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Santaris Presents Data on Tiny LNAs for miRNA Family Inhibition

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By Doug Macron

While Santaris Pharma's efforts with microRNA-targeting therapeutics have primarily been based on its standard locked nucleic acid technology, the company is also testing smaller LNA molecules, dubbed tiny LNAs, designed to inhibit entire miRNA families.

LNAs are nucleic acid analogs in which the ribose ring is locked by a methylene bridge connecting the 2'-O atom with the 4'-O atom. While the technology is being used to inhibit mRNA targets, Santaris is also currently testing miravirsen, a 15 nucleotide-long LNA that targets miR-122, in a phase II study as a treatment for hepatitis C (GSN 9/23/2010).

Last week on the sidelines of the sixth annual Oligonucleotide Therapeutics Society meeting in Dana Point, Calif., Santaris CSO Henrik Orum told Gene Silencing News that while LNAs the size of miravirsen have therapeutic potential, the company doesn't envision a "one-size-fits-all" solution for miRNA-targeting drugs.

Although there are indications for which inhibiting a single miRNA is ideal, he said, in other cases knocking down an entire family of the small, non-coding RNAs will be required to see a therapeutic benefit. To address this issue, the company has been developing 8-nucleotide-long LNAs that are perfectly complementary to an miRNA seed region, which would allow the oligo to block all the miRNAs within a particular family.

At the OTS meeting, Santaris researcher Susanna Obad presented data demonstrating that tiny LNAs not only work in vitro, but can also silence miRNAs in vivo with no apparent off-target effects.

In initial experiments, Santaris investigators cloned the perfect-match target to miR-21, which has been associated with cancer and cardiac disease, into a luciferase sensor. They then measured luciferase levels in HeLa cells that contained the miRNA endogenously and repress the sensor.

Addition of the miR-21-targeting tiny LNAs triggered a concentration-dependent de-repression of the sensor, achieving complete de-repression at the five-nanomolar level, while control sequences had no effect, Obad said.

To test the tiny LNAs' specificity, "we re-designed [the molecule] and introduced single mismatches or double mismatches" and found only partial or no de-repression, she said. Further experimentation revealed that significant de-repression could only be achieved when the miRNA's entire seed region was targeted.

"We also looked at protein levels" and found that levels of the miR-21 target Pdcd4 increased compared to controls with the addition of the tiny LNAs, she said, which also was found to reduce colony formation.

"Seeing that we could target single microRNAs with these [tiny LNAs], we continued [on] to look into a family," starting first with the miR-221/miR-222 family, which has a highly conserved seven-nucleotide seed region, Obad said.

She and colleagues cloned miR-221 and miR-222 luciferase sensors, which they transfected either separately or together into PC3 cells, which endogenously express both miRNAs, she said during her presentation.

When treated with tiny LNAs, a concentration-dependent de-repression of the sensors was achieved, while LNA controls had no effect. Meanwhile, levels of p27, a target of the miRNA family, increased with higher concentrations of the tiny LNAs.

The team then turned its attention to a bigger miRNA family, let-7, which has nine members with an eight nucleotide-long seed region, Obad explained.

"We cloned the 3' UTR of [the let-7 target HMGA2] … into Huh7 cells, along with pri-miRs for each one of the different [family] members," she said.

After treating cells with let-7 tiny LNAs, "you get a de-repression of the HMGA2 3' UTR sensor, but you don't get anything with an LNA control," while protein levels of HMGA2 increased in a concentration-dependent manner as compared to controls.

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To test the tiny LNAs in vivo, the Santaris researchers radioactively labeled tiny LNAs against miR-21 and injected them into mice, finding the oligos accumulating in a number of tissues including bone marrow, kidney cortex, lung, myocardium, liver, lymph node, and spleen.

To confirm delivery of the tiny LNAs, as well as to establish that they are functional, "we did Western blot of [miR-21 target] BTG2 and we saw an up-regulation of BTG2 in the tiny [LNA]-treated mice compared to saline and LNA control," Obad said.

In collaboration with Cold Spring Harbor Laboratory researcher Greg Hannon, Santaris then injected the miR-21-targeting tiny LNAs into a mouse model of breast cancer. Analyses revealed that the tiny LNAs were delivered to the tumor cells and inhibited miR-21, according to Obad.

Looking to test the tiny LNA technology head-to-head with Santaris' more established LNA approach, Obad and her colleagues developed tiny LNAs targeting miR-122, which is associated with HCV replication and cholesterol regulation, and compared the effects to miravirsen.

"The oligos were injected into mice three times over seven days" in doses of either 5 mg/kg or 20 mg/kg, she said. Analyses revealed that both agents were able to up-regulate targets of miR-21, while both miravirsen and the tiny LNAs both dose-dependently lowered cholesterol levels.

"In order to further compare [the two], we did microarray expression profiling on the microRNA," she said. The results indicated that both oligos "have similar effects on the liver transcriptome.

"In order to validate these results, we identified all genes that were differentially expressed between … treated mice and control mice," she added. "Cluster analysis shows a high level of concordance between the 8-mer and 15-mer oligos."

Recognizing that the tiny LNAs may have "many predicted perfect-match binding sites in the transcriptome" given their small size, Santaris examined their potential for off-target effects, Obad said.

"We … looked at all [the] potential binding sites to see if there was an effect on the mRNA on something that was not our target" using Sylamer, a system for identifying over- or under-represented words in gene lists, she explained. "We don't see any effects," she said.

The team also constructed a sensor vector containing the open reading frame of renilla luciferase, and designed 8-mer tiny LNAs against it. "If these 8-mers were to bind and have an effect on the renilla, you would see … a decrease of luciferase activity," she said. "They do not have a significant effect."

"So even if there are lots of potential binding sites out there for these tinies, with the method we have used so far, we don't see that they have" any impact, she noted.

Following up on these data, Santaris is in the process of testing a variety of tiny LNAs in different mouse disease models," Obad noted.

In an e-mail, Orum said that Santaris is "quite excited" about the technology and believes "it will have a place in the future of miRNA therapeutics."

He added that the company aims to publish additional details about tiny LNAs in "the near future."

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