Compositions and methods for inhibiting expression of IKK-B gene

• US 7,888,498 B2
• Filed: 05/21/2007
• Issued: 02/15/2011
• Est. Priority Date: 05/22/2006
• Status: Active Grant

• Current Assignee: Alnylam Pharmaceuticals Incorporated, Imperial College of Science Technology & Medicine (Imperial College London)
• Original Assignee: Alnylam Pharmaceuticals Incorporated, Imperial College of Science Technology & Medicine (Imperial College London)
• Inventors: Copeland Catley, Matthew, Harborth, Jens, Sprague, Andrew, Belvisi, Maria Gabriela, Birrell, Mark Andrew, Hardaker, Elizabeth Louise, De Fougerolles, Antonin
• Primary Examiner(s): ANGELL, JON E
• Application Number:
  US11/751,283
• Publication Number:
  US 20080108584A1
• Time in Patent Office: 1,366 Days
• Field of Search: 536/24.5, 514/44.A
• US Class Current: 536/24.5
• CPC Class Codes: A61P 29/00 Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• CPC Class Codes: C12N 15/1137 against enzymes viral enzymes C12N15/1131; receptors C12N15/1138
• CPC Class Codes: C12N 2310/111 spanning the whole gene, or a large part of it
• 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 2310/3515 Lipophilic moiety, e.g. cholesterol
• CPC Class Codes: C12N 2310/3521 Methyl
• CPC Class Codes: C12Y 207/1101 IkappaB kinase (2.7.11.10)

Abstract

The invention relates to a doable-stranded ribonucleic acid (dsRNA) for inhibiting the expression of the IKK-B gene, comprising an antisense strand having a nucleotide sequence which is less that 30 nucleotides in length, generally 19-25 nucleotides in length, and which is substantially complementary to at least a part of the IKK-B gene. The invention also relates to a pharmaceutical composition comprising the dsRNA together with a pharmaceutically acceptable carrier; methods for treating diseases, caused by the expression or activation of the IKK-B gene using the pharmaceutical composition; and methods for inhibiting the expression of the IKK-B gene in a cell.

Claims

1. A double-stranded ribonucleic acid (dsRNA) for inhibiting the expression of a human IKK-B gene, wherein said dsRNA comprises a sense strand and an antisense strand and a duplex structure between 15 and 30 base pairs in length, the antisense strand comprising at least 17 contiguous nucleotides of the nucleotide sequence of SEQ ID NO:

24.

2. The dsRNA of claim 1, wherein said dsRNA comprises at least one modified nucleotide.

3. The dsRNA of claim 2, wherein said modified nucleotide is chosen from the group of a 2′

-O-methyl modified nucleotide, a nucleotide comprising a 3′

-phosphorothioate group, and a terminal nucleotide linked to a cholesteryl derivative or dodecanoic acid bisdecylamide group.

4. The dsRNA of claim 2, wherein said modified nucleotide is chosen from the group of a 2′

-deoxy-2′

-fluoro modified nucleotide, a 2′

-deoxy-modified nucleotide, a locked nucleotide, an abasic nucleotide, 2′

-amino-modified nucleotide, 2′

-alkyl-modified nucleotide, morpholino nucleotide, a phosphoramidate, and a non-natural base comprising nucleotide.

5. A cell comprising the dsRNA of claim 1.

6. A pharmaceutical composition for inhibiting the expression of the IKK-B gene, comprising the dsRNA of claim 1 and a pharmaceutically acceptable carrier.

7. A method for inhibiting the expression of the IKK-B gene in a cell, the method comprising:

(a) introducing into the cell the dsRNA of claim 1; and

(b) maintaining the cell produced in step (a) for a time sufficient to obtain degradation of the mRNA transcript of the IKK-B gene, thereby inhibiting expression of the IKK-B gene in the cell.

8. A method of treating inflammation comprising administering to a patient in need thereof a therapeutically effective amount of the dsRNA of claim 1.

9. A vector for inhibiting the expression of the IKK-B gene, said vector comprising a regulatory sequence operably linked to a nucleotide sequence that encodes at least one strand of the dsRNA of claim 1.

10. A cell comprising the vector of claim 9.

11. The dsRNA of claim 1, wherein the sense strand comprises at least 15 contiguous nucleotides of the nucleotide sequence of SEQ ID NO:

23.

12. The dsRNA of claim 11, wherein the antisense strand comprises the nucleotide sequence of SEQ ID NO:

24 and the sense strand comprises the nucleotide sequence of SEQ ID NO;

23.

13. The dsRNA of claim 11, wherein said sense strand consists of SEQ ID NO:

23 and said antisense strand consists of SEQ ID NO;

24.

14. The dsRNA of claim 1, wherein the antisense strand comprises at least 17 contiguous nucleotides of the nucleotide sequence CCCUGAUCCmAGCUCCUUGGTT (SEQ ID NO:

30) wherein the trailing lower case “

m”

denotes a nucleotide containing a 2′

O-Methyl substitution and the last “

T”

has a phosphorothioate modification.

15. The dsRNA of claim 1, wherein the antisense strand comprises the nucleotide sequence CCCUGAUCCmAGCUCCUUGGTT (SEQ ID NO:

30) wherein the trailing lower case “

m”

denotes a nucleotide containing a 2′

O-Methyl substitution and the last “

T”

has a phosphorothioate modification.

16. The dsRNA of claim 1, wherein the antisense strand consistsof the nucleotide sequence CCCUGAUCCmAGCUCCUUGGTT (SEQ ID NO:

30) wherein the trailing lower case “

m”

denotes a nucleotide containing a 2′

O-Methyl substitution and the last “

T”

has a phosphorothioate modification.

17. The dsRNA of claim 1, wherein the sense strand comprises at least 15 contiguous nucleotides of the nucleotide sequence CmCmAAGGAGCmUmGGAUmCmAGGGTT (SEQ ID NO:

29) wherein a trailing lower case “

m”

denotes a nucleotide containing a 2′

O-Methyl substitution and the last “

T”

has a phosphorothioate modification.

18. The dsRNA of claim 1, wherein the sense strand comprises the nucleotide sequence CmCmAAGGAGCmUmGGAUmCmAGGGTT (SEQ ID NO:

29) wherein a trailing lower case “

m”

denotes a nucleotide containing a 2′

O-Methyl substitution and the last “

T”

has a phosphorothioate modification.

19. The dsRNA of claim 1, wherein the sense strand consists of the nucleotide sequence CmCmAAGGAGCmUmGGAUmCmAGGGTT (SEQ ID NO:

29) wherein a trailing lower case “

m”

denotes a nucleotide containing a 2′

O-Methyl substitution and the last “

T”

has a phosphorothioate modification.

20. The dsRNA of claim 1, wherein the antisense strand comprises the nucleotide sequence CCCUGAUCCmAGCUCCUUGGTT (SEQ ID NO:

30) and the sense strand comprises the nucleotide sequence CmCmAAGGAGCmUmGGAUmCmAGGGTT (SEQ ID NO;

     29) wherein in both the sense and antisense sequences, a trailing lower case “

m”

denotes a nucleotide containing a 2′

O-Methyl substitution and the last “

T”

has a phosphorothioate modification.

21. The dsRNA of claim 1, wherein the antisense strand consists of the nucleotide sequence CCCUGAUCCmAGCUCCUUGGTT (SEQ ID NO:

30) and the sense strand consists of the nucleotide sequence CmCmAAGGAGCmUmGGAUmCmAGGGTT (SEQ ID NO;

     29) wherein in both the sense and antisense sequences, a trailing lower case “

m”

denotes a nucleotide containing a 2′

O-Methyl substitution and the last “

T”

has a phosphorothioate modification.

22. The dsRNA of claim 1, wherein said dsRNA, upon contact with a cell expressing said IKK-B, inhibits expression of said IKK-B gene by at least 25%.

23. The dsRNA of claim 1, wherein said dsRNA, upon contact with a cell expressing said IKK-B, inhibits expression of said IKK-B gene by at least 40%.

24. The dsRNA of claim 1, comprising at least one 2′

-O-methyl modified nucleotide.

25. The dsRNA of claim 1, comprising at least one single-stranded nucleotide overhang of 1 to 4 nucleotides.

26. The dsRNA of claim 1 conjugated to at least one non-ligand group.

27. The pharmaceutical composition of claim 6, wherein the carrier is a lipid carrier.

28. The dsRNA of claim 1, wherein the antisense strand comprises at least 18 contiguous nucleotides of the nucleotide sequence of SEQ ID NO:

24.

29. The dsRNA of claim 1, wherein the antisense strand comprises at least 19 contiguous nucleotides of the nucleotide sequence of SEQ ID NO:

24.

30. The dsRNA of claim 1, wherein the antisense strand comprises at least 20 contiguous nucleotides of the nucleotide sequence of SEQ ID NO:

24.