First there was gene splicing. Next came monoclonal antibodies. And now, RNA interference, or gene silencing, has taken the scientific community by storm. Reportedly, RNA interference (RNAi) technology has revolutionized the practice of molecular biology – and the excitement it has generated continues to mount. By allowing scientists to knock out gene expression selectively – not to mention easily and quickly -- RNAi promises to deliver data on what each and every gene does in just a few short years. Moreover, the technology can be used (at least theoretically) to create new drugs that target disease-causing genes – a goal that’s proved difficult to achieve so far by other methods. (For a detailed discussion of this technology, including its comparison to antisense RNA methods, please refer to the Signals article, “Shoot The Messenger.”)
With all the hoopla surrounding RNA interference – including seminal publications in peer-reviewed journals, conferences dedicated to the topic and scads of coverage in the press -- it seems only natural that entrepreneurial scientists would be founding new companies right and left to capitalize on the technology’s use in drug discovery and/or development. And, given the near-euphoric state that RNAi’s promises induce in so many individuals, you’d predict that these fledgling firms would be magnets for venture capital. Yet, in the last year and a half, surprisingly few gene silencing outfits have actually emerged from stealth mode -- although there could be a much larger number of start-ups still under wraps. When they finally debut, however, they’ll be faced with plenty of competition – from huge pharma companies like Abbott Laboratories to major biotech firms like Genentech Inc.; from already established, dedicated players like Benitec Ltd., Intradigm Corp. and Sequitur Inc. to newly refocused biotech companies like Sirna Therapeutics Inc. and CytRx Corp. And that’s just for starters, for many firms are joining in this great adventure. Of course, they’re also staking out their intellectual property claims – a situation that could spell trouble down the road as RNA interference patent-holders wrangle with interference suits and counter-suits.


There are a dozen or so fairly new RNAi firms that have sprung to life in recent years. Many of these -- including Dharmacon Inc. of Lafayette, CO, Benitec of St. Lucia, Australia and Mirus Corp. of Madison, WI -- are primarily focused on developing the tools and reagents that researchers need to carry out their experiments. The others – including Sequitur of Natick, MA, Cenix Bioscience GmbH of Dresden, Germany, Ribopharma AG of Kulmbach, Germany and Alnylam Pharmaceuticals Inc. of Cambridge, MA – intend to develop RNAi-based therapeutics.

Of them all, only four companies have raised venture capital since January 2002 – but when Alnylam Pharmaceuticals came out of stealth mode in July 2002 with $15 million in venture backing and an all-star roster of founding fathers, it caught everyone’s attention. Formed in 2002 by Nobel Laureate Phillip Sharp, National Academy of Sciences member Paul Schimmel and three academic pioneers in RNAi research – Dave Bartel of MIT, Tom Tuschl of Rockefeller University (and formerly of the Max Planck Institute) and Phil Zamore of the University of Massachusetts Medical School – Alnylam appeared to hold the keys that could unlock the therapeutic potential of RNA interference. And, in February this year, the company took the necessary steps to secure its position in the burgeoning field by licensing fundamental founder patents from Max Planck, MIT, UMass Medical School (UMMS) and The Whitehead Institute. Basically, these are “broad, fundamental patents issued by the U.S. PTO and the EPO covering the use of siRNAs as therapeutics,” according to Alnylam’s president and CEO John Maraganore.


But, even though RNA interference technology is full of promise, researchers still have a long way to go to turn it into a bona fide method for creating drugs. In order to silence a particular gene, scientists first construct a double stranded RNA (dsRNA) molecule that matches the gene sequence of interest and then introduce it into the target cell (or organism) where it is digested into small interfering RNAs (siRNAs, 19-21 base pair duplexes). These siRNAs then bind to a nuclease complex to form a silencing complex, which targets the homologous transcript by base pairing and cleaves the messenger RNA. In mammalian cells, however, introducing long dsRNA initiates an antiviral response, so instead scientists introduce – or cause the production of –shorter siRNAs.

It sounds straightforward enough, but significant challenges remain. “It’s not going to be a slam dunk,” Maraganore explained. “If we can solve the key hurdles we may be able to produce a new broad class of therapeutics, the first since [recombinant] proteins and monoclonals.”

“The number one hurdle for making siRNA drugs is delivery,” he continued. “We need to optimize the ability of 21 nucleotide, dsRNA to get into the cell’s cytoplasm.” Luckily, researchers do have some guidelines to follow: “We can take advantage of 15 years’ work in antisense, as well as the use of plasmid DNA in non-viral vector gene therapy.” In those cases, the putative therapeutics had to be delivered into the cell’s nucleus. But, since siRNAs need only to reach the cytoplasm to do their work, “it’s a more tractable problem.”

The other hurdles, Maraganore said, are “relatively straightforward.” First of all, “we have to avoid toxicity.” Again, scientists can take lessons from earlier work in antisense technology. “The chemistries used in antisense had side effects,” including activation of both innate immunity and the complement cascade. Moreover, “We have to optimize specificity and potency,” challenges that can be addressed “largely through informatics,” he added. “RNAi is already a highly specific and selective mechanism, but we clearly need to make sure we explore [its effects] genome-wide” to ascertain that only the target RNA has been silenced.

And the last hurdle? To put together the right partnerships, Maraganore said. “There’s lots of interest in this technology by a group of pharma companies that clearly see the potential and have the courage and interest to take this on.” Alnylam hasn’t got a collaborative partner yet, but the company could be announcing its first deal any day now.


Another new entrant in the RNAi race – Los Angeles-based CytRx -- has already lined up a collaboration that will prove extremely important to the firm’s ability to become a major player in this field. And it’s licensed some key patents from UMass Medical School to establish a proprietary position. This is a radical shift in direction for the firm, which was founded in 1985 to develop therapeutics based on copolymer technology.

The transition began in July 2002, when CytRx – then based in Atlanta – merged with Global Genomics Capital Inc. of LA and the latter’s chairman and founder Steven Kriegsman took over. He shifted the company’s focus away from internal R&D to licensing and alliances to develop its products Flocor (an intravenous agent for treating sickle cell disease) and TranzFect, a delivery technology for DNA-based vaccines.

Kriegsman also gathered an all-star scientific advisory board to help set a new direction, and by April this year, CytRx had signed a number of exclusive license agreements with UMMS to develop RNAi-based drug candidates for treating obesity, diabetes and cancer. In the process, UMMS became the company’s second largest stockholder. Moreover, CytRx will be working with UMMC researchers in a sponsored drug development program focused on obesity and diabetes and led by Michael Czech, professor and chair of molecular medicine at UMMC. The broad alliance, plus the sponsored research program, marked a turning point for CytRx.

CytRx’s Kreigsman said that he’d been aware of the “RNAi explosion” for quite some time, and once he’d taken over CytRx and “cleaned it out,” he met with advisors and concluded that RNAi research was the place to be. And, since one of his advisors was already familiar with the RNAi program at UMMS, the connection was a natural. “We want to be ahead of the curve rather than behind the 8 ball,” he explained. And, he added, CytRx has already identified drug targets in major markets.

So has Sirna Therapeutics, which has programs in hepatitis C virus and macular degeneration, both of which are in the lead identification stage of development. Earlier this year, Ribozyme Pharmaceuticals Inc. transformed itself from a ribozyme-based firm to one focused on RNA interference-based drug development. The Boulder, CO company changed its name to Sirna Therapeutics and raised $48 million in venture capital to effect the shift. But in this case, the transformation wasn’t truly radical, since the firm had already developed a strong base in nucleic acid technology, backed by a decade of research into RNA chemistry and pharmacology. Sirna also has filed about 50 patents in RNAi covering various aspects of the technology, including chemically stabilized RNAi constructs, methods of synthesizing RNAi constructs, application of RNAi to specific therapeutic targets, and for target discovery.

While Sirna has built its substantial portfolio from within, so to speak, other RNAi players have in-licensed technology from universities or research institutions. As mentioned earlier, both Alnylam and CytRx have licensed RNAi patents from UMMC. CytRx’s are based on work performed by Craig Mello, who, in collaboration with Andrew Fire of the Carnegie Institution of Washington, is credited with discovering the gene-silencing properties of dsRNA. (Last month, CytRx also licensed the exclusive rights to a DNA-based HIV vaccine from UMMS.)

Alnylam’s UMMC connection is Phil Zamore, who was a co-inventor on the Sharp patent, which relates to the use of siRNA in mammalian cells, Maraganore said. “UMass has a very broad portfolio of patents that relate to RNAi,” he explained. For instance, many are for use in gene therapy, while others apply to plants. And while Alnylam gets exclusive use of the Sharp patent (together with the Tuschl patent) in the therapeutics arena, Dharmacon, Proligo LLC, Ambion Inc. and Qiagen N.V. have the rights for developing reagents, he said. “UMass is looking to aggressively find partners for much of that portfolio.”

Plenty of other companies are jumping on the RNAi bandwagon, too. For instance, OSI Pharmaceuticals Inc. signed a research agreement with Cold Spring Harbor Laboratory early this year to use the latter’s RNAi technology platform in its cancer drug discovery programs. And in early May, Acuity Pharmaceuticals, which was formed in late 2002 to develop siRNA therapeutics for serious diseases of the eye and retina, garnered an exclusive license to RNAi technologies invented by researchers at the University of Pennsylvania.

Will the claims in these various patents overlap? Well, apparently the UMMS patents do not, meaning that Alnylam and CytRx shouldn’t find themselves in a litigious position in the future. But it’s probably not an exaggeration to say that hundreds upon hundreds of RNAi-based patents have already been filed – and more are surely in the works. If history proves out, some of them will certainly end up in the courts.