Hepatitis B virus (HBV) dsRNA agent compositions and methods of use thereof

1. A double stranded ribonucleic acid (dsRNA) agent, comprising a sense strand and an antisense strand forming a double stranded region, wherein the antisense strand and the sense strand comprise the modified nucleotide sequences as set forth in, respectively:

(a)

(SEQ ID NO: 16)

5′-usGfsuga(Agn)gCfGfaaguGfcAfcacsusu-3′

and

(SEQ ID NO: 29)

5′-gsusguGfcAfCfUfucgcuucaca-3′;

(b)

(SEQ ID NO: 18)

5′-usGfsuga(Agn)gcgaaguGfdCAfcacsusu-3′

and

(SEQ ID NO: 29)

5′-gsusguGfcAfCfUfucgcuucaca-3′;

(c)

(SEQ ID NO: 20)

5′-usGfsudGa(Agn)gCfGfaaguGfcAfcacsusu-3′

and

(SEQ ID NO: 29)

5′-gsusguGfcAfCfUfucgcuucaca-3′;

(d)

(SEQ ID NO: 23)

5′-usGfsudGadAgdCGfaaguGfcAfcacsusu-3′

and

(SEQ ID NO: 29)

5′-gsusguGfcAfCfUfucgcuucaca-3′;

(e)

(SEQ ID NO: 24)

5′-usGfsuga(Agn)dGCfGfaaguGfcAfcacsusu-3′

and

(SEQ ID NO: 29)

5′-gsusguGfcAfCfUfucgcuucaca-3′;

(f)

(SEQ ID NO: 25)

5′-usGfsudGadAgdCGfaaguGfcAfdCacsusu-3′

and

(SEQ ID NO: 29)

5′-gsusguGfcAfCfUfucgcuucaca-3′;

or

(g)

(SEQ ID NO: 28)

5′-usGfsuga(Agn)gCfGfaaguGfdCAfcacsusu-3′

and

(SEQ ID NO: 29)

5′-gsusguGfcAfCfUfucgcuucaca-3′;

wherein a, c, g, and u are 2′-O-methyladenosine-3′-phosphate, 2′-O-methylcytidine-3′-phosphate, 2′-O-methylguanosine-3′-phosphate, and 2′-O-methyluridine-3′-phosphate, respectively;

Af, Cf, Gf, and Uf are 2′-fluoroadenosine-3′-phosphate, 2′-fluorocytidine-3′-phosphate, 2′-fluoroguanosine-3′-phosphate, and 2′-fluorouridine-3′-phosphate, respectively;

dA, dC, dG, and dT are 2′-deoxyadenosine-3′-phosphate, 2′-deoxycytidine-3′-phosphate, 2′-deoxyguanosine-3′-phosphate, and 2′-deoxythymidine-3′-phosphate, respectively;

(Agn) is adenosine-glycol nucleic acid (GNA); and

s is a phosphorothioate linkage.

2. The dsRNA agent of claim 1 , wherein at least one strand comprises a 3′ overhang of at least 1 nucleotide.

3. The dsRNA agent of claim 1 , wherein the double stranded region is 19-21 nucleotide pairs in length.

4. The dsRNA agent of claim 1 , wherein each strand independently has 19-23 nucleotides.

5. The dsRNA agent of claim 1 , further comprising a ligand.

6. The dsRNA agent of claim 5 , wherein the ligand is conjugated to the 3′ end of the sense strand of the dsRNA agent.

7. The dsRNA agent of claim 6 , wherein the ligand is an N-acetylgalactosamine (GalNAc) derivative.

8. The dsRNA agent of claim 7 , wherein the ligand is

9. The dsRNA agent of claim 8 , wherein the ligand is conjugated to the 3′ end of the sense strand of the dsRNA agent according to the following structure:

wherein X is O or S.

10. The dsRNA agent of claim 9 , wherein the X is O.

11. The dsRNA agent of claim 1 , wherein the antisense strand and the sense strand consist of the modified nucleotide sequences as set forth in, respectively:

(a)

(SEQ ID NO: 16)

5′-usGfsuga(Agn)gCfGfaaguGfcAfcacsusu-3′

and

(SEQ ID NO: 10)

5′-gsusguGfcAfCfUfucgcuucacaL96-3′;

(b)

(SEQ ID NO: 18)

5′-usGfsuga(Agn)gcgaaguGfdCAfcacsusu-3′

and

(SEQ ID NO: 10)

5′-gsusguGfcAfCfUfucgcuucacaL96-3′;

(c)

(SEQ ID NO: 20)

5′-usGfsudGa(Agn)gCfGfaaguGfcAfcacsusu-3′

and

(SEQ ID NO: 10)

5′-gsusguGfcAfCfUfucgcuucacaL96-3′;

(d)

(SEQ ID NO: 23)

5′-usGfsudGadAgdCGfaaguGfcAfcacsusu-3′

and

(SEQ ID NO: 10)

5′-gsusguGfcAfCfUfucgcuucacaL96-3′;

(e)

(SEQ ID NO: 24)

5′-usGfsuga(Agn)dGCfGfaaguGfcAfcacsusu-3′

and

(SEQ ID NO: 10)

5′-gsusguGfcAfCfUfucgcuucacaL96-3′;

(f)

(SEQ ID NO: 25)

5′-usGfsudGadAgdCGfaaguGfcAfdCacsusu-3′

and

(SEQ ID NO: 10)

5′-gsusguGfcAfCfUfucgcuucacaL96-3′;

or

(g)

(SEQ ID NO: 28)

5′-usGfsuga(Agn)gCfGfaaguGfdCAfcacsusu-3′

and

(SEQ ID NO: 10)

5′-gsusguGfcAfCfUfucgcuucacaL96-3′;

wherein a, c, g, and u are 2′-O-methyladenosine-3′-phosphate, 2′-O-methylcytidine-3′-phosphate, 2′-O-methylguanosine-3′-phosphate, and 2′-O-methyluridine-3′-phosphate, respectively;

Af, Cf, Gf, and Uf are 2′-fluoroadenosine-3′-phosphate, 2′-fluorocytidine-3′-phosphate, 2′-fluoroguanosine-3′-phosphate, and 2′-fluorouridine-3′-phosphate, respectively;

dA, dC, dG, and dT are 2′-deoxyadenosine-3′-phosphate, 2′-deoxycytidine-3′-phosphate, 2′-deoxyguanosine-3′-phosphate, and 2′-deoxythymidine-3′-phosphate, respectively;

(Agn) is adenosine-glycol nucleic acid (GNA);

s is a phosphorothioate linkage; and

L96 is N-[tris(GalNAc-alkyl)-amidodecanoyl)]-4-hydroxyprolinol.

12. The dsRNA agent of claim 1 , wherein the antisense strand and the sense strand consist of the modified nucleotide sequences as set forth in, respectively:

(SEQ ID NO: 16)

5′-usGfsuga(Agn)gCfGfaaguGfcAfcacsusu-3′

and

(SEQ ID NO: 10)

5′-gsusguGfcAfCfUfucgcuucacaL96-3′;

wherein a, c, g, and u are 2′-O-methyladenosine-3′-phosphate, 2′-O-methylcytidine-3′-phosphate, 2′-O-methylguanosine-3′-phosphate, and 2′-O-methyluridine-3′-phosphate, respectively;

Af, Cf, Gf, and Uf are 2′-fluoroadenosine-3′-phosphate, 2′-fluorocytidine-3′-phosphate, 2′-fluoroguanosine-3′-phosphate, and 2′-fluorouridine-3′-phosphate, respectively;

dA, dC, dG, and dT are 2′-deoxyadenosine-3′-phosphate, 2′-deoxycytidine-3′-phosphate, 2′-deoxyguanosine-3′-phosphate, and 2′-deoxythymidine-3′-phosphate, respectively;

(Agn) is adenosine-glycol nucleic acid (GNA);

s is a phosphorothioate linkage; and

L96 is N-[tris(GalNAc-alkyl)-amidodecanoyl)]-4-hydroxyprolinol.

13. An isolated cell containing the dsRNA agent of claim 1 .

14. A pharmaceutical composition comprising the dsRNA agent of claim 1 and a pharmaceutical excipient.

15. A method of inhibiting HBV gene expression or inhibiting replication of a HBV in a cell, the method comprising contacting the cell with the dsRNA agent of claim 1 .

16. The method of claim 15 , wherein the cell is within a human subject.

17. The method of claim 15 , wherein the cell is in vitro.

18. A method of reducing the level of a Hepatitis B virus (HBV) antigen or reducing the viral load of HBV in a subject infected with HBV, comprising administering to the subject a therapeutically effective amount of the dsRNA agent of claim 1 .

19. The method of claim 18 , wherein the HBV antigen is HBsAg or HBeAg.

20. The method of claim 18 , wherein the subject is HBeAg positive.

21. The method of claim 18 , wherein the subject is HBeAg negative.

22. A method of treating a subject having a HBV infection, comprising administering to the subject a therapeutically effective amount of the dsRNA agent of claim 1 , thereby treating the subject.

23. A method of treating a subject having a HBV-associated disorder, comprising administering to the subject a therapeutically effective amount of the dsRNA agent of claim 1 , thereby treating the subject.

24. The method of claim 23 , wherein the HBV-associated disorder is chronic hepatitis and the subject is HBeAg positive.

25. The method of claim 23 , wherein the HBV-associated disorder is chronic hepatitis and the subject is HBeAg negative.

26. The method of claim 23 , wherein the HBV-associated disorder is hepatitis D virus (HDV) infection.

27. The method of claim 22 , wherein the dsRNA agent comprises a antisense strand comprising the modified nucleotide sequence as set forth in 5′-usGfsuga(Agn)gCfGfaaguGfcAfcacsusu-3′ (SEQ ID NO:16), and a sense strand comprising the modified nucleotide sequence as set forth in 5′-gsusguGfcAfCfUfucgcuucacaL96-3′ (SEQ ID NO:10);

wherein a, c, g, and u are 2′-O-methyladenosine-3′-phosphate, 2′-O-methylcytidine-3′-phosphate, 2′-O-methylguanosine-3′-phosphate, and 2′-O-methyluridine-3′-phosphate, respectively;

Af, Cf, Gf, and Uf are 2′-fluoroadenosine-3′-phosphate, 2′-fluorocytidine-3′-phosphate, 2′-fluoroguanosine-3′-phosphate, and 2′-fluorouridine-3′-phosphate, respectively;

dA, dC, dG, and dT are 2′-deoxyadenosine-3′-phosphate, 2′-deoxycytidine-3′-phosphate, 2′-deoxyguanosine-3′-phosphate, and 2′-deoxythymidine-3′-phosphate, respectively;

(Agn) is adenosine-glycol nucleic acid (GNA);

s is a phosphorothioate linkage; and

L96 is N-[tris(GalNAc-alkyl)-amidodecanoyl)]-4-hydroxyprolinol.

28. The method of claim 23 , wherein the dsRNA agent comprises a antisense strand comprising the modified nucleotide sequence as set forth in 5′-usGfsuga(Agn)gCfGfaaguGfcAfcacsusu-3′ (SEQ ID NO:16), and a sense strand comprising the modified nucleotide sequence as set forth in 5′-gsusguGfcAfCfUfucgcuucacaL96-3′ (SEQ ID NO:10);

wherein a, c, g, and u are 2′-O-methyladenosine-3′-phosphate, 2′-O-methylcytidine-3′-phosphate, 2′-O-methylguanosine-3′-phosphate, and 2′-O-methyluridine-3′-phosphate, respectively;

Af, Cf, Gf, and Uf are 2′-fluoroadenosine-3′-phosphate, 2′-fluorocytidine-3′-phosphate, 2′-fluoroguanosine-3′-phosphate, and 2′-fluorouridine-3′-phosphate, respectively;

dA, dC, dG, and dT are 2′-deoxyadenosine-3′-phosphate, 2′-deoxycytidine-3′-phosphate, 2′-deoxyguanosine-3′-phosphate, and 2′-deoxythymidine-3′-phosphate, respectively;

(Agn) is adenosine-glycol nucleic acid (GNA);

s is a phosphorothioate linkage; and

L96 is N-[tris(GalNAc-alkyl)-amidodecanoyl)]-4-hydroxyprolinol.

29. The method of claim 28 wherein the HBV-associated disorder is chronic hepatitis and the subject is HBeAg positive.

30. The method of claim 28 , wherein the HBV-associated disorder is chronic hepatitis and the subject is HBeAg negative.

31. The method of claim 28 , wherein the HBV-associated disorder is hepatitis D virus (HDV) infection.

32. The method of claim 22 , wherein the dsRNA agent comprises a antisense strand consisting of the modified nucleotide sequence as set forth in 5′-usGfsuga(Agn)gCfGfaaguGfcAfcacsusu-3′ (SEQ ID NO:16), and a sense strand consisting of the modified nucleotide sequence as set forth in 5′-gsusguGfcAfCfUfucgcuucacaL96-3′ (SEQ ID NO:10);

wherein a, c, g, and u are 2′-O-methyladenosine-3′-phosphate, 2′-O-methylcytidine-3′-phosphate, 2′-O-methylguanosine-3′-phosphate, and 2′-O-methyluridine-3′-phosphate, respectively;

Af, Cf, Gf, and Uf are 2′-fluoroadenosine-3′-phosphate, 2′-fluorocytidine-3′-phosphate, 2′-fluoroguanosine-3′-phosphate, and 2′-fluorouridine-3′-phosphate, respectively;

dA, dC, dG, and dT are 2′-deoxyadenosine-3′-phosphate, 2′-deoxycytidine-3′-phosphate, 2′-deoxyguanosine-3′-phosphate, and 2′-deoxythymidine-3′-phosphate, respectively;

(Agn) is adenosine-glycol nucleic acid (GNA);

s is a phosphorothioate linkage; and

L96 is N-[tris(GalNAc-alkyl)-amidodecanoyl)]-4-hydroxyprolinol.

33. The method of claim 23 , wherein the dsRNA agent comprises a antisense strand consisting of the modified nucleotide sequence as set forth in 5′-usGfsuga(Agn)gCfGfaaguGfcAfcacsusu-3′ (SEQ ID NO:16), and a sense strand consisting of the modified nucleotide sequence as set forth in 5′-gsusguGfcAfCfUfucgcuucacaL96-3′ (SEQ ID NO:10);

wherein a, c, g, and u are 2′-O-methyladenosine-3′-phosphate, 2′-O-methylcytidine-3′-phosphate, 2′-O-methylguanosine-3′-phosphate, and 2′-O-methyluridine-3′-phosphate, respectively;

Af, Cf, Gf, and Uf are 2′-fluoroadenosine-3′-phosphate, 2′-fluorocytidine-3′-phosphate, 2′-fluoroguanosine-3′-phosphate, and 2′-fluorouridine-3′-phosphate, respectively;

dA, dC, dG, and dT are 2′-deoxyadenosine-3′-phosphate, 2′-deoxycytidine-3′-phosphate, 2′-deoxyguanosine-3′-phosphate, and 2′-deoxythymidine-3′-phosphate, respectively;

(Agn) is adenosine-glycol nucleic acid (GNA);

s is a phosphorothioate linkage; and

L96 is N-[tris(GalNAc-alkyl)-amidodecanoyl)]-4-hydroxyprolinol.

34. The method of claim 33 wherein the HBV-associated disorder is chronic hepatitis and the subject is HBeAg positive.

35. The method of claim 33 , wherein the HBV-associated disorder is chronic hepatitis and the subject is HBeAg negative.

36. The method of claim 33 , wherein the HBV-associated disorder is hepatitis D virus (HDV) infection.