LIGAND-MODIFIED DOUBLE-STRANDED NUCLEIC ACIDS
The invention provides for double stranded nucleic acid molecules comprising a 5′ extension of the sense or antisense strand and further comprising a plurality of nucleotides that are conjugated to a ligand and methods of using the double-stranded nucleic acid molecules. Ligand-modified oligomers where the sense stands form a tetraloop provide new potent and stable RNA interference agents. These dsNA molecules are synthesized using a plurality of nucleotides that include ligand-modified monomers, nucleotide analog monomers, modified nucleotide monomers and the like, using standard nucleotide synthetic methods and systems.
1 .- 89 . (canceled)
90 . An oligomer comprising:
a) a first oligonucleotide strand having a length of 15-35 nucleic acid monomers;
b) a second oligonucleotide strand having a length of 15-85 nucleic acid monomers;
c) a duplex formed by said first and second strands, said duplex comprising 15-35 paired nucleic acid monomers; and
d) a single-stranded 5′-extension, extending from the 5′-end of the first strand or the 5′-end of the second strand, said 5′-extension having a length of about 1 to about 50 nucleic acid monomers;
wherein the oligomer comprises at least one ligand-conjugated nucleic acid monomer located within the single-stranded 5′-extension.
91 . The oligomer of claim 90 , wherein the 5′-extension comprises 5-30 nucleic acid monomers.
92 . The oligomer of claim 90 , comprising 1, 2, 3, or 4 ligand-conjugated nucleic acid monomers.
93 . The oligomer of claim 92 , wherein each ligand-conjugated nucleic acid monomer is separated from other nucleic acid monomers by one or more spacer nucleic acid monomers.
94 . The oligomer of claim 90 , wherein the at least one ligand-conjugated monomer is positioned within said 5′-extension of the second strand.
95 . The oligomer of claim 90 , wherein at least one of the ligands is conjugated to the nucleic acid monomer through a 2′-hydroxyl group on a ribose of the nucleic acid monomer.
96 . The oligomer of claim 90 , wherein each ligand is conjugated to a nucleic acid monomer of the oligomer through a 2′-hydroxyl group on a ribose of the nucleic acid monomer.
97 . The oligomer of claim 90 , wherein said ligand is selected from the group consisting of N-acetyl galactosamine, cholesterol, cholic acid, adamantine acetic acid, 1-pyrene butyric acid, dihydrotestosterone, 1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine), bile acid, PEG, folate, vitamin A, vitamin E, biotin, pyridoxal, a peptide, peptide mimic, mannose, galactose, fructose, ribose, xylose, arabinose, lyxose, allose, altrose, gulose, iodose, glucose, talose, disaccharide, trisaccharide, tetrasaccharide, oligosaccharide, polysaccharide, an endosomolytic component, uvaol, hecigenin, diosgenin, triterpenesarsasapogenin, Friedelin, epifriedelanol-derivatized lithocholic acid, a cationic lipid, and antibody.
98 . The oligomer of claim 90 , wherein said ligand is N-acetylgalactosamine.
99 . The oligomer of claim 90 , wherein said ligand is attached to said nucleic acid monomer via a linker.
100 . The oligomer of claim 90 , wherein said linker is a releasable linker.
101 . The oligomer of claim 90 , wherein said 5′-extension comprises 10-30 nucleic acid monomers.
102 . The oligomer of claim 90 , wherein said oligomer comprises at least one modified nucleic acid monomer.
103 . The oligomer of claim 90 , wherein at least one strand of said oligomer comprises a 3′-overhang of 1 to 4 nucleic acid monomers.
104 . The oligomer of claim 103 , wherein at least one nucleic acid monomer of said 3′-overhang comprises a sugar or backbone modification, or both.
105 . The oligomer of claim 90 , wherein said nucleic acid monomer is a DNA or RNA nucleotide monomer, or an abasic or inverted abasic nucleotide monomer.
106 . The oligomer of claim 90 , wherein one or more nucleic acid monomers comprises a sugar or backbone modification, or both modifications.
107 . The oligomer of claim 106 , wherein said sugar modification comprises a 2′-O-methyl, 2′-methoxyethoxy, 2′-fluoro, 2′-allyl, 2′-O-[2-(methylamino)-2-oxoethyl], 4′-thio, 4′-CH 2 —O-2′-bridge, 4′-(CH 2 ) 2 —O-2′-bridge, 2′-LNA, 2′-amino, or 2′-O—(N-methylcarbamate) modification.
108 . The oligomer of claim 106 , wherein said backbone modification comprises a phosphate backbone modification selected from phosphonate, phosphorothioate, phosphotriester, methylphosphonate, morpholino or bicyclic furanose analog modification.
109 . The oligomer of claim 90 , wherein the duplex comprises a region containing at least nine consecutive phosphorothioate linkages.
110 . The oligonucleotide of claim 90 , wherein said first or second strand comprises a modified nucleic acid monomer that directs orientation of Dicer cleavage.
111 . The oligomer of claim 90 , wherein said oligomer is a Dicer cleavage substrate.
112 . The oligomer of claim 90 , wherein said oligomer is not a Dicer cleavage substrate.
113 . The oligomer of claim 90 , wherein said 5′-extension comprises a DNA region comprising 10 or more deoxyribonucleotide monomers.
114 . The oligomer of claim 90 , wherein said 5′-extension comprises at least nine contiguous phosphorothioate modified nucleic acid monomers.
115 . The oligomer of claim 90 , wherein said first and second strands are aligned for maximal complementarity in the duplex region.
116 . The oligomer of claim 90 , wherein said second strand is sufficiently complementary to a target RNA along at least 15 nucleic acid monomers to reduce target gene expression when introduced into a mammalian cell.
117 . The oligomer of claim 90 , comprising a structure represented by formula I, wherein:
a) D is the duplex;
b) E is the 5′-extension;
c) O is the oligomer;
d) M is a nucleic acid monomer;
e) X is a ligand;
f) L is an optional linker joining M and X;
g) S1 and S2 are nucleic acid monomer spacers;
h) P is a unit formed of ligand-modified nucleic acid monomer (XLM) and S1 spacer;
i) a is independently 1-4 for each P;
j) n is 1-10; each z is independently 0-10 for each P; and
k) each of b and c is 0-35, wherein one of b and c is 0.
118 . The oligomer of claim 117 , wherein said linker (L) comprises a backbone selected from the group consisting of: an alkyl, an alkenyl, an aromatic, a heterocycl, a substituted alkyl, and a substituted alkenyl, wherein one of more methylenes can be interrupted or terminated by one or more of O, S, S(O), SO2, N, NH, NH 2 , NH(CO), P, P(O 4 ), C≡C or C(O), and PEG.
119 . The oligomer of claim 117 , wherein said oligomer comprises a plurality of nucleic acid monomers each independently selected from the group consisting of: ribonucleotides, deoxyribonucleotides, modified nucleotides, abasic nucleotides, inverted abasic nucleotides, nucleotide analogs, and non-nucleoside analogs.
120 . The oligomer of claim 117 , comprising a phosphate backbone selected from the group consisting of: phosphonate, phosphorothioates, phosphotriester, methylphosphonate, locked nucleic acid (LNA), a morpholino, SATE (S-acyl-2-thioethyl) modified phosphate & BMEG (Isobutyryl Mercapto Ethyl Glycol) modified phosphate and a bicyclic furanose analog.
121 . The oligomer of claim 117 , wherein the linker L comprises the structure:
wherein i may
be 1-10 and k may be 1-10 carbon atoms.
122 . The oligomer of claim 99 , wherein the linker L is selected from the group consisting of:
wherein n ranges from 0
to 20;
b)
c)
wherein n ranges from 0 to 20; and
d)
wherein n ranges from 0 to 20; and
wherein said ligand is conjugated to the sugar or base of said nucleotide.
123 . The oligomer of claim 99 , wherein the linker is an acetal linker.
124 . The oligomer of claim 90 , wherein the at least one ligand is conjugated to a sugar of a nucleic acid monomer via a linker as set forth in Formula VII:
wherein:
B is a nucleobase;
m and n each independently range from 1 to 20; and
X is the ligand.
125 . The oligomer of claim 117 , wherein said ligand is selected from the group consisting of a lipophile, steroid, protein, vitamin, carbohydrate, and terpene.
126 . The oligomer of claim 117 , wherein said ligand is selected from the group consisting of N-acetyl galactosamine, cholesterol, cholic acid, adamantine acetic acid, 1-pyrene butyric acid, dihydrotestosterone, 1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine), bile acid, PEG, folate, vitamin A, vitamin E, biotin, pyridoxal, a peptide, peptide mimic, mannose, galactose, fructose, ribose, xylose, arabinose, lyxose, allose, altrose, gulose, iodose, glucose, talose, disaccharide, trisaccharide, tetrasaccharide, oligosaccharide, polysaccharide, an endosomolytic component, uvaol, hecigenin, diosgenin, triterpenesarsasapogenin, Friedelin, epifriedelanol-derivatized lithocholic acid, a cationic lipid, and antibody.
127 . The oligomer of claim 117 , wherein said oligomer comprises at least one modified nucleic acid monomer comprising a modified base and/or a modified sugar moiety.
128 . The oligomer of claim 127 , wherein the modified sugar moiety is selected from the group consisting of 2′-hydroxyl, 3′-hydroxyl, 2′-O-methyl, 2′-methoxyethoxy, 2′-fluoro, 2′-allyl, 2′-O[2-(methylamino)-2-oxoethyl], 4′-thio, 4′-CH2-O-2′-bridge, 4′-(CH2)2-O-2′-bridge, 2′-LNA, 2′-amino and 2′-O—(N-methylcarbamate).
129 . The oligomer of claim 127 , wherein said at least one modified nucleic acid monomer comprises a base analog selected from the group consisting of: hypoxanthine (I), xanthine (X), 3β-D-ribofuranosyl-(2,6-diaminopyrimidine) (K), 3β-D-ribofuranosyl-(1-methyl-pyrazolo[4,3-d]pyrimidine-5,7(4H,6H)-dione) (P), iso-cytosine (iso-C), iso-guanine (iso-G), 1-β-D-ribofuranosyl-(5-nitroindole), 1-β-D-ribofuranosyl-(3-nitropyrrole), 5-bromouracil, 2-aminopurine, 4-thio-dT, 7-(2-thienyl)-imidazo[4,5-b]pyridine (Ds), pyrrole-2-carbaldehyde (Pa), 2-amino-6-(2-thienyl)purine (S), 2-oxopyridine (Y), difluorotolyl, 4-fluoro-6-methylbenzimidazole, 4-methylbenzimidazole, 3-methyl isocarbostyrilyl, 5-methyl isocarbostyrilyl, 3-methyl-7-propynyl isocarbostyrilyl, 7-azaindolyl, 6-methyl-7-azaindolyl, imidizopyridinyl, 9-methyl-imidizopyridinyl, pyrrolopyrizinyl, isocarbostyrilyl, 7-propynyl isocarbostyrilyl, propynyl-7-azaindolyl, 2,4,5-trimethylphenyl, 4-methylindolyl, 4,6-dimethylindolyl, phenyl, napthalenyl, anthracenyl, phenanthracenyl, pyrenyl, stilbenzyl, tetracenyl and pentacenyl.
130 . The oligomer of claim 117 , wherein D is covalently bonded to P at the 5′-end of said first strand or said second strand wherein b and c are each zero; a is 1; n is 4, and z is 2, 2, 2, and 1, respectively for each unit of P.
131 . The oligomer of claim 127 , wherein D is covalently bonded to P at the 5′-end of said first strand or said second strand wherein a is 1, n is 4, and each of b, c, z is zero.
132 . The oligomer of claim 127 , wherein D is covalently bonded to P at the 5′-end of said first strand or said second strand wherein a is 1; n is 4; each of c and z is zero; and b is 1-11.
133 . The oligomer of claim 90 , conjugated to a non-nucleic acid moiety.
134 . The oligomer of claim 133 , wherein the non-nucleic acid moiety is a peptide or an organic compound.
135 . The oligomer of claim 134 , wherein said organic compound is a dye.
136 . The oligomer of claim 134 , wherein said organic compound is cholesterol.
137 . The oligomer of claim 133 , wherein said non-nucleic acid moiety comprises a detectable label.
138 . An oligomer of claim 90 , wherein said first and second strands form a duplex of at least 21 base pairs.
139 . The oligomer of claim 90 , wherein said ligand enhances binding affinity of said oligomer to asialoglycoprotein-receptor (ASGPr) as compared to a double stranded nucleic acid molecule lacking said ligand.
140 . The oligomer of claim 90 , wherein said ligand enhances delivery of said oligomer to target cells as compared to a double stranded nucleic acid lacking said ligand.
141 . The oligomer of claim 117 , wherein said P moiety increases the cellular uptake of the said oligomer as compared to an oligomer that does not contain moiety P.
142 . A method for reducing expression of a target gene in a cell, comprising contacting a cell with the oligomer of claim 90 in an amount effective to reduce expression of a target gene in said cell.
143 . A method for reducing expression of a target gene in an animal, comprising:
administering to said animal in the oligomer of claim 90 in an amount effective to reduce expression of a target gene in said animal.
144 . A pharmaceutical composition for reducing expression of a target gene in a cell of a subject, said composition comprising the oligomer of claim 90 in an amount effective to reduce expression of said target gene in said cell or animal, and a pharmaceutically acceptable carrier.
145 . A kit comprising the oligomer of claim 90 and instructions for its use.