Chemically modified oligonucleotides for use in modulating micro RNA and uses thereof

Abstract

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 (miRNA) in a cell of the central nervous system (CNS) in a mammal, the method comprising administering an antagomir to the mammal, wherein the antagomir 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 SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4.  
     
     
         2 . The method of  claim 1 , wherein the antagomir comprises a sequence selected from the group consisting of SEQ ID NOS: 5-39.  
     
     
         3 . The method of  claim 2 , wherein the antagomir further comprises a phosphorothioate backbone modification.  
     
     
         4 . The method of  claim 1 , wherein the target sequence is SEQ ID NO: 2.  
     
     
         5 . The method of  claim 2 , wherein the antagomir is at least nineteen nucleotides in length.  
     
     
         6 . The method of  claim 2 , wherein the antagomir is stabilized against nucleolytic degradation.  
     
     
         7 . The method of  claim 3 , wherein the phosphorothioate modification is at least at the first two internucleotide linkage at the 5′ end of the nucleotide sequence.  
     
     
         8 . The method of  claim 3 , wherein the phosphorothioate modification is at least at the first four internucleotide linkage at the 3′ end of the nucleotide sequence.  
     
     
         9 . The method of  claim 3 , wherein the phosphorothioate modification is at the first two internucleotide linkage at the 5′ end of the nucleotide sequence, and at the first four internucleotide linkage at the 3′ end of the nucleotide sequence.  
     
     
         10 . The method of  claim 2 , wherein the antagomir further comprises a 2′-modified nucleotide.  
     
     
         11 . The method 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 method of  claim 11 , wherein the 2′-modified nucleotide comprises a 2′-O-methyl.  
     
     
         13 . The method of  claim 2 , wherein the antagomir further comprises a cholesterol molecule attached to the 3′ end of the agent.  
     
     
         14 . A method of treating a mammal suffering from a disease, disorder or condition of the central nervous system, the method comprising administering an antagomir to the mammal, wherein the antagomir 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 SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4, further wherein the presence of the antagomir in the central nervous system effects treatment of the disease, disorder or condition.  
     
     
         15 . The method of  claim 14 , wherein the antagomir comprises a sequence selected from the group consisting of SEQ ID NOS: 5-39.  
     
     
         16 . The method of  claim 15 , wherein the antagomir further comprises a phosphorothioate backbone modifications.  
     
     
         17 . The method of  claim 14 , wherein the target sequence is SEQ ID NO: 2.  
     
     
         18 . The method of  claim 15 , wherein the antagomir is at least nineteen nucleotides in length.  
     
     
         19 . The method of  claim 15 , wherein the antagomir is stabilized against nucleolytic degradation.  
     
     
         20 . The method of  claim 16 , wherein the phosphorothioate modification is at least at the first two internucleotide linkage at the 5′ end of the nucleotide sequence.  
     
     
         21 . The method of  claim 16 , wherein the phosphorothioate modification is at least at the first four internucleotide linkage at the 3′ end of the nucleotide sequence.  
     
     
         22 . The method of  claim 16 , wherein the phosphorothioate modification is at the first two internucleotide linkage at the 5′ end of the nucleotide sequence, and at the first four internucleotide linkage at the 3′ end of the nucleotide sequence.  
     
     
         23 . The method of  claim 15 , wherein the antagomir further comprises a 2′-modified nucleotide.  
     
     
         24 . The method of  claim 23 , 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).  
     
     
         25 . The method of  claim 24 , wherein the 2′-modified nucleotide comprises a 2′-O-methyl.  
     
     
         26 . The method of  claim 15 , wherein the antagomir further comprises a cholesterol molecule attached to the 3′ end of the agent.  
     
     
         27 . The method of  claim 14 , wherein the mammal is a human.  
     
     
         28 . The method of  claim 14 , wherein said disease, disorder or condition of the central nervous system is selected from the group consisting of a genetic disease, a disease associated with unregulated expression of miR-16.