RNA interference mediated inhibition of muscarinic colinergic receptor gene expression using short interfering nucleic acid (siNA)
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 modified short interfering nucleic acid molecule (siNA), wherein:
(a) the siNA molecule comprises a sense strand and a separate antisense strand, each strand having one or more pyrimidine nucleotides and one or more purine nucleotides;
(b) each strand is independently 18 to 27 nucleotides in length, and together comprise a duplex having between 18 and 23 base pairs;
(c) the antisense strand is complementary to a human cholinergic receptor muscarinic 3 (CHRM3) RNA sequence comprising SEQ ID NO: 305;
(d) each pyrimidine nucleotide of the sense strand has a 2′-fluoro modification, each purine nucleotide of the sense strand has a 2′-deoxy modification, and both the 5′- and 3′-ends of the sense strand have a terminal cap moiety;
(e) each pyrimidine nucleotide of the antisense strand has a 2′-fluoro modification, each purine nucleotide of the antisense strand has a 2′-deoxy modification, and the antisense strand has a phosphorothioate internucleotide linkage at the 3′-end; and,
(f) wherein the siNA molecule mediates RNA interference.
2. The siNA molecule of claim 1 , wherein the terminal cap moiety is an inverted deoxy abasic moiety.
3. The siNA molecule of claim 1 , wherein said nucleic acid molecule includes two or more phosphorothioate internucleotide linkages.
4. The siNA molecule of claim 1 , wherein the 5′-end of the antisense strand includes a terminal phosphate group.
5. The siNA molecule of claim 1 , wherein the sense strand, the antisense strand, or both the sense strand and the antisense strand include a 3′-overhang.
6. A composition comprising the siNA molecule of claim 1 , in a pharmaceutically acceptable carrier or diluent.
7. The siNA molecule of claim 5 , wherein both the sense and the antisense strand include a 3′-overhang.
8. The siNA molecule of claim 5 , wherein the 3′-overhang comprises 1-3 nucleotides in length.
9. The siNA molecule of claim 5 wherein the overhang comprises ribonucleotides that are chemically modified at a nucleic acid sugar, base, or backbone.