RNAi agents, compositions and methods of use thereof for treating transthyretin (TTR) associated diseases

1. A double stranded RNAi agent comprising a sense strand complementary to an antisense strand, wherein said antisense strand comprises a region complementary to nucleotides 504 to 526 of the transthyretin (TTR) gene (SEQ ID NO:1), wherein each strand independently has 14 to 30 nucleotides, wherein said double stranded RNAi agent is represented by formula (III):

sense:

5′ n p  -N a  -(X X X) i -N b  - Y Y Y -N b -(Z Z Z) j -N a  -

n q  3′

antisense:

3′ n p ′-N a ′-(X′X′X′) k -N b ′-Y′Y′Y′-N b ′-(Z′Z′Z′) l -N a ′-

n q ′ 5′

(III)

wherein:

i, j, k, and 1 are each independently 0 or 1, provided that at least one of i, j, k, and 1 is 1;

p, p′, q, and q′ are each independently 0-6;

each N a and N a ′ independently represents an oligonucleotide sequence comprising 2-20 nucleotides which are modified, wherein the modifications on the nucleotides are 2′-O-methyl, 2′-fluoro or both;

each N b and N b ′ independently represents an oligonucleotide sequence comprising 1-10 nucleotides which are modified, wherein the modifications on the nucleotides are 2′-O-methyl, 2′-fluoro or both;

each n p , n p ′, n q , and n q ′ independently represents an overhang nucleotide;

XXX, YYY, ZZZ, X′X′X′, Y′Y′Y′, and Z′Z′Z′ each independently represent one motif of three identical modifications on three consecutive nucleotides; and

wherein the sense strand is conjugated to at least one ligand.

2. The RNAi agent of claim 1 , wherein j is 1; or wherein 1 is 1; or wherein both j and 1 are 1.

3. The RNAi agent of claim 1 , wherein XXX is complementary to X′X′X′, YYY is complementary to Y′Y′Y′, and ZZZ is complementary to Z′Z′Z′.

4. The RNAi agent of claim 1 , wherein the YYY motif occurs at or near the cleavage site of the sense strand; or wherein the Y′Y′Y′ motif occurs at the 11, 12 and 13 positions of the antisense strand from the 5′-end.

5. The RNAi agent of claim 4 , wherein the Y′ is 2′-O-methyl.

6. The RNAi agent of claim 1 , wherein formula (III) is represented as formula (IIIa):

sense:

5′ n p  -N a  -Y Y Y -N b  -Z Z Z -N a -n q  3′

antisense:

3′ n p ′-N a ′-Y′Y′Y′-N b ′-Z′Z′Z′-N a ′n q ′ 5′

(IIIa)

wherein each N b and N b ′ independently represents an oligonucleotide sequence comprising 1-5 modified nucleotides; or wherein

formula (III) is represented as formula (IIIb):

sense:

5′ n p  -N a  -X X X -N b  -Y Y Y -N a -n q  3′

antisense:

3′ n p ′-N a ′-X′X′X′-N b ′-Y′Y′Y′-N a ′-n q ′ 5′

(IIIb)

wherein each N b and N b ′ independently represents an oligonucleotide sequence comprising 1-5 modified nucleotides; or wherein

formula (III) is represented as formula (IIIc):

sense:

5′ n p -N a -X X X -N b -Y Y Y -N b -Z Z Z-N a -

n q  3′

antisense:

3′ n p ′-N a ′-X′X′X′-N b ′-Y′Y′Y′-N b ′-Z′Z′

Z′-N a ′-n q ′ 5′

(IIIc)

wherein each N b and N b ′ independently represents an oligonucleotide sequence comprising 1-5 modified nucleotides and each N a and N a ′ independently represents an oligonucleotide sequence comprising 2-10 modified nucleotides.

7. The RNAi agent of claim 1 , wherein the sense strand and the antisense strand form a duplex region which is 15-30 nucleotide pairs in length.

8. The RNAi agent of claim 7 , wherein the duplex region is 17-25 nucleotide pairs in length.

9. The RNAi agent of claim 1 , wherein each strand independently has 19-25 nucleotides.

10. The RNAi agent of claim 1 , wherein the ligand is one or more GalNAc derivatives attached through a bivalent or trivalent branched linker.

11. The RNAi agent of claim 1 , wherein the ligand is

12. The RNAi agent of claim 1 , wherein the ligand is attached to the 3′ end of the sense strand.

13. The RNAi agent of claim 12 , wherein the RNAi agent is conjugated to the ligand as shown in the following schematic

wherein X is O or S.

14. The RNAi agent of claim 1 further comprising at least one phosphorothioate or methylphosphonate internucleotide linkage.

15. The RNAi agent of claim 14 , wherein the phosphorothioate or methylphosphonate internucleotide linkage is at the 3′-terminal of one strand.

16. The RNAi agent of claim 15 , wherein said strand is the antisense strand.

17. The RNAi agent of claim 1 , wherein the Y nucleotides contain a 2′-fluoro modification and wherein the Y′ nucleotides contain a 2′-O-methyl modification.

18. The RNAi agent of claim 1 , wherein p′=2.

19. The RNAi agent of claim 18 , wherein q′=0, p=0, q=0, and p′ overhang nucleotides are complementary to the target mRNA.

20. The RNAi agent of claim 18 , wherein at least one np′ is linked to a neighboring nucleotide via a phosphorothioate linkage.

21. The RNAi agent of claim 18 , wherein q′=0, p=0, q=0, and p′ overhang nucleotides are non-complementary to the target mRNA.

22. The RNAi agent of claim 1 , wherein the double stranded RNAi agent comprises a double stranded RNAi agent selected from the group consisting of AD-51546 (5′-UfgGfGfAfuUfuCfAfUfgUfaAfcCfAfAfgAfL96-3′ (SEQ ID NO: 2210) and 5′-uCfuugGfuUfaCfaugAfaAfuccCfasUfsc-3′ (SEQ ID NO: 2216)), and

AD-51547 (5′-UfgGfgAfuUfuCfAfUfgUfaacCfaAfgAfL96-3′ (SEQ ID NO: 2211) and 5′-uCfuUfgGfUfUfaCfaugAfaAfuCfcCfasUfsc-3′ (SEQ ID NO: 2217)),

wherein a, g, c and u are 2′-O-methyl (2′-OMe) A, G, C, and U; Af, Gf, Cf, and Uf are 2′-fluoro A, G, C and U; s is a phosphorothioate linkage; and L96 is a GalNAc3 ligand.

23. The RNAi agent of claim 1 , wherein each of the sense strand and the antisense strand independently have 21 to 23 nucleotides.

24. The RNAi agent of claim 1 , wherein the sense strand has a total of 21 nucleotides and the antisense strand has a total of 23 nucleotides.

25. The RNAi agent of claim 1 , wherein the Z nucleotides contain a 2′-O-methyl modification.

26. The RNAi agent of claim 1 , wherein the RNAi agent is AD-51547 (5′-UfgGfgAfuUfuCfAfUfgUfaacCfaAfgAfL96-3′ (SEQ ID NO: 2211) and 5′-uCfuUfgGfUfUfaCfaugAfaAfuCfcCfasUfsc-3′ (SEQ ID NO: 2217)),

wherein a, g, c and u are 2′-O-methyl (2′-OMe) A, G, C, and U; Af, Gf, Cf, and Uf are 2′-fluoro A, G, C and U; s is a phosphorothioate linkage; and L96 is a GalNAc3 ligand.

27. An isolated cell containing the RNAi agent of claim 1 .

28. A pharmaceutical composition comprising the RNAi agent of claim 1 .

29. A double stranded RNAi agent for inhibiting expression of TTR in a cell,

wherein said double stranded RNAi agent comprises a sense strand and an antisense strand forming a double stranded region;

wherein the sense strand comprises the nucleotide sequence 5′-UGGGAUUUCAUGUAACCAAGA 3′ (SEQ ID NO:2211) and the antisense strand comprises the nucleotide sequence 5′-UCUUGGUUACAUGAAAUCCCAUC-3′ (SEQ ID NO:2217);

wherein substantially all of the nucleotides of said sense strand comprise a modification selected from the group consisting of a 2′-O-methyl modification and a 2′-fluoro modification;

wherein substantially all of the nucleotides of said antisense strand comprise a modification selected from the group consisting of a 2′-O-methyl modification and a 2′-fluoro modification; and

wherein said sense strand is conjugated to one or more GalNAc derivatives attached through a branched bivalent or trivalent linker at the 3′-terminus.

30. A method of inhibiting expression of a transthyretin (TTR) in a cell comprising contacting said cell with the RNAi agent of claim 1 in an amount effective to inhibit expression of said TTR in said cell, thereby inhibiting expression of said transthyretin (TTR) in said cell.

31. A method of treating a TTR-associated disease in a subject, comprising administering to said subject a therapeutically effective amount of the RNAi agent of claim 1 , thereby treating said TTR-associated disease in said subject.

32. The method of claim 31 , wherein said subject is a human.

33. The method of claim 31 , wherein said subject carries a TTR gene mutation that is associated with the development of a TTR-associated disease.

34. The method of claim 31 , wherein said TTR-associated disease is selected from the group consisting of senile systemic amyloidosis (SSA), systemic familial amyloidosis, familial amyloidotic polyneuropathy (FAP), familial amyloidotic cardiomyopathy (FAC), leptomeningeal/Central Nervous System (CNS) amyloidosis, and hyperthyroxinemia.

35. The method of claim 31 , wherein said subject has a TTR-associated amyloidosis and said method reduces an amyloid TTR deposit in said subject.

36. The method of claim 31 , wherein said RNAi agent is administered to the subject subcutaneously.

37. A kit for performing the method of claim 30 or 31 , comprising

a) said RNAi agent, and

b) instructions for use.

38. The kit for performing the method of claim 37 , further comprising means for administering said RNAi agent to said subject.

39. A method of inhibiting production of TTR protein in a cell, comprising contacting the cell with the RNAi agent of claim 1 , thereby inhibiting production of TTR protein in the cell.

40. The method of claim 39 , wherein the cell is within a subject.

41. The method of claim 40 , wherein the subject is a human subject.

42. The method of claim 41 , wherein said subject carries a TTR gene mutation that is associated with the development of a TTR-associated disease.

43. The method of claim 42 , wherein the subject has a TTR-associated amyloidosis and the method reduces an amyloid TTR deposit in the subject.

44. A method of inhibiting production of an amyloid TTR deposit in a subject carrying a mutation that is associated with development of a TTR-associated amyloidosis, comprising administering to the subject the RNAi agent of claim 1 , thereby inhibiting production of the amyloid TTR deposit in the subject.

45. The method of claim 44 , wherein the subject is a human subject.