Antisense modulation of BCL-X expression

• US 6,172,216 B1
• Filed: 10/07/1998
• Issued: 01/09/2001
• Est. Priority Date: 10/07/1998
• Status: Expired due to Term

• Current Assignee: Ionis Pharmaceuticals, Inc.
• Original Assignee: ISIS Pharmaceuticals Incorporated
• Inventors: Nickoloff, Brian J., Zhang, QingQing, Monia, Brett P., Dean, Nicholas M., Bennett, C. Frank
• Primary Examiner(s): Schwartzman, Robert A.
• Assistant Examiner(s): EPPS -SMITH, JANET L
• Application Number:
  US09/167,921
• Time in Patent Office: 825 Days
• Field of Search: 536/23.1, 536/24.5, 536/24.3, 536/24.33, 536/25.31, 435/91.1, 435/325, 435/6, 514/44
• US Class Current: 536/24.5
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: C12N 15/1135 against oncogenes or tumor suppressor genes
• CPC Class Codes: C12N 2310/11 Antisense
• CPC Class Codes: C12N 2310/315 Phosphorothioates
• CPC Class Codes: C12N 2310/3181 Peptide nucleic acid, PNA
• CPC Class Codes: C12N 2310/321 2'-O-R Modification
• CPC Class Codes: C12N 2310/3233 Morpholino-type ring
• CPC Class Codes: C12N 2310/334 Modified C
• CPC Class Codes: C12N 2310/3341 5-Methylcytosine
• CPC Class Codes: C12N 2310/341 Gapmers, i.e. of the type ===---===
• CPC Class Codes: C12N 2310/345 having at least two different backbone modifications
• CPC Class Codes: C12N 2310/346 having a combination of backbone and sugar modifications
• CPC Class Codes: C12N 2310/3525 MOE, methoxyethoxy

Abstract

Compositions and methods are provided for modulating the expression of bcl-x. Antisense compounds, particularly antisense oligonucleotides, targeted to nucleic acids encoding bcl-x are preferred. Methods of using these compounds for modulation of bcl-x expression and for treatment of diseases associated with expression of bcl-x are also provided.

Claims

1. An antisense compound 8 to 30 nucleotides in length targeted to a nucleic acid molecule encoding a human bcl-x, wherein said antisense compound modulates the expression of human bcl-x, and wherein said antisense compound is an antisense oligonucleotide which comprises at least an 8 nucleobase portion of SEQ ID NO:

3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 31, 32, 33, 34, 36, 37, 38, 39, or 40.

2. The antisense compound of claim 1 which is an antisense oligonucleotide comprises at least one modified internucleoside linkage.

3. The antisense compound of claim 2 wherein the modified internucleoside linkage of the antisense oligonucleotide is a phosphorothioate linkage.

4. The antisense compound of claim 1 wherein the antisense oligonucleotide comprises at least one modified sugar moiety.

5. The antisense compound of claim 4 wherein the modified sugar moiety of the antisense oligonucleotide is a 2′

-O-methoxyethyl sugar moiety.

6. The antisense compound of claim 5 wherein substantially all sugar moieties of the antisense oligonucleotide are 2′

-O-methoxyethyl sugar moieties.

7. The antisense compound of claim 1 wherein the antisense oligonucleotide comprises at least one modified nucleobase.

8. The antisense compound of claim 7 wherein the modified nucleobase of the antisense oligonucleotide is a 5-methylcytosine.

9. The antisense compound of claim 5 wherein each 2′

-O-methoxyethyl modified cytosine nucleobase of the antisense oligonucleotide is a 5-methylcytosine.

10. The antisense compound of claim 1 which is a chimeric oligonucleotide.

11. A pharmaceutical composition comprising the antisense compound of claim 1 and a pharmaceutically acceptable carrier or diluent.

12. The pharmaceutical composition of claim 11 further comprising a colloidal dispersion system.

13. The antisense compound of claim 1 which is targeted 20 to bcl-xl.

14. The antisense compound of claim 1 which is targeted to a nucleic acid molecule encoding bcl-xl and which preferentially inhibits the expression of bcl-xl.

15. The antisense compound of claim 14 which is targeted to a region of a nucleic acid molecule encoding bcl-xl which is not found in a nucleic acid molecule encoding bcl-xs.

16. The antisense compound of claim 14 which promotes apoptosis.

17. The antisense compound of claim 1 which is targeted to a region of a nucleic acid molecule encoding bcl-xs and which reduces the expression of bcl-xs.

18. The antisense compound of claim 17 which inhibits apoptosis.

19. The antisense compound of claim 1 which alters the ratio of bcl-x isoforms expressed by a cell or tissue.

20. The antisense compound of claim 19 which increases the ratio of bcl-xl to bcl-xs expressed.

21. The antisense compound of claim 19 which decreases the ratio of bcl-xl to bcl-xs expressed.

32. A method of inhibiting the expression of bcl-x in human cells or tissues in vitro comprising contacting said cells or tissues with the antisense compound of claim 1 so that expression of bcl-x is inhibited.

33. A method of altering the ratio of the isoforms of bcl-x in human cells or tissues in vitro comprising contacting said cells or tissues with the antisense compound of claim 1 so that the ratio of the bcl-x isoforms is altered.

34. The pharmaceutical composition of claim 11 further comprising a chemotherapeutic agent for the treatment of cancer.

35. The antisense compound of claim 2 wherein the modified internucleoside linkage of the antisense oligonucleotide is a morpholino linkage.

22. An antisense compound 8 to 30 nucleotides in length which is targeted to a bcl-x RNA transcript containing a first and second alternative splice site, which, when contacted with the transcript, reduces the relative frequency of splicing at said second splice site such that the ratio of resulting RNA splice products is altered and wherein said antisense compounds is targeted to at least a portion of the RNA region within 20 nucleotides to the 5′

side of said second splice site.

23. The antisense compound of claim 22 which is an antisense oligonucleotide.

24. The antisense compound of claim 23 wherein the antisense oligonucleotide comprises at least one modified sugar moiety.

25. The antisense compound of claim 24 wherein the modified sugar moiety of the antisense oligonucleotide is a 2′

-O-methoxyethyl sugar moiety.

26. The antisense compound of claim 25 wherein substantially all sugar moieties of the antisense oligonucleotide are 2′

-O-methoxyethyl sugar moieties.

27. An antisense compound which alters splicing of an RNA encoding human bcl-x, such that the ratio of bcl-xs to bcl-xl is altered, and which is targeted to nucleotides 664 to 698 of SEQ ID NO:

1, a nucleic acid encoding human bcl-x.

28. The antisense compound of claim 27 which is an antisense oligonucleotide.

29. The antisense compound of claim 28 wherein the antisense oligonucleotide comprises at least one 2′

-O-methoxyethyl sugar moiety.

30. The antisense compound of claim 29 wherein substantially all sugar moieties of the antisense oligonucleotide are 2′

-O-methoxyethyl sugar moieties.

31. An antisense oligonucleotide comprising SEQ ID No:

33, 34 or 46.