City of Hope's Beckman Research Institute has exclusively licensed its Dicer-substrate RNAi technology to oligo manufacturer Integrated DNA Technologies for research and functional genomics applications, COH said this week.
COH is also on the lookout for parties interested in licensing the technology for therapeutic applications, and negotiations are underway with at least one RNAi-based drug developer, RNAi News has learned.
The technology — which was developed by Beckman Research Institute scientist John Rossi in collaboration with colleagues from the COH and IDT's vice president of molecular genetics Mark Behlke — uses synthetic RNA duplexes between 25 and 30 nucleotides long to trigger RNAi silencing.
According to a paper by Rossi et al. published in a February issue of Nature Biotechnology, initial work with the technology indicated that 27-nucleotide long RNA duplexes could be up to 100 times more effective at silencing genes than conventional 21 nucleotide-long siRNAs without inducing an interferon response or activating protein kinase R in cells.
"The reason [for this] is [the 27-mer oligo] enters into a pathway where there's concerted handoff of the process product to the Ago2 complex, and that facilitates the whole RNAi process," Rossi told RNAi News this week. "It … also may direct the antisense strand selection process."
"It's a great improvement [over the earlier work detailed in Nature Biotechnology] because now we can find the potent 21-mer, incorporate it into the 27-mer that's going to be diced, and increase its potency even more."
During the course of this early experimentation, "our favorite compounds were blunt 27-mers — we didn't find any specific advantage to having any specific overhangs," Behlke told RNAi News. "But it turns out that some of these things are actually sequence context-dependent. In subsequent studies we found out that sometimes these blunt molecules [don't] work as well as the 21-mers, and [they sometimes] … can be processed … into as many as four or five different 21-mers — and it wasn't necessarily the best 21-mer" that the researchers were ending up with.
Since the active compound of the 27-mers "is, in fact, still a 21-mer of the traditional design, and it requires processing by Dicer," Behlke said, "we realized that we needed to gain greater control over the system."
To do so, the researchers looked to naturally occurring microRNAs, "which use the two-base 3' overhang as their entry point for Dicer," Rossi said. "We had seen in our earlier studies that, if we had DNA on the ends of … the blunt-ended molecules, it actually inhibited dicing," Rossi said. "So we just basically combined this idea of a two-base 3' overhang on one end and a couple of dioxynucleotides on the blunt end, and we directed Dicer to a particular 21-mer: the first 21 to 22-base sequence from the two-base 3' overhang."
This approach "allows us to predict what the actual products will be, and therefore gain control over the dicing reaction," Behlke said.
"It's a great improvement [over the earlier work detailed in Nature Biotechnology] because now we can find the potent 21-mer, incorporate it into the 27-mer that's going to be diced, and increase its potency even more," Rossi added.
A paper detailing the fine-tuning of the Dicer-substrate technology has been accepted for publication by Nucleic Acids Research, Behlke noted.
With the rights to use the technology for research and functional genomics applications in hand, IDT has begun offering for sale the 27-mers "to pretty much anyone who wants to try them," Behlke said. "We have a design tool on our website that facilitates the site selection, as well as incorporates all of our latest design parameters that have come out of the ongoing research collaboration between City of Hope and IDT."
He noted that in August, the company expects to introduce sets of the 27-mers targeting all the genes in the human, mouse, and rat genomes, "which can be directly ordered off a bioinformatics interface."
COH continues to own the rights to the technology for therapeutic, diagnostic, and plant-based applications. However, according to Brian Clark, director of the office of technology licensing at COH, a number of drug developers are interested in possible licensing arrangements.
"Our plan is to both field inquiries and deal with inquires we already have in this area, and also make it well-known that City of Hope has these rights in therapeutic and diagnostic areas," he told RNAi News, adding that success with the technology in basic research is expected to fuel interest in using the technology in other areas, such as drug development.
Clark said that COH is still debating how to best out-license the technology for therapeutic applications, but "our current thinking is along the lines of broad, non-exclusive licensing.
"This is a platform technology, and we believe people will come to the table with specific formulations or targets that they already have … and then want access to our … technology," he said. "When people come with their 21-mer that they've already identified, we think it will make a lot of sense for people to explore wrapping it up in this 27-mer approach and then get the rights from City of Hope."
According to Rossi, RNAi drugs startup Calando Pharmaceuticals, which he co-founded (see RNAi News, 2/25/2005), is "definitely" interested in using the Dicer-substrate technology. "There're negotiations ongoing," he said, referring additional questions to Clark, who declined to comment.
Clark also declined to indicate whether there are active negotiations with companies aside from Calando. Even if there are none, it seems likely that COH is bound to get inquiries from other parties interested in using the technology in RNAi drug development given the positive early data coming in.
Rossi noted that COH researchers Edouard Cantin and Patric Lundberg are in the process of conducting in vivo work using the technology, and that they have had some promising early results.
In Cantin's and Lundberg's experiments, a version of the 27-mers administered to mice intraperitoneally in a lipid complex has proven to be "incredibly potent at protecting mice from lipopolysaccharide-induced inflammatory responses," Rossi said. "They induced mice with lipopolysaccharide to get an inflammatory response, and when you come back the next morning these mice are dead. But if they treat them with this 27-mer, the mice are still alive and the liver pathology shows that they're getting marked protection against inflammatory responses."
While these results haven't been compared with 21-mer siRNAs, in vitro work in a macrophage cell line indicates that 27-mers targeting TNF alpha are "several-fold better" than the conventional siRNAs "at the lowest concentrations tested," Rossi added.
Behlke also noted that two IDT collaborators at the University of Iowa — Jeanne Snyder and Paul McCray — are experimenting with delivering the 27-mers intratracheally to reach pulmonary targets in mice. He declined to provide more detail about this work.
Assuming the Dicer-substrate technology is effective, an even more attractive aspect of the technology for drug developers is its use of 27 nucleotide-long RNA duplexes, which would appear to be clear of the so-called Tuschl-1 and Tuschl-2 patent applications that loom on the RNAi IP horizon.
This IP, named for Rockefeller University researcher and Alnylam Pharmaceutical co-founder Tom Tuschl, essentially covers the use of short interfering RNAs, 21 to 23 nucleotides in length, to induce RNAi in mammalian cells, as well as the two-to-three nucleotide 3' overhanging ends on the end of certain siRNAs.
The Tuschl-1 patent application has been licensed to both Alnylam and its rival Sirna Therapeutics, which have committed to out-licensing it at fair commercial terms (see RNAi News, 9/12/2003). The Tuschl-2 application is held solely by Alnylam, but is also eligible for out-licensing (see RNAi News, 12/19/2003).
According to Richard Warburg, a partner at the law firm of Foley & Lardner, by using the 27-mers for RNAi-based drugs, instead of standard 21-mers, a drug developer "can pay less attention to the Tuschl [IP]."
However, he warned that "it doesn't open up the landscape with respect to all the other patents that might be out there. You've got 20 patents out there you have to worry about," he told RNAi News. "You've taken out one or two — that's nice, but it's just a step in the right direction."
— Doug Macron ([email protected])