Compositions for inhibiting gene expression and uses thereof

Abstract

The inventors have examined the means for providing more efficacious gene expression blocking compounds. The inventors have discovered new structural features that surprisingly improve the efficacy of gene expression 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 technologies.

1 . A synthetic oligonucleotide-based compound comprising two or more oligonucleotides that are complementary to one or more single-stranded RNA sequence, wherein the oligonucleotide forms a sufficient number of hydrogen bonds through Watson-Crick interactions of its nucleobases with nucelobases of the single-stranded RNA sequence to form a double helix with the single-stranded RNA sequence under physiological conditions, wherein the oligonucleotides are linked at their 5′-ends, such that the oligonucleotide-based compound has two or more accessible 3′-ends and the oligonucleotide-based compound specifically hybridize to and inhibit the expression of the one or more single-stranded RNA sequence. 
     
     
         2 . The oligonucleotide-based compound of  claim 1 , wherein the oligonucleotides are independently 15 to 40 nucleotides in length. 
     
     
         3 . The oligonucleotide-based compound of  claim 1 , wherein the oligonucleotides are linked to each other through a nucleotide linkage. 
     
     
         4 . The oligonucleotide-based compound of  claim 1 , wherein the oligonucleotides are linked to each other through a linker. 
     
     
         5 . The oligonucleotide-based compound of  claim 4 , wherein the linker is a non-nucleotide linker. 
     
     
         6 . The oligonucleotide-based compound of  claim 1 , wherein the oligonucleotides comprise one or more ribonucleotides, deoxyribonucleotides, locked nucleic acids, arabino sugar nucleotides or a combination thereof. 
     
     
         7 . The oligonucleotide-based compound of  claim 1 , wherein at least one of the oligonucleotides is modified. 
     
     
         8 . The oligonucleotide-based compound of  claim 7 , wherein the modified oligonucleotide has at least one intemucleotide linkage selected from the group consisting of alkylphosphonate, phosphorothioate, phosphorodithioate, methylphosphonate, and non-nucleotide linker. 
     
     
         9 . The oligonucleotide-based compound of  claim 7 , wherein the modified oligonucleotide comprises at least one 2′-O-substituted nucleotide. 
     
     
         10 . The oligonucleotide-based compound of  claim 9 , wherein the 2′-O-substitution is selected from 2′-O-methyl, 2′ -O-methoxy, 2′-O-ethoxy, 2′-O-methoxyethyl, 2′-O-aryl, and 2′-O-allyl. 
     
     
         11 . The oligonucleotide-based compound according to  claim 1 , wherein the oligonucleotide-based compound is selectively bound and/or cleaved by a protein involved in RNAi-mediated inhibition of gene expression. 
     
     
         12 . The oligonucleotide-based compound according to  claim 11 , wherein the protein involved in RNAi-mediated inhibition of gene expression is selected from Dicer, Argonaut, and RISC. 
     
     
         13 . The oligonucleotide-based compound according to  claim 1 , wherein the oligonucleotide-based compound is selectively bound and/or cleaved by a protein involved in RNaseH-mediated inhibition of gene expression. 
     
     
         14 . A composition comprising an oligonucleotide-based compound according to  claim 1  and a physiologically acceptable carrier. 
     
     
         15 . An oligonucleotide-based compound comprising two oligonucleotides that are complementary to one or more single-stranded RNA sequence, wherein the oligonucleotide forms a sufficient number of hydrogen bonds through Watson-Crick interactions of its nucleobases with nucelobases of the single-stranded RNA sequence to form a double helix with the single-stranded RNA sequence under physiological conditions, wherein the oligonucleotides are linked at their 5′-ends, such that the oligonucleotide-based compound has two accessible 3′-ends and the oligonucleotide-based compound specifically hybridizes to and inhibit the expression of the one or more single-stranded RNA sequence, the oligonucleotide-based compound having the structure: 
       
         
           
           
               
               
           
         
       
       wherein at least one of Domain A or Domain B is an oligoribonucelotide that is hybridized to a complementary oligoribonucleotide to form an siRNA. 
     
     
         16 . An oligonucleotide-based compound comprising three oligonucleotides that are complementary to one or more single-stranded RNA sequence, wherein the oligonucleotide forms a sufficient number of hydrogen bonds through Watson-Crick interactions of its nucleobases with nucelobases of the single-stranded RNA sequence to form a double helix with the single-stranded RNA sequence under physiological conditions, wherein the oligonucleotides are linked at their 5′-ends, such that the oligonucleotide-based compound has two or more accessible 3′-ends and the oligonucleotide-based compound specifically hybridizes to and inhibit the expression of the one or more single-stranded RNA sequence, the oligonucleotide-based compound having the structure: 
       
         
           
           
               
               
           
         
       
       wherein at least one of Domain A, Domain B, or Domain C is an oligoribonucleotide that is hybridized to a complementary oligoribonucleotide to form an siRNA molecule. 
     
     
         17 . An oligonucleotide-based compound comprising three oligonucleotides that are complementary to one or more single-stranded RNA sequence, wherein the oligonucleotide forms a sufficient number of hydrogen bonds through Watson-Crick interactions of its nucleobases with nucelobases of the single-stranded RNA sequence to form a double helix with the single-stranded RNA sequence under physiological conditions, wherein two of the oligonucleotides are linked at their 5′-ends, such that the oligonucleotide-based compound has two accessible 3′-ends and the oligonucleotide-based compound specifically hybridizes to and inhibit the expression of the one or more single-stranded RNA sequence, the oligonucleotide-based compound having the structure: 
       
         
           
           
               
               
           
         
       
       wherein at least one of Domain A, Domain B, or Domain C is an oligoribonucleotide that is hybridized to a complementary oligoribonucleotide to form an siRNA molecule. 
     
     
         18 . An oligonucleotide-based compound comprising four oligonucleotides that are complementary to one or more single-stranded RNA sequence, wherein the oligonucleotide forms a sufficient number of hydrogen bonds through Watson-Crick interactions of its nucleobases with nucelobases of the single-stranded RNA sequence to form a double helix with the single-stranded RNA sequence under physiological conditions, wherein the oligonucleotides are linked at their 5′-ends, such that the oligonucleotide-based compound has two accessible 3′-ends and the oligonucleotide-based compound specifically hybridizes to and inhibits the expression of the one or more single-stranded RNA sequence, the oligonucleotide-based cnmnnund having the structure: 
       
         
           
           
               
               
           
         
       
       wherein at least one of Domain A or Domain B or Domain C or Domain D is an oligoribonucleotide that is hybridized to a complementary oligoribonucleotide to form an siRNA molecule. 
     
     
         19 . An oligonucleotide-based compound comprising three oligonucleotides, wherein the oligonucleotide forms a sufficient number of hydrogen bonds through Watson-Crick interactions of its nucleobases with nucelobases of the single-stranded RNA sequence to form a double helix with the single-stranded RNA sequence under physiological conditions, wherein two of the oligonucleotides are linked at their 5′-ends, such that the oligonucleotide-based compound has two accessible 3′-ends, the oligonucleotide-based compound having the structure: 
       
         
           
           
               
               
           
         
       
       wherein at least one of Domain A, Domain B, or Domain C is an oligonucleotide that is complementary to a single-stranded RNA sequence such that the oligonucleotide-based compound specifically hybridizes to and inhibit the expression of at least one single-stranded RNA sequence, and wherein at least one of Domain A, Domain B, or Domain C is an oligonucleotide that is an antagonist of an intracellular or extracellular receptor. 
     
     
         20 . The oligonucleotide-based compound according to  claim 19 , wherein the receptor is a TLR.