Antisense modulation of apolipoprotein B-expression

• US RE44,760 E1
• Filed: 09/27/2012
• Issued: 02/11/2014
• Est. Priority Date: 11/13/2002
• Status: Expired due to Fees

• Current Assignee: Kastle Therapeutics, LLC.
• Original Assignee: Genzyme Corporation (Sanofi )
• Inventors: Crooke, Rosanne M., Graham, Mark J., Freier, Susan M.
• Primary Examiner(s): EPPS -SMITH, JANET L
• Application Number:
  US13/629,214
• Time in Patent Office: 502 Days
• Field of Search: None
• US Class Current: 435/455
• CPC Class Codes: Y02P 20/582 Recycling of unreacted starting or intermediate materials

Abstract

Antisense compounds, compositions and methods are provided for modulating the expression of apolipoprotein B. The compositions comprise antisense compounds, particularly antisense oligonucleotides, targeted to nucleic acids encoding apolipoprotein B. Methods of using these compounds for modulation of apolipoprotein B expression and for treatment of diseases associated with expression of apolipoprotein B are provided.

Claims

1. An antisense compound 12 to 30 nucleobases in length, wherein said antisense compound is fully complementary within the range of nucleotides 5562-5625 of SEQ ID NO:

3, and comprises at least one modified sugar moiety.

2. The antisense compound of claim 1, wherein said antisense compound is fully complementary within the range of nucleotides 5582-5625 of SEQ ID NO:

3.

3. The antisense compound of claim 1, wherein said antisense compound is targeted to the range of nucleotides 5589-5608 of SEQ ID NO:

3 and is fully complementary over its length to SEQ ID NO;

3.

4. The antisense compound of claim 1, wherein said antisense compound comprises at least 8 contiguous nucleobases of SEQ ID NO:

224.

5. The antisense compound of claim 1, wherein said antisense compound comprises SEQ ID NO:

224.

6. The antisense compound of claim 1, wherein the antisense compound comprises an antisense oligonucleotide.

7. The antisense compound of claim 1, wherein the antisense compound further comprises one or more modifications selected from the group consisting of a modified backbone and a modified nucleobase.

8. The antisense compound of claim 1, wherein the modified sugar moiety is selected from the group consisting of a 2′

-methoxyethoxy modified sugar moiety, a 2′

-methoxy modified sugar moiety, a 2′

-O-alkyl modified sugar moiety, and a bicyclic nucleic acid sugar moiety.

9. The antisense compound of claim 1, wherein the antisense compound is single-stranded.

10. The antisense compound of claim 1, wherein the antisense compound is double-stranded.

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

12. The antisense compound of claim 1, wherein the antisense compound is covalently linked to a lipid moiety.

13. The antisense compound of claim 12 wherein the lipid moiety comprises a cholesterol.

14. The antisense compound of claim 1, wherein the antisense compound is 20 to 30 nucleobases in length.

15. The antisense compound of claim 1, wherein the antisense compound is 20 nucleobases in length.

16. The antisense compound of claim 1, wherein the antisense compound is a chimeric antisense compound.

17. An antisense compound 20 to 30 nucleobases in length comprising at least 13 contiguous nucleobases of SEQ ID NO:

224 and at least one modified sugar moiety. wherein said antisense compound specifically hybridizes to SEQ ID NO;

3.

18. The antisense compound of claim 17. wherein the antisense compound is 20 nucleobases in length.

19. The antisense compound of claim 17, wherein the modified sugar moiety is a 2′

-methoxyethoxy modified sugar moiety, a 2′

-methoxy modified sugar moiety, a 2′

-O-alkyl modified sugar moiety, or a bicyclic nucleic acid sugar moiety.

20. The antisense compound of claim 17, wherein the antisense compound is an antisense oligonucleotide.

21. The antisense compound of claim 20, wherein the antisense oligonucleotide is a chimeric antisense oligonucleotide.

22. The antisense compound of claim 21, wherein the chimeric antisense oligonucleotide comprises a gap segment of 2′

-deoxynucleotides positioned between wing segments, wherein each nucleotide of each wing segment comprises a 2′

-methoxyethoxy sugar moiety.

23. The antisense compound of claim 22, wherein the gap segment is ten 2′

-deoxynucleotides in length and each wing segment comprises from one to nine 2′

-methoxyethyoxy nucleotides.

24. The antisense compound of claim 22, wherein the gap segment is ten 2′

-deoxynucleotides and each wing segment comprises five 2′

-methoxyethoxy nucleotides.

25. The antisense compound of claim 17, wherein each internucleoside linkage is a phosphorothioate internucleoside linkage.

26. The antisense compound of claim 17, wherein each cytosine is a 5-methylcytosine.

27. The antisense compound of claim 17, wherein the antisense compound is covalently linked to a lipid moiety.

28. The antisense compound of claim 27, wherein the lipid moiety is cholesterol.

29. The antisense compound of claim 17, wherein the antisense compound is fully complementary to SEQ ID NO:

3.

30. The antisense compound of claim 17, wherein the antisense compound comprises SEQ ID NO:

224.

31. The antisense compound of claim 17, wherein the antisense compound consists of SEQ ID NO:

224.

35. A composition comprising the antisense oligonucleotide of any of claim 17, 32, 33 or 34 and a pharmaceutically acceptable carrier or diluent.

36. The antisense compound of claim 17, wherein said antisense compound inhibits expression of apolipoprotein B mRNA in HepG2 cells by at least 40% when contacted with the HepG2 cells at a concentration of 50 nM.

32. An antisense oligonucleotide consisting of SEQ ID NO:

224, wherein nucleobases 1-5 and 16-20 comprise 2′

-methoxyethoxy nucleotides, nucleobases 6-15 are 2′

-deoxynucleotides, each internucleoside linkage is a phosphorothioate internucleoside linkage, and each cytosine is a 5-methylcytosine.

33. The antisense oligonucleotide of claim 32, wherein the antisense oligonucleotide is in a salt form.

34. The antisense oligonucleotide of claim 33, wherein the salt is sodium salt.

37. A method of inhibiting the expression of apolipoprotein B in human cells or tissues in vivo comprising contacting said cells or tissues with an antisense compound 12 to 30 nucleobases in length, wherein said antisense compound is fully complementary within the range of nucleotides 5562-5625 of SEQ ID NO:

3, and comprises at least one modified sugar moiety.

38. A method of inhibiting the expression of apolipoprotein B in human cells or tissues in vivo comprising contacting said cells or tissues with an antisense compound 20 to 30 nucleobases in length comprising at least 13 contiguous nucleobases of SEQ ID NO:

224 and at least one modified sugar moiety, wherein said antisense compound specifically hybridizes to SEQ ID NO;

3.

39. A method of inhibiting the expression of apolipoprotein B in human cells or tissues in vivo comprising contacting said cells or tissues with an antisense oligonucleotide consisting of SEQ ID NO:

224, wherein nucleobases 1-5 and 16-20 comprise 2′

-methoxyethoxy nucleotides, nucleobases 6-15 are 2′

-deoxynucleotides, each internucleoside linkage is a phosphorothioate internucleoside linkage, and each cytosine is a 5-methylcytosine.

40. A method of inhibiting the expression of apolipoprotein B mRNA in HepG2 cells by at least 40% comprising contacting said cells with an antisense compound 20 to 30 nucleobases in length comprising at least 13 contiguous nucleobases of SEQ ID NO:

224 and at least one modified sugar moiety, wherein said antisense compound specifically hybridizes to SEQ ID NO;

3, wherein the antisense compound is at a concentration of 50 nM, and wherein said mRNA is inhibited by at least 40%.

41. An antisense compound 12 to 30 nucleobases in length, wherein said antisense compound is fully complementary within the range of nucleotides 5562-5625 of SEQ ID NO:

3, wherein the antisense compound is chemically modified.

42. An antisense compound 20 to 30 nucleobases in length comprising at least 13 contiguous nucleobases of SEQ ID NO:

224, wherein the antisense compound is chemically modified, and wherein said antisense compound specifically hybridizes to SEQ ID NO;

3.

43. An oligomeric compound 12 to 30 nucleobases in length, wherein said oligomeric compound is fully complementary within the range of nucleotides 5562-5625 of SEQ ID NO:

3, and comprises at least one modified sugar moiety.

44. An oligomeric compound 20 to 30 nucleobases in length comprising at least 13 contiguous nucleobases of SEQ ID NO:

224 and at least one sugar moiety, and wherein said oligomeric compound specifically hybridizes to SEQ ID NO;

3.

45. A method of inhibiting the expression of apolipoprotein B in human cells or tissues in vivo comprising contacting said cells or tissues with an antisense compound 12 to 30 nucleobases in length, wherein said antisense compound is fully complementary within the range of nucleotides 5562-5625 of SEQ ID NO:

3, wherein the antisense compound is chemically modified.

46. A method of inhibiting the expression of apolipoprotein B in human cells or tissues in vivo comprising contacting said cells or tissues with an antisense compound 20 to 30 nucleobases in length comprising at least 13 contiguous nucleobases of SEQ ID NO:

224, wherein the antisense compound is chemically modified, and wherein said antisense compound specifically hybridizes to SEQ ID NO;

3.

47. A method of inhibiting the expression of apolipoprotein B in human cells or tissues in vivo comprising contacting said cells or tissues with an oligomeric compound 12 to 30 nucleobases in length, wherein said oligomeric compound is fully complementary within the range of nucleotides 5562-5625 of SEQ ID NO:

3, and comprises at least one modified sugar moiety.

48. A method of inhibiting the expression of apolipoprotein B in human cells or tissues in vivo comprising contacting said cells or tissues with an oligomeric compound 20 to 30 nucleobases in length comprising at least 13 contiguous nucleobases of SEQ ID NO:

224 and at least one sugar moiety, wherein said oligomeric compound specifically hybridizes to SEQ ID NO;

3.

49. A method of inhibiting the expression of apolipoprotein B in human cells or tissues in vivo comprising contacting said cells or tissues with a conjugate compound, wherein said conjugate compound comprises an antisense compound 12 to 30 nucleobases in length, wherein said antisense compound is fully complementary within the range of nucleotides 5562-5625 of SEQ ID NO:

3, and wherein said antisense compound is conjugated to an active drug substance.

50. A method of inhibiting the expression of apolipoprotein B in human cells or tissues in vivo comprising contacting said cells or tissues with a conjugate compound, wherein said conjugate compound comprises an antisense compound 20 to 30 nucleobases in length comprising at least 13 contiguous nucleobases of SEQ ID NO:

224, wherein said antisense compound specifically hybridizes to SEQ ID NO;

3, and wherein said antisense compound is conjugated to an active drug substance.

51. A method of inhibiting the expression of apolipoprotein B in human cells or tissues in vivo comprising contacting said cells or tissues with a conjugate compound, wherein said conjugate compound comprises an oligomeric compound 12 to 30 nucleobases in length, wherein said oligomeric compound is fully complementary within the range of nucleotides 5562-5625 of SEQ ID NO:

3, and wherein said oligomeric compound is conjugated to an active drug substance.

52. A method of inhibiting the expression of apolipoprotein B in human cells or tissues in vivo comprising contacting said cells or tissues with a conjugate compound, wherein said conjugate compound comprises an oligomeric compound 20 to 30 nucleobases in length comprising at least 13 contiguous nucleobases of SEQ ID NO:

224, wherein said oligomeric compound specifically hybridizes to SEQ ID NO;

3, and wherein said oligomeric compound is conjugated to an active drug substance.

53. A method of inhibiting the expression of apolipoprotein B in human cells or tissues in vivo comprising contacting said cells or tissues with an antisense compound 12 to 30 nucleobases in length, wherein said antisense compound is fully complementary within the range of nucleotides 5562-5625 of SEQ ID NO:

3, and comprises a modified backbone or modified nucleobase.

54. A method of inhibiting the expression of apolipoprotein B in human cells or tissues in vivo comprising contacting said cells or tissues with an antisense compound 20 to 30 nucleobases in length comprising at least 13 contiguous nucleobases of SEQ ID NO:

224 and a modified backbone or modified nucleobase, wherein said antisense compound specifically hybridizes to SEQ ID NO;

3.

55. A method of inhibiting the expression of apolipoprotein B mRNA in HepG2 cells by at least 40% comprising contacting said cells with an antisense compound at a concentration of 50 nM, wherein said mRNA is inhibited by at least 40%, wherein said antisense compound is 20 to 30 nucleobases in length comprising at least 13 contiguous nucleobases of SEQ ID NO:

224 and a modified backbone or modified nucleobase, and wherein said antisense compound specifically hybridizes to SEQ ID NO;

3.

56. A method of inhibiting the expression of apolipoprotein B in human cells or tissues in vivo comprising contacting said cells or tissues with an oligomeric compound 12 to 30 nucleobases in length, wherein said oligomeric compound is fully complementary within the range of nucleotides 5562-5625 of SEQ ID NO:

3, and comprises a modified backbone or modified nucleobase.

57. A method of inhibiting the expression of apolipoprotein B in human cells or tissues in vivo comprising contacting said cells or tissues with an oligomeric compound 20 to 30 nucleobases in length comprising at least 13 contiguous nucleobases of SEQ ID NO:

224 and a modified backbone or modified nucleobase, wherein said oligomeric compound specifically hybridizes to SEQ ID NO;

3.

58. A method of inhibiting the expression of apolipoprotein B mRNA in HepG2 cells by at least 40% comprising contacting said cells with an oligomeric compound at a concentration of 50 nM, wherein said mRNA is inhibited by at least 40%, wherein said oligomeric compound is 20 to 30 nucleobases in length comprising at least 13 contiguous nucleobases of SEQ ID NO:

224 and a modified backbone or modified nucleobase, and wherein said oligomeric compound specifically hybridizes to SEQ ID NO;

3.