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RNAi Roundup: Ambion Gets Possible Edge from City of Hope PCR IP; Sirna Describes Preclinical Programs in Online Seminar

NEW YORK, July 23  - In a field where IP position is often more important than underlying technology, Ambion has made another inroad, licensing an invention where PCR is used to create siRNA expression cassettes.


The exclusive, worldwide research market license "expands our range of siRNA-related products and applications," said Bruce Leander, president of Ambion. "If someone wants to work with siRNA and doesn't want to work with RNA, they can work with DNA."


The underling IP is contained in an unpublished US patent application, SN 60/399,718, owned by the City of Hope Comprehensive Cancer Center in Duarte, Calif., and Beckman Research Institute. It covers an invention by John Rossi, chair of the division of Molecular Biology at City of Hope, and an associate, Daniella Castanotto, which is designed to save time and cost in making siRNA.

"In contrast to siRNA expression vectors, which require cloning and

sequencing prior to use and thus take one to two weeks to prepare, siRNA expression cassettes, which are created by PCR, can be prepared in less than a day," said Leander.


Rossi, who told GenomeWeb he has been using a form of this technology for eight years, said that it "gives a fair amount of flexibility for looking for good siRNA target sites." Given that siRNAs do not always work against the target for which they are designed, multiple siRNAs must usually be designed for a given target, and cost can be a factor. "The cost of doing the PCR reaction is a fifth or less vs. the chemical synthesis of siRNA," Rossi said.


The PCR cassette also has the advantage of enabling users to make mutational controls quickly, and has a longer lasting effect than chemically synthesized siRNA, according to Rossi. The silencing effects of these siRNA PCR cassettes have "gone on to six days," and "we still see no dropoff." He attributed this stability to the fact that the PCR products are DNA, not RNA.


Leander said these cassettes can also be used with expression vectors. "By incorporating restriction sites at their ends, [siRNA expression cassettes] found to effectively elicit gene silencing can be readily cloned into a plasmid to create an siRNA expression vector with an

antibiotic resistance gene or other marker. The siRNA expression vector can then be used for stable expression and long-term studies."


This license could give Ambion an edge in the highly competitive market to provide siRNA for research uses. Ambion is the co-licensee, along with Dharmacon, of Lafayette, Colo., Qiagen, and Proligo, of Boulder, Colo., of patent applications held by MIT and describing the mechanism of short interfering RNA. 


But none of these other companies makes the PCR cassettes, according to information on their websites.


The only other company to have IP for a similar technology is Promega, which holds an exclusive sublicense for research uses to Australian company Benitec's granted patents on ddRNAi technology. In this technology, DNA constructs are introduced into the cells and trigger the synthesis of RNAi.


But given the early stage of IP in this industry, the picture could quickly change.



Sirna Therapeutics' preclinical work with siRNA therapeutics was the major topic of a July 15 online seminar on siRNA, Exploring the Applications of RNA Interference (RNAi) Technologies.


In the seminar, which was pay-per-view and is still available in recorded form, Nassim Usman, chief scientific officer of Sirna, and Barry Polisky, vice president of research, discussed the company's work in chemically altering siRNA with proprietary molecules to stabilize it, as well as their early work to develop siRNA-based therapeutics for HCV and macular degeneration.


While little of this information was new to those who have seen Sirna's presentations at any RNAi conferences, the executives provided some useful commentary on the current obstacles to using siRNA as a therapeutic, and the way they are addressing them.


Usman offered arguments for the company's work in chemically stabilized siRNA. "Unstabilized siRNA -- this is the type of siRNA that the world uses now in cell culture systems -- if you put this into an animal, it is rapidly cleared renally following a therapeutic route of administration, an intravenous or subcutaneous bolus injection, a normal injection, not a hydrodynamic injection to the liver."  Sirna, he said, has been able to design siRNA that is stable even after 72 hours.


In the company's HCV work, its researchers have been able to get around the fact that the virus does not replicate in cell culture after the company in-licensed an HCV cell culture model invented by Charlie Rice at Rockefeller University, that uses a subgenomic replicon of the single-stranded HCV virus. The researchers have found that the siRNA is highly active in this replicon system without any general toxic effects, Usman said.


The company chose the eye disease macular degeneration as its other therapeutic area, in part in order to avoid the issue of general toxicity, said Usman, since its siRNA compound is administered locally through the eye. So far, the company has identified siRNAs that are effective against the VEGF pathway, which is the pathway responsible for growth of abnormal blood vessels that cause blindness in this disease. They have also, according to Usman, identified active siRNAs against VEGF receptor 1, and 2, and are testing this an in vivo model of choroidal neovascularization (which mimics macular degeneration) with a collaborator.


In a question-and-answer session following the Sirna presentation, Polisky addressed two of the hot issues surrounding siRNA: design of oligos and non-specific effects.


As far as oligo design, Sirna is in the simple-and-quick camp. "It's not a very sophisticated approach," said Polisky. "We avoid runs of Gs, we try to keep the A-T and G-C balance reasonable, we Blast everything to make sure that we're not cross-reacting with other genes that could potentially confuse the issues."


But he believes that microarrays are critical to make sure that an siRNA is not having a non-specific effect. "If you don't do something like microarray analysis, you are basically looking under the lamppost for your lost keys," Polisky said. "You've got to look globally if you're concerned about off-target knockdown, [and] the only way to really do that is by microarray analysis."



In the conference arena, IBC Life Sciences announced Thursday that it would be holding the second international conference on RNAi, November 3rd and 4th, at the Hilton Boston Logan Airport in Boston.

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