After nearly two years of work, Sirna Therapeutics has begun animal testing of a novel type of RNAi molecule, termed "multifunctional" siRNAs, designed to knock down expression of two or more target genes with a single oligo.
According to Barry Polisky, senior vice president of research and chief scientific officer at Sirna, in vivo work is currently being conducted in mice, and manuscripts detailing both in vitro and in vivo work with the multifunctional siRNAs are being prepared for submission to peer-reviewed journals.
As reported by RNAi News earlier this month, multifunctional siRNAs are essentially siRNAs modified in such a way that both the sense and antisense strands can be used to each silence a distinct gene (see RNAi News, 11/11/2005).
"Standard siRNAs have one 5' terminus that you are interested in and one 5' terminus you are not interested in," Polisky told RNAi News last week. With this kind of traditional siRNA, which targets a single gene, "you do the best you can to bias the entry of the strand you want into the RISC complex."
But researchers at Sirna, he said, figured that "since there are two 5' ends there, if they were of equal thermodynamic stability, [one may] create a situation where either strand would have an interesting biological effect.
"We wanted to see if we could design siRNAs that actually targeted two completely sequence-unrelated targets … and we figured out a way to do that," Polisky said. "It takes advantage of sequence-design features that all targets tend to have in common, even if they are sequence-unrelated. We can find short regions of sequence relatedness that permit us to design a bifunctional siRNA where one 5' end targets one [gene] and the other 5' end targets another [gene either] in the same or different pathway."
"We wanted to see if we could design siRNAs that actually targeted two completely sequence-unrelated targets … and we figured out a way to do that."
Using this technique, he added, Sirna has been able to develop RNAi oligos that essentially hit two molecular targets at once. "From a therapeutics-development [standpoint] that his big implications because it means that instead of making a cocktail of two standard siRNAs and making four stands and putting together two duplexes, we can make two strands and accomplish the same thing," he said.
Additionally, multifunctional siRNAs can be used to target more than two genes if there are appropriate levels of sequence homology. "It's possible to imagine knocking down three targets under favorable conditions," Polisky said. "It's not really feasible to start making longer and longer versions of these … but at least two targets [per strand is] certainly [possible], and we've been able to show this in vitro."
As an example, Bharat Chowrira, vice president of legal affairs and chief patent counsel at Sirna, pointed to the VEGF pathway. "VEGF receptor 1 and receptor 2 have some regions of sequence homology," he told RNAi News last week. "So you can design multifunctional siRNAs where one strand recognizes the conserved region between [receptor] 1 and [receptor] 2, and the second strand can VEGF, so you are targeting three targets … with the same molecule."
According to Polisky, one of the biggest benefits of the multifunctional approach is co-localization. "We're delivering these to the exact same intracellular environment, so in principle … we don't have to worry about the delivered siRNAs somehow being in the cell at different concentrations."
Another benefit lies within the US Food and Drug Administration submission process. Although it remains unclear how the FDA will deal with therapies composed of multiple siRNAs, many believe the agency will treat each component of the cocktail as a separate drug. But as for multifunctional siRNAs, "there's no question that the FDA will be able to look at [multifunctional siRNAs] in terms of one entity," he said.
"We're delivering these to the exact same intracellular environment, so in principle … we don't have to worry about the delivered siRNAs somehow being in the cell at different concentrations."
Additionally, Sirna expects multifunctional siRNAs to help streamline manufacturing. If a company wanted to use an siRNA cocktail therapeutically to hit multiple targets at once, it "would be faced with all of the challenges of making those [oligos], purifying them, characterizing them, looking at metabolites, and so on, which is a big deal," Polisky said. In the case of multifunctionals, "we avoid those problems."
According to Chowrira, Sirna has filed US and international patent applications on the multifunctional technology, and two of the applications, US 20050233329 and WO 0578097, have already been published.
Since this technology is "home-grown" and "not covered by any other patents out there except Sirna's," Chowrira said that he expects the company to be able to develop and commercialize the multifunctional siRNAs without any concerns about intellectual property infringement • which is no small thing in the world of RNAi where the IP landscape has proven to be highly litigious.
As for when the multifunctional siRNAs might be ready for primetime, Polisky declined to provide any specific timelines.
"We're in the process of evaluating these in our viral systems and other in vivo models that we have, so it's very early stage," he said. "But these things can move quickly because the models are all set up, the designs are straightforward, [and] synthesis is straightforward.
"We're actually in the process of comparing the efficacy of a number of these … to monofunctional siRNAs and looking to see what the in vivo performance is," Polisky added. "I don't know that I want to say when these things will be in the clinic, but they've [been] … moved to the front of our preclinical pipeline."
• Doug Macron ([email protected])