The molecular function of an individual miRNA can be discovered by inhibiting it and measuring the resulting changes in the levels of each mRNA or protein in the cell or by evaluating other phenotypic changes, such as developmental defects, cell proliferation, organ function, lipid metabolism, or behavior. ASOs engineered to withstand degradation by extra- and intracellular nucleases can effectively inhibit miRNAs in whole animals34,35
. The first ASOs used to inhibit miRNAs were composed of 22-O
-methyl ribose-modified RNA. Such 2′-O
-methyl oligonucleotides proved to be effective miRNA inhibitors when introduced by lipid-mediated transfection into cultured human cells or by injection in whole nematodes (Caenorhabditis elegans
). Dextran-conjugated ASOs can be injected into the C. elegans
germ line and block the function of a specific miRNA function in the progeny36
“Antagomirs” were the first miRNA inhibitors demonstrated to work in mammals (). Because the amount of pre-miRNA was unchanged by the antagomir, ASOs likely target the mature miRNA37,38
. These synthetic ASOs contain 2′-O
-methyl modified ribose sugars, terminal phosphorothioates, and at the 3′ end a cholesterol group, which helps deliver the antagomir to cells. Cholesterol conjugation causes cellular uptake of the modified nucleic acid oligonucleotide by promoting its association with high-and low-density lipoproteins that can bind cell surface membrane receptors: the Scavenger receptor BI for HDL and the LDL receptor for LDL. Intravenous injection of 80 mg of antagomir per kg mouse body weight on each of three successive days inhibited the corresponding miRNA in mouse liver, lung, kidney, heart, intestine, fat, skin, bone marrow, muscle, ovaries and adrenal glands. Nonetheless, antagomirs are unlikely to be used clinically, as they require higher doses to achieve the same efficacy as other ASO strategies.
Alternative RNA chemistries () such as 2′-O
-methoxyethyl (2′-MOE), 2′-fluoro, and 2′,4′ methylene (“locked nucleic acids” or LNAs) have greater affinity to bind and inhibit miRNA function in vivo than 2′-O
-methyl RNA oligonucleotides39-41
. Alternative chemistries are also more resistant to degradation. In a test of stability of modified RNAs in 10% fetal bovine serum, 2′-fluoro RNA with LNA ends was less degraded after 24 hours than a 2′-O
-methyl RNA with LNA ends or a DNA/LNA oligonucleotide, which was degraded within 2 hours35
. Phosphorothioates substitute a non-bridging oxygen atom on the phosphate group with a sulfur. Phosphorothioate bonds promote serum protein binding, thereby increasing the in vivo distribution and bioavailability of the ASO. A direct comparison of anti-miRNA oligonucleotide chemistries in vitro revealed that combining 2′-O
-methyl and LNA with phosphorothioate ends was ~10 times more effective than the 2′-O
-methyl or phosphorothioate modifications alone and twice as effective as the 2′-O
-methyl with LNA modifications35
Figure 4 Strategies for delivery of anti-miRNA oligonucleotides to cells in vivo. (A) Modification. Black filled circles represent 2′-O-methyl, 2′-O-methoxyethyl, or 2′-fluoro modified nucleotides. (B) Conjugation. Antagomirs are 2′- (more ...)
The 2′,4′ methylene bridge in LNAs constrains the ribose to the C3′ endo conformation present in RNA:RNA and DNA:RNA helices. (DNA:DNA helices are C2′ endo.) LNAs cannot interconvert between the C3′ endo conformation, which favors pairing with an RNA, and the C2′ endo conformation, which does not. Consequently, an LNA modification increases RNA:RNA melting temperature by 2.4□ per modification and confers high specificity for their target sequences. Moreover, locked nucleic acids are resistant to many endonucleases. ASOs containing LNAs are effective probes for accurate detection of miRNAs by Northern blotting, in situ hybridization and, most importantly, are potent miRNA inhibitors in vivo.
The unique target mRNA-binding properties of a miRNA bound to an Argonaute protein—nearly all the binding specificity comes from the seed sequence—allow antisense miRNA inhibitors to be shorter than the miRNA itself. A 16 nt LNA-modified oligonucleotide complementary to miRNA-122 injected intravenously each day for five successive days at 10 mg/kg, lowered plasma cholesterol levels for more than 20 days in African green monkeys42
. This pioneering non-human primate study established that LNA-modified anti-miRNA oligonucleotides are specific, stable, and non-toxic when administered intravenously. A subsequent study showed that Hepatitis C virus (HCV) replication could be inhibited in chimpanzees by a 15 nt LNA oligonucleotide targeting miRNA-12243
. Two chimpanzees were injected intravenously with 5 mg per kg of LNA inhibitor each week for 12 weeks. Two weeks after treatment ended, viral titer was 400- and 200-times lower in serum and liver, respectively. Free anti-miR-122 LNA was detected in liver for 8 weeks after treatment ceased, until week 25, at which point the drug had declined significantly and the level of miR-122 had increased. No liver toxicity was detected, and treatment was associated with improved liver histology, presumably due to prolonged suppression of viremia and normalization of the interferon pathway. No viral escape was detected by sequence analysis of the HCV RNA target sites for miR-122 at the 16th week, in contrast to treatment with an antiviral non-nucleoside polymerase inhibitor with which resistance mutations occur after 2 days of treatment. miRNA-122 inhibition by LNA-modified oligonucleotides is now being tested in humans. A successful phase 1 trial has paved the way for a phase 2 study that will assess the safety and tolerability of weekly or bi-weekly subcutaneous injections of the anti-miR-122 LNA in 55 patients with chronic HCV genotype 1 infection.