissues other than the liver,” he added. “But I think [delivery is] still the big limitation so far.”