By Doug Macron
Following years of excitement over the potential of RNAi, 2010 saw the gene-silencing technology become much more a part of the mainstream, especially as a research tool, according to a number of academic and industry players.
Still, there remains much work to be done on the basic biology side if RNAi is to become a therapeutic modality. Meantime, advances in the microRNA field continue to generate interest amid growing evidence of the role of these molecules in human health.
According to Bill Marshall, president and CEO of Miragen Therapeutics and a scientific founder of Dharmacon, the evolution of RNAi from a newly discovered phenomenon to an accepted technology for research applications continued in earnest during 2010 "in terms of standardizing the systems people use and the way they interpret results.
"There was a period of time where [RNAi] was such a hot, new, exciting thing that people wanted to use it … [but found it] more challenging that what they thought," he told Gene Silencing News. "Because of a lot of people's contributions … it's become much more of a standardized molecular biology tool and people should be a lot more comfortable in its use."
At the same time, RNAi's use as a "broad-ranging, genomics-wide screening tool has also developed nicely" during last year, Marshall said. In the early days of RNAi, the hope was that "the tool would develop from something that was an art form into something that was much more standardly applicable, and I think we are there today."
Despite this widened acceptance of RNAi as a research tool, however, the potential for off-target effects to skew data remains a significant issue, according to Art Krieg, CSO of Pfizer's Research Technology Center.
"I've certainly seen more published reports using RNAi where I'm very skeptical of the data than [ones] where I think, 'This is really solid. They know exactly what they're going and I can really take their conclusions home,'" he said.
Mark Kay, professor and head of human gene therapy at Stanford University, agrees.
"I've seen studies where people who aren't that familiar with RNAi don't do the right controls, and you can get non-specific effects depending on how you use your RNAi [molecule], what lipids you use, and what conditions you get these non-specifics," he told Gene Silencing News. "As a result, sometimes the conclusions that are reached are wrong."
Even researchers familiar with RNAi and its limitations can encounter hurdles not previously seen or appreciated, he noted, but they would be more likely to address these issues.
"If people use the reagents incorrectly or the experiments aren't well controlled for, it can lead to misinformation," Kay said. "It's easy to order [RNAi reagents] now and it's easy to complex [them] with lipids. But as with any experiment, there are lots of variables and sometimes people just don't appreciate those."
Overcoming these problems, which will also help those using the technology to develop therapeutics, will require a greater understanding of the fundamental mechanisms of RNAi, John Rossi, professor and chair of molecular and cellular biology at the City of Hope, said.
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"There are four Argonautes," he said. "What are they all doing? Is there really RNAi going on in the nucleus? Some people don't believe it, some people fully believe it. There are definitely Arognautes in the nucleus, [and] there are definitely small RNAs in the nucleus, [but] what are they doing? Are they regulating chromatin structure?
"There are tons of questions to be asked," he told Gene Silencing News.
And sometimes science has taken a backseat to commercial interests, according to Johannes Fruehauf, vice president of research and development at Aura Biosciences and former vice president of Cequent Pharmaceuticals.
With the excitement surrounding RNAi around the time its discoverers won the Nobel Prize in 2006, "there were many things that were pushed into [preclinical] development that may have benefitted from more basic science research," he said.
This is not to say that there hasn't been "some very nice progress" in areas such as siRNA design, and RISC assembly and function, he said. "But is that sufficient? Certainly not. There will have to be more understanding of the RNAi mechanism."
As RNAi marches forward in the lab and the clinic, miRNAs continue to garner attention with the "continual emergence of the importance of microRNA-mediated gene regulation … in biology, and soon in medicine," Rossi said. "There have been increasing numbers of associations of microRNA over-expression or under-expression that correlate with various diseases. I think this is just getting stronger as time goes on."
Marshall, whose new company develops miRNA-based therapeutics, is optimistic about the therapeutic potential of the small, non-coding RNAs, and expects "we can skirt some of the inherent delivery issues you find in double-stranded triggers with [single-stranded antagonist] approaches. I think that's going to allow us to move more quickly to the clinic."
In addition, the lessons learned in RNAi should help those in the miRNA therapeutics space "because you're hitting the same kind of machinery in the opposite direction," he explained.
"With siRNA triggers, you're trying to load up RISC and get it to work," he said. "Understanding the issues there around off-targeting and all that sort of stuff piggybacks onto some of your considerations around microRNA targeting, how many different related family members there are, how that affects the potential for off-targets, [and] your tissue distribution."
"If we don't really pay attention and use the knowledge that has been developed from a historical precedent, we're re-inventing the wheel," Marshall said.
Nonetheless, repeating the mistakes of the past is not uncommon, and the miRNA field may very well suffer from the same kind of build-up and letdown that both antisense and RNAi experienced, Krieg warned.
"Having been through the ups and downs of antisense, when RNAi came along, it was like deja vu all over again," he said. "Those of us who had been in the antisense field for a long time found the science of RNAi incredibly exciting, but we found some of the investors rushing into the field were not aware of the history of antisense.
"I wouldn't be surprised if there are investors in the microRNA field who don't internalize all of the lessons from RNAi or believe that those lessons are not going to apply to them for one reason or another," he said.
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