CHIRAL CONTROL

• US 20150211006A1
• Filed: 07/12/2013
• Published: 07/30/2015
• Est. Priority Date: 07/13/2012
• Status: Active Grant

• Current Assignee: WAVE Life Sciences Ltd.
• Original Assignee: Wave Life Sciences Pte. Ltd.
• Inventors: Butler, David, Iwamoto, Naoki, Meena, Meena, Verdine, Gregory L., Zlatev, Ivan, Svrzikapa, Nenad
• Granted Patent: US 9,982,257 B2
• Time in Patent Office: Days
• US Class Current: 1/1
• 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: C07C 317/28 with sulfone or sulfoxide groups bound to acyclic carbon atoms of the carbon skeleton
• CPC Class Codes: C07D 295/088 to an acyclic saturated chain
• CPC Class Codes: C07H 1/02 Phosphorylation
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C07H 21/02 with ribosyl as saccharide radical
• CPC Class Codes: C12N 15/11 DNA or RNA fragments; Modified forms thereof DNA or RNA not used in recombinant technology, C07H21/00; Non-coding nucleic acids having a biological activity
• CPC Class Codes: C12N 15/111 General methods applicable to biologically active non-coding nucleic acids
• 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 2310/14 interfering N.A.
• CPC Class Codes: C12N 2310/31 of the backbone
• CPC Class Codes: C12N 2310/315 Phosphorothioates
• CPC Class Codes: C12N 2330/30 chemically synthesised
• CPC Class Codes: C12Q 1/6876 Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes

Abstract

The present invention relates to chirally controlled oligonucleotides, chirally controlled oligonucleotide compositions, and the method of making and using the same. The invention specifically encompasses the identification of the source of certain problems with prior methodologies for preparing chiral oligonucleotides, including problems that prohibit preparation of fully chirally controlled compositions, particularly compositions comprising a plurality of oligonucleotide types. In some embodiments, the present invention provides chirally controlled oligonucleotide compositions. In some embodiments, the present invention provides methods of making chirally controlled oligonucleotides and chirally controlled oligonucleotide compositions.

Claims

1. A chirally controlled oligonucleotide composition comprising a plurality of oligonucleotides of at least one type, wherein each type is defined by:

1) base sequence;

2) pattern of backbone linkages;

3) pattern of backbone chiral centers; and

4) pattern of backbone X-moieties.

2. The composition of claim 1, wherein the oligonucleotide of at least one type is a unimer.

3. The composition of claim 2, wherein the oligonucleotide of at least one type is a stereounimer, P-modification unimer, or linkage unimer.

4. The composition of claim 1, wherein the oligonucleotide of at least one type is an altmer.

5. The composition of claim 4, wherein the oligonucleotide of at least one type is a stereoaltmer, P-modification altmer, or linkage altmer.

6. The composition of claim 1, wherein the oligonucleotide of at least one type is a blockmer.

7. The composition of claim 6, wherein the oligonucleotide of at least one type is a stereoblockmer, P-modification blockmer, or linkage blockmer.

8. The composition of claim 1, wherein the oligonucleotide of at least one type is a gapmer.

9. The composition of claim 1, wherein the oligonucleotide of at least one type is a skipmer

10. The composition of claim 1, wherein the oligonucleotide of at least one type is an antisense oligonucleotide, an antagomir, a microRNA, a pre-microRNA, an antimir, a supermir, a ribozyme, a Ul adaptor, RNA activator, RNAi agent, a decoy oligonucleotide, a triplex forming oligonucleotide, or an aptamer.

11. The composition of claim 1, wherein the oligonucleotide of at least one type is useful in regulating the level of a protein, or an RNA.

12. The composition of claim 11, wherein the oligonucleotide of at least one type is useful in regulating the level of an mRNA.

13. The composition of claim 11, wherein the oligonucleotide of at least one type is useful in regulating the level of a microRNA.

14. The composition of claim 1, wherein the at least one oligonucleotide type comprises one or more phosphorothioate triester internucleotide linkages.

15. The composition of claim 1, wherein the at least one oligonucleotide type comprises at least 2, at least 3, at least 4, at least 5, or at least 10 phosphorothioate triester internucleotide linkages.

16. The composition of claim 1, wherein the at least one oligonucleotide type comprises at least 10, at least 12, or at least 15 phosphorothioate triester internucleotide linkages.

17. The composition of claim 1, wherein the at least one oligonucleotide type comprises one or more modified internucleotide linkages independently having the structure of formula I:

18. The composition of claim 1, wherein the at least one oligonucleotide type comprises one or more modified internucleotide linkages independently having the structure of formula I-a, I-b, or I-c.

19. The composition of claim 18, wherein the at least one oligonucleotide type comprises one or more modified internucleotide linkages independently having the structure of formula I-c.

20. The composition of any of claims 1-19, wherein the at least one oligonucleotide type further comprises one or more phosphate diester linkage.

26. The oligonucleotide composition of claim 1 or claim 21, wherein one or more oligonucleotide types is selected from Table 2

65. A method comprising steps of administering to a subject suffering from or susceptible to cancer an amount of an oligonucleotide composition of any of claims 1-26 and 64, sufficient to partially or completely alleviate, ameliorate, relieve, inhibit, prevent, delay onset of, reduce severity of, and/or reduce incidence of one or more symptoms or features of cancer.

100. The method of claim 98, wherein the second composition is a composition of any one of claims 1-26 and 95-96.

21. An oligonucleotide composition that is chirally controlled in that the composition contains predetermined levels of one or more individual oligonucleotide types, wherein an oligonucleotide type is defined by:

1) base sequence;

2) pattern of backbone linkages;

3) pattern of backbone chiral centers; and

4) pattern of backbone X-moieties.

22. The oligonucleotide composition of claim 21, wherein the composition is chirally pure.

23. The oligonucleotide composition of claim 22, wherein the composition is chirally uniform.

24. The oligonucleotide composition of claim 22, wherein the composition contains predetermined levels of at least two oligonucleotide types.

25. The oligonucleotide composition of claim 22, wherein the composition contains predetermined levels of at least three or at least four oligonucleotide types.

27. A chirally controlled oligonucleotide comprising one or more phosphorothioate triester internucleotide linkages.

28. The composition of claim 27, comprising at least 2, at least 3, at least 4, at least 5, or at least 10 phosphorothioate triester internucleotide linkages.

33. The chirally controlled oligonucleotide of any of claims 27-32, wherein the at least one oligonucleotide type further comprises one or more phosphate diester linkage.

34. The chirally controlled oligonucleotide of any of claims 27-33 comprising at least 5, at least 6, or at least 7 consecutive modified internucleotidic linkages, and at least one of the consecutive modified internucleotidic linkages is not a phosphorothioate diester linkage.

35. The chirally controlled oligonucleotide of any of claims 27-34, comprising at least one internucleotidic linkage having the structure of

64. A pharmaceutical composition for treating a disease comprising a therapeutically effective amount of the chirally controlled oligonucleotide of any of claims 27-36, and a pharmaceutically acceptable excipient.

66. The chirally controlled oligonucleotide of any of claims 27-35, wherein the chirally controlled oligonucleotide comprises a sequence found in GCCTCAGTCTGCTTCGCACC.

67. The chirally controlled oligonucleotide of claim 66, selected from Table 4.

68. The chirally controlled oligonucleotide of any of the claims 27-36, 66 and 67, wherein the chirally controlled oligonucleotide has a purity of >

50%.

69. The chirally controlled oligonucleotide of claim 68, wherein the chirally controlled oligonucleotide has a purity of >

60%.

70. The chirally controlled oligonucleotide of claim 68, wherein the chirally controlled oligonucleotide has a purity of >

70%.

71. The chirally controlled oligonucleotide of claim 68, wherein the chirally controlled oligonucleotide has a purity of >

80%.

72. The chirally controlled oligonucleotide of claim 68, wherein the chirally controlled oligonucleotide has a purity of >

85%.

73. The chirally controlled oligonucleotide of claim 68, wherein the chirally controlled oligonucleotide has a purity of >

90%.

74. The chirally controlled oligonucleotide of claim 68, wherein the chirally controlled oligonucleotide has a purity of >

95%.

75. The chirally controlled oligonucleotide of claim 68, wherein the chirally controlled oligonucleotide has a purity of >

98%.

76. The chirally controlled oligonucleotide of claim 68, wherein the chirally controlled oligonucleotide has a purity of >

99%.

95. The chirally controlled oligonucleotide of any one of claims 27-36 and 66-94, wherein the chirally controlled oligonucleotide is not linked to a solid support.

97. A chirally controlled oligonucleotide composition comprising a chirally controlled oligonucleotide of any one of claims 27-36 and 66-95.

29. A chirally controlled oligonucleotide comprising at least 10, at least 12, or at least 15 phosphorothioate triester internucleotide linkages.

30. A chirally controlled oligonucleotide comprising one or more modified internucleotide linkages independently having the structure of formula I:

31. A chirally controlled oligonucleotide, comprising one or more modified internucleotide linkages independently having the structure of formula I-a, I-b, or I-c.

32. A chirally controlled oligonucleotide, comprising one or more modified internucleotide linkages independently having the structure of formula I-c.

36. A chirally controlled oligonucleotide selected from Table 2.

37. A method for making a chirally controlled oligonucleotide comprising steps of:

(1) coupling;

(2) capping;

(3) modifying;

(4) deblocking; and

(5) repeating steps (1)-(4) until a desired length is achieved.

38. The method of claims 37, wherein the step of coupling comprises the use of CMPT and

39. The method of claim 37 or 38, wherein the step of capping comprises steps of capping of the amino group in the chiral auxiliary and the capping of unreacted 5′

-OH.

40. The method of claims 39, wherein the step of capping comprises using of (Pac)₂O.

41. The method of claims 37, wherein the modifying step comprises sulfurization.

42. The method of claim 41, wherein the sulfurization step comprises using a sulfurization reagent is of formula S-I or S-II.

43. The method of claim 41, wherein the cycle comprising the said sulfurization step forms an internucleotidic linkage having the structure of formula I.

44. The method of claim 37, wherein the step of deblocking comprises steps of using an acid.

45. The method of claim 37, wherein all oligonucleotides that are one nucleotide shorter than the desired length, when combined, are less than 10% of the crude product.

46. The method of claim 45, wherein all oligonucleotides that are one nucleotide shorter than the desired length, when combined, are less than 5% of the crude product.

47. The method of claim 45, wherein all oligonucleotides that are one nucleotide shorter than the desired length, when combined, are less than 4% of the crude product.

48. The method of claim 45, wherein all oligonucleotides that are one nucleotide shorter than the desired length, when combined, are less than 3% of the crude product.

49. The method of claim 45, wherein all oligonucleotides that are one nucleotide shorter than the desired length, when combined, are less than 2% of the crude product.

50. The method of claim 45, wherein all oligonucleotides that are one nucleotide shorter than the desired length, when combined, are less than 1% of the crude product.

51. The method of claim 45, wherein all oligonucleotides that are one nucleotide shorter than the desired length, when combined, are less than 0.5% of the crude product.

53. The method of claim 37, wherein a phosphorothioate diester precursor is used for each phosphorothioate diester linkage.

54. The method of claim 53, wherein the phosphorothioate diester precursor is

55. The method of claim 53 or 54, wherein each phosphorothioate diester precursor is converted to a phosphorothioate diester linkage after the desired oligonucleotide length is achieved.

56. The method of any of claims 37-55, wherein at least one modifying step is replaced by an oxidation step.

90. A chirally controlled oligonucleotide prepared by a method of any one of claims 37-60.

52. A method for making a chirally controlled oligonucleotide composition, comprising providing one or more oligonucleotide types in a pre-determined amount.

57. A method for modifying an oligonucleotide comprising steps of:

(1) providing a chirally controlled oligonucleotide;

(2) providing a modification reagent; and

(3) reacting the chirally controlled oligonucleotide with the modification reagent under conditions suitable to effect modification of the linkage phosphorus of the chirally controlled oligonucleotide.

58. The method of claim 57, wherein the modification reagent is of formula S-I or S-II.

59. The method of claim 57, wherein at least one phosphorothioate diester precursor is converted to a phosphorothioate diester linkage.

60. The method of claim 59, wherein each phosphorothioate diester precursor is converted to a phosphorothioate diester linkage.

61. A sulfurization reagents having the formula of S-I.

62. A sulfurization reagents having the formula of S-II.

63. A sulfurization reagent selected from Table 5.

77. A chirally controlled oligonucleotide of one of the structures depicted in FIG. 26.

78. A chirally controlled oligonucleotide of one of the structures depicted in FIG. 27.

79. A chirally controlled oligonucleotide of one of the structures depicted in FIG. 28.

80. A chirally controlled oligonucleotide of one of the structures depicted in FIG. 29.

81. A chirally controlled oligonucleotide of one of the structures depicted in FIG. 30.

82. A chirally controlled oligonucleotide composition comprising one or more of the oligonucleotides depicted in FIG. 26.

83. A chirally controlled oligonucleotide composition comprising one or more of the oligonucleotides depicted in FIG. 27.

84. A chirally controlled oligonucleotide composition comprising one or more of the oligonucleotides depicted in FIG. 28.

85. A chirally controlled oligonucleotide composition comprising one or more of the oligonucleotides depicted in FIG. 29.

86. A chirally controlled oligonucleotide composition comprising one or more of the oligonucleotides depicted in FIG. 30.

87. A chirally controlled oligonucleotide of any of the structures depicted in WO 2012/030683.

88. A chirally controlled oligonucleotide composition comprising an oligonucleotide of any of the structures depicted in WO 2012/030683.

89. A chirally controlled oligonucleotide having any of the base sequences described or depicted in WO 2012/030683.

91. A chirally controlled oligonucleotide prepared by a method comprising one or more cycles depicted in Scheme I.

92. A chirally controlled oligonucleotide prepared by a method comprising one or more cycles depicted in Scheme I-b.

93. A chirally controlled oligonucleotide prepared by a method comprising one or more cycles depicted in Scheme I-c.

94. A chirally controlled oligonucleotide prepared by a method comprising one or more cycles depicted in Scheme I-d.

96. A chirally controlled oligonucleotide composition comprising an oligonucleotide having any of the base sequences described or depicted in WO 2012/030683.

98. A method comprising steps of:

a) performing a first analysis of a first composition, which first composition comprises a plurality of different types of an oligonucleotide; and

b) comparing the performed first analysis with a second analysis, under comparable conditions as the first analysis, of a second composition, which second composition is a chirally controlled composition of the oligonucleotide, where differences between the first and second analyses reflect differences in presence or level of at least one type of the oligonucleotide in the first as compared with the second composition.

99. The method of claim 98, wherein the second composition contains only a single type of the oligonucleotide.