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Market Unfamiliarity Prompts Mirus to Debut Veteran RNAi Gene-Function Tool as New

Mirus Bio last week announced the availability of its hydrodynamic delivery technology through licensing and service deals for RNAi research applications.
While the company billed the delivery approach as a “new tool” for gene function analysis, the technique has actually been available for years. According to David Lewis, RNAi program director at Mirus, the decision to call the technology new stemmed from a lack of awareness among researchers.
“Hydrodynamic [delivery] through the tail vein has been around since 1999, [and for siRNAs, the approach] was first demonstrated in 2002,” he told RNAi News this week. “But, when we talk to people, it’s amazing how many people haven’t heard about hydrodynamic injection or what it’s for or what it allows you to do.
“So … we called it new just because we realized there is a big audience of people who are thinking about using RNAi in vivo but may not be familiar with [the] technology,” he said.
Richard Schifreen, vice president of research products at Mirus, added that the time seemed right to launch a concerted effort to commercialize the technology after the company received the go-ahead from partner Rosetta Inpharmatics to publish data from work the companies did comparing the hydrodynamic delivery method with traditional gene knockout.
“For some time, we’ve had a number of different collaborations and service-type work we’ve done for people … [but] with a lot of the work we’ve done and continue to do, our partner or customer wants to keep the work proprietary,” he said.
But with Rosetta open to publishing the data, “the demonstration is now out there for the public … [and] we’re ramping up in terms of our commercialization,” Schifreen added. Mirus is “trying to do more to get the word out that this will work, exactly what it can do, and our willingness to work with people in a flexible way to allow them to use this technology.”
The Paper
The research with Rosetta, published on Aug. 31 in Nucleic Acids Research through the journal’s advanced access service, demonstrates that naked siRNAs delivered to mice via hydrodynamic tail vein injection produced molecular and phenotypic changes similar to those in knockout mice.
The researchers targeted the gene peroxisome proliferator-activated receptor alpha, or Ppar alpha, which is associated with the regulation of fatty acid metabolism. According to Lewis, the gene was an ideal target because it is well-characterized and Ppar alpha knockout mice already exist. Additionally, the gene is “known to be expressed in the liver, which is one of our target organs” for hydrodynamic delivery of RNAi molecules.
Ppar alpha knockdown in the liver using three different siRNAs resulted in a transcript profile and metabolic phenotype that is “comparable to those of [Ppar alpha knockout] mice,” the Mirus and Rosetta researchers wrote in Nucleic Acids Research.
“Combining the profiles from mice treated with the [Ppar alpha] agonist fenofibrate, we confirmed the specificity of the RNAi response and identified candidate genes proximal to [Ppar alpha] regulation,” they noted. Ppar alpha “knockdown animals developed hypoglycemia and hypertriglyceridemia, phenotypes observed in [the knockout] mice. In contrast to [Ppar alpha knockout] mice, fasting was not required to uncover these phenotypes.”
The paper’s authors note that although RNAi provides a fast and inexpensive method to analyze gene function compared with the creation of knockout animals, the gene-silencing technology has limitations. These include the widely known potential for off-target effects, incomplete knockdown, and non-targeting of splice variants by the selected siRNA sequence.
As such, the researchers suggest that RNAi be used to complement knockout animals rather than replace them.
“One potential application of the siRNA approach would be as a screening method to gain insight on the phenotypes of large numbers of genes quickly,” they wrote. “The function of the genes identified in the RNAi screen could then be verified or analyzed in more detail by creating knockout strains.”
Lewis noted that RNAi might also provide information about a gene’s function unavailable with knockout animals when the two technologies are used together.
“Despite the very comparable phenotypes, we did see some differences between the knockdown and knockout mice,” he said. “We’re not sure why these differences exist; it could simply be that [with siRNA-treated mice] the knockdown is never 100 percent like it would be in a knockout.
“But it might be that what we’re seeing are phenotypes that are caused by an abrupt knockdown when you deliver siRNA versus a knockout where the gene is missing throughout growth and development of the animal,” he added. “In humans, we know that most diseases are the result of changes in gene expression in a point in time, not throughout development. I think in this way RNAi may more closely mirror what is going on in human disease and may be more relevant to the study of the genetic basis of disease.”
Schifreen noted that the work detailed in Nucleic Acids Research was conducted under a partnership with Rosetta where “they had interest in this technology and its applications,” but he declined to provide additional details on the status of the collaboration.
What’s Old is New
With a more formal commercialization effort for its hydrodynamic delivery technology underway, Mirus is looking to build upon an “active licensing program” that has already yielded a number of pharma/biotech partnerships including one with French gene-therapy firm Transgene.
“There are two types of potential users” for the technology, Jim Hagstrom, Mirus’ vice president of scientific operations, explained. “One is large pharma looking at using this as a research tool,” and which the company hopes will see the Nucleic Acids Research paper as evidence of the delivery method’s efficacy.
For this group, Mirus aims to sign licensing deals for the hydrodynamic delivery technology, as well as contracts under which it would provide delivery and/or analysis services for customers.
“The other group of people that are interested is people with a longer-term look to therapeutics,” Hagstrom said. “There is still a big debate whether hydrodynamic [delivery] can be used therapeutically — we believe in numerous contexts it can.”
About two years ago, Mirus unveiled the Pathway IV delivery technology, used to deliver genes and siRNA molecules into the muscle tissue of a limb via an intravenous injection (see RNAi News, 6/11/2004). The approach, based on hydrodynamic delivery, involves using a blood pressure cuff or tourniquet to occlude the upper portion of a limb, then rapidly delivering nucleic acids into the bloodstream through a catheter-based injection in the distal portion of the limb.

“When we talk to people, it’s amazing how many people haven’t heard about hydrodynamic injection or what it’s for or what it allows you to do. So … we called it new just because we realized there is a big audience of people who are thinking about using RNAi in vivo but may not be familiar with [the] technology.”

The Pathway IV approach is currently being developed as part of a gene therapy for muscular dystrophy with Transgene. Mirus is also independently exploring the use of Pathway IV as part of a gene therapy for peripheral ischemia.
Although the company is on the lookout for other partners interested in the therapeutic potential of hydrodynamic delivery, Mirus’ Schifreen said he expects that potential partners will require a demonstration of efficacy in humans before signing any deals — and this demonstration is most likely going to come from the company’s partnership with Transgene.
“While it’s not an RNAi-based program, what [the muscular dystrophy project] is going to do is get us into the clinic in a phase I [trial] where we can show safety for the technology,” he said. “We think that’s going to be a validation of the technology and is going to increase both our own interest to try different things, [as well as that of] potential collaborators and partners. Once they see the demonstration of safety, they will feel more confident using the technology in their own programs.”
Mirus and Transgene’s muscular dystrophy therapy is expected to enter phase I studies by the end of next year.
As for users of the hydrodynamic technology outside of big pharma/biotech and research organizations, Schifreen said that Mirus maintains a policy that encourages use of the technology by not-for-profit institutions, which are not required to take a license.
“We do require licenses from for-profit institutions that want to use the technology in their research,” he said, adding that “I can’t comment on the companies that have actually taken licenses [or] those that we might be looking at to take licenses.”

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