NEW YORK (GenomeWeb News) – For the first time, researchers have reported specifically silencing a microRNA in a non-human primate, the African green monkey.
Scientists from the Danish biopharmaceutical company Santaris Pharma and elsewhere used an engineered, “locked” RNA oligonucleotide to silence a liver-specific miRNA — miR-122 — that is involved in lipid metabolism and hepatitis C infection. As expected, the researchers found a dose-dependent decrease in blood cholesterol in the treated animals, without any obvious side effects. The results, published online today in the journal Nature, are a promising step towards developing miRNA-based therapeutics.
Santaris CFO and Vice President of Finance and Administration Henrik Stage announced similar results at the Acumen Biofin Rodman & Renshaw Healthcare conference last November.
MicroRNAs are small, non-coding RNAs that bind to messenger RNAs and hinder their expression. Various miRNAs have been tied to everything from development to disease, including cancer, cardiovascular disease, and viral infections.
For this study, Santaris researchers, along with collaborators at Stanford University, RxGen, UB-in situ, and the University of Copenhagen, evaluated an miRNA antagonist that targets miR-122, a liver expressed miRNA that seems to influence both lipid and cholesterol metabolism and hepatitis C virus replication.
The putative therapeutic was developed using so-called locked-nucleic-acid-modified oligonucleotides — modified nucleic acids with enhanced specificity to their RNA or DNA targets and increased thermal stability. Specifically, Santaris’ locked nucleic acid technology is based on an RNA analogue with an altered ribose sugar that potently binds RNA.
Before they tested this LAN-antimiR in primates, though, the researchers did several experiments in Huh-7 human hepatoma and HeLa cell lines and mouse models to evaluate various sequence-specific miR-122 antagonists and to test the effectiveness of their main LAN-antimiR of interest.
For instance, the team injected mice with the LAN-antimiR daily for five days, comparing it to saline or a mismatched LNA control. They saw increased expression of a gene that’s suppressed by miR-122 and dose-dependent decreases in plasma cholesterol. The expression of other mouse liver genes, assessed using Affymetrix Mouse Genome 430 2.0 arrays, also changed when the mice were injected with the miR-122 specific LAN.
In an effort to determine whether the miRNA antagonist had similar effects in non-human primates, the researchers intravenously injected it into thirty female African green monkeys. After giving three injections two days apart, at doses ranging from one to ten milligrams per kilogram, Northern blot analysis indicated that the targeted miRNA, miR-122, was indeed being bound by the LAN-antimiR and depleted.
And the team saw a dose-dependent decrease in total plasma cholesterol in the monkeys — up to 40 percent in the high-dose group — though there were fluctuations over time. This decrease lasted up to seven weeks (again in the high-dose group) and levels returned to baseline after about three months.
Based on clinical chemistry, blood coagulation profiles, and histopathology of biopsied livers, the researchers did not detect LAN-antimirR related toxicity in the monkeys. The LAN-antimiR treated animals remained in good health at least ten months after receiving the treatment, the authors reported.
“Although additional studies will be needed to optimize the dosing regimen and to assess the biodistribution and safety of LNA-antimiR compounds after long-term treatment, our findings highlight the utility of LNA-mediated silencing in studying miRNA function in rodents and primates as well as in the future developments of miRNA-based therapeutics,” the authors wrote.
Even so, despite the promising results for hyperlipidemia, Santaris’ focus has long been on applying the miRNA antagonist to treating hepatitis C.
The company holds worldwide exclusive pharmaceutical rights to a three-dimensional locked nucleic acid analogue. Last December, the company entered into an agreement giving GlaxoSmithKline exclusive options to license antiviral, locked nucleic acid-based RNA antagonist drug candidates developed by Santaris that have completed Phase IIa trials.
At the end of December, Santaris’ Vice President and CSO Henrik Orum told GenomeWeb Daily News sister publication RNAi News that the company expects to undertake a Phase I trial of its miR-122 targeting molecule, this year, probably in Europe. Last month, at the annual BIO-CEO and Investor conference, President and CEO Keith McCullagh said the company is on track to start that trial in the first half of 2008.