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Isis Studies Suggest Therapeutic Potential of Single-Stranded RNAi Molecules


Researchers from Isis Pharmaceuticals last month published new data demonstrating that unformulated single-stranded antisense oligos can trigger an RNAi effect and the subsequent down-regulation of target genes in animals, including a mouse model of Huntington's disease, in a variety of tissues.
The findings, which appeared in two separate papers in Cell, suggest that these so-called single-stranded siRNAs, or ss-siRNAs, have therapeutic potential, according to Isis Chairman and CEO Stanley Crooke, who was also a co-author of both studies.
However, it seems unlikely that Isis will have an ss-siRNA drug under formal development any time soon.
The technology “is not our central focus,” Crooke told Gene Silencing News this week, adding that it reflects Isis' longstanding effort to pursue “all the antisense mechanisms.”
The ss-siRNA molecules “may have some advantages and disadvantages compared to other cellular means of degrading RNA,” he said. “The balancing act for us will be to decide how we feel about the off-target effects, how we feel about the behavior of these molecules relative to [others] … the durability of the effects — all kinds of things need to be sorted out” before drug-development work could begin in earnest.
The single-stranded RNAi approach “is another arrow in our quiver,” Crooke said, “and if we have a target we think is going to be more attractively reduced using this mechanism, we'll do it.” However, he declined to comment on whether Isis had any such targets in mind.
According to Crooke, the interest in ss-siRNAs stems from Isis' concerns over the difficulties and expense of creating double-stranded RNA drugs formulated with cationic lipids — an approach employed by partner Alnylam Pharmaceuticals with its siRNA drug candidates.

Notably, Alnylam had once been helping to develop Isis' ss-siRNA technology and had agreed to contribute $3 million annually to the effort under a 2009 deal (GSN 4/30/2009), but pulled out of the arrangement a little over a year later as part of a move to trim costs (GSN 11/18/2010).
Single-stranded antisense molecules, however, are more easily administered and distributed to tissues beyond the liver without the need for formulations, he noted, while the lack of a sense strand reduces the potential for off-target effects.
In the Cell papers, Isis and collaborators at the University of Texas Southwestern Medical Center have demonstrated “an approach to use [Argonaute 2] with oligos we know distribute [beyond the liver] without special formulations,” Crooke said. “We think that's a big step.”
In the first paper, a group of Isis investigators wrote that previous work with single-stranded oligos designed to activate the RNAi pathway had shown that potency — on par with siRNAs — requires “the identification of chemical modifications that enhance the metabolic stability and other pharmacokinetic properties of the ssRNAs and interactions with key components of the RISC mechanism.”
To address these issues, the team examined various chemically modified ss-siRNAs for their ability to bind to human Ago2 and “support cleavage activity,” they wrote. “Having defined the sites that tolerate modifications and the structure-activity relationships of productive interactions with Ago2, we then evaluated the modified ss-siRNAs for stability, other pharmacokinetic properties, and activity in cultured cells.”
The oligos were specifically designed to inhibit a variety of disease-relevant targets including PTEN, which is linked to certain cancers; factor VII, which is involved in hemophilia; and apolipoprotein C3, which has been associated with coronary heart disease.
Rodent studies confirmed that the ss-siRNAs do not require a sense strand to activate RNAi, and that guide-strand activity requires Ago2, “demonstrating action through the RNAi pathway,” the researchers stated.
They noted that the molecules require a 5′phosphate for in vivo activity, and that they developed a metabolically stable 5′-(E)-vinylphosphonate with conformation and sterioelectronic properties “similar to the natural phosphate.” Meantime, the addition of lipophilic modifications enhanced the ss-siRNAs distribution to the liver, as well as to muscle, lung, and fat tissues.
“A challenge of delivering drugs using cationic lipids is basically that you deliver to the liver,” Crooke said. “This paper shows we're able to get decent activity in fat and other tissues, and in our more recent work we're continuing to see very good activity in other tissues.”
Overall, the paper shows that the ss-siRNAs “work through the Ago mechanism … [and] work in tissues well beyond the liver,” he added. And while Isis has achieved greater potency with the molecules in more recent studies, “the doses we see in [in the paper] are sufficiently potent that we think we can make drugs out of these molecules.”
In the second Cell paper, a group led by UTSWMC researcher David Corey and supported by Isis investigators developed chemically modified, mismatch-containing ss-siRNAs modeled on microRNAs that silence a mutant form of the huntingtin gene responsible for Huntington's disease.
In fibroblasts derived from Huntington's disease patients, the oligos were able to inhibit expression of their target by more than 80 percent for up to eight days. Moving over to a mouse model of the disease, the researchers found that intrathecal administration of the ss-siRNAs led to a reduction in mutant huntingtin levels in a number of brain regions including the contralateral cortex, thalamus, ipsilateral striatum, contralateral striatum, cerebellum, and brainstem.
The results, the team reported, “introduce ss-siRNAs as a strategy for treating neurodegenerative disease that provides an alternative to [antisense oligos] or dsRNAs.
“Chemically, ss-siRNAs are similar to [antisense oligos] because they both possess a single chemically modified antisense strand,” they wrote. “Mechanistically, they resemble duplex RNAs that function through RNAi.”
Therefore, they “combine strengths of the two existing approaches, possess unique advantages, and provide a distinctive new strategy for silencing gene expression,” Corey and his colleagues concluded. “By further optimizing the type of chemical modification, placement of mismatched bases, or other design features, it is likely that subsequent generations of inhibitory ss-siRNAs will possess even more favorable properties.”

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