CHEMICALLY MODIFIED OLIGONUCLEOTIDES FOR USE IN MODULATING MICRO RNA AND USES THEREOF
This invention relates generally to chemically modified oligonucleotides useful for modulating expression of microRNAs and pre-microRNAs. More particularly, the invention relates to single stranded chemically modified oligonucleotides for inhibiting microRNA and pre-microRNA expression and to methods of making and using the modified oligonucleotides. Also included in the invention are compositions and methods for silencing microRNAs in the central nervous system.
1 . A method of reducing the amount of a microRNA in a cell in a subject comprising the step of administering an antagomir to the subject, wherein the oligonucletide agent is substantially single-stranded, comprises a sequence which is substantially complementary to 12 to 23 contiguous nucleotides of a target sequence, and wherein the target sequence differs by no more than 1, 2, or 3 nucleotides from a sequence selected from the group consisting of those provided in Table 1.
2 . The method of claim 1 , wherein said antagomir is selected from the group consisting of those provided in Tables 2a-e and Table 4.
3 . An isolated oligonucleotide agent, comprising a nucleotide sequence sufficiently complementary to a microRNA target sequence of about 12 to 23 nucleotides, wherein the target sequence differs by no more than 1, 2, or 3 nucleotides from a sequence selected from the group consisting of those provided in Table 1.
4 . The antagomir of claim 3 , comprising a nucleotide sequence sufficiently complementary to a target sequence of about 15 to 23 nucleotides of the microRNA nucleotide sequence, and wherein the target sequence differs by no more than 1, 2, or 3 nucleotides from a sequence selected from the group consisting of those provided in Table 1.
5 . The antagomir of claim 3 , wherein the antagomir further comprises a non-nucleotide moiety.
6 . The antagomir of claim 3 , wherein the antagomir is stabilized against nucleolytic degradation.
7 . The antagomir of claim 3 , further comprising a phosphorothioate at the first internucleotide linkage at the 5′ end of the nucleotide sequence.
8 . The antagomir of claim 3 , further comprising a phosphorothioate at the first internucleotide linkage at the 3′ end of the nucleotide sequence.
9 . The antagomir of claim 3 , further comprising a phosphorothioate at the first internucleotide linkage at the 5′ end of the nucleotide sequence, and a phosphorothioate at the first internucleotide linkage at the 3′ end of the nucleotide sequence.
10 . The antagomir of claim 3 , further comprising a 2′-modified nucleotide.
11 . The antagomir of claim 10 , wherein the 2′-modified nucleotide comprises a modification selected from the group consisting of: 2′-deoxy, 2′-deoxy-2′-fluoro, 2′-O-methyl, 2′-O-methoxyethyl (2′-O-MOE), 2′-O-aminopropyl (2′-O-AP), 2′-O-dimethylaminoethyl (2′-O-DMAOE), 2′-O-dimethylaminopropyl (2′-O-DMAP), 2′-O-dimethylaminoethyloxyethyl (2′-O-DMAEOE), and 2′-O—N-methylacetamido (2′-O-NMA).
12 . The antagomir of claim 10 , wherein the 2′-modified nucleotide comprises a 2′-O-methyl.
13 . The antagomir of claim 3 , further comprising a cholesterol molecule attached to the 3′ end of the agent.
14 . The antagomir of claim 3 , wherein the antagomir is selected from the group consisting of those provided in Table 2a-e and Table 4.
15 . A method of reducing the amount of miR-122 in a cell, comprising contacting the cell with an antagomir of claim 3 , wherein the sequence selected from the group consisting of those provided in Table 1 is miR-122.
16 . A method of making an antagomir of claim 3 , the method comprising the synthesis of the oligonucleotide agent, wherein the nucleotide sequence comprises at least one modification that stabilizes the antagomir against nucleolytic degradation.
17 . A pharmaceutical composition comprising an antagomir of claim 3 and a pharmaceutically acceptable carrier.
18 . A method of inhibiting miR-122 expression in a cell, comprising contacting the cell with an effective amount of an antagomir of claim 3 , wherein the sequence selected from the group consisting of those provided in Table 1 is miR-122.
19 . A method of increasing aldolase-A protein levels in a cell comprising contacting the cell with an effective amount of an antagomir of claim 3 , wherein the sequence selected from the group consisting of those provided in Table 1 is miR-122.