Compositions for Inhibiting Gene Expression and Uses Thereof

Abstract

The inventors have examined the means for providing more efficacious miRNA blocking compounds. The inventors have discovered new structural features that surprisingly improve the efficacy of miRNA blocking molecules. These features include the presence of multiple 3′ ends and a linker at the 5′ ends. Surprisingly, these features improve the efficacy of the gene expression blocking compounds in a manner that decreases the compound's biologic instability. Even more surprisingly, this effect has been found to be applicable to both DNA and RNA oligonucleotide-based compounds and to have application in traditional antisense and RNAi technology.

What is claimed is: 
     
         1 . A method for therapeutically treating a mammal having a disease or disorder mediated by a miRNA, the method comprising administering to the mammal a synthetic oligonucleotide-based compound comprising two single stranded oligonucleotides that are complementary to a miRNA sequence, wherein the oligonucleotides are linked at their 5′-ends such that the oligonucleotide-based compound has two accessible 3′-ends, and the oligonucleotides specifically hybridize to and inhibit the activity of the miRNA sequence. 
     
     
         2 . A method for preventing a disease or disorder mediated by one or more miRNA in a mammal at risk for developing the disease or disorder, the method comprising administering to the mammal a synthetic oligonucleotide-based compound comprising two single stranded oligonucleotides that are complementary to a miRNA sequence, wherein the oligonucleotides are linked at their 5′-ends such that the oligonucleotide-based compound has two accessible 3′-ends, and the oligonucleotides specifically hybridize to and inhibit the activity of the miRNA sequence. 
     
     
         3 . The method according to  claim 1  or  2  wherein the disease or disorder is cancer. 
     
     
         4 . The method according to  claim 1  or  2 , wherein the mammal is a human. 
     
     
         5 . The method according to  claim 1  or  2  wherein the miRNA sequence is miR-21. 
     
     
         6 . The method according to  claim 1  or  2 , wherein the oligonucleotides are independently 15 to 22 nucleotides in length. 
     
     
         7 . The method according to  claim 1  or  2 , wherein the oligonucleotides comprise one or more ribonucleotides, deoxyribonucleotides, locked nucleic acids, arabino sugar nucleotides or a combination thereof. 
     
     
         8 . The method according to  claim 1  or  2 , wherein at least one of the oligonucleotides is modified. 
     
     
         9 . The method according to  claim 8 , wherein the modified oligonucleotide has at least one internucleotide linkage selected from the group consisting of alkylphosphonate, phosphorothioate, phosphorodithioate, methylphosphonate, and non-nucleotide linker. 
     
     
         10 . The method according to  claim 8 , wherein the modified oligonucleotide comprises at least one 2′-O-substituted nucleotide. 
     
     
         11 . The method according to  claim 10 , wherein the 2′-O-substitution is selected from 2′-β-methyl, 2′-O-methoxy, 2′-O-ethoxy, 2′-O-methoxyethyl, 2′-O-alkyl, 2′-O-aryl, and 2′-O-allyl. 
     
     
         12 . The method according to  claim 1  or  2 , further comprising administering one or more vaccines, antigens, antibodies, cytotoxic agents, allergens, antibiotics, antisense oligonucleotides, TLR agonist, TLR antagonist, siRNA molecules, miRNA molecules, antisense oligonucleotides, aptamers, proteins, peptides, gene therapy vectors, adjuvants, kinase inhibitors, chemotherapeutic agents, targeted therapeutic agents, activated cells, co-stimulatory molecules or combinations thereof. 
     
     
         13 . The method according to  claim 12 , wherein the vaccine is a DNA vaccine, peptide vaccine, or protein vaccine. 
     
     
         14 . A method for inhibiting in vivo miRNA activity, the method comprising administering to a mammal an oligonucleotide-based compound comprising two single-stranded oligonucleotides that are complementary to a miRNA sequence, wherein the oligonucleotides are linked at their 5′-ends such that the oligonucleotide-based compound has two accessible 3′-ends, wherein at least one of the oligonucleotides is modified, and the oligonucleotides specifically hybridize to and inhibit the activity of the miRNA sequence. 
     
     
         15 . The method according to  claim 14 , wherein the oligonucleotides are independently 15 to 22 nucleotides in length. 
     
     
         16 . The method according to  claim 14 , wherein the oligonucleotides comprise one or more ribonucleotides, deoxyribonucleotides, locked nucleic acids, arabino sugar nucleotides or a combination thereof. 
     
     
         17 . The method according to  claim 14 , wherein the modified oligonucleotide has at least one internucleotide linkage selected from the group consisting of alkylphosphonate, phosphorothioate, phosphorodithioate, methylphosphonate, and non-nucleotide linker. 
     
     
         18 . The method according to  claim 14 , wherein the modified oligonucleotide comprises at least one 2′-O-substituted nucleotide. 
     
     
         19 . The method according to  claim 18 , wherein the 2′-O-substitution is selected from 2′-β-methyl, 2′-O-methoxy, 2′-O-ethoxy, 2′-O-methoxyethyl, 2′-O-alkyl, 2′-O-aryl, and 2′-O-allyl. 
     
     
         20 . The method according to  claim 14 , further comprising administering one or more vaccines, antigens, antibodies, cytotoxic agents, allergens, antibiotics, antisense oligonucleotides, TLR agonist, TLR antagonist, siRNA molecules, miRNA molecules, antisense oligonucleotides, aptamers, proteins, peptides, gene therapy vectors, adjuvants, kinase inhibitors, chemotherapeutic agents, targeted therapeutic agents, activated cells, co-stimulatory molecules or combinations thereof. 
     
     
         21 . The method according to  claim 20 , wherein the vaccine is a DNA vaccine, peptide vaccine, or protein vaccine.