LNA-mediated microRNA silencing in non-human primates

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Abstract
The realization that microRNAs play a central role in gene regulation in both normal development and disease, such as cancer, cardiac disease and metabolic disorders, suggests that they might be viable targets for therapeutic intervention. Now, for the first time, efficient, long-lasting and reversible microRNA silencing has been achieved in non-human primates. African green monkeys received intraperitoneal injections of a short modified DNA sequence that binds to and blocks the function of miR-122, a microRNA that regulates genes that influence cholesterol levels. Dose-dependent lowering of blood cholesterol followed, with no signs of toxicity. It is still a giant leap to therapeutic applications and, as microRNAs can act on many genes, toxicity risks are considerable. Small interfering RNAs (siRNAs) can be systemically administered in non-human primates and significantly reduce expression from the mRNA against which they are directed. A therapeutic effect can also be achieved by targeting a microRNA (miRNA). A modified oligonucleotide directed against miR-122 was administered to monkeys, resulting in a reduction in serum cholesterol without detectable toxicity. microRNAs (miRNAs) are small regulatory RNAs that are important in development and disease1,2,3 and therefore represent a potential new class of targets for therapeutic intervention4. Despite recent progress in silencing of miRNAs in rodents5,6, the development of effective and safe approaches for sequence-specific antagonism of miRNAs in vivo remains a significant scientific and therapeutic challenge. Moreover, there are no reports of miRNA antagonism in primates. Here we show that the simple systemic delivery of a unconjugated, PBS-formulated locked-nucleic-acid-modified oligonucleotide (LNA-antimiR) effectively antagonizes the liver-expressed miR-122 in non-human primates. Acute administration by intravenous injections of 3 or 10 mg kg-1 LNA-antimiR to African green monkeys resulted in uptake of the LNA-antimiR in the cytoplasm of primate hepatocytes and formation of stable heteroduplexes between the LNA-antimiR and miR-122. This was accompanied by depletion of mature miR-122 and dose-dependent lowering of plasma cholesterol. Efficient silencing of miR-122 was achieved in primates by three doses of 10 mg kg-1 LNA-antimiR, leading to a long-lasting and reversible decrease in total plasma cholesterol without any evidence for LNA-associated toxicities or histopathological changes in the study animals. Our findings demonstrate the utility of systemically administered LNA-antimiRs in exploring miRNA function in rodents and primates, and support the potential of these compounds as a new class of therapeutics for disease-associated miRNAs.