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Researchers Develop Novel Oligos for miRNA Knock Down; Alnylam Snaps Up Drug Rights

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Researchers from Rockefeller University claim to have developed a new class of oligonucleotides, termed antagomirs, that can be used to selectively inhibit microRNAs in vivo.

The oligos, which were developed in the lab of Rockefeller professor Markus Stoffel, could open the door for researchers to determine in a high-throughput fashion the function of the ever-growing population of miRNAs. Previously, Stoffel said, the only way to determine miRNA function in vivo was with "a traditional knockout approach, [which is] tough and, most of all, slow."

The oligos hold the potential to act as therapeutic agents, selectively suppressing the expression of disease-associated miRNAs, Stoffel said. This possibility has caught the attention of RNAi drug developer Alnylam Pharmaceuticals, and Stoffel • a member of Alnylam's scientific advisory board • said the company has taken an exclusive license from Rockefeller on the use of antagomirs in all therapeutic indications.

"There are more and more microRNAs being discovered • there are probably 250 to 300 in mammalian systems • but we know very little about their function," Stoffel told RNAi News last week.

Further complicating matters is the fact that often an miRNA is encoded by several genes, rather than just one. "In order to do a knockout of one particular microRNA, in many cases you have to knock out three different genes," Stoffel said. Additionally, miRNAs can sometimes "appear … as clusters … [encoded] in polycistronic transcripts. It's very hard to, within a cluster, knock out a single [miRNA], most likely because you interfere with the primary structure and you don't get splicing of the other microRNAs," he said.


"There are more and more microRNAs being discovered • there are probably 250 to 300 in mammalian systems • but we know very little about their function."

"Lastly … gene regulation is subtle • at least for many genes," Stoffel said. "There are lots of data out suggesting that there are several microRNAs regulating a particular target. So if you wanted to … knock out several different microRNAs, you again get into that problem of having to knock out many genes or cross individual knockout [animals] together, which is very slow."

In looking for a better way to selectively knock down miRNA expression, Stoffel and colleagues developed chemically modified single-stranded RNA analogues complementary to a specific miRNA. In order to enhance the antagomirs' uptake by cells and improve target degradation, they took a page out of Alnylam's playbook and linked the oligos to a cholesterol molecule.

The antagomir research, which appears in this week's Nature, comes about a year after Alnylam researchers published their own research in the journal describing how the conjugation of cholesterol to the 3' ends of an siRNA's sense strand could improve the pharmacokinetic properties of the RNAi molecule (see RNAi News, 11/12/2004).

"We thought, 'How about doing this to chemically modified single-stranded RNAs?'," Stoffel said, and found that "the cholesterol conjugate worked amazingly." He noted that Alnylam synthesized the materials for his experiments and acted as advisors on his work. Also contributing to the new Nature paper was Rockefeller researcher Thomas Tuschl, who co-founded Alnylam.

In his experiments, Stoffel intravenously administered to mice small volumes of antagomirs targeting four miRNAs: miR-16, a ubiquitously expressed miRNA that would allow the researchers to determine into which organs the antagomirs could and could not enter; miR-122, which is the mostly highly expressed miRNA in the liver and therefore a good candidate for the researchers to target in order to see how powerful a knockdown effect they can achieve; and miR-192 and miR-194, which are part of an miRNA cluster, providing Stoffel and his lab the opportunity to try to knock down one part of a cluster without affecting other parts.

The antagomirs were administered once every morning for three days, and the researchers found that the antagomirs were able to knock down their target miRNAs and penetrate the liver, lung, kidney, heart, intestine, fat, skin, bone marrow, muscle, ovaries, and adrenals. They were not able to enter the brain, Stoffel said, most likely because they cannot cross the blood-brain barrier.

The antagomirs also proved to be very specific, he said, "in that when we knock out a single member of [an miRNA] cluster, we do not affect the expression of the other microRNAs."

The effect also proved to be potent, lasting for at least 23 days. "We are now waiting … to see when we lose the effect," he said, adding that "we also have data where just a single injection will markedly reduce the expression of [a target] microRNA."

He noted that in Alnylam's Nature paper last year, which used dsRNA, the knockdown effect was lost after a few days. He said it is unclear why the antagomirs act for so long, but that "we think that as new microRNAs are being synthesized by the cells, they are being captured by the antagomirs and immediately degraded • but we don't know the mechanism. We are studying it now."

Biological significance of the knockdown was evaluated using an antagomir for miR-122. "Gene expression and bioinformatic analysis of messenger RNA from antagomir-treated animals revealed that the 30 untranslated regions of upregulated genes are strongly enriched in miR-122 recognition motifs, whereas downregulated genes are depleted in these motifs," the researchers wrote in the Nature paper. "Analysis of the functional annotation of downregulated genes specifically predicted that cholesterol biosynthesis genes would be affected by miR-122." In line with this, Stoffel found that plasma cholesterol levels in miR-122-treated mice dropped 40 percent, he said.

Stoffel noted that no toxicities were observed in the mice.

"Our novel pharmacological approach to silence miRNAs specifically will allow the rapid generation of mice lacking specific miRNAs or combinations of miRNAs for further functional studies," Stoffel and colleagues wrote in this week's Nature paper. Also, "because it has been shown that miRNAs are involved in cancer, cell growth and differentiation, insulin secretion, and viral infection, silencing of miRNAs with antagomirs could become a therapeutic strategy for diseases such as cancer, hepatitis and diabetes, and others almost certain to be discovered, in which miRNAs participate in disease etiology."

Stoffel noted that researchers at non-profit organizations are free to synthesize antagomirs for use in their experiments. As for those looking to investigate the oligos' therapeutic potential, they'll have to discuss possible licensing arrangements with Alnylam.

In a statement issued this week, Alnylam said that it has "taken an exclusive license to all of the Rockefeller University's interest in antagomir technology." Company officials were not available to comment on whether that license was limited to therapeutics or also included the rights to use antagomirs for commercial research applications, or whether it would out-license the technology in areas outside of its core focus under its InterfeRx program (see RNAi News, 12/19/2003).

- Doug Macron ([email protected])

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