Antisense modulation of bcl-x expression

• US 6,214,986 B1
• Filed: 06/02/1999
• Issued: 04/10/2001
• Est. Priority Date: 10/07/1998
• Status: Expired due to Term

• Current Assignee: Ionis Pharmaceuticals, Inc.
• Original Assignee: ISIS Pharmaceuticals Incorporated (Ionis Pharmaceuticals, Inc.)
• Inventors: Monia, Brett P., Dean, Nicholas M., Bennett, C. Frank, Nickoloff, Brian J., Zhang, QingQing
• Primary Examiner(s): Schwartzman, Robert A.
• Assistant Examiner(s): EPPS -SMITH, JANET L
• Application Number:
  US09/323,743
• Time in Patent Office: 678 Days
• Field of Search: 536/23.1, 536/24.3, 536/24.5, 536/24, 536/24.33, 435/6, 435/325, 435/375, 435/91.1, 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: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• 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. Methods of sensitizing cells to apoptotic stimuli 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:

43, 44, 46, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, or 66.

2. The antisense compound of claim 1 wherein the 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, a morpholino linkage or a peptide-nucleic acid 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 or a 2′

-dimethylaminooxyethoxy sugar moiety.

6. The antisense compound of claim 4 wherein substantially all sugar moieties of the antisense oligonucleotide are modified 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 composition comprising the antisense compound of claim 1 and a pharmaceutically acceptable carrier or diluent.

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

13. The antisense compound of claim 1 which is targeted 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.

29. 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.

30. 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 compo claim 1 so that the ratio of the bcl-x isoforms is altered.

31. A method of sensitizing a cell in vitro to an apoptotic stimulus comprising treating the cell with an inhibitor of bcl-x, wherein said inhibitor is the antisense compound of claim 1.

32. The method of claim 31 wherein the apoptotic stimulus is radiation.

33. The method of claim 32 wherein the radiation is ultraviolet radiation.

34. The method of claim 31 wherein the apoptotic stimulus is a cancer chemotherapeutic drug.

35. The method of claim 34 wherein the cancer chemotherapeutic drug is VP-16, cisplatinum or taxol.

36. The method of claim 31 wherein the apoptotic stimulus is a cellular signaling molecule.

37. The method of claim 36 wherein the apoptotic stimulus is ceramide or a cytokine.

40. The method of claim 31 wherein said apoptotic stimulus causes mitochondrial dysfunction.

41. The method of claim 40 wherein said mitochondrial dysfunction is loss of mitochondrial membrane potential.

42. A method of inducing mitochondrial dysfunction in a cell comprising treating the cell with an inhibitor of bcl-xl, wherein said inhibitor is the antisense compound of claim 1.

43. The method of claim 42 further comprising exposing the cell to an apoptotic stimulus.

44. The method of claim 43 wherein the apoptotic stimulus is radiation.

45. The method of claim 44 wherein the radiation is ultraviolet radiation.

46. The method of claim 43 wherein the apoptotic stimulus is a cancer chemotherapeutic drug.

47. The method of claim 46 wherein the cancer chemotherapeutic drug is VP-16, cisplatinum or taxol.

48. The method of claim 43 wherein the apoptotic stimulus is ceramide or staurosporine.

50. The method of claim 42 wherein the mitochondrial dysfunction is loss of mitochondrial membrane potential.

22. 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 at least a portion of the region from nucleotides 638 to 698 of SEO ID NO 1, a nucleic acid encoding human bcl-x.

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 2′

-O-methoxyethyl sugar moiety or 2′

-dimethylaminooxyethoxy sugar moiety.

25. The antisense compound of claim 23 wherein substantially all sugar moieties of the antisense oligonucleotide are modified sugar moieties.

26. An antisense compound which comprises SEQ ID NO:

33, 34, 46, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65 or 66.

27. The antisense compound of claim 26, wherein said antisense compound is an antisense oligonucleotide 8 to 30 nucleotides in length which alters splicing of an RNA encoding a bcl-x gene product, wherein said oligonucleotide is targeted to said bcl-x gene product and comprises at least one 2′

-O-methoxyethyl or 2′

-dimethylaminooxyethoxy sugar moiety.

28. The antisense oligonucleotide of claim 27 wherein substantially all sugar moieties are 2′

-O-methoxyethyl or 2′

-dimethylaminooxyethoxy sugar moieties.

38. A method of sensitizing a cell in vitro to an apoptotic stimulus comprising treating the cell with an antisense inhibitor of bcl-xl wherein the antisense inhibitor of bcl-xl has a sequence comprising at least an 8-nucleobase portion of SEQ ID NO:

31 or 33.

39. The method of claim 38 wherein the apoptotic stimulus is ceramide is staurosporine.

49. A method of inducing mitochondrial dysfunction in a cell comprising treating the cell in vitro with an antisense inhibitor of bcl-xl wherein the antisense inhibitor of bcl-xl has a sequence comprising at least an 8-nucleobase portion of SEQ ID NO:

31 or 33.