Chimeric single-stranded antisense polynucleotides and double-stranded antisense agent

Abstract

Chimeric single-stranded polynucleotides and double-stranded antisense agents useful for modifying the expression of a target gene by means of an antisense effect are disclosed. The chimeric single-stranded antisense polynucleotide and double-stranded antisense agents comprise a central nucleotide region flanked by a first 5′-wing region and a first 3′-wing region of modified nucleotides, which are themselves flanked by a second 5′-wing region and/or a second 3′-wing region of nucleotides that have a low affinity for proteins and/or that have higher resistance to DNase or RNase than a natural DNA or RNA and are missing in a cell when the chimeric polynucleotide delivered. The double-stranded antisense agent further comprises a complementary strand annealed to the antisense strand. The polynucleotide can be used to modify RNA transcription levels, miRNA activity, or protein levels in cells.

1 . A chimeric antisense polynucleotide comprising:
 a center nucleotide region comprising at least 5 nucleotides;   a first 5′-wing region joined to the 5′ end of the center nucleotide region comprising 1-10 nucleotides wherein at least 1 is a nucleotide analog;   a first 3′-wing region joined to the 3′ end of the center nucleotide region comprising 1-10 nucleotides wherein at least 1 is a nucleotide analog; and   a second 5′-wing region and/or a second 3′-wing region, wherein:   the second 5′-wing region is joined to the 5′ end of the first 5′-wing region and comprises at least 1 low protein-affinity nucleotide; and   the second 3′-wing region is joined to the 3′ end of the first 3′-wing region and comprises at least 1 low protein-affinity nucleotide;   wherein the total number of nucleotides, nucleotide analogs, and low protein-affinity nucleotides is no more than 100 nucleotides.   
     
     
         2 . A chimeric antisense polynucleotide comprising:
 a center nucleotide region comprising at least 5 nucleotides;   a first 5′-wing region joined to the 5′ end of the center nucleotide region comprising 1-10 nucleotides wherein at least 1 is a nucleotide analog;   a first 3′-wing region joined to the 3′ end of the center nucleotide region comprising 1-10 nucleotides wherein at least 1 is a nucleotide analog; and   a second 5′-wing region and/or a second 3′-wing region, wherein:   the second 5′-wing region is joined to the 5′ end of the first 5′-wing region, has higher resistance to DNase or RNase than a natural DNA or RNA and is missing in a cell when the chimeric polynucleotide delivered; and   the second 3′-wing region is joined to the 3′ end of the first 3′-wing region, has higher resistance to DNase or RNase than a natural DNA or RNA and is missing in a cell when the chimeric polynucleotide delivered,   
       wherein the total number of nucleotides and nucleotide analogs is no more than 100 nucleotides. 
     
     
         3 . The chimeric antisense polynucleotide of  claim 1 , wherein the center nucleotide region comprises nucleotides that, when hybridized to an RNA polynucleotide, the center nucleotide region/RNA polynucleotide duplex is recognized by RNase H. 
     
     
         4 . The chimeric antisense polynucleotide of  claim 3 , wherein the nucleotides of the center nucleotide region are independently selected from DNA and phosphorothioate DNA nucleotides. 
     
     
         5 . The chimeric antisense polynucleotide of  claim 4 , wherein the center nucleotide region comprises 5-20 DNA nucleotides. 
     
     
         6 . The chimeric antisense polynucleotide of  claim 4 , wherein the center nucleotide region comprises 5-12 DNA nucleotides. 
     
     
         7 . The chimeric antisense polynucleotide of  claim 1 , wherein the center nucleotide region comprises nucleotides independently selected from RNA nucleotides and nucleotide analogs. 
     
     
         8 . The chimeric antisense polynucleotide of  claim 7 , wherein the nucleotide analogs are independently selected from LNA nucleotides, BNA nucleotides, 2′-O-Me RNA nucleotides, 2′-O-methoxyethyl RNA nucleotides. 
     
     
         9 . The chimeric antisense polynucleotide of  claim 8 , wherein the center nucleotide region comprises nucleotides independently selected from 2′-O-Me RNA nucleotides and 2′-O-methoxyethyl RNA nucleotides. 
     
     
         10 . The chimeric antisense polynucleotide of  claim 1 , wherein at least one of the nucleotides in the center nucleotide region is phosphorothioated. 
     
     
         11 . The chimeric antisense polynucleotide of  claim 10 , wherein all nucleotides in the center nucleotide region are phosphorothioated. 
     
     
         12 . The chimeric antisense polynucleotide of  claim 1 , wherein the nucleotides in the first wing regions are bridged nucleotides. 
     
     
         13 . The chimeric antisense polynucleotide of  claim 1 , wherein the bridged nucleotides are independently selected from LNA, cEt BNA, amideBNA, and cMOE ENA. 
     
     
         14 . The chimeric antisense polynucleotides of  claim 1 , wherein at least one of the nucleotide analogs in the center nucleotide region and the first wing region(s) are phosphorothioated. 
     
     
         15 . The chimeric antisense polynucleotide of  claim 1 , wherein the low protein-affinity nucleotides are independently selected from 2′-O-methyl RNA nucleotides, 2′-O-methoxyethyl RNA nucleotides, LNA, cMOE BNA, 2-fluoro RNA nucleotides, boranophosphate nucleotides, methylphosphonate nucleotides, phosphoramidite nucleotides, 5-methylcytosine, UNA and 5-propynyluridine. 
     
     
         16 . The chimeric antisense polynucleotide of  claim 1 , wherein the chimeric antisense polynucleotide includes the second 5′-wing region. 
     
     
         17 . The chimeric antisense polynucleotide of  claim 1 , wherein the chimeric antisense polynucleotide includes the second 3′-wing region. 
     
     
         18 . The chimeric antisense polynucleotide of  claim 1 , wherein the chimeric antisense polynucleotide includes the second 5′-wing region and the second 3′-wing region. 
     
     
         19 . The chimeric antisense polynucleotide of  claim 1 , wherein the chimeric antisense polynucleotide further comprises a functional moiety joined to the 3′-end and/or the 5′-end of the chimeric antisense polynucleotide. 
     
     
         20 . The chimeric antisense polynucleotide of  claim 19 , wherein the functional moiety has a function selected from a labeling function, a purification function, and a targeted delivery function. 
     
     
         21 . The chimeric antisense polynucleotide of  claim 19 , wherein the functional moiety is joined to the chimeric antisense polynucleotide via a cleavable linker moiety. 
     
     
         22 . The chimeric antisense polynucleotide of  claim 19 , wherein the functional moiety is a molecule selected from a lipid, a peptide, and a protein. 
     
     
         23 . The chimeric antisense polynucleotides of  claim 22 , wherein the lipid is selected from cholesterol, a fatty acid, a lipid-soluble vitamin, a glycolipid, and a glyceride. 
     
     
         24 . The chimeric antisense polynucleotides of  claim 22 , wherein the lipid is selected from cholesterol, a tocopherol, and a tocotrienol. 
     
     
         25 . The chimeric antisense polynucleotides of  claim 22 , wherein the peptide is selected from a receptor ligand fragment and an antibody fragment. 
     
     
         26 . The chimeric antisense polynucleotide of  claim 22 , wherein the protein is selected from a receptor ligand and an antibody. 
     
     
         27 . The chimeric antisense polynucleotide of  claim 1 , wherein the chimeric antisense polynucleotide can hybridize to a cellular transcription product in a 100 mM sodium chloride, 10 mM sodium phosphate buffer, pH 7.2, at 25° C. 
     
     
         28 . The chimeric antisense polynucleotide of  claim 27 , wherein the center region is fully complementary to the cellular transcription product to which the chimeric antisense polynucleotide can hybridize. 
     
     
         29 . The chimeric antisense polynucleotide of  claim 27 , wherein the second 5′-wing region and/or the second 3′-wing region comprise at least one mismatched base when the chimeric antisense polynucleotide hybridizes to the cellular transcription product to which the chimeric antisense polynucleotide can hybridize. 
     
     
         30 . The chimeric antisense polynucleotide of  claim 29 , wherein all bases of the second 5′-wing region and/or the second 3′-wing region are mismatched. 
     
     
         31 . A double-stranded antisense agent comprising the chimeric antisense polynucleotide of  claim 1  and a complementary strand annealed to the chimeric antisense polynucleotide. 
     
     
         32 . The double-stranded antisense agent comprising the chimeric antisense polynucleotide of  claim 31 , wherein the complementary strand further comprises a functional moiety joined to the 3′-end and/or the 5′-end. 
     
     
         33 . The double-stranded antisense agent of  claim 32 , wherein the functional moiety has a function independently selected from a labeling function, a purification function, and a targeted delivery function. 
     
     
         34 . The double-stranded antisense agent of  claim 32 , wherein the functional moiety is independently joined to the chimeric antisense polynucleotide and/or the complementary strand via a cleavable linker moiety. 
     
     
         35 . The double-stranded antisense agent of  claim 32 , wherein the functional moiety is a molecule independently selected from a lipid, a peptide, and a protein. 
     
     
         36 . The double-stranded antisense agent of  claim 35 , wherein the lipid is independently selected from cholesterol, a fatty acid, a lipid-soluble vitamin, a glycolipid, and a glyceride. 
     
     
         37 . The double-stranded antisense agent of  claim 35 , wherein the lipid is independently selected from cholesterol, a tocopherol, and a tocotrienol. 
     
     
         38 . The double-stranded antisense agent of  claim 35 , wherein the peptide is independently selected from a receptor ligand fragment and an antibody fragment. 
     
     
         39 . The double-stranded antisense agent of  claim 35 , wherein the protein is independently selected from a receptor ligand and an antibody. 
     
     
         40 . The double-stranded antisense agent comprising the chimeric antisense polynucleotide of  claim 31 , wherein the chimeric antisense polynucleotide can hybridize to a cellular transcription product in a 100 mM sodium chloride, 10 mM sodium phosphate buffer, pH 7.2, at 25° C. 
     
     
         41 . A pharmaceutical composition comprising the chimeric antisense polynucleotide of  claim 1 , and a pharmaceutically acceptable carrier. 
     
     
         42 . A method of modifying the function of a transcription product in a cell comprising the step of administering to the cell a composition comprising the chimeric antisense polynucleotide of  claim 1 . 
     
     
         43 . A method of changing the expressed level of a protein in a cell comprising the step of administering to the cell a composition comprising the chimeric antisense polynucleotide of  claim 1 . 
     
     
         44 . A method of changing a protein structure in a cell comprising the step of administering to the cell a composition comprising the chimeric antisense polynucleotide of  claim 1 . 
     
     
         45 . A method for treating a patient having a condition characterized by changing expression level, function or editing of a target gene, comprising:
 administering to said patient a therapeutically effective amount of a pharmaceutical composition comprising   (a) at least one chimeric antisense polynucleotide of  claim 1 ; and   (b) a pharmaceutically acceptable carrier.