After Science named RNA interference its breakthrough of 2002 a little more than a year ago, many eyes turned to the fledgling technology to see if it would live up to the hype. According to a number of those working with RNAi, it has, though often in unexpected ways.
“It’s been an incredible year for RNA interference,” Alnylam Pharmaceuticals president and CEO John Maraganore told RNAi News, noting that work conducted by his company, as well as academics and industry competitors such as Sirna Therapeutics and Acuity Pharmaceuticals, is “pointing to the ability for siRNAs to work in vivo.”
It is perhaps no accident, then, that RNAi showed up for a second year in a row on Science’s breakthrough list (although as a runner up, not the winner, this time around).
“RNAi is starting to mature,” Chris Echeverri, CEO and CSO of Cenix BioScience, told RNAi News. “The black box is starting to be opened up. I’d say this year is the year we started moving beyond the initial hype and really learned to deal with the reality of the data.”
Examples of dealing with the data, Echeverri said, include the establishment of new approaches to creating siRNAs and improved design algorithms, as well as gaining an improved understanding of the RNAi mechanism itself.
Echeverri cited a paper by Phil Zamore and colleagues from the University of Massachusetts Medical School published in Cell, which described the asymmetry of two strands that make up dsRNA and their assembly into RISC, as a “beautiful” example of research this year that has expanded our understanding of the fundamental aspects of RNAi. (See Cell, Oct 17, 2003; 115: 2, 199-208).
Maraganore agreed. “Phil Zamore’s Cell paper was a tour de force of well-done characterization of the basic underlying mechanisms of how sRNAs are recognized by the RISC complex,” he said, adding that a paper by Harvard Medical School researcher Judy Lieberman published in March by Nature Medicine “was [also] an important publication.” (See Nature Medicine, March 2003; 9: 3, 347-351). In that article, Lieberman and colleagues reported that intravenous injection of Fas siRNA specifically reduced Fas mRNA levels and expression of Fas protein in mouse hepatocytes.
For advancing basic RNAi research, Maraganore also cited as groundbreaking work by Rockefeller University’s Thomas Tuschl on the role of microRNAs, and “the fact that our genome contains a significant number of genes that encode for these microRNAs.”
Tuschl himself told RNAi News that “the discovery of new micro-RNA genes and pathways in which they are involved … certainly has been a huge activity this year.”
Advances on the miRNA front stand out as a highlight of 2003 for Echeverri, as well. “The whole side of microRNAs is just wonderful. To find out that [there] does seem to be a whole system of gene regulation that had been essentially missed until now, and that people like Tom Tuschl and others are characterizing — that, from a scientific point of view, is just a pleasure to watch,” he said.
“It certainly feels like the original idea that [RNAi] evolved primarily as a defense mechanism was short-sighted,” Echeverri added. “Now it smells a little bit more like a broader gene regulation system.”
An additional key development is the “descriptions of two classes of small RNAs that occurred in plants and Drosophila, where one was linked to the transcriptional silencing or DNA methylation, and the other one was linked to post-transcriptional cleavage,” Tuschl said.
This work ties into the notion of the broader three-armed phenomenon termed RNA silencing. According to Tuschl, RNA interference is one branch of RNA silencing in which “mRNA degradation [is] initiated by double-stranded RNA.” The other two branches are miRNA-mediated translational regulation and transcriptional regulation, “which must start with modifying the chromatin and then sometimes followed up by methylation,” he said.
“Some organisms only have one arm, some have two arms, some have three arms,” Tuschl said. “It just adds enormous complexity … and it’s only just starting with the microRNAs, the leader in sorting out the natural functions of the RNA silencing processes.”
Tuschl added, however, that thus far there hasn’t been much work “addressing the natural function of RNA silencing, especially not in mammalian systems.”
Beyond the Basics
The year 2003 also saw RNAi becoming well established for use in target validation and gene function analysis, according to Dmitry Samarsky, director of business development at Sequitur. With this has come the sense that viable in vivo applications of the technology are not too far off.
As a result, Samarsky told RNAi News that during the year he also saw an increased interest in the technology by big pharma. “It’s a natural thing. Target validation occurs on two levels: One is in vitro, where you take a cell culture and knock down genes and monitor the effect,” he said.
“In terms of disease model systems … in vivo target validation is a very important step,” he said. “So far, the most powerful tool was the knockout mouse, but this is expensive and very time consuming, and in many cases it can’t be applied.”
If viable in vivo, Samarsky said, RNAi would be extremely useful for target validation and for relieving a major bottleneck in the drug development process. “That’s why pharma is interested in having [RNAi, and] there is a lot of talk there.”
The collaboration that Merck and Alnylam initiated this September certainly indicates that big pharma’s interest is piqued by RNA interference, and this appears to have given the investment community an added shot of confidence in the potential of the technology to succeed.
“We shouldn’t forget the fin- ancing environment out there,” Maraganore said. Despite a promising performance in 2003, the investment market for drug discovery tools and biotech still remains far from the highs experienced in the boom years of the late 1990’s and 2000. However, RNAi continues to be well received by investors, despite its newness.
“The continued enthusiasm from venture investors in this space has really been very encouraging,” he said. As just two examples, Alnylam raised about $35 million and Sirna pulled in about $53 million in financing in 2003.
Not a Perfect Picture
Despite RNAi’s successes in 2003, reviews of the technology’s progress were not uniformly positive.
In Samarsky’s view, the performance of academia in the RNAi space during 2003 was disappointing. He said that he is surprised so many questions about the fundamentals of RNAi remain despite the level of talent and effort found in academic institutions.
As an example, he cited the recent Cell paper from Phil Zamore. “It certainly was a wonderful discovery, but I would [expect] it to be made earlier and that by now [we’d] know the mechanism of the entire thing,” Samarsky said.
Samarsky added that he feels academia has been “looking at RNAi more like a source of income rather than a curious natural phenomenon. There are cases when academic people are trying to commercialize [technology] — that’s a little weird to me,” he said.
On the other hand, researchers from the biotech industry are seemingly picking up the slack, Samarsky said, citing the discovery of strand asymmetry by researchers from Amgen and Dharmacon independ- ently from, but concurrent with, Zamore’s group. The findings of the two companies’ researchers were published in the same issue of Cell as the paper from Zamore et al. (See Cell, Oct. 17, 2003; 115: 2, 209-216).
“That’s industry asking basic things,” Samarsky said. Important work on off-target effects of RNAi also came out from industry sources this year, namely Abbott Laboratories and Merck’s Rosetta Inpharmatics, he noted. (See Proceedings of the National Academy of Sciences, May 27, 2003; 100: 11, 6347-6352 and Nature Biotechnology, June 2003; 21: 6, 635-637, respectively).
For John Rossi, of the Beckman Research Institute of the City of Hope Cancer Center, the work by the teams at Rosetta and Abbott/Dharmacon, and the growing awareness of RNAi’s potential non-specific effects, in fact, stand out as the defining aspects of RNAi in 2003.
“The thing that was most striking to me was the alarm signals that went up,” he told RNAi News. “Immediately, there was concern about off-targeting effects, and that continues to be a concern. [There was also] concern about interferon induction.”
And while these are indeed roadblocks to the development of RNAi, especially in the therapeutics area, their discovery and ongoing elucidation should be considered an advance.
“It really boils down to gaining a better knowledge of the mechanisms, understanding the rules that apply biologically to this phenomenon,” Rossi said. “We’re understanding more about how the specificity of the silencing effect is determined,” Echeverri added. “Yes, we are finding some caveats, but that’s fine — we need to find those things and we need to be able to know what the caveats are so we can design around them.”