MicroRNA Compounds and Methods for Modulating MIR-21 Activity

1 . A compound having the structure:

L n -linker-X-N m -X-MO

wherein each L is, independently, a ligand and n is from 1 to 10; each N is, independently, a modified or unmodified nucleoside and m is from 1 to 5; each X is, independently, a phosphodiester linkage or a phosphorothioate linkage; and MO is a modified oligonucleotide, wherein

a. the modified oligonucleotide comprises at least 8 contiguous nucleosides of the following nucleoside pattern I in the 5′ to 3′ orientation:

(R) X -N B -N Q -N Q -N B -(N Q -N Q -N Q -N B ) 3 -N Q -N Z

wherein each R is, independently, a non-bicyclic nucleoside; X is from 1 to 4;

each N B is, independently, a bicyclic nucleoside;

each N Q is, independently, a non-bicyclic nucleoside; and

each N Z is, independently, a modified nucleoside;

b. the modified oligonucleotide consists of 8 to 19 linked nucleosides, and wherein the modified oligonucleotide comprises at least 8 contiguous nucleosides of the following nucleoside pattern II in the 5′ to 3′ orientation:

N M -N B -N Q -N Q -N B -(N Q -N Q -N Q -N B ) 3 -N Q -N Z

wherein N M is, independently, a modified nucleoside that is not a bicyclic nucleoside;

each N B is, independently, a bicyclic nucleoside;

each N Q is, independently, a non-bicyclic nucleoside; and

N Z is, independently, a modified nucleoside;

c. the modified oligonucleotide comprises at least 8 contiguous nucleosides of the following nucleoside pattern III in the 5′ to 3′ orientation:

(R) X -N B -N Q -N Q -N B -(N Q -N Q -N Q -N B ) 3 -N Y -N Z

wherein each R is a non-bicyclic nucleoside; X is from 1 to 4;

each N B is a bicyclic nucleoside;

each N Q is a non-bicyclic nucleoside;

N Y is a modified nucleoside or an unmodified nucleoside; and

each N Z is a modified nucleoside;

d. the modified oligonucleotide consists of 8 to 19 linked nucleosides, and wherein the modified oligonucleotide comprises at least 8 contiguous nucleosides of the following nucleoside pattern IV in the 5′ to 3′ orientation:

N M -N B -N Q -N Q -N B -(N Q -N Q -N Q -N B ) 3 -N Y -N Z

wherein N M is a modified nucleoside that is not a bicyclic nucleoside;

each N B is a bicyclic nucleoside;

each N Q is a non-bicyclic nucleoside;

N Y is a modified nucleoside or an unmodified nucleoside; and

N Z is a modified nucleoside;

e. the modified oligonucleotide consists of 8 to 19 linked nucleosides, and wherein the modified oligonucleotide comprises at least 8 contiguous nucleosides of the following nucleoside pattern V in the 5′ to 3′ orientation:

N M -N B -(N Q -N Q -N B -N B ) 4 -N Z

wherein N M is a modified nucleoside that is not a bicyclic nucleoside;

each N B is a bicyclic nucleoside;

each N Q is a non-bicyclic nucleoside; and

N Z is a modified nucleoside;

f. the modified oligonucleotide consists of 8 to 15 linked nucleosides, and wherein the modified oligonucleotide comprises at least 8 contiguous nucleosides of the following nucleoside pattern VI in the 5′ to 3′ orientation:

N Q -N B -N B -N Q -(N B -N B -N Q -N Q ) 2 -N B -N Q -N B

wherein each N Q is a non-bicyclic nucleoside; and

each N B is a bicyclic nucleoside; and/or

g. the modified oligonucleotide consists of 8 to 19 linked nucleosides, and wherein the modified oligonucleotide comprises at least 8 contiguous nucleosides of the following nucleoside pattern VII in the 5′ to 3′ orientation:

N M -(N B -N M -N M ) 2 -N M -(N B -N Q -N Q -N Q ) 2 -N B -N B -N Z

wherein each N M is a modified nucleoside that is not a bicyclic nucleoside;

each N B is a bicyclic nucleoside;

each N Q is a non-bicyclic nucleoside; and

N Z is a modified nucleoside.

2 . (canceled)

3 . (canceled)

4 . The compound of claim 1 , wherein if n is greater than 1, L n -linker has the structure:

wherein each L is, independently, a ligand; n is from 1 to 10; S is a scaffold; and Q′ and Q″ are, independently, linking groups.

5 . (canceled)

6 . The compound of claim 4 , wherein the scaffold links 2, 3, 4, or 5 ligands to a modified oligonucleotide.

7 . (canceled)

8 . The compound of claim 1 , comprising the structure:

wherein:

B is selected from —O—, —S—, —N(R N )—, —Z—P(Z′)(Z″)O—, —Z—P(Z′)(Z″)O—N m -X—, and —Z—P(Z′)(Z″)O—N m —Y—;

MO is the modified oligonucleotide;

R N is selected from H, methyl, ethyl, propyl, isopropyl, butyl, and benzyl;

Z, Z′, and Z″ are each independently selected from O and S;

each N is, independently, a modified or unmodified nucleoside;

m is from 1 to 5;

X is selected from a phosphodiester linkage and a phosphorothioate linkage;

Y is a phosphodiester linkage; and

the wavy line indicates the connection to the rest of the linker and ligand(s).

9 . The compound of claim 1 , wherein n is from 1 to 5, 1 to 4, 1 to 3, or 1 to 2.

10 . (canceled)

11 . (canceled)

12 . (canceled)

13 . The compound of claim 1 , wherein at least one ligand is selected from N-acetylgalactosamine, galactose, galactosamine, N-formylgalactosamine, N-propionyl-galactosamine, N-n-butanoylgalactosamine, and N-iso-butanoyl-galactosamine.

14 . (canceled)

15 . The compound of claim 1 , wherein the compound has the structure:

wherein each N is, independently, a modified or unmodified nucleoside and m is from 1 to 5; X 1 and X 2 are each, independently, a phosphodiester linkage or a phosphorothioate linkage; and MO is the modified oligonucleotide.

16 . The compound of claim 0 , wherein at least one of X 1 and X 2 is a phosphodiester linkage.

17 . The compound of claim 0 , wherein each of X 1 and X 2 is a phosphodiester linkage.

18 . The compound of claim 1 , wherein m is 1, 2, 3, 4 or 5.

19 - 44 . (canceled)

45 . The compound of claim 1 , wherein each bicyclic nucleoside is independently selected from an LNA nucleoside, a cEt nucleoside, and an ENA nucleoside.

46 . (canceled)

47 . (canceled)

48 . The compound of claim 1 , wherein each non-bicyclic nucleoside is independently selected from a β-D-deoxyribonucleoside, a 2′-O-methyl, and a 2′-O-methoxyethyl nucleoside.

49 - 51 . (canceled)

52 . The compound of claim 1 wherein:

i. the compound is a compound of claim 1 (a), wherein:

a. R consists of four linked nucleosides N R1 -N R2 -N R3 -N R4 , wherein N R1 is a 2′-O-methoxyethyl nucleoside and each of NR 2 —NR 3 —N R4 is a β-D-deoxyribonucleoside;

each N B is an S-cEt nucleoside;

each N Q is a β-D-deoxyribonucleoside; and

N Z is a 2′-O-methoxyethyl nucleoside;

b. each R is a 2′-O-methoxyethyl nucleoside; X is 1;

each N B is an S-cEt nucleoside;

each N Q is a β-D-deoxyribonucleoside; and

N Z is a 2′-O-methoxyethyl nucleoside;

c. each R is a 2′-O-methoxyethyl nucleoside; X is 1;

each N B is an S-cEt nucleoside;

each N Q is a 2′-O-methoxyethyl nucleoside; and

N Z is a 2′-O-methoxyethyl nucleoside;

d. each R is a 2′-O-methoxyethyl nucleoside; X is 1;

each N B is an S-cEt nucleoside;

each N Q is a β-D-deoxyribonucleoside; and

N Z is an S-cEt nucleoside;

e. each R is a 2′-O-methoxyethyl nucleoside; X is 1;

each N B is an LNA nucleoside;

each N Q is a β-D-deoxyribonucleoside; and

N Z is a 2′-O-methoxyethyl nucleoside; or

f. each R is a 2′-O-methoxyethyl nucleoside; X is 1;

each N B is an LNA nucleoside;

each N Q is a β-D-deoxyribonucleoside; and

N Z is an LNA nucleoside;

ii. the compound is a compound of claim 1 (b), wherein:

a. N M is a 2′-O-methoxyethyl nucleoside;

each N B is an S-cEt nucleoside;

each N Q is a β-D-deoxyribonucleoside; and

N Z is a 2′-O-methoxyethyl nucleoside;

b. N M is a 2′-O-methoxyethyl nucleoside;

each N B is an S-cEt nucleoside;

each N Q is a 2′-O-methoxyethyl nucleoside; and

N Z is a 2′-O-methoxyethyl nucleoside;

c. N M is a 2′-O-methoxyethyl nucleoside;

each N B is an S-cEt nucleoside;

each N Q is a β-D-deoxyribonucleoside;

each N is a β-D-deoxyribonucleoside; and

N Z is an S-cEt nucleoside;

d. N M is a 2′-O-methoxyethyl nucleoside;

each N B is an LNA nucleoside;

each N Q is a β-D-deoxyribonucleoside; and

N Z is a 2′-O-methoxyethyl nucleoside; or

e. N M is a 2′-O-methoxyethyl nucleoside;

each N B is an LNA nucleoside;

each N Q is a β-D-deoxyribonucleoside; and

N Z is an LNA nucleoside;

iii. the compound is a compound of claim 1 (c), wherein:

a. each R is a 2′-O-methoxyethyl nucleoside; X is 1;

each N B is an S-cEt nucleoside;

each N Q is a β-D-deoxyribonucleoside;

N Y is a β-D-deoxyribonucleoside; and

N Z is a 2′-O-methoxyethyl nucleoside;

b. each R is a 2′-O-methoxyethyl nucleoside; X is 1;

each N B is an S-cEt nucleoside;

each N Q is a β-D-deoxyribonucleoside;

N Y is a β-D-deoxyribonucleoside; and

N Z is an S-cEt nucleoside; or

c. each R is a 2′-O-methoxyethyl nucleoside; X is 1;

each N B is an S-cEt nucleoside;

each N Q is a β-D-deoxyribonucleoside;

N Y is an S-cEt nucleoside; and

N Z is an S-cEt nucleoside;

d. each R is a 2′-O-methoxyethyl nucleoside; X is 1;

each NB is an S-cEt nucleoside;

each NQ is independently selected from a β-D-deoxyribonucleoside and a 2′-O-methoxyethyl nucleoside;

N Y is selected from an S-cEt nucleoside and a β-D-deoxyribonucleoside; and

N Z is an S-cEt nucleoside;

iv. the compound is a compound of claim 1 (d), wherein:

a. N M is a 2′-O-methoxyethyl nucleoside;

each N B is an S-cEt nucleoside;

each N Q is a β-D-deoxyribonucleoside;

N Y is a β-D-deoxyribonucleoside;

N Z is a 2′-O-methoxyethyl nucleoside; and

b. N M is a 2′-O-methoxyethyl nucleoside;

each N B is an S-cEt nucleoside;

each N Q is a β-D-deoxyribonucleoside;

N Y is a β-D-deoxyribonucleoside; and

N Z is an S-cEt nucleoside;

c. N M is a 2′-O-methoxyethyl nucleoside;

each N B is an S-cEt nucleoside;

each N Q is a β-D-deoxyribonucleoside;

N Y is an S-cEt nucleoside; and

N Z is an S-cEt nucleoside;

d. N M is a 2′-O-methoxyethyl nucleoside;

each N B is an S-cEt nucleoside;

each N Q is independently selected from a β-D-deoxyribonucleoside and a 2′-O-methoxyethyl nucleoside;

N Y is selected from an S-cEt nucleoside and a β-D-deoxyribonucleoside; and

N Z is an S-cEt nucleoside;

v. the compound is a compound of claim 1 (e), wherein:

a. N M is a 2′-O-methoxyethyl nucleoside;

each N B is an S-cEt nucleoside;

each N Q is a β-D-deoxyribonucleoside; and

N Z is a 2′-O-methoxyethyl nucleoside;

vi. the compound is a compound of claim 1 (f), wherein:

a. each N B is an S-cEt nucleoside; and

each N Q is a 2′-O-methoxyethyl nucleoside;

b. each N B is an S-cEt nucleoside; and

each N Q is a β-D-deoxyribonucleoside;

vii. the compound is a compound of claim 1 (g), wherein:

a. each N M is a 2′-O-methoxyethyl nucleoside;

each N B is an S-cEt nucleoside;

each N Q is independently selected from a 2′-O-methyl nucleoside and a β-D-deoxyribonucleoside; and

N Z is selected from an S-cEt nucleoside and a 2′-O-methoxyethyl nucleoside;

b. each N M is a 2′-O-methoxyethyl nucleoside;

each N B is an S-cEt nucleoside;

each N Q is a β-D-deoxyribonucleoside; and

N Z is an S-cEt nucleoside;

c. each N M is a 2′-O-methoxyethyl nucleoside;

each N B is an S-cEt nucleoside;

each N Q is independently selected from a 2′-O-methyl nucleoside and a β-D-deoxyribonucleoside; and

N Z is an S-cEt nucleoside; or

d. each N M is a 2′-O-methoxyethyl nucleoside;

each N B is an S-cEt nucleoside;

each N Q is independently selected from a 2′-O-methyl nucleoside and a β-D-deoxyribonucleoside; and

N Z is a 2′-O-methoxyethyl nucleoside.

53 - 57 . (canceled)

58 . The compound of claim 1 having the structure:

(SEQ ID NO: 3)

A E C S A E T E C S A E G E T E C S TGAU S AAGC S TA S ; 

(SEQ ID NO: 3)

A E C S A E T E C S A E G E T E C S TGAU S AAGC S U S A S [[.]];

(SEQ ID NO: 8)

A E C S ATC S A S GTC S U S GAU S A S AGC S U S A E ;

or

(SEQ ID NO: 7)

Me C E A S A S T E C S U S A E A E U S A S A E G E C S T E A S ;

wherein nucleosides not followed by a subscript are β-D-deoxyribonucleosides; nucleosides followed by a subscript “E” are 2′-MOE nucleosides; nucleosides followed by a subscript “S” are S-cEt nucleosides; and Me C is 5-methyl cytosine.

59 - 69 . (canceled)

70 . The compound of claim 1 , wherein the modified oligonucleotide has 0, 1, 2, or 3 mismatches with respect to the nucleobase sequence of miR-21.

71 - 74 . (canceled)

75 . The compound of claim 1 , wherein the modified oligonucleotide has a structure selected from the structures in Table 1.

76 . A method of inhibiting the activity of miR-21 comprising contacting a cell with a compound of claim 1 .

77 - 79 . (canceled)

80 . A method of treating, preventing or delaying the onset of a disease associated with miR-21 comprising administering to a subject having a disease associated with miR-21 a compound of claim 1 .

81 . The method of claim 80 , wherein the disease is fibrosis.

82 - 88 . (canceled)

89 . The method of claim 80 , wherein the disease is cancer.

90 - 106 . (canceled)