RNA interference mediated inhibition of gene expression using short interfering nucleic acids (siNA)

• US 9,260,471 B2
• Filed: 10/25/2011
• Issued: 02/16/2016
• Est. Priority Date: 10/29/2010
• Status: Active Grant

• Current Assignee: Sirna Therapeutics, Inc. (Alnylam Pharmaceuticals Incorporated)
• Original Assignee: Sirna Therapeutics, Inc. (Alnylam Pharmaceuticals Incorporated)
• Inventors: Cancilla, Mark, Cunningham, James J., Flanagan, Michael W., Haringsma, Henry J., Stanton, Matthew G., Stirdivant, Steven M., Willingham, Aarron, Kenski, Denise
• Primary Examiner(s): Bowman, Amy
• Application Number:
  US13/881,415
• Publication Number:
  US 20130217756A1
• Time in Patent Office: 1,575 Days
• US Class Current: 1/1
• CPC Class Codes: A61K 31/713 Double-stranded nucleic acids or oligonucleotides
• CPC Class Codes: C07H 21/02 with ribosyl as saccharide radical
• CPC Class Codes: C12N 15/111 General methods applicable to biologically active non-coding nucleic acids
• CPC Class Codes: C12N 15/113 Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing when used in plants C12N15/8218
• CPC Class Codes: C12N 2310/14 interfering N.A.
• CPC Class Codes: C12N 2310/315 Phosphorothioates
• CPC Class Codes: C12N 2310/321 2'-O-R Modification
• CPC Class Codes: C12N 2310/322 2'-R Modification
• CPC Class Codes: C12N 2310/3231 having an additional ring, e.g. LNA, ENA
• CPC Class Codes: C12N 2310/331 Universal or degenerate base
• CPC Class Codes: C12N 2310/344 Position-specific modifications, e.g. on every purine, at the 3'-end
• CPC Class Codes: C12N 2310/346 having a combination of backbone and sugar modifications
• CPC Class Codes: C12N 2310/351 Conjugate
• CPC Class Codes: C12N 2310/3521 Methyl
• CPC Class Codes: C12N 2310/3527 Other alkyl chain
• CPC Class Codes: C12N 2310/3533 Halogen
• CPC Class Codes: C12N 2320/32 Special delivery means, e.g. tissue-specific
• CPC Class Codes: C12N 2320/51 modulating the chemical stability, e.g. nuclease-resistance

Abstract

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.

Claims

1. A compound comprising:

I) a double-stranded short interfering nucleic acid (siNA) molecule that inhibits the expression of a target gene via RNA interference, comprising a sense strand and an antisense strand, said siNA molecule comprising formula (A);

B—

N_X3—

(N)_X2B-3′

B(N)_X1—

N_X4—

[N]_X5-5′

 

 

(A)

wherein the upper strand is the sense strand and the lower strand is the antisense strand of the siNA molecule;

wherein the antisense strand comprises a sequence having at least 15 nucleotides that are complementary to a target RNA sequence encoded by the target gene and the sense strand comprises a sequence that is complementary to the antisense strand;

each N is independently a nucleotide which is unmodified or chemically modified, or is a non-nucleotide;

each B is independently a terminal cap that is present or absent;

(N) represents overhanging nucleotides, each of which is independently unmodified or chemically modified;

[N] represents nucleotides at the 5′

-terminus of the antisense strand;

X1 and X2 are independently integers from 0 to 4;

X3 is an integer from 15 to 30;

X4 is an integer from 12 to 27; and

X5 is an integer from 1-6, provided that the sum of X4 and X5 is an integer from 15-30; and

wherein

(a) five or more pyrimidine nucleotides in N_X4 positions are a combination of two of;

2′

-deoxy-2′

-fluoro nucleotides, 2′

-O-alkyl nucleotides, 2′

deoxy nucleotides, or ribonucleotides;

(b) five or more purine nucleotides in N_X4 positions are a combination of two of;

2′

-deoxy-2′

-fluoro nucleotides, 2′

-O-alkyl nucleotides, 2′

deoxy nucleotides, or ribonucleotides;

(c) five or more pyrimidine nucleotides in N_X3 positions are a combination of two of;

2′

-deoxy-2′

-fluoro nucleotides, 2′

-O-alkyl nucleotides, 2′

deoxy nucleotides, or ribonucleotides;

(d) five or more purine nucleotides in N_X3 positions are a combination of two of;

2′

-deoxy-2′

-fluoro nucleotides, 2′

-O-alkyl nucleotides, 2′

deoxy nucleotides, or ribonucleotides;

(e) [N] position nucleotide(s) are ribonucleotides, deoxyribonucleotides, 2′

-O-alkyl nucleotides, or 2′

-halo nucleotides, or any combination thereof irrespective of purine or pyrimidine content; and

(f) the nucleotide at position 14 from the 5′

-end of the antisense strand is a 2′

-deoxy-2′

-fluoro nucleotide regardless of whether it is a purine or pyrimidine; and

II) a ligand.

2. The compound according to claim 1, wherein:

(a) five or more pyrimidine nucleotides in N_X4 positions are 2′

-O-methyl nucleotides;

(b) five or more purine nucleotides in N_X4 positions are 2′

-deoxy-2′

-fluoro nucleotides;

(c) five or more pyrimidine nucleotides in N_X3 positions are 2′

-O-methyl nucleotides;

(d) five or more purine nucleotides in N_X3 positions are 2′

-deoxy-2′

-fluoro nucleotides; and

(e) [N] position nucleotide(s) are any combination of ribonucleotides, deoxyribonucleotides, 2′

-O-alkyl nucleotides, or 2′

-halo nucleotides.

3. The compound according to claim 1, wherein:

(a) 5, 6, 7, 8, 9, 10 or more pyrimidine nucleotides in N_X4 positions are 2′

-O-methyl nucleotides;

(b) 5, 6, 7, 8, 9, 10 or more purine nucleotides in N_X4 positions are 2′

-deoxy-2′

-fluoro nucleotides;

(c) 5, 6, 7, 8, 9, 10 or more pyrimidine nucleotides in N_X3 positions are 2′

-O-methyl nucleotides;

(d) 5, 6, 7, 8, 9, 10 or more purine nucleotides in N_X3 positions are 2′

-deoxy-2′

-fluoro nucleotides; and

(e) [N] position nucleotide(s) are any combination of ribonucleotides, deoxyribonucleotides, 2′

-O-alkyl nucleotides, or 2′

-halo nucleotides.

4. The compound according to claim 1, wherein X5 is 3.

5. The compound according to claim 4, wherein the three [N] nucleotides of formula (A) are represented as 5′

-[N1, N2, N3]-3′

, wherein;

a. each N1, N2, and N3 is a ribonucleotide;

or

b. each N1, N2, and N3 is a 2′

-deoxy-2′

-fluoro nucleotide;

or

c. each N1, N2, and N3 is a 2′

-deoxy nucleotide;

or

d. each N1, N2, and N3 is a 2′

-O-alkyl nucleotide; and

e. any of N1, N2, or N3 optionally comprises a phosphorothioate internucleotide linkage.

6. The compound according to claim 4, wherein the three [N] nucleotides of formula (A) are represented as 5′

-[N1, N2, N3]-3′

, wherein;

a. N1 is a 2′

-deoxy-2′

-fluoro nucleotide, N2 is 2′

-deoxy-2′

-fluoro nucleotide, and N3 is a 2′

-deoxynucleotide; and

b. any of N1, N2, or N3 optionally comprises a phosphorothioate internucleotide linkage.

7. The compound according to claim 4, wherein the three [N] nucleotides of formula (A) are represented as 5′

-[N1, N2, N3]-3′

, wherein;

a. N1 is a 2′

-deoxy nucleotide, N2 is 2′

-deoxy-2′

-fluoro nucleotide, and N3 is a 2′

-O-methyl nucleotide; and

b. any of N1, N2, or N3 optionally comprises a phosphorothioate internucleotide linkage.

8. The compound according to claim 4, wherein the three [N] nucleotides of formula (A) are represented as 5′

-[N1, N2, N3]-3′

, wherein;

a. N1 is a 2′

-deoxy nucleotide, N2 is 2′

-deoxy-2′

-fluoro nucleotide, and N3 is a 2′

-deoxy nucleotide; and

b. any of N1, N2, or N3 optionally comprises a phosphorothioate internucleotide linkage.

9. The compound according to claim 4, wherein the three [N] nucleotides of formula (A) are represented as 5′

-[N1, N2, N3]-3′

, wherein;

a. N1 is a 2′

-deoxy-2′

-fluoro nucleotide, N2 is 2′

-deoxy-2′

-fluoro nucleotide, and N3 is a 2′

-deoxy-2′

-fluoro nucleotide; and

b. any of N1, N2, or N3 optionally comprises a phosphorothioate internucleotide linkage.

10. The compound according to claim 1, wherein X1 is 2.

11. The compound according to claim 1, wherein X5 is 3, X1 is 2 and X2 is 2.

12. The compound according to claim 1, wherein said siNA molecule comprises one or more universal base substitutions.

13. The compound according to claim 1, wherein said siNA molecule comprises one or more locked nucleic acid (LNA) substitutions.

14. The compound according to claim 1, wherein one or more overhanging nucleotides of (N) of said siNA molecule is a 2′

-O-methyl nucleotide.

15. The compound according to claim 1, wherein said siNA molecule comprises at least one phosphorothioate internucleotide linkage.

16. The compound according to claim 1, wherein X5=3;

each X1 and X2=1 or 2;

X3=18, 19, 20, 21, 22, or 24, and X4=17, 18, 19, 20, 21, 22, or 23.

17. The compound according to claim 1, wherein X5=3;

each X1 and X2=2;

X3=19, and X4=16.

22. The compound of claim 1, wherein the 2′

-O-alkyl nucleotide is a 2′

-O-methyl modification.

23. The compound of claim 1, wherein the 2′

-halo nucleotide is a 2′

-deoxy-2′

-fluoro nucleotide.

24. The compound of claim 1, wherein five or more purine nucleotides in the N_X4 positions are 2′

-O-methyl nucleotides.

25. The compound of claim 1, wherein five or more pyrimidines in the N_X4 positions are 2′

-fluoro nucleotides.

26. The compound of claim 1, wherein five or more purine nucleotides in the N_X3 positions are 2′

-O-methyl nucleotides.

27. The compound of claim 1, wherein five or more purine nucleotides in the N_X3 positions are 2′

-fluoro nucleotides.

28. The compound of claim 1, wherein five or more pyrimidine nucleotides in the N_X3 positions are 2′

-fluoro nucleotides.

29. The compound of claim 1, wherein:

(a) 5, 6, 7, 8, 9, 10 or more of the pyrimidine and purine nucleotides in the N_X4 positions are 2′

-O-methyl nucleotides and 2′

-deoxy-2′

-fluoro nucleotides;

(b) 5, 6, 7, 8, 9, 10 or more of the pyrimidine and purine nucleotides in the N_X3 positions are 2′

-O-methyl nucleotides and 2′

-deoxy-2′

-fluoro nucleotides; and

(c) [N] position nucleotide(s) are any combination of ribonucleotides, deoxyribonucleotides, 2′

-O-alkyl nucleotides, 2′

-halo nucleotides, or any combination thereof irrespective of the purine or pyrimidine content.

30. The compound of claim 1, wherein one or both strands comprise:

(i) at least 5 pyrimidine nucleotides that are 2′

-deoxy-2′

-fluoro pyrimidine nucleotides and (ii) at least 5 purine nucleotides that are 2′

-O-methyl purine nucleotides.

31. The compound of claim 1, wherein one or both strands comprise (i) at least 5 pyrimidine nucleotides that are 2′

-O-methyl pyrimidine nucleotides and (ii) at least 5 purine nucleotides that are 2′

-deoxy-2′

-fluoro purine nucleotides.

32. The compound of claim 1, wherein said siNA molecule comprises one or more acyclic nucleotides.

33. The compound of claim 1, further comprising one or more phosphorothioate linkages between the first terminal (N) and the adjacent nucleotide on the 3′

end of the sense strand.

34. The compound of claim 1, wherein X1=2 overhanging nucleotide positions with a phosphorothioate internucleotide linkage.

35. The compound of claim 1, wherein the ligand is chosen from a steroidal compound, a galactosamine, a vitamin, a protein, a peptide, or an antibody.

36. The compound of claim 1, wherein the ligand is a cholesterol.

37. The compound of claim 1, wherein the ligand comprises one or more N-acetylgalactosamine.

38. The compound of claim 35, wherein the ligand is covalently attached to the siNA molecule.

39. The compound of claim 35, wherein the ligand is covalently attached to the siNA molecule via a linker.

40. The compound of claim 39, wherein the linker is chosen from a phosphate ester based linker, an amino based linker, a disulfide based linker, a succinyl based linker, an alkyl or substituted alkyl based linker, or an amide based linker.

41. A composition comprising the siNA molecule according to claim 1 and a pharmaceutically acceptable carrier or diluents.

42. The compound of claim 1, wherein 5, 6, 7, 8, 9, 10 or more of the pyrimidine or purine nucleotides in the N_X3 positions are chosen from a combination of 2′

-deoxy-2′

-fluoro nucleotides and ribonucleotides, 2′

-O-methyl nucleotides and ribonucleotides, 2′

-deoxy-2′

-fluoro nucleotides and deoxyribonucleotides, or 2′

-deoxy-2′

-fluoro nucleotides and 2′

-O-methyl nucleotides.

43. The compound of claim 1, wherein 5, 6, 7, 8, 9, 10 or more of the pyrimidine or purine nucleotides in the N_X4 positions are chosen from a combination of 2′

-deoxy-2′

-fluoro nucleotides and ribonucleotides, 2′

-O-methyl nucleotides and ribonucleotides, 2′

-deoxy-2′

-fluoro nucleotides and deoxyribonucleotides, or 2′

-deoxy-2′

-fluoro nucleotides and 2′

-O-methyl nucleotides.

44. The compound of claim 1, wherein (N)_X1 or (N)_X2 comprises one or more of 2′

-O-methyl nucleotide, 2′

-deoxy-2′

-fluoro nucleotide, deoxyribonucleotide or a locked nucleic acid (LNA).

45. The compound of claim 44, wherein (N)_X1 further comprises at least one phosphorothioate internucleotide linkage.

46. The compound of claim 1, wherein:

(i) 5, 6, 7, 8, 9, 10 or more of the pyrimidine or purine nucleotides in the N_X3 positions are chosen from a combination of 2′

-deoxy-2′

-fluoro nucleotides and ribonucleotides, 2′

-O-methyl nucleotides and ribonucleotides, 2′

-deoxy-2′

-fluoro nucleotides and deoxyribonucleotides, or 2′

-deoxy-2′

-fluoro nucleotides and 2′

-O-methyl nucleotides;

(ii) 5, 6, 7, 8, 9, 10 or more of the pyrimidine or purine nucleotides in the N_X4 positions are chosen from a combination of 2′

-deoxy-2′

-fluoro nucleotides and ribonucleotides, 2′

-O-methyl nucleotides and ribonucleotides, 2′

-deoxy-2′

-fluoro nucleotides and deoxyribonucleotides, or 2′

-deoxy-2′

-fluoro nucleotides and 2′

-O-methyl nucleotides; and

(iii) (N)_X1 comprises one or more of 2′

-O-methyl nucleotide, 2′

-deoxy-2′

-fluoro nucleotide, deoxyribonucleotide or a locked nucleic acid (LNA), and at least one phosphorothioate internucleotide linkage.

18. A double-stranded short interfering nucleic acid (siNA) molecule that inhibits the expression of a target gene via RNA interference comprising a sense strand and an antisense strand and comprising formula (A):

B—

N_X3—

(N)_X2B-3′

B(N)_X1—

N_X4—

[N]_X5-5′

 

 

(A)

wherein, the upper strand is the sense strand and the lower strand is the antisense strand of the siNA molecule;

wherein the antisense strand comprises a sequence having at least 15 nucleotides that are complementary to a target RNA sequence encoded by the target gene and the sense strand comprises a sequence that is complementary to the antisense strand;

each N is independently a nucleotide which is unmodified or chemically modified, or a non-nucleotide;

each B is independently a terminal cap that is present or absent;

(N) represents overhanging nucleotides, each of which is independently unmodified or chemically modified;

[N] represents nucleotides at the 5′

-terminus of the antisense strand;

X1 and X2 are independently integers from 0 to 4;

X3 is an integer from 15 to 30;

X4 is an integer from 12 to 27; and

X5 is an integer from 1-6, provided that the sum of X4 and X5 is an integer from 15-30; and

wherein

(a) all pyrimidine nucleotides in N_x4 positions are 2′

-O-alkyl nucleotides;

(b) all purine nucleotides in N_x4 positions are 2′

-halo nucleotides;

(c) all pyrimidine nucleotides in N_x3 positions are 2′

-O-alkyl nucleotides;

(d) all purine nucleotides in N_x4 positions are 2′

-halo nucleotides; and

(e) [N] position nucleotide(s) are any combination of ribonucleotides, deoxyribonucleotides, 2′

-O-alkyl nucleotides, or 2′

-halo nucleotides;

(f) the nucleotide at position 14 from the 5′

-end of the antisense strand is a 2′

-deoxy-2′

-fluoro nucleotide regardless of whether it is a purine or pyrimidine; and

(g) [N] nucleotides of formula (A) are represented as 5′

-[N1, N2, N3]-3′

, wherein

i) N1 is a 2′

-deoxy nucleotide, N2 is 2′

-deoxy-2′

-fluoro nucleotide, and N3 is a 2′

-deoxy nucleotide;

or

ii) N1 is a 2′

-deoxy-2′

-fluoro nucleotide, N2 is 2′

-deoxy-2′

-fluoro nucleotide, and N3 is a 2′

-deoxy-2′

-fluoro nucleotide;

or

iii) N1 is a 2′

-deoxy-2′

-fluoro nucleotide, N2 is 2′

-deoxy-2′

-fluoro nucleotide, and N3 is a 2′

-deoxynucleotide;

or

iv) N1 is a 2′

-deoxy nucleotide, N2 is 2′

-deoxy-2′

-fluoro nucleotide, and N3 is a 2′

-O-methyl nucleotide;

or

v) N1, N2, and N3 are all ribonucleotides having phosphorothioate internucleotide linkages.

19. A polymer comprising the double-stranded short interfering nucleic acid (siNA) molecule according to claim 18.

20. A compound comprising the double-stranded short interfering nucleic acid (siNA) molecule according to claim 18 covalently attached to a ligand.

21. A composition comprising the double-stranded short interfering nucleic acid (siNA) molecule according to claim 18 and a pharmaceutically acceptable carrier or diluent.

47. A conjugate comprising a double-stranded short interfering nucleic acid (siNA) molecule that inhibits the expression of a target gene, said siNA molecule comprising a sense strand and an antisense strand of formula (A):

B—

N_X3—

(N)_X2B-3′

B(N)_X1—

N_X4—

[N]_X5-5′

 

 

(A)

wherein the upper strand is the sense strand and the lower strand is the antisense strand of the siNA molecule;

wherein the antisense strand comprises a sequence having at least 15 nucleotides that are complementary to a target RNA sequence encoded by the target gene and the sense strand comprises a sequence that is complementary to the antisense strand;

each N is independently a nucleotide which is unmodified or chemically modified, or is a non-nucleotide;

each B is independently a terminal cap that is present or absent;

(N) represents overhanging nucleotides, each of which is independently unmodified or chemically modified;

[N] represents nucleotides at the 5′

-terminus of the antisense strand;

X1 and X2 are independently integers from 0 to 4;

X3 is an integer from 15 to 30;

X4 is an integer from 12 to 27; and

X5 is an integer from 0-6, provided that the sum of X4 and X5 is an integer from 15-30; and

wherein

(a) five or more pyrimidine nucleotides in N_X4 positions a combination of two of;

2′

-deoxy-2′

-fluoro nucleotides, 2′

-O-alkyl nucleotides, 2′

deoxy nucleotides, or ribonucleotides;

(b) five or more purine nucleotides in N_X4 positions are a combination of two of;

2′

-deoxy-2′

-fluoro nucleotides, 2′

-O-alkyl nucleotides, 2′

deoxy nucleotides, or ribonucleotides;

(c) five or more pyrimidine nucleotides in N_X3 positions are a combination of two of;

2′

-deoxy-2′

-fluoro nucleotides, 2′

-O-alkyl nucleotides, 2′

deoxy nucleotides, or ribonucleotides;

(d) five or more purine nucleotides in N_X3 positions are a combination of two of;

2′

-deoxy-2′

-fluoro nucleotides, 2′

-O-alkyl nucleotides, 2′

deoxy nucleotides, or ribonucleotides;

(e) [N] position nucleotide(s) are ribonucleotides, deoxyribonucleotides, 2′

-O-alkyl nucleotides, or 2′

-halo nucleotides, or any combination thereof irrespective of purine or pyrimidine content; and

(f) the nucleotide at position 14 from the 5′

-end of the antisense strand is a 2′

-deoxy-2′

-fluoro nucleotide regardless of whether it is a purine or pyrimidine.

48. The conjugate of claim 47, wherein:

(i) 5, 6, 7, 8, 9, 10 or more of the pyrimidine or purine nucleotides in the N_X3 positions are chosen from a combination of 2′

-deoxy-2′

-fluoro nucleotides and ribonucleotides, 2′

-O-methyl nucleotides and ribonucleotides, 2′

-deoxy-2′

-fluoro nucleotides and deoxyribonucleotides, or 2′

-deoxy-2′

-fluoro nucleotides and 2′

-O-methyl nucleotides;

(ii) 5, 6, 7, 8, 9, 10 or more of the pyrimidine or purine nucleotides in the N_X4 positions are chosen from a combination of 2′

-deoxy-2′

-fluoro nucleotides and ribonucleotides, 2′

-O-methyl nucleotides and ribonucleotides, 2′

-deoxy-2′

-fluoro nucleotides and deoxyribonucleotides, or 2′

-deoxy-2′

-fluoro nucleotides and 2′

-O-methyl nucleotides; and

(iii) (N)_X1 comprises one or more of 2′

-O-methyl nucleotide, 2′

-deoxy-2′

-fluoro nucleotide, deoxyribonucleotide or a locked nucleic acid (LNA), and at least one phosphorothioate internucleotide linkage.

49. A conjugate comprising a double-stranded short interfering nucleic acid (siNA) molecule that inhibits the expression of a target gene, said siNA molecule comprising a sense strand and an antisense strand of formula (A):

B—

N_X3—

(N)_X2B-3′

B(N)_X1—

N_X4—

[N]_X5-5′

 

 

(A)

wherein;

(a) the upper strand is the sense strand and the lower strand is the antisense strand of the siNA molecule;

(b) the antisense strand comprises a sequence having at least 15 nucleotides that are complementary to a target RNA sequence encoded by the target gene and the sense strand comprises a sequence that is complementary to the antisense strand;

(c) each N is independently a nucleotide or a non-nucleotide;

(d) X1 and X2 are independently integers from 0 to 4;

(e) X3 is an integer from 15 to 30;

(f) X4 is an integer from 12 to 27;

(g) X5 is an integer from 0-6, provided that the sum of X4 and X5 is an integer from 15-30;

(h) each (N) is independently a 2′

-O-methyl nucleotide, a 2′

-deoxy-2′

-fluoro nucleotide, a 2′

-deoxy-nucleotide or a locked nucleic acid (LNA), or a combination thereof;

(i) N_x3 is any combination of a 2′

-O-methyl nucleotide, a 2′

-deoxy-2′

-fluoro nucleotide, or a 2′

-deoxy-nucleotide;

(j) N_x4 is any combination of a 2′

-O-methyl nucleotide, a 2′

-deoxy-2′

-fluoro nucleotide, or a 2′

-deoxy-nucleotide;

(k) each [N] is independently a 2′

-O-methyl nucleotide, a 2′

-deoxy-2′

-fluoro nucleotide, a 2′

-deoxy-nucleotide, or a combination thereof;

(l) B is independently a terminal cap that is present or absent; and

(m) the nucleotide at position 14 from the 5′

-end of the antisense strand is a 2′

-deoxy-2′

-fluoro nucleotide regardless of whether it is a purine or pyrimidine.

50. The conjugate of claim 49, which comprises a phosphorothioate linkage between one or more of (N), (N);

N,N;

(N),N;

N,[N];

or [N],[N].

51. The conjugate of claim 49, wherein X1=2 overhanging nucleotide positions with a phosphorothioate internucleotide linkage.

52. The conjugate of claim 49, which comprises a ligand chosen from a steroidal compound, a galactosamine, a vitamin, a protein, a peptide, or an antibody.

53. The conjugate of claim 49, which comprises one or more N-acetylgalactosamine.