SHORT INTERFERING NUCLEIC ACID (siNA) COMPOSITIONS
The present invention relates to compounds, compositions, and methods for the study, diagnosis, and treatment of traits, diseases and conditions that respond to the modulation of gene expression and/or activity, and/or modulate a gene expression pathway. Specifically, the invention relates to double-stranded nucleic acid molecules including small nucleic acid molecules, such as short interfering nucleic acid (siNA) molecules that are capable of mediating or that mediate RNA interference (RNAi) against target gene expression.
1 . A composition comprising:
a double-stranded short interfering nucleic acid (siNA) molecule that modulates expression of a target RNA sequence via RNA interference, wherein the molecule has a passenger strand and a guide strand and comprises structure selected from the structures represented by
wherein,
(a) the upper strand is the passenger strand and the lower strand is the guide strand of the double-stranded nucleic acid molecule; the guide strand is complementary to the target sequence and the passenger strand is complementary to the guide strand, wherein one or more mismatches between the guide strand and passenger strand and/or between the guide strand and the target sequence are tolerated so long as RNAi activity is maintained;
(b) each N is independently a nucleotide wherein one or more Ns may be substituted with a non-nucleotide moiety so long as RNAi activity is maintained;
(c) each B 1 , B 2 , and B 3 is independently a terminal cap optionally including a ligand, polymer, protein or peptide transduction domain, nuclear localization sequence, cell penetrating peptide, receptor, steroid, vitamin, antibody, protamine, and/or hormone, optionally attached via a linker, wherein any of B 1 , B 2 , and/or B 3 is optionally absent;
(d) x is an integer from 0 to 4, provided that when x is 1, 2, 3, or 4, one or more of the (N) x nucleotides can be complementary to nucleotides in target sequence, and one or more phosphorothioate internucleotide linkage(s) “s” can be present in the (N) x region when x is 1, 2, 3, or 4;
(e) y is an integer from 0 to 4, provided that when y is 1, 2, 3, or 4, one or more of the (N) y nucleotides can be complementary to nucleotides in target sequence, and one or more phosphorothioate internucleotide linkage(s) “s” can be present in the (N) y region when y is 1, 2, 3, or 4;
(f) N nucleotides of the guide strand are 2′-deoxy-2′-fluoro nucleotides and N nucleotides of the guide strand are 2′-O-methyl nucleotides, with an optional variance of 1 or 2 N or N nucleotides being tolerated provided that as RNAi activity is maintained;
(g) S=a phosphorothioate or phosphorodithioate internucleotide linkage wherein S 1 is required and S 2 and S 3 are optional; and
(h) N nucleotides of the passenger strand are independently selected from ribonucleotide, 2′-O-alkyl nucleotide, 2′-deoxy-2′-fluoro nucleotide, 2′-deoxy nucleotide, and LNA.
2 - 10 . (canceled)
11 . The composition according to claim 1 , wherein each (N) of the passenger strand is independently selected from 2′-O-alkyl, 2′-deoxy-2′-fluoro, 2′-deoxy, and/or LNA modified nucleotide.
12 . The composition according to claim 1 , wherein each (N) of the passenger strand is a 2′-O-alkyl modified nucleotide.
13 . The composition according to claim 1 , wherein each (N) of the passenger strand is a 2′-O-methyl modified nucleotide.
14 . The composition according to claim 1 , wherein x=2 and y=2.
15 . The composition according to claim 1 , wherein each N nucleotide of the guide strand is a 2′-deoxy-2′-fluoro nucleotide and each N nucleotide of the guide strand is a 2′-O-methyl nucleotide.
16 . The composition according to claim 1 , wherein 5, 6, 7, 8, 9, or 10 or more pyrimidine N nucleotides of the passenger strand are 2′-O-methyl nucleotides and 5, 6, 7, 8, 9, or 10 or more purine N nucleotides of the passenger strand are 2′-deoxy-2′-fluoro nucleotides.
17 . The composition according to claim 1 , wherein the 5′-terminal N position comprises a terminal phosphate group.
18 . The composition according to claim 1 , wherein B 1 and B 2 each comprise an inverted abasic 2′-deoxyribose moiety and B 3 is optionally absent.
19 . The composition according to claim 1 , wherein B 1 comprises a ligand, polymer, protein or peptide transduction domain, nuclear localization sequence, cell penetrating peptide, receptor, steroid, vitamin, antibody, protamine, and/or hormone, optionally attached via a linker to an inverted abasic 2′-deoxyribose moiety, B 2 comprises an inverted abasic 2′-deoxyribose moiety, and B 3 is optionally absent.
20 . The composition according to claim 1 , wherein B 1 comprises one or more galactosamine moieties optionally attached via a linker to an inverted abasic 2′-deoxyribose moiety, B 2 comprises an inverted abasic 2′-deoxyribose moiety, and B 3 is optionally absent.
21 . The composition according to claim 1 , wherein B 1 comprises one or more N-acetylgalactosamine moieties optionally attached via a linker to an inverted abasic 2′-deoxyribose moiety, B 2 comprises an inverted abasic 2′-deoxyribose moiety, and B 3 is optionally absent.
22 . The composition according to claim 1 , wherein B 1 comprises one or more folate moieties optionally attached via a linker to an inverted abasic 2′-deoxyribose moiety, B 2 comprises an inverted abasic 2′-deoxyribose moiety, and B 3 is optionally absent.
23 . The composition according to claim 1 , wherein B 1 comprises one or more cholesterol moieties optionally attached via a linker to an inverted abasic 2′-deoxyribose moiety, B 2 comprises an inverted abasic 2′-deoxyribose moiety, and B 3 is optionally absent.
24 . The composition according to claim 1 , wherein B 1 comprises a ligand, polymer, protein or peptide transduction domain, nuclear localization sequence, cell penetrating peptide, receptor, steroid, vitamin, antibody, protamine, and/or hormone, optionally attached via a linker to an inverted abasic 2′-deoxyribose moiety, B 2 comprises a ligand, polymer, protein or peptide transduction domain, nuclear localization sequence, cell penetrating peptide, receptor, steroid, vitamin, antibody, protamine, and/or hormone, optionally attached via a linker to an inverted abasic 2′-deoxyribose moiety, and B 3 is optionally absent.
25 . The composition according to claim 1 , wherein B 1 comprises one or more galactosamine moieties optionally attached via a linker to an inverted abasic 2′-deoxyribose moiety, B 2 comprises a ligand, polymer, protein or peptide transduction domain, nuclear localization sequence, cell penetrating peptide, receptor, steroid, vitamin, antibody, protamine, and/or hormone, optionally attached via a linker to an inverted abasic 2′-deoxyribose moiety, and B 3 is optionally absent.
26 . The composition according to claim 1 , wherein B 1 comprises one or more N-acetylgalactosamine moieties optionally attached via a linker to an inverted abasic 2′-deoxyribose moiety, B 2 comprises a ligand, polymer, protein or peptide transduction domain, nuclear localization sequence, cell penetrating peptide, receptor, steroid, vitamin, antibody, protamine, and/or hormone, optionally attached via a linker to an inverted abasic 2′-deoxyribose moiety, and B 3 is optionally absent.
27 . The composition according to claim 1 , wherein B 1 comprises one or more folate moieties optionally attached via a linker to an inverted abasic 2′-deoxyribose moiety, B 2 comprises a ligand, polymer, protein or peptide transduction domain, nuclear localization sequence, cell penetrating peptide, receptor, steroid, vitamin, antibody, protamine, and/or hormone, optionally attached via a linker to an inverted abasic 2′-deoxyribose moiety, and B 3 is optionally absent.
28 . The composition according to claim 1 , wherein B 1 comprises one or more cholesterol moieties optionally attached via a linker to an inverted abasic 2′-deoxyribose moiety, B 2 comprises a ligand, polymer, protein or peptide transduction domain, nuclear localization sequence, cell penetrating peptide, receptor, steroid, vitamin, antibody, protamine, and/or hormone, optionally attached via a linker to an inverted abasic 2′-deoxyribose moiety, and B 3 is optionally absent.
29 . The composition according to claim 1 , wherein B 1 comprises a ligand, polymer, protein or peptide transduction domain, nuclear localization sequence, cell penetrating peptide, receptor, steroid, vitamin, antibody, protamine, and/or hormone, optionally attached via a linker to an inverted abasic 2′-deoxyribose moiety, B 2 comprises any of SEQ ID NOS: 54-528 optionally attached via a linker to an inverted abasic 2′-deoxyribose moiety, and B 3 is optionally absent.
30 . The composition according to claim 1 , wherein B 1 comprises one or more galactosamine moieties optionally attached via a linker to an inverted abasic 2′-deoxyribose moiety, B 2 comprises any of SEQ ID NOS: 54-528 optionally attached via a linker to an inverted abasic 2′-deoxyribose moiety optionally attached via a linker to an inverted abasic 2′-deoxyribose moiety, and B 3 is optionally absent.
31 . The composition according to claim 1 , wherein B 1 comprises one or more N-acetylgalactosamine moieties optionally attached via a linker to an inverted abasic 2′-deoxyribose moiety, B 2 comprises any of SEQ ID NOS: 54-528 optionally attached via a linker to an inverted abasic 2′-deoxyribose moiety optionally attached via a linker to an inverted abasic 2′-deoxyribose moiety, and B 3 is optionally absent.
32 . The composition according to claim 1 , wherein B 1 comprises one or more folate moieties optionally attached via a linker to an inverted abasic 2′-deoxyribose moiety, B 2 comprises any of SEQ ID NOS: 54-528 optionally attached via a linker to an inverted abasic 2′-deoxyribose moiety optionally attached via a linker to an inverted abasic 2′-deoxyribose moiety, and B 3 is optionally absent.
33 . The composition according to claim 1 , wherein B 1 comprises one or more cholesterol moieties optionally attached via a linker to an inverted abasic 2′-deoxyribose moiety, B 2 comprises any of SEQ ID NOS: 54-528 optionally attached via a linker to an inverted abasic 2′-deoxyribose moiety optionally attached via a linker to an inverted abasic 2′-deoxyribose moiety, and B 3 is optionally absent.
34 . The composition according to claim 1 , wherein S 1 and S 2 are required and S 3 is optional.
35 . The composition according to claim 1 , wherein S 1 and S 3 are required and S 2 is optional.
36 . The composition according to claim 1 , wherein S 1 , S 2 , and S 3 are required.
37 . The composition according to claim 1 , wherein B 1 comprises:
wherein each L is independently a linker and GalNAc is an N-acetyl galactosamine moiety.
38 . The composition according to claim 1 , wherein B 1 comprises:
39 . The composition according to claim 1 , wherein B 1 comprises:
wherein n is an integer from 1 to 20.
40 . The composition according to claim 1 , wherein B 1 or B 2 comprises:
wherein X is —O—, —S—, —CH 2 — or —NH—; n is 1, 2, 3, or 4; and the bond with “ ” indicates the point of attachment optionally including one or more linkers, which may be the same or different; and further optionally including one or more targeting ligands, solubilizing agents, pharmacokinetics enhancing agents, lipids, and/or masking agents.
41 . A composition comprising:
a double-stranded short interfering nucleic acid (siNA) molecule that modulates expression of a target RNA sequence via RNA interference, wherein the molecule has a passenger strand and a guide strand and comprises structure selected from the structures represented by
wherein,
(a) the upper strand is the passenger strand and the lower strand is the guide strand of the double-stranded nucleic acid molecule; the guide strand is complementary to the target sequence and the passenger strand is complementary to the guide strand, wherein one or more mismatches between the guide strand and passenger strand and/or between the guide strand and the target sequence are tolerated so long as RNAi activity is maintained;
(b) B 1 and B 2 , are each independently a terminal cap optionally including a ligand, polymer, protein or peptide transduction domain, nuclear localization sequence, cell penetrating peptide, receptor, steroid, vitamin, antibody, protamine, and/or hormone, optionally attached via a linker;
(c) every N nucleotide is a 2′-deoxy-2′-fluoro nucleotide and every N nucleotide is a 2′-O-methyl nucleotide;
(d) each S is a phosphorothioate or phosphorodithioate internucleotide linkage.
42 - 46 . (canceled)
47 . The composition according to claim 41 , wherein B 1 comprises:
wherein each L is independently a linker and GalNAc is an N-acetyl galactosamine moiety.
48 . The composition according to claim 41 , wherein B 1 comprises:
49 . The composition according to claim 41 , wherein B 1 comprises:
wherein n is an integer from 1 to 20.
50 . The composition according to claim 41 , wherein B 1 or B 2 comprises:
wherein X is —O—, —S—, —CH 2 — or —NH—; n is 1, 2, 3, or 4; and the bond with “ ” indicates the point of attachment optionally including one or more linkers, which may be the same or different; and further optionally including one or more targeting ligands, solubilizing agents, pharmacokinetics enhancing agents, lipids, and/or masking agents.
51 . The composition according to claim 1 , further comprising a terminal phosphate group at the 5′-end of the guide strand.
52 . A composition comprising the double-stranded short interfering nucleic acid (siNA) molecule according to claim 1 , and a pharmaceutically acceptable carrier or diluent.
53 . The composition according to claim 41 , further comprising a terminal phosphate group at the 5′-end of the guide strand.
54 . A composition comprising the double-stranded short interfering nucleic acid (siNA) molecule according to claim 41 , and a pharmaceutically acceptable carrier or diluent.