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Isis Shows Targeting miRNA with Antisense Benefits Obese Mice; More Work Remains Before the Clinic

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Isis Pharmaceuticals this week released preclinical data from its microRNA drug-discovery program showing that antisense-based inhibition of miR-122 improved the health of obese mice without significant side effects.

While the findings support further development of drugs targeting miR-122, Isis Senior Vice President of Research Frank Bennett noted that the science of microRNAs is still new, and that additional research is needed to fully elucidate their role before clinical trials could proceed.

In the study, mice genetically engineered to be obese -- so-called ob/ob mice -- were given once-weekly intraperitoneal injections of either an miR-122-targeting antisense agent or a control in saline, both at 25 mg/kg, for 4.5 weeks.

According to Bennett, without treatment the mice "end up developing type II diabetes, very high blood glucose, accumulate fat in the liver … and start showing a lot of liver damage." However, with antisense treatment, the animals' experienced a significant drop in plasma cholesterol levels and a decrease in hepatic triglyceride levels, he said. The researchers also observed a reduction in steatosis through a decrease in liver enzymes, namely plasma ALT and AST, which indicates improved liver function.


Without treatment the mice "end up developing type II diabetes, very high blood glucose, accumulate fat in the liver … and start showing a lot of liver damage."

The antisense treatment did not result in changes in plasma glucose levels or glucose tolerance, and no overt toxicities, change in food intake, or change in body weight were observed, Isis said. In fact, rather than causing adverse reactions, antisense treatment "made the animals healthier," Bennett added.

The data, which were presented earlier this week at the American Diabetes Association's 66th Annual Scientific Sessions in Washington, DC, are an extension of work Isis had published earlier this year in Cell Metabolism, Bennett said.

In that paper, company researchers showed that inhibiting miR-122 with antisense reduced plasma cholesterol levels, increased hepatic fatty-acid oxidation, and decreased hepatic fatty-acid and cholesterol synthesis rates in normal mice -- again without side effects.

"We extended the work into mice that were placed on a high-fat diet," Bennett said. "In those animals, the liver becomes very intoxicated with fat accumulation until you see a large amount of triglycerides in the liver, very high cholesterol, very high circulating triglycerides, and then you end up [with] liver injury. [In Cell Metabolism] we demonstrated that we got a clearing of fat out of the liver, decreased serum triglycerides, and an improved cholesterol effect in the [high-fat] animals."

Taken together, the preclinical data from Cell Metabolism and the ADA presentation indicate that miR-122 plays a role in regulating cholesterol and lipid metabolism, and that it may make a good therapeutic target. As such, Bennett said, Isis is advancing the program with the ultimate goal of investigating whether antisense could be used to target the miRNA in a clinical setting.

"The next step is to continue to flesh out the pharmacology, and then conduct toxicology studies with it," he said. Additionally, "there is some [structure-activity relationship work] that we're continuing to do to further optimize … where you place the chemistries in the oligonucleotide."

Bennett noted that the company is using a second-generation antisense technology, which incorporates a chemistry called 2'-methoxyethyl and has significant benefits over its first generation of antisense compounds.

According to Isis, the 2'-methoxyethyl modification has been added to the sulfur modification that characterized the company's first-generation compounds, which improves target binding affinity.

"Second-generation drugs are composed of both RNA-like and DNA-like nucleotides, while first-generation drugs are entirely DNA-like," the company noted. "Because RNA hybridizes more tightly to RNA than to DNA, the second-generation drugs have a greater affinity for their RNA targets and, therefore, greater potency." The second-generation compounds also show greater resistance to nuclease degradation, which slows their clearance from the body, and have an improved side-effect profile.


"MicroRNAs are a new area of science, and it's really not known what the different functions microRNAs are playing in normal physiology, [let alone] pathophysiology. We want to proceed cautiously so that we don't put patients at risk."

Currently, Isis has 13 second-generation antisense compounds in the clinic for various indications.

Although "with antisense-based technologies, you can very rapidly go from concept to the clinic," Bennett cautioned that an miRNA-targeting antisense drug isn't something that will reach human testing anytime soon.

"What's [mostly] missing now for microRNA drug discovery is identifying clinical utility for modulating a microRNA," he told RNAi News. Although the work presented at ADA is a step in the right direction, "microRNAs are a new area of science, and it's really not known what the different functions microRNAs are playing in normal physiology, [let alone] pathophysiology."

He added that in its miRNA drug-discovery program, Isis is primarily using antisense "as a vehicle to identify which microRNAs produce meaningful effects when you inhibit their function -- basically as a target-validation tool. Once we've validated it, scientists can pursue it therapeutically."

According to Bennett, the scientific community is only "beginning to define in more detail what this particular microRNA, [miR-122,] is doing."

Aside from Isis' findings, he noted that Stanford University researcher Peter Sarnow has also published data linking miR-122 to hepatitis C infection. In the September 2005 issue of Science, Sarnow and colleagues reported that miR-122 appears to facilitate replication of the hepatitis C virus, raising the possibility that it may make a target for antiviral intervention.

As the most abundant miRNA in the liver, miR-122 has also been the subject of research of several industry groups, as well. Rockefeller University researcher and Alnylam Pharmaceuticals advisor Markus Stoffel recently published in Nature data describing the use of novel miRNA-targeting agents to knock down miR-122 (see RNAi News, 11/4/2005). Additionally, Santaris Pharma has experimented with inhibiting the miRNA using its proprietary locked nucleic acids (see RNAi News, 5/4/2006).

"The data we're seeing thus far is encouraging, [and miR-122 is] a very attractive target for us," Bennett said. But miRNAs are "a new area of biology that we don't understand. We want to proceed cautiously so that we don't put patients at risk."

-- Doug Macron ([email protected])

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