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Santaris, Mass General Team Report Non-human Primate Efficacy Data on miR-33 Inhibitor

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A team led by researchers from Massachusetts General Hospital and Santaris Pharma this week published new data showing that inhibition of the microRNA-33 family using short locked nucleic acids could impact cholesterol levels in non-human primates without triggering any adverse effects.

According to the study's authors, the data suggest that targeting an entire miRNA family could have therapeutic potential — a key finding for Santaris, which originated the so-called "tiny" LNAs and holds a license from Mass General to intellectual property relating to miR-33 as a drug target.

Amid growing evidence of their role in a host of biological processes, miRNAs have become the focus of a number of drug developers. Santaris, for example, is testing an LNA-based antagonist of miR-122 in phase II studies for hepatitis C, while Mirna Therapeutics recently began phase I testing of a miR-34 mimic for cancer.

However, a potential limitation of therapeutic miRNA inhibitors is the "redundancy and compensatory action among miRNA family members and other miRNAs sharing the same seed sequence," the Mass General/Santaris group wrote in Science Translational Medicine this week.

In humans, they noted, 47 percent of highly conserved miRNA families contain two or more family members that exhibit the same seed sequence. At the same time, a number of the non-coding RNAs share seed sequences with miRNAs outside of their family, although they differ from them in the sequences outside of the seed region.

"This highlights the challenge of sequence-specific antisense targeting of potentially redundant miRNAs," they stated.

To address this, Santaris and its academic collaborators have been working on tiny LNAs — 8-nucleotide-long LNAs that are perfectly complementary to an miRNA seed, which allows the oligos to block all members within a particular miRNA's family.

In late 2010, Santaris presented data at the Oligonucleotide Therapeutics Society's annual meeting showing that tiny LNAs targeting miR-21 can work both in vitro and in vivo without toxicity.

The next year, the company and scientists at Cold Spring Harbor Laboratory published a paper demonstrating the ability of tiny LNAs to knock down a number of different miRNA families in cell culture, and to inhibit the miR-21 family in breast cancer mouse models without side effects.

In another step toward advancing tiny LNAs to the clinic, Santaris and Mass General have now shown that these molecules can have a therapeutic effect in non-human primates without any safety issues.

To do so, Santaris and Mass General investigators focused on miR-33, which they previously showed can affect cholesterol hemeostasis by working with sterol regulatory element-binding proteins, or SREBPs.

Specifically, miR-33a and miR-33b, which differ by two nucleotides outside the seed sequence, were found in introns of SREBP genes. They were also discovered to target the adenosine triphosphate-binding cassette transporter A1, or ABCA1, which is an "important regulator of high-density lipoprotein synthesis and reverse cholesterol transport, for post-transcriptional repression."

In the earlier study, antisense inhibition of miR-33 in mice and human cells upregulated ABCA1 and boosted cholesterol efflux. Meanwhile, animals that were injected with the oligo displayed elevated plasma high-density lipoprotein levels.

To extend these data to primates, the Mass General/Santaris group ran screening and in vivo experiments and chose three oligos for testing: two 16-mer LNAs that targeted miR-33a and miR-33b, respectively, and a tiny LNA that inhibited both miRNAs.

The researchers then selected a population of 20 obese African green monkeys, which were fed a high-fat diet with excess cholesterol for 12 weeks in order to induce the abnormalities observed in humans with metabolic syndrome such as insulin resistance and abnormal low-density lipoprotein levels.

The animals were divided into four groups of five, and then treated with subcutaneous doses of either the miR-33a-targeting LNA, the miR-33b-targeting LNA, the tiny LNA against both miRNAs, or a control vehicle. The monkeys all received an initial dose of 20 mg/kg and then weekly doses of 5 mg/kg for a total of 108 days.

After eight weeks of treatment, the animals were shifted to a high-sugar diet in order to mimic the effects of a high-carbohydrate diet.

To assess the effects of the LNAs, levels of HDL and LDL cholesterol were measured weekly, as were total cholesterol and triglycerides.

The investigators found that treatment with the tiny LNA increased HDL levels by as much as 39 percent compared with controls, while the longer LNAs had little effect on circulating HDL — a finding that the team said highlights the redundancy of miR-33a and miR-33b and the importance of targeting both family members simultaneously.

Liver biopsies showed strong derepression of ABCA1 by the tiny LNAs, while qRT-PCR revealed a "modest increase" in the hepatic expression of selected miR-33 targets, according to the Science Translational Medicine paper. In contrast, the miRNA-specific 16-mer LNAs were "less efficacious and inconsistent in their effects on direct miR-33 targets."

Looking to determine the safety of tiny LNA treatment, the research group analyzed the oligo's plasma stability after the last dose, finding that its terminal half-life was 17.5 days — comparable to the half-life of other LNA-modified antisense molecules between 12 and 16 nucleotides.

"No evidence of injection site reactions was observed after loading dose or maintenance dose administration," the team wrote in its paper. "In addition, no adverse reactions were noted during routine cage-side observations, indicating a lack of local toxicity."

An analysis of blood samples showed that markers of liver function, such as alanine aminotransferase and aspartate aminotransferase, were "not markedly changed" after treatment with the tiny LNAs, suggesting the absence of hepatic toxicity. Kidney toxicity parameters also remained in the normal range.

Finally, liver biopsy analysis indicated no hepatic steatosis among any of the treated animals, and no inflammation or fibrosis was observed, all of which indicated that tiny LNA treatment for over 100 days was well tolerated in the primates.

Taken together, the study results open the door to "clinical studies of seed-targeting antimiR-33a/b inhibitors for the treatment of cardiovascular disease in humans, as well as the potential use of seed-targeting 8-mer LNA-modified antimiRs for the inhibition of other miRNA families in human diseases," the authors concluded.

Despite its ongoing interest in tiny LNAs and its rights to the miR-33 IP from Mass General, however, Santaris has been tightlipped on any expansion of its miRNA therapeutics efforts beyond miravirsen.

Company officials were not available for comment.

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