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In 2004, RNAi Reaches Key Milestones; More Work Left to Be Done in 2005


The field of RNA interference reached innumerable milestones during 2004, but one thing the technology did not do was show up as a Science breakthrough of the year (unlike in 2003 and 2002, when it topped the list).

RNAi’s absence from the magazine’s list was not so much an oversight, however, as a reminder of the technology’s maturation.

With growth comes greater visibility and higher expectations, and RNAi appears in many ways to have lived up to the hype it generated over the past couple of years — although not without hitting a few bumps along the way — based on the comments from a number of researchers and industry players who spoke with RNAi News recently.

Among the most important events for the RNAi industry in 2004 was the initiation of clinical trials with siRNA-based drugs, which came in just before the year ended, Cenix BioScience CEO and CSO Chris Echeverri said.

Last year “was the year that RNAi therapeutics getting into the clinic started becoming a reality,” he told RNAi News. “Even though that’s not my primary interest, I have to admit that a few years from now, looking back, we’ll all note that [2004] is the year [clinical development] started happening.”

Specifically, Acuity Pharmaceuticals announced in early November that it had begun dosing patients with Cand5, an siRNA-based treatment for age-related macular degeneration that is administered directly into the eye (see RNAi News, 11/12/2004). Shortly thereafter, Sirna Therapeutics reported that it had begun a phase I study of its own siRNA drug for AMD (see RNAi News, 11/26/2004).

Douglas Fambrough, a principal at Boston-based Oxford Bioscience Partners (which holds a stake in Sirna), agreed. “The first use in humans … is very much a milestone,” and one made even more important by the quickness with which the technology developed, he said. “I suppose it’s a hurry-up world and these things can never come too soon, but it really is a remarkably short period of time from the first landmark publication on the use of siRNAs in human cells in culture to actually having a product that is being given to a human in a clinical-trial setting.”

That said, Fambrough noted that he considers the start of the clinical trials more of a “visceral” milestone than anything else. “I’m not sure it’s commercially that important at the end of the day, given how long human testing takes. And, the current formulations and specific molecules that are going into humans may not be the first molecules and formulations that are approved … so I’m not sure that it’s going to be the most important commercial milestone.”

Echeverri also pointed out that the clinical testing of directly administered RNAi drugs still represents the pursuit of “the low-hanging fruit — the indications where delivery is not much of a problem.”

But while the start of the phase I trials might not offer much in the way of support for the notion of systemic RNAi-based therapies, Echeverri said, a Nature paper published in November by a number of researchers from Alnylam Pharmaceuticals does.

In that paper, the researchers wrote that aopB was targeted using synthetic siRNAs with partial phosphorothioate backbone and 2’-O-methyl sugar modifications on the sense and antisense strands. The siRNAs were further modified through the conjugation of cholesterol to the 3’ ends of their sense strands using a pyrrolidine linker. The resultant chol-siRNAs showed improved in vivo pharmacokinetic properties after being administered intravenously into rats, but did not impair gene-silencing activity in cell culture, according to the researchers.

Alnylam officials told RNAi News at the time that this was the first demonstration of “in vivo silencing of an endogenous gene using a clinically relevant route of administration with confirmation of RNAi-mediated cleavage of mRNA using 5’ RACE.”

“The paper from Alnylam … with the cholesterol-based delivery stands out in my view as something that provides the first strong indication of a potentially more universal solution for systemic delivery of siRNAs in animals,” Echeverri said.

The Nature paper also stood out in the mind of David Dorris, director of RNAi technologies at Ambion. “I think [the paper] was very important,” he said. Although there were some issues with the research, such as the high doses that were used, these are “irrelevant,” he added.

According to Dorris, “the whole point is not to get every detail right, the whole point is to move the field. Who’s going to look at the original Kary Mullis paper and say, ‘I think your taq-enzyme concentration was 25 percent too high, therefore PCR doesn’t work.’? [The Alnylam paper is] just really nice … in the broad sense,” he said.

The year 2004 also saw a jump in the number of scientists looking to apply RNAi to their research, Dorris told RNAi News. “The number of people using RNAi has just absolutely exploded,” he said. “People in the industry a year ago [would say], ‘Can any more people use this?’ — [the fact is] we have just scratched the surface.

“My prediction is that this time next year, there will be significantly more people using this technology,” Dorris added. “Almost every molecular biology lab has used RNAi, [but] there’re still a lot of cell biology labs, neurobiology labs, and other labs that have not. So the penetration is increasing, and I think the results [people] are getting are almost unbelievable … in that five years ago, you couldn’t even think about doing these kinds of experiments.”

Fambrough noted, however, that the growing adoption of RNAi seen this past year failed to materialize among large pharmaceutical and biotechnology companies.

Early in 2004, Eli Lilly did jump into the RNAi waters through an 18-month deal with Sirna to evaluate siRNAs against undisclosed oncology targets (see RNAi News, 1/30/2004). Additionally, over the summer Merck expanded its relationship with Alnylam, committing itself more fully to the development of RNAi-based drugs (see RNAi News, 7/2/2004). But other than this, established drugmakers have kept their distance from RNAi-based therapeutics.

“I expected more deal activity [by RNAi drug firms] with pharmaceutical companies [in 2004], but [big pharma] took more of the wait-and-see attitude than I expected,” Fambrough said. “I don’t know if it’s going to take the form of ‘The Big Deal,’ or whether it’s going to take the form of a number of companies sticking their toes in the water, but I do think you’re going to see an increased number of pharmaceutical companies [embracing RNAi in 2005].”

Howard Robin, president and CEO of Sirna, expressed a similar sentiment. “I think 2005 will be a year where large pharmaceutical companies and large biotech companies finally understand the value of siRNAs, because they’ll see more and more data coming out,” he said.

“I think there will be a growing interest on the part of large companies to start engaging in siRNA research and development,” Robin told RNAi News. “Everybody’s been a bit cautious simply because the technology is very new. But if you look at the progress that’s been made in 2004 and add to that what I expect to happen in 2005, I think siRNAs will become much more mainstream and much more comfortable for the larger companies.”

A number of people speaking with RNAi News also noted that 2004 saw the first disputes in the high-profile area of RNAi intellectual property.

“Of course, [2004 was] the year … that the patent management started showing a little more aggression,” Echeverri said. “It had been relatively civil until [2004] when … some of the players started playing more aggressively. It was impressive to see that, until now, everything had been settled with licensing agreements … but it’s a bit inevitable that some [IP] will start getting challenged — the honeymoon’s over.”

Last year, Benitec fired the opening salvo in a patent dispute with Nucleonics, GenScript, and Ambion (see RNAi News, 4/2/2004). While the last two companies settled the matter out of court, Nucleonics has continued to fight and challenge the validity of Benitec’s patents in the US and Australia (see RNAi News, 9/10/2004 and 10/8/2004). Additionally, Benitec’s relationship with Promega, which may or may not continue to be the exclusive licensee of Benitec’s RNAi technology, has deteriorated amid an ongoing legal battle over royalty payments (see RNAi News, 7/30/2004 and 11/5/2004).

Despite these legal wranglings, Dmitry Samarsky, who handles business and technology development at Invitrogen, is sanguine about the future. “So far, it doesn’t seem like much is happening, and based on my communication with people, I don’t think people are really willing to start battles on the IP field,” he told RNAi News. “There is still a lot of confusion about who owns what and what will happen … so people at this point are cautious. I don’t think there is a real fighting mood.”

Despite any reservations people in the industry might have about starting up an IP battle, there are bound to be some conflicts, but Fambrough doesn’t anticipate that future ones will take the same form as those involving Benitec.

“I think the IP focus [in the RNAi drugs field] is going to shift … away from who owns RNAi to who owns RNAi against a specific target,” he said. “The field has to move out of RNAi in general to an RNAi therapeutic for a particular target in a particular disease with a particular market size. It has to shift from a mechanism and general promise to the specific opportunity that’s going to make people money in the future.” Once this shift is made, Fambrough said, the question becomes: Who owns the IP related to the use of siRNAs against a specific target?

“There is an extremely analogous relationship between the IP in the antibody space and the IP in the RNAi space, and that’s a prediction in and of itself because none of the IP has been issued in the siRNA space,” he said. “But [IP has] been issued in the antibody space where you can own an antibody against a target, and you can keep others from practicing. My prediction is that that is going to hold [true with] siRNAs.”


Among all the advancements made in the science of RNAi during 2004, those in the field of microRNAs seem to be somewhere on just about everyone’s list of the most significant.

“I would argue that [2004] was the coming-out party for microRNAs, in a way,” Echeverri said. “People who are in the know were already recognizing the importance of these things late [in 2003], but [in 2004] it really became much more mainstream. The full-range of biological processes that apparently involve microRNAs is now becoming more obvious, and the microRNA explosion started” in 2004, he said.

“People have already made great progress in understanding what microRNAs look like, their structure, which portions of the sequence have importance in binding and conferring specificity, etcetera,” Echeverri added. “The big thing now is to figure out which are the genes that get controlled.”

According to John Rossi, professor and chair of molecular biology at the City of Hope’s Beckman Research Institute, “some of the findings with microRNAs were really important,” including Rockefeller University’s Markus Stoffel’s paper in Nature identifying a microRNA regulator of insulin secretion. “This [paper] points to [the fact that we’re seeing] the tip of the iceberg of the roles the small RNAs are playing in normal cellular processes,” he noted.

“Academically, one of the really big, exciting developments [in 2004] was the expansion of the amount of biology surrounding microRNAs and … things that look like siRNAs but don’t meet the definition of a microRNA,” Phillip Zamore, associate professor of biochemistry and molecular pharmacology at the University of Massachusetts Medical School, told RNAi News.

“The two really exciting pieces of work were: a whole series of papers by [Columbia University’s Oliver] Hobert on neuronal asymmetry in C. elegans being controlled by small numbers of microRNAs, which act in complementary ways to turn one set of genes on in left cells and a complementary set of genes on in right cells; [and] also [the insulin secretion] work from Markus Stoffel,” he said. “I think we’re going to see tons of stuff like that in the coming year because there are now lots of tools, many of them very traditional, for simply looking at where microRNAs are made and thinking about how they regulate gene expression.”

MicroRNA research was also a highlight of 2004 to Carl Novina, assistant professor at the Dana-Farber Cancer Institute.

“Our knowledge of microRNA biology has increased — we’ve cloned several more microRNAs and we’re starting to get a better sense of what the microRNA/mRNA pairs are,” he told RNAi News. “We are learning more … about some transcription and regulation of microRNAs, and that’s something that we will be hearing more about in 2005.

“Another important thing we’ve discovered through microRNA profiling is that microRNAs seem to be lost in certain cancers, and so we’re starting to get some better tissue profiles of microRNAs — that happened in 2004 and will increase with this new technology of microRNAs on a chip in 2005,” Novina added. “I think what we’ll begin to see, at least as a sort of beta experiment, [is researchers] looking at microRNA profiles in certain transformed cells to see if there is some concordance between microRNAs [that] are lost or over-expressed [and] certain malignancies.”

But Not Just microRNAs

Looking at key developments in 2004 beyond microRNAs, Zamore pointed to the publication of a Science paper authored by Steve Jacobsen and colleagues at the University of California, Los Angeles, which dealt with the relationship of the RNAi pathway with DNA methylation in plants.

“Of course, everyone knew that double-stranded RNA could direct DNA methylation from the pioneering work of Marjori Matzke, but for me [Jacobsen’s paper] was a real highlight of 2004,” he said.

Another highlight of the past year for Zamore was “the demonstration from Tom Tuschl [at Rockefeller University] [and] Greg Hannon [at Cold Spring Harbor Laboratory], but most importantly Leemor Joshua-Tor [also of Cold Spring Harbor Lab] and David Barford [from Chester Beatty Laboratories], that argonaute proteins are endonucleases if they have the right amino acids at the catalytic site,” he said.

“I don’t know if your average cell biologist not working on the RNAi pathway was that interested, but to people in the community this issue of how do you prove whether or not argonaute proteins are actually Slicer was huge, especially the two crystal structures, [which] finally provided a way out of this problem,” Zamore said. “Purifying the protein to ever-higher levels of purity, from a purely intellectual standpoint, could never answer the question because you can always argue that there’s some trace impurity you can’t detect that is an enzyme. But seeing in three dimensions the remarkable structural identity between RNase H and argonaute in the Piwi domain — that was amazing.”

— DM

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