Claims for Patent: 8,106,022

Summary for Patent: 8,106,022

Title:	Carbohydrate conjugates as delivery agents for oligonucleotides
Abstract:	The present invention provides iRNA agents comprising at least one subunit of the formula (I): ##STR00001## wherein: A and B are each independently for each occurrence O, N(R.sup.N) or S; X and Y are each independently for each occurrence H, OH, a hydroxyl protecting group, a phosphate group, a phosphodiester group, an activated phosphate group, an activated phosphite group, a phosphoramidite, a solid support, --P(Z')(Z'')O-nucleoside, --P(Z')(Z'')O-oligonucleotide, a lipid, a PEG, a steroid, a lipophile, a polymer, --P(Z')(Z'')O-Linker-OP(Z''')(Z'''')O-oligonucleotide, a nucleotide, an oligonucleotide, --P(Z')(Z'')-formula (I), --P(Z')(Z'')-- or -Linker-R; R is L.sup.G, -Linker-L.sup.G, or has the structure shown below: ##STR00002## L.sup.G is independently for each occurrence a carbohydrate, e.g., monosaccharide, disaccharide, trisaccharide, tetrasaccharide, oligosaccharide, polysaccharide; R.sup.N is independently for each occurrence H, methyl, ethyl, propyl, isopropyl, butyl, or benzyl; and Z', Z'', Z''' and Z'''' are each independently for each occurrence O or S.
Inventor(s):	Manoharan; Muthiah (Weston, MA), Rajeev; Kallanthottathil G. (Wayland, MA), Narayanannair; Jayaprakash K. (Wakefield, MA), Maier; Martin (Belmont, MA)
Assignee:	Alnylam Pharmaceuticals, Inc. (Cambridge, MA)
Application Number:	12/328,528
Patent Claims:	1. An iRNA agent comprising a compound having the structure shown in formula (I) ##STR00124## wherein: A and B are each independently for each occurrence O, N(R.sup.N) or S; R.sup.N is independently for each occurrence H or C.sub.1-C.sub.6 alkyl; X and Y are each independently for each occurrence H, a protecting group, a phosphate group, a phosphodiester group, an activated phosphate group, an activated phosphite group, a phosphoramidite, a solid support, --P(Z')(Z'')O-nucleoside, --P(Z')(Z'')O-oligonucleotide, a lipid, a PEG, a steroid, a polymer, a nucleotide, a nucleoside, --P(Z')(Z'')O-Linker-OP(Z''')(Z'''')O-oligonucleotide, an oligonucleotide, --P(Z')(Z'')-formula (I), --P(Z')(Z'')- or -Linker-R; R is L.sup.G or has the structure shown below: ##STR00125## L.sup.G is independently for each occurrence a ligand; and Z', Z'', Z''' and Z'''' are each independently for each occurrence O or S. 2. An iRNA agent comprising a compound having the structure shown in formula (I'): ##STR00126## wherein: A and B are each independently for each occurrence O, N(R.sup.N) or S; X and Y are each independently for each occurrence H, a protecting group, a phosphate group, a phosphodiester group, an activated phosphate group, an activated phosphite group, a phosphoramidite, a solid support, --P(Z')(Z'')O-nucleoside, --P(Z')(Z'')O-oligonucleotide, a lipid, a PEG, a steroid, a polymer, a nucleotide, annucleoside, --P(Z')(Z'')O--R.sup.1-Q'--R.sup.2--OP(Z''')(Z'''')O-oligonucleotide, or an oligonucleotide, --P(Z')(Z'')-formula (I), --P(Z')(Z'')- or -Q-R; R is L.sup.1or has the structure shown in formula (II)-(V) ##STR00127## q.sup.2A, q.sup.2B, q.sup.3A, q.sup.3B, q4.sup.A, q.sup.4B, q.sup.5A, q.sup.5B and q.sup.5C represent independently for each occurrence 0-20 and wherein the repeating unit can be the same or different; Q and Q' are independently for each occurrence absent, --(P.sup.7-Q.sup.7-R.sup.7).sub.p-T.sup.7- or -T.sup.7-Q.sup.7-T.sup.7'-B-T8'-Q.sup.8-T.sup.8; P.sup.2A, P.sup.2B, P.sup.3A, P.sup.3B, P.sup.4A, P.sup.4B, P.sup.5A, P.sup.5B, P.sup.5C, P.sup.7, T.sup.2A, T.sup.2B, T.sup.3A, T.sup.3B, T.sup.4A, T.sup.4B, T.sup.4A, T.sup.5B, T.sup.5C, T.sup.7, T.sup.7', T.sup.8 and T.sup.8' are each independently for each occurrence absent, CO, NH, O, S, OC(O), NHC(O), CH.sub.2, CH.sub.2NH or CH.sub.2O; B is --CH.sub.2--N(B.sup.L)--CH.sub.2--; B.sup.L is -T.sup.B-Q.sup.B-T.sup.B'-R.sup.x; Q.sup.2A, Q.sup.2B, Q.sup.3A, Q.sup.3B, Q.sup.4A, Q.sup.4B, Q.sup.5A, Q.sup.5B, Q.sup.5C, Q.sup.7, Q.sup.8 and Q.sup.B are independently for each occurrence absent, alkylene, substituted alkylene wherein one or more methylenes can be interrupted or terminated by one or more of O, S, S(O), SO.sub.2, N(R.sup.N), C(R').dbd.C(R''), C.ident.C or C(O); T.sup.B and T.sup.B' are each independently for each occurrence absent, CO, NH, O, S, OC(O), OC(O)O, NHC(O), NHC(O)NH, NHC(O)O, CH.sub.2, CH.sub.2NH or CH.sub.2O; R.sup.x is a lipophile, or a cationic lipid; R.sup.1, R.sup.2, R.sup.2A, R.sup.2B, R.sup.3A, R.sup.3B, R.sup.4A, R.sup.4B, R.sup.5A, R.sup.5B, R.sup.5C, R.sup.7 are each independently for each occurrence absent, NH, O, S, CH.sub.2, C(O)O, C(O)NH, NHCH(R.sup.a)C(O), --C(O)--CH(R.sup.a)--NH--, CO, CH.dbd.N--O, ##STR00128## or heterocyclyl; L.sup.1, L.sup.2A, L.sup.2B, L.sup.3A, L.sup.3B, L.sup.4A, L.sup.4B, L.sup.5A, L.sup.5B and L.sup.5C are each independently for each occurrence a carbohydrate; R' and R'' are each independently H, C.sub.1-C.sub.6 alkyl, OH, SH or N(R.sup.N).sub.2; R.sup.N is independently for each occurrence methyl, ethyl, propyl, isopropyl, butyl or benzyl; R.sup.a is H or amino acid side chain; Z', Z'', Z''' and Z'''' are each independently for each occurrence O or S; p represent independently for each occurrence 0-20. 3. The iRNA agent of claim 1, wherein said iRNA agent is double stranded. 4. The iRNA agent of claim 3, wherein said compound is at the 3'-end of one of the strands. 5. The iRNA agent of claim 4, wherein said compound is at the 3'-end of the sense strand. 6. The iRNA agent of claim 1, wherein the iRNA agent further comprises at least one structure of formula (VI) ##STR00129## wherein X.sup.6 and Y.sup.6 are each independently H, a hydroxyl protecting group, a phosphate group, a phosphodiester group, an activated phosphate group, an activated phosphite group, a phosphoramidite, a solid support, --P(Z')(Z'')O-nucleoside, --P(Z')(Z'')O-oligonucleotide, a lipid, a PEG, a steroid, a polymer, --P(Z')(Z'')O--R.sup.1-Q'--R.sup.2--OP(Z''')(Z'''')O-oligonucleotide, a nucleotide, or an oligonucleotide, --P(Z')(Z'')-formula (I) or --P(Z')(Z'')-; Q.sup.6 is absent or --(P.sup.6-Q.sup.6A-R.sup.6).sub.v-T.sup.6-; P.sup.6 and T.sup.6 are each independently for each occurrence absent, CO, NH, O, S, OC(O), NHC(O), CH.sub.2, CH.sub.2NH or CH.sub.2O; Q.sup.6A is independently for each occurrence absent, alkylene, substituted alkylene wherein one or more methylenes can be interupted or terminated by one or more of O, S, S(O), SO.sub.2, N(R.sup.N), C(R').dbd.C(R''), C.ident.C or C(O); R.sup.6 is independently for each occurrence absent, NH, O, S, CH.sub.2, C(O)O, C(O)NH, NHCH(R.sup.a)C(O), --C(O)--CH(R.sup.a)--NH--, CO, CH.dbd.N--O, ##STR00130## or heterocyclyl; R' and R'' are each independently H, C.sub.1-C.sub.6 alkyl OH, SH, N(R.sup.N).sub.2; R.sup.N is independently for each occurrence H, methyl, ethyl, propyl, isopropyl, butyl or benzyl; R.sup.a is H or amino acid side chain; Z', Z'', Z''' and Z'''' are each independently for each occurrence O or S; v represent independently for each occurrence 0-20; R.sup.L is a lipophile or a cationic lipid. 7. The iRNA agent of claim 6, wherein R.sup.L is a lipophile. 8. The iRNA agent of claim 6, wherein R.sup.L is cholesterol. 9. The iRNA agent of claim 6, wherein R.sup.L is ##STR00131## 10. The iRNA agent of claim 2, wherein R is one of formula (II), formula (III), formula (IV) or formula (V). 11. The iRNA agent of claim 10, wherein R has the structure of formula (V): ##STR00132## 12. The iRNA agent of claim 11, wherein R is ##STR00133## 13. The iRNA agent of claim 11, wherein R is ##STR00134## 14. The iRNA agent of claim 2, wherein said compound has the structure ##STR00135## 15. The iRNA agent of claim 2, wherein said compound has the structure ##STR00136## 16. The iRNA agent of claim 2, wherein said compound has the structure ##STR00137## 17. A method of modulating the expression of a target gene in a cell, comprising providing to said cell an iRNA agent of claim 1. 18. The method of claim 17, wherein the target gene is selected from the group consisting of Factor VII, Eg5, PCSK9, TPX2, apoB, SAA, TTR, RSV, PDGF beta gene, Erb-B gene, Src gene, CRK gene, GRB2 gene, RAS gene, MEKK gene, JNK gene, RAF gene, Erk1/2 gene, PCNA (p21) gene, MYB gene, JUN gene, FOS gene, BCL-2 gene, Cyclin D gene, VEGF gene, EGFR gene, Cyclin A gene, Cyclin E gene, WNT-1 gene, beta-catenin gene, c-MET gene, PKC gene, NFKB gene, STAT3 gene, survivin gene, Her2/Neu gene, topoisomerase I gene, topoisomerase II alpha gene, mutations in the p73 gene, mutations in the p21(WAF1/CIP1) gene, mutations in the p27(KIP1) gene, mutations in the PPM1D gene, mutations in the RAS gene, mutations in the caveolin I gene, mutations in the MIB I gene, mutations in the MTAI gene, mutations in the M68 gene, mutations in tumor suppressor genes, and mutations in the p53 tumor suppressor gene. 19. A pharmaceutical composition comprising an iRNA agent of claim 1 alone or in combination with a pharmaceutically acceptable carrier or excipient. 20. The iRNA agent of claim 1, wherein L.sup.G is carbohydrate. 21. The iRNA agent of claim 20, wherein the carbohydrate is selected from the group consisting of monosaccharide, disaccharide, trisaccharide, tetrasaccharide and polysaccharide.