RNA interference mediated inhibition of muscarinic cholinergic receptor gene expression using short interfering nucleic acid (siNA)

Abstract

The present invention relates to compounds, compositions, and methods useful for modulating the expression of genes associated with respiratory and pulmonary disease, such as cholinergic muscarinic receptor genes, using short interfering nucleic acid (siNA) molecules. This invention also relates to compounds, compositions, and methods useful for modulating the expression and activity of cholinergic muscarinic receptor genes, or other genes involved in pathways of cholinergic muscarinic receptor gene expression and/or activity by RNA interference (RNAi) using small nucleic acid molecules. In particular, the instant invention features small nucleic acid molecules, such as short interfering nucleic acid (siNA), short interfering RNA (siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA), and short hairpin RNA (shRNA) molecules and methods used to modulate the expression of M3 muscarinic acetylcholine receptor or cholinergic receptor muscarinic 3 (CHRM3).

1 . A chemically synthesized double stranded short interfering nucleic acid (siNA) molecule that directs cleavage of a cholinergic receptor muscarinic 3 (CHRM3) RNA via RNA interference, wherein: 
 a. each strand of said siNA molecule is about 19 to about 23 nucleotides in length;    b. one strand of said siNA molecule comprises nucleotide sequence having sufficient complementarity to said CHRM3 RNA for the siNA molecule to direct cleavage of the CHRM3 RNA via RNA interference; and    c. said siNA molecule does not require the presence of nucleotides having a 2′-hydroxy group for mediating RNA interference.    
     
     
         2 . The siNA molecule of  claim 1 , wherein said siNA molecule comprises no ribonucleotides.  
     
     
         3 . The siNA molecule of  claim 1 , wherein said siNA molecule comprises ribonucleotides.  
     
     
         4 . The siNA molecule of  claim 1 , wherein one strand of said double-stranded siNA molecule comprises a nucleotide sequence that is complementary to a nucleotide sequence of a CHRM3 gene or a portion thereof, and wherein a second strand of said double-stranded siNA molecule comprises a nucleotide sequence substantially similar to the nucleotide sequence or a portion thereof of said CHRM3 RNA.  
     
     
         5 . The siNA molecule of  claim 4 , wherein each strand of the siNA molecule comprises about 19 to about 23 nucleotides, and wherein each strand comprises at least about 19 nucleotides that are complementary to the nucleotides of the other strand.  
     
     
         6 . The siNA molecule of  claim 1 , wherein said siNA molecule comprises an antisense region comprising a nucleotide sequence that is complementary to a nucleotide sequence of a CHRM3 gene or a portion thereof, and wherein said siNA further comprises a sense region, wherein said sense region comprises a nucleotide sequence substantially similar to the nucleotide sequence of said CHRM3 gene or a portion thereof.  
     
     
         7 . The siNA molecule of  claim 6 , wherein said antisense region and said sense region comprises about 19 to about 23 nucleotides, and wherein said antisense region comprises at least about 19 nucleotides that are complementary to nucleotides of the sense region.  
     
     
         8 . The siNA molecule of  claim 1 , wherein said siNA molecule comprises a sense region and an antisense region, and wherein said antisense region comprises a nucleotide sequence that is complementary to a nucleotide sequence of RNA encoded by a CHRM3 gene, or a portion thereof, and said sense region comprises a nucleotide sequence that is complementary to said antisense region.  
     
     
         9 . The siNA molecule of  claim 6 , wherein said siNA molecule is assembled from two separate oligonucleotide fragments wherein one fragment comprises the sense region and a second fragment comprises the antisense region of said siNA molecule.  
     
     
         10 . The siNA molecule of claim  claim 6 , wherein said sense region is connected to the antisense region via a linker molecule.  
     
     
         11 . The siNA molecule of  claim 10 , wherein said linker molecule is a polynucleotide linker.  
     
     
         12 . The siNA molecule of  claim 10 , wherein said linker molecule is a non-nucleotide linker.  
     
     
         13 . The siNA molecule of  claim 6 , wherein pyrimidine nucleotides in the sense region are 2′-O-methyl pyrimidine nucleotides.  
     
     
         14 . The siNA molecule of  claim 6 , wherein purine nucleotides in the sense region are 2′-deoxy purine nucleotides.  
     
     
         15 . The siNA molecule of  claim 6 , wherein pyrimidine nucleotides present in the sense region are 2′-deoxy-2′-fluoro pyrimidine nucleotides.  
     
     
         16 . The siNA molecule of  claim 9 , wherein the fragment comprising said sense region includes a terminal cap moiety at the 5′-end, the 3′-end, or both of the 5′ and 3′ ends of the fragment comprising said sense region.  
     
     
         17 . The siNA molecule of  claim 16 , wherein said terminal cap moiety is an inverted deoxy abasic moiety.  
     
     
         18 . The siNA molecule of  claim 6 , wherein pyrimidine nucleotides of said antisense region are 2′-deoxy-2′-fluoro pyrimidine nucleotides  
     
     
         19 . The siNA molecule of  claim 6 , wherein purine nucleotides of said antisense region are 2′-O-methyl purine nucleotides.  
     
     
         20 . The siNA molecule of  claim 6 , wherein purine nucleotides present in said antisense region comprise 2′-deoxy-purine nucleotides.  
     
     
         21 . The siNA molecule of  claim 18 , wherein said antisense region comprises a phosphorothioate internucleotide linkage at the 3′ end of said antisense region.  
     
     
         22 . The siNA molecule of  claim 6 , wherein said antisense region comprises a glyceryl modification at the 3′ end of said antisense region.  
     
     
         23 . The siNA molecule of  claim 9 , wherein each of the two fragments of said siNA molecule comprise 21 nucleotides.  
     
     
         24 . The siNA molecule of  claim 23 , wherein about 19 nucleotides of each fragment of the siNA molecule are base-paired to the complementary nucleotides of the other fragment of the siNA molecule and wherein at least two 3′ terminal nucleotides of each fragment of the siNA molecule are not base-paired to the nucleotides of the other fragment of the siNA molecule.  
     
     
         25 . The siNA molecule of  claim 24 , wherein each of the two 3′ terminal nucleotides of each fragment of the siNA molecule are 2′-deoxy-pyrimidines.  
     
     
         26 . The siNA molecule of  claim 25 , wherein said 2′-deoxy-pyrimidine is 2′-deoxy-thymidine.  
     
     
         27 . The siNA molecule of  claim 23 , wherein all 21 nucleotides of each fragment of the siNA molecule are base-paired to the complementary nucleotides of the other fragment of the siNA molecule.  
     
     
         28 . The siNA molecule of  claim 23 , wherein about 19 nucleotides of the antisense region are base-paired to the nucleotide sequence of the RNA encoded by a CHRM3 gene or a portion thereof.  
     
     
         29 . The siNA molecule of  claim 23 , wherein 21 nucleotides of the antisense region are base-paired to the nucleotide sequence of the RNA encoded by a CHRM3 gene or a portion thereof.  
     
     
         30 . The siNA molecule of  claim 9 , wherein the 5′-end of the fragment comprising said antisense region optionally includes a phosphate group.  
     
     
         31 . A pharmaceutical composition comprising the siNA molecule of  claim 1  in an acceptable carrier or diluent.