Antisense modulation of apolipoprotein B expression

• US 7,407,943 B2
• Filed: 05/15/2002
• Issued: 08/05/2008
• Est. Priority Date: 08/01/2001
• Status: Expired due to Fees

• Current Assignee: Kastle Therapeutics, LLC.
• Original Assignee: ISIS Pharmaceuticals Incorporated
• Inventors: Graham, Mark J., Crooke, Rosanne M.
• Primary Examiner(s): MCGARRY, SEAN
• Application Number:
  US10/147,196
• Publication Number:
  US 20030215943A1
• Time in Patent Office: 2,274 Days
• Field of Search: 536/23.1, 536/24.3, 536/24.31, 536/24.33, 536/24.5, 435/6, 435/41.31, 435/325, 435/375, 514/44
• US Class Current: 514/44.00A
• 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 3/00 Drugs for disorders of the metabolism of the blood or the extracellular fluid A61P7/00
• CPC Class Codes: A61P 3/04 Anorexiants; Antiobesity agents
• CPC Class Codes: A61P 3/06 Antihyperlipidemics
• CPC Class Codes: A61P 3/10 for hyperglycaemia, e.g. antidiabetics
• CPC Class Codes: A61P 9/00 Drugs for disorders of the cardiovascular system
• CPC Class Codes: A61P 9/10 for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
• CPC Class Codes: C12N 15/113 Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing when used in plants C12N15/8218
• CPC Class Codes: C12N 2310/11 Antisense
• CPC Class Codes: C12N 2310/314 Phosphoramidates
• CPC Class Codes: C12N 2310/315 Phosphorothioates
• CPC Class Codes: C12N 2310/321 2'-O-R Modification
• CPC Class Codes: C12N 2310/3341 5-Methylcytosine
• CPC Class Codes: C12N 2310/341 Gapmers, i.e. of the type ===---===
• CPC Class Codes: C12N 2310/346 having a combination of backbone and sugar modifications
• CPC Class Codes: C12N 2310/3525 MOE, methoxyethoxy
• 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. 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 targeted to a nucleic acid molecule encoding apolipoprotein B wherein said compound is 100% complementary to the nucleic acid sequence set forth in SEQ ID NO:

3, and wherein said compound is not a ribozyme.

5. The method of claim 1, wherein said antisense compound comprises one or more modifications selected from the group consisting of:

a modified sugar moiety, a modified internucleoside linkage, and a modified nucleobase.

6. The method of claim 5, wherein said modifications comprise a modified sugar moiety selected from the group consisting of:

a 2′

-O-methoxyethyl modified sugar moiety, a 2′

-methoxy modified sugar moiety, a 2′

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

7. The method of claim 5, wherein said modifications comprise a phosphorothioate internucleoside linkage.

8. The method of claim 5, wherein said modifications comprise a 5-methylcytosine.

9. The method of claim 1, wherein said antisense compound is 20 nucleobases in length.

10. The method of claim 1, wherein said antisense compound is not targeted to the start codon region of SEQ ID NO:

3.

11. The method of claim 1, wherein said antisense compound is targeted to nucleotides 1-6530 of SEQ ID NO:

3.

12. The method of claim 1, wherein said antisense compound is targeted to nucleotides 6531-14121 of SEQ ID NO:

3.

13. The method of claim 1, wherein contacting said cells or tissues with a antisense compound comprises parenteral administration of said antisense compound.

14. The method of claim 13, wherein said parenteral administration comprises intravenous administration or subcutaneous administration.

15. The method of claim 1, wherein said antisense compound is a gapmer comprising a gap segment comprised of linked 2′

-deoxynucleotides positioned between 5′ and

3′

wing segments.

16. The method of claim 15, wherein said wing segments are comprised of at least one modified sugar moiety selected from the group consisting of a 2′

-O-methoxyethyl modified sugar moiety, a 2′

-methoxy modified sugar moiety, a 2′

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

17. The method of claim 16, wherein said gap segment is ten 2′

-deoxynucleotides in length and each of said wing segments is five 2′

-O-methoxyethyl nucleotides in length.

62. The method of claim 1 wherein the antisense compound is an antisense oligonucleotide.

2. A method of decreasing serum cholesterol levels in a human comprising administering to a human having elevated serum cholesterol levels an antisense compound 12 to 30 nucleobases in length targeted to a nucleic acid molecule encoding apolipoprotein B wherein said compound is 100% complementary to the nucleic acid sequence set forth in SEQ ID NO:

3, and wherein said compound is not a ribozyme.

18. The method of claim 2, wherein said antisense compound is 20 nucleobases in length.

19. The method of claim 2, wherein said antisense compound is not targeted to the start codon region of SEQ ID NO:

3.

20. The method of claim 2, wherein said antisense compound is targeted to nucleotides 1-6530 of SEQ ID NO:

3.

21. The method of claim 2, wherein said antisense compound is targeted to nucleotides 6531-14121 of SEQ ID NO:

3.

22. The method of claim 2, wherein administering said antisense compound comprises parenteral administration of said antisense compound.

23. The method of claim 22, wherein said parenteral administration comprises intravenous administration or subcutaneous administration.

24. The method of claim 2, wherein said serum cholesterol levels are LDL-cholesterol levels.

25. The method of claim 2, wherein said serum cholesterol levels are VLDL-cholesterol levels.

26. The method of claim 2, wherein said serum cholesterol levels are total cholesterol levels.

27. The method of claim 2, further comprising measuring said serum cholesterol levels following administration of said antisense compound.

28. The method of claim 2 wherein the antisense compound comprises at least one modification selected from the group consisting of:

a modified sugar moiety, a modified internucleoside linkage, and a modified nucleobase.

29. The method of claim 28, wherein said modification comprises a modified sugar moiety selected from the group consisting of:

a 2′

-O-methoxyethyl modified sugar moiety, a 2′

-methoxy modified sugar moiety, a 2′

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

30. The method of claim 28, wherein said modification comprises a phosphorothioate internucleoside linkage.

31. The method of claim 28, wherein said modification comprises a 5-methylcytosine.

32. The method of claim 2, wherein said antisense compound is a gapmer comprising a gap segment comprised of linked 2′

-deoxynucleotides positioned between 5′ and

3′

wing segments.

33. The method of claim 32, wherein said wing segments are comprised of at least one modified sugar moiety selected from the group consisting of a 2′

-O-methoxyethyl modified sugar moiety, a 2′

-methoxy modified sugar moiety, a 2′

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

34. The method of claim 33, wherein said gap segment is ten 2′

-deoxynucleotides in length and each of said wing segments is five 2′

-O-methoxyethyl nucleotides in length.

63. The method of claim 2 wherein the antisense compound is an antisense oligonucleotide.

3. A method of decreasing lipoprotein levels in a human comprising administering to a human having elevated lipoprotein levels an antisense compound 12 to 30 nucleobases in length targeted to a nucleic acid molecule encoding apolipoprotein B wherein said compound is 100% complementary to the nucleic acid sequence set forth in SEQ ID NO:

3, and wherein said compound is not a ribozyme.

35. The method of claim 3, wherein said antisense compound is 20 nucleobases in length.

36. The method of claim 3, wherein said antisense compound is not targeted to the start codon region of SEQ ID NO:

3.

37. The method of claim 3, wherein said antisense compound is targeted to nucleotides 1-6530 of SEQ ID NO:

3.

38. The method of claim 3, wherein said antisense compound is targeted to nucleotides 6531-14121 of SEQ ID NO:

3.

39. The method of claim 3, wherein administering said antisense compound comprises parenteral administration of said antisense compound.

40. The method of claim 39, wherein said parenteral administration comprises intravenous administration or subcutaneous administration.

41. The method of claim 3, wherein said lipoprotein levels are lipoprotein(a) levels.

42. The method of claim 3 wherein said antisense compound comprises at least one modification selected from the group consisting of:

a modified sugar moiety, a modified internucleoside linkage, and a modified nucleobase.

43. The method of claim 42, wherein said modification is a modified sugar moiety is selected from the group consisting of:

a 2′

-O-methoxyethyl modified sugar moiety, a 2′

-methoxy modified sugar moiety, a 2′

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

44. The method of claim 42, wherein said modification comprises a phosphorothioate internucleoside linkage.

45. The method of claim 42, wherein said modification comprises a 5-methylcytosine.

46. The method of claim 3, wherein said antisense compound is a gapmer comprising a gap segment comprised of linked 2′

-deoxynucleotides positioned between 5′ and

3′

wing segments.

47. The method of claim 46, wherein said wing segments are comprised of at least one modified sugar moiety selected from the group consisting of a 2′

-O-methoxyethyl modified sugar moiety, a 2′

-methoxy modified sugar moiety, a 2′

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

48. The method of claim 47, wherein said gap segment is ten 2′

-deoxynucleotides in length and each of said wing segments is five 2′

-O-methoxyethyl nucleotides in length.

64. The method of claim 3 wherein the antisense compound is an antisense oligonucleotide.

4. A method of decreasing serum triglyceride levels in a human comprising administering to a human having elevated serum triglyceride levels an antisense compound 12 to 30 nucleobases in length targeted to a nucleic acid molecule encoding apolipoprotein B wherein said compound is 100% complementary to the nucleic acid sequence set forth in SEQ ID NO:

3, and wherein said compound is not a ribozyme.

49. The method of claim 4, wherein said antisense compound is 20 nucleobases in length.

50. The method of claim 4, wherein said antisense compound is not targeted to the start codon region of SEQ ID NO:

3.

51. The method of claim 4, wherein said antisense compound is targeted to nucleotides 1-6530 of SEQ ID NO:

3.

52. The method of claim 4, wherein said antisense compound is targeted to nucleotides 6531-14121 of SEQ ID NO:

3.

53. The method of claim 4, wherein administering said antisense compound comprises parenteral administration of said antisense compound.

54. The method of claim 53, wherein said parenteral administration comprises intravenous administration or subcutaneous administration.

55. The method of claim 4 wherein said antisense compound comprises at least one modification selected from the group consisting of:

a modified sugar moiety, a modified internucleoside linkage, and a modified nucleobase.

56. The method of claim 55, wherein said modification is a modified sugar moiety is selected from the group consisting of:

a 2′

-O-methoxyethyl modified sugar moiety, a 2′

-methoxy modified sugar moiety, a 2′

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

57. The method of claim 55, wherein said modification comprises a phosphorothioate internucleoside linkage.

58. The method of claim 55, wherein said modification comprises a 5-methylcytosine.

59. The method of claim 4, wherein said antisense compound is a gapmer comprising a gap segment comprised of linked 2′

-deoxynucleotides positioned between 5′ and

3′

wing segments.

60. The method of claim 59, wherein said wings segment are comprised of at least one modified sugar moiety selected from the group consisting of a 2′

-O-methoxyethyl modified sugar moiety, a 2′

-methoxy modified sugar moiety, a 2′

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

61. The method of claim 60, wherein said gap segment is ten 2′

-deoxynucleotides in length and each of said wing segments is five 2′

-O-methoxyethyl nucleotides in length.

65. The method of claim 4 wherein the antisense compound is an antisense oligonucleotide.