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 sequence that is complementary to nucleotides 504 to 526 of the transthyretin (TTR) gene (SEQ ID NO:1), wherein the sense strand is 21 nucleotides in length and the antisense strand is 23 nucleotides in length, wherein said double stranded RNAi agent is represented by formula (III):

(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′

wherein:

j=1; and i, k, and l are 0;

p′ is 2; p, q, and q′ are 0;

each N a and N a ′ independently represents an oligonucleotide sequence comprising 2-10 nucleotides which are modified nucleotides;

each N b and N b ′ independently represents an oligonucleotide sequence comprising 0-7 nucleotides which are modified nucleotides;

n p ′ represents an overhang nucleotide;

YYY, ZZZ, and Y′Y′Y′, each independently represent one motif of three identical modifications on three consecutive nucleotides, wherein the Y nucleotides contain a 2′-fluoro modification, the Y′ nucleotides contain a 2′-O-methyl modification, and the Z nucleotides contain a 2′-O-methyl modification;

and

wherein the sense strand is conjugated to at least one ligand, wherein the ligand is one or more GalNAc derivatives attached through a bivalent or trivalent branched linker.

2. 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.

3. The RNAi agent of claim 1 , wherein the modifications on the N a , N a ′, N b and N b ′ nucleotides are each independently selected from the group consisting of LNA, HNA, CeNA, 2′-methoxyethyl, 2′-O-alkyl, 2′-O-allyl, 2′-C-allyl, 2′-fluoro, 2′-deoxy, 2′-hydroxyl, and combinations thereof.

4. The RNAi agent of claim 3 , wherein the modifications on the N a , N a ′, N b and N b ′ nucleotides are 2′-O-methyl, 2′-fluoro or both.

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

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

7. The RNAi agent of claim 6 , wherein the RNAi agent is conjugated to the ligand as shown in the following schematic

wherein X is O or S.

8. The RNAi agent of claim 7 , wherein the RNAi agent is conjugated to the ligand as shown in the following schematic

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

10. The RNAi agent of claim 9 , wherein the phosphorothioate or methylphosphonate internucleotide linkage is at the 3′-terminal of one strand.

11. The RNAi agent of claim 10 , wherein said strand is the antisense strand or the sense strand.

12. The RNAi agent of claim 1 , wherein the base pair at the 1 position of the 5′-end of the antisense strand of the duplex is an AU base pair.

13. The RNAi agent of claim 1 , wherein the p′ overhang nucleotides are complementary to the target mRNA or wherein the p′ overhang nucleotides are non-complementary to the target mRNA.

14. The RNAi agent of claim 1 , wherein at least one np′ is linked to a neighboring nucleotide via a phosphorothioate linkage.

15. The RNAi agent of claim 14 , wherein all np′ are linked to neighboring nucleotides via phosphorothioate linkages.

16. The RNAi agent of claim 1 , wherein said RNAi agent is selected from the group consisting of AD-51546 and AD-51547.

17. The RNAi agent of claim 1 , wherein the RNAi agent is AD-51547.

18. The double stranded RNAi agent of claim 1 , wherein the sense strand comprises the nucleotide sequence 5′-UGGGAUUUCAUGUAACCAAGA-3′ (SEQ ID NO:2211).

19. The double stranded RNAi agent of claim 1 , wherein the sense strand comprises the nucleotide sequence 5′-UfgGfgAfuUfuCfAfUfgUfaacCfaAfgAfL96-3′ (SEQ ID NO:2) and the antisense strand comprises the nucleotide sequence 5′-uCfuUfgGfUfUfaCfaugAfaAfuCfcCfasUfsc-3′ (SEQ ID NO:3),

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.

20. A double stranded RNAi agent, comprising a sense strand and an antisense strand, wherein the sense strand comprises the nucleotide sequence 5′-UfgGfgAfuUfuCfAfUfgUfaacCfaAfgAfL96-3′ (SEQ ID NO:2) and the antisense strand comprises the nucleotide sequence 5′-uCfuUfgGfUfUfaCfaugAfaAfuCfcCfasUfsc-3′ (SEQ ID NO:3),

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.

21. An isolated cell containing an RNAi agent of any one of claims 1 , 17 , or 20 .

22. A pharmaceutical composition comprising an RNAi agent of any one of claims 1 , 17 , or 20 .

23. The pharmaceutical composition of claim 22 , wherein the RNAi agent is administered in an unbuffered solution.

24. The pharmaceutical composition of claim 23 , wherein said unbuffered solution is saline or water.

25. The pharmaceutical composition of claim 22 , wherein said RNAi agent is administered with a buffer solution.

26. The pharmaceutical composition of claim 25 , wherein said buffer solution comprises acetate, citrate, prolamine, carbonate, or phosphate or any combination thereof.

27. The pharmaceutical composition of claim 26 , wherein said buffer solution is phosphate buffered saline (PBS).

28. A method of inhibiting expression of a transthyretin (TTR) in a cell comprising contacting said cell with an RNAi agent of any one of claims 1 , 17 , or 20 in an amount effective to inhibit expression of said TTR in said cell, thereby inhibiting expression of said transthyretin (TTR) in said cell.

29. A kit for performing the method of claim 28 , comprising

a) said RNAi agent, and

b) instructions for use.

30. A method of treating a TTR-associated disease in a subject, comprising administering to said subject a therapeutically effective amount of an RNAi agent of any one of claims 1 , 17 , or 20 , thereby treating said TTR-associated disease in said subject.

31. The method of claim 30 , wherein said subject is a human.

32. The method of claim 30 , wherein said subject carries a TTR gene mutation that is associated with the development of a TTR-associated disease.

33. The method of claim 30 , 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.

34. The method of claim 30 , wherein said subject has a TTR-associated amyloidosis and said method reduces an amyloid TTR deposit in said subject.

35. The method of claim 30 , wherein said RNAi agent is administered to the subject subcutaneously.

36. A kit for performing the method of claim 30 , comprising

a) said RNAi agent,

b) instructions for use, and

c) optionally, means for administering said RNAi agent to said subject.