Ionizable amine lipids

Abstract

This disclosure provides ionizable lipid and lipid nanoparticle (LNP) compositions comprising ionizable lipids, helper lipids, neutral lipids, and PEG lipids, which are useful for the delivery of biologically active drugs, for example, for the preparation of manipulated cells. The LNP compositions disclosed herein are useful in gene editing methods, methods for delivering biologically active drugs, and methods for modifying or cleaving DNA.

Claims

1. Compounds represented by structural formula I: 【Chemistry 1】 or its salt. [In the formula, A is O or NH, X １ is C １－５ It is alkylene, R １ and R ２ Each is independently C １－３ Alkyl, or R １ R ２ And together with the nitrogen atoms to which they are bonded, they form a 5, 6, or 7-membered ring. Z １ is C １－５ alkylene, and Y １ and Y ２ Each is independently C ３－１０ Alkoxyl or O(C) ３－１０ Alkinyl) is Z ２ C １－５ Alkylene or direct bond Y ３ and Y ４ Each is independently C ３－１０ Alkoxyl or O(C) ３－１０ Alkinyl) or Y ３ and Y ４ Each is independently C ３－１０ Alkyl or C ３－１０ It is alkinyl, However, Y １ , Y ２ , Y ３ , and Y ４ However, each is independent of C ３－１０ It is an alkoxy, R １ and R ２ C ２ R, not alkyl １ However, R ２ [and provided that they do not form a six-membered ring together with the nitrogen atom to which they are bonded.]

2. The compound according to claim 1, wherein A is O.

3. The compound according to claim 1, wherein A is NH.

4. X １ C ２－３ The compound according to any one of claims 1 to 3, which is an alkylene.

5. Z １ C ２－３ The compound according to any one of claims 1 to 4, which is an alkylene.

6. Z ２ C ２－３ The compound according to any one of claims 1 to 5, which is an alkylene.

7. Z ２ The compound according to any one of claims 1 to 5, wherein the bond is a direct bond.

8. Z １ C ２－３ It is alkylene, Z ２ The compound according to any one of claims 1 to 4, wherein the bond is a direct bond.

9. Y １ and Y ２ However, each is independent of C ６－９ A compound according to any one of claims 1 to 8, wherein the compound is an alkoxyl.

10. Y ３ and Y ４ However, each is independent of C ６－９ A compound according to any one of claims 1 to 9, wherein the compound is an alkoxyl.

11. Y ３ C ６－９ It is alkyl, Y ４ C ３－５ A compound according to any one of claims 1 to 9, wherein the compound is alkyl.

12. R １ and R ２ However, each is independent of C １－３ A compound according to any one of claims 1 to 11, wherein it is alkyl.

13. The compound according to claim 1, wherein the compound is represented by one of the following structural formulas. 【Chemistry 2】 or its salt.

14. Compounds represented by structural formula II 【Transformation 3】 or its salt. [In the formula, Q is CR ３ or 【Chemistry 4】 And, A is O, NH, or a direct bond. X １ is C １－５ It is alkylene, R １ and R ２ Each is independently C １－３ Alkyl, or R １ The nitrogen atom and X to which it is bonded. １ Together with 1 to 3 carbon atoms, they form a 4, 5, or 6-membered ring, or R １ R ２ And together with the nitrogen atoms to which they are bonded, they form a 5, 6, or 7-membered ring. R ３ is H or C １－３ It is alkyl, Z １ and Z ２ Each is independently C １－５ It is alkylene, Z ３ and Z ４ Each of them is independent and is -C(=O)O- in either direction, Z ５ and Z ６ Each is independently directly bonded or C １－３ It is alkylene, Y １ H, C １－１０ Alkyl, C ３－１０ Alkenyl and C ３－１０ Selected from Alkinil, Y ２ , Y ３ , and Y ４ Each is independently C ３－１０ Alkyl, C ３－１０ Alkenyl and C ３－１０ Selected from Alkinil, n is either 0 or 1.

15. Q is 【Transformation 5】 The compound according to claim 14.

16. The compound according to claim 14, wherein the compound is represented by structural formula IIa. 【Transformation 6】

17. The compound according to any one of claims 14 to 16, wherein n is 1.

18. R ３ The compound according to claim 16 or 17, wherein is H.

19. The compound according to any one of claims 14 to 18, wherein A is O.

20. The compound according to any one of claims 14 to 18, wherein A is NH.

21. X １ C ２－３ The compound according to any one of claims 14 to 20, which is an alkylene.

22. X １ is C ２ The compound according to claim 21, wherein the alkylene is C

23. Z １ and Z ２ However, each is independent of C ３ The compound according to any one of claims 14 to 22, wherein it is an alkylene.

24. Z １ and Z ２ are each independently C ４ alkylene, the compound according to any one of claims 14 to 22.

25. Z １ C ２ It is alkylene, Z ２ C ３ The compound according to any one of claims 14 to 22, wherein it is an alkylene.

26. Z １ and Z ２が , each independently C ３ It is alkylene, A is NH, and X １ C ２ The compound according to any one of claims 14 to 18, wherein it is an alkylene.

27. Z １ and Z ２ However, each is independent of C ５ The compound according to any one of claims 14 to 21, which is an alkylene.

28. Z ３ and Z ４ Each 【Transformation 7】 And a is Z １ and Z ２ A compound according to any one of claims 14 to 27, which shows a bonding site with

29. Z ３ but 【Transformation 8】 And b is Z １ The connection point with Z ４ but 【Chemistry 9】 And b is Z ２ A compound according to any one of claims 14 to 27, which shows a bonding site with

30. Z ５ and Z ６ The compound according to any one of claims 14 to 29, wherein each is independently directly bonded.

31. Z ５ C １ It is alkylene, Z ６ The compound according to any one of claims 14 to 29, wherein the bond is a direct bond.

32. Y １ , Y ２ , Y ３ , and Y ４ However, each is independent of C ７－９ The compound according to any one of claims 14 to 31, wherein it is alkyl.

33. Y １ , Y ２ , Y ３ , and Y ４ However, each is independent of C ８ The compound according to claim 32, wherein it is alkyl.

34. Y １ and Y ２ However, each is independent of C ５－７ It is alkyl, Y ３ and Y ４ However, each is independent of C ３－５ The compound according to any one of claims 14 to 31, wherein it is alkyl.

35. R １ and R ２ However, each is independent of C １－３ The compound according to any one of claims 14 to 34, wherein it is alkyl.

36. R １ and R ２ Each C ２ The compound according to claim 35, wherein it is alkyl.

37. R １ However, R ２ The compound according to any one of claims 14 to 34, wherein the compounds and the nitrogen atoms to which they are bonded together form a 5, 6, or 7-membered ring.

38. R １ However, R ２ The compound according to claim 37, wherein the nitrogen atoms to which they are bonded together form a five-membered ring.

39. The compound according to claim 14, wherein the compound is represented by one of the following structural formulas. 【Chemistry 10-1】 【Chemistry 10-2】 【Chemistry 10-3】 [Chemistry 10-4] or its salt.

40. Compounds represented by structural formula III 【Chemistry 11】 or its salt. [In the formula, Q is CH or 【Chemistry 12】 And, A is O or NH, X １ is C １－５ It is alkylene, R １ and R ２ Each is independently C １－３ Alkyl, or R １ The nitrogen atom and X to which it is bonded. １ Together with 1 to 3 carbon atoms, they form a 4, 5, or 6-membered ring, or R １ R ２ And together with the nitrogen atoms to which they are bonded, they form a 5, 6, or 7-membered ring. Z １ is C ２－９ It is alkylene, Z ２ C １－３ Alkylene or direct bond Y １ and Y ２ Each of them is independent of C ３－１０ Alkyl, C ３－１０ Alkenyl and C ３－１０ [Selected from Alkinil]

41. Q is 【Chemistry 13】 The compound according to claim 40.

42. The compound according to claim 40, wherein the compound is represented by structural formula IIIa. 【Chemistry 14】

43. The compound according to any one of claims 40 to 42, wherein A is O.

44. A compound according to any one of claims 40 to 42, wherein A is NH.

45. X １ C ２－３ The compound according to any one of claims 40 to 44, which is an alkylene.

46. X １ C ３ The compound according to any one of claims 40 to 45, which is an alkylene.

47. Z １ However, C ３－５ Alkylene, C ５－７ Alkylene, or C ７－９ The compound according to any one of claims 40 to 46, which is an alkylene.

48. Z ２ The compound according to any one of claims 40 to 47, wherein the bond is a direct bond.

49. Y １ and Y ２ However, each is independent of C ３－５ Alkyl, C ５－７ Alkyl, or C ７－９ The compound according to any one of claims 40 to 48, wherein it is alkyl.

50. The compound according to claim 40, wherein the compound is represented by one of the following structural formulas. 【Chemistry 15-1】 【Chemistry 15-2】 【Chemistry 15-3】 【Chemistry 15-4】 or its salt.

51. Compounds represented by structural formula IV 【Chemistry 16】 or its salt. [In the formula, Q is CH or 【Chemistry 17】 And, A is O, NH, or a direct bond. X １ and X ２ Each is independently C １－５ It is alkylene, R １ C ３－９ Alkyl, C ３－９ Alkenyl and C ３－９ Selected from Alkinil, R ２ and R ３ Each is independently C １－３ Alkyl, or R ２ R ３ And together with the nitrogen atoms to which they are bonded, they form a 5, 6, or 7-membered ring. Z １ is C ６－１０ It is alkylene, Y １ and Y ２ Each of them is independent of C ３－１０ Alkyl, C ３－１０ Alkenyl and C ３－１０ [Selected from Alkinil]

52. Q is [Chemistry 18] The compound according to claim 51.

53. The compound according to claim 51, wherein the compound is represented by the structural formula IVa. 【Chemistry 19】

54. The compound according to any one of claims 51 to 53, wherein A is NH.

55. X １ C ２－３ The compound according to any one of claims 51 to 54, which is an alkylene.

56. X ２ C ２－３ The compound according to any one of claims 51 to 55, which is an alkylene.

57. Z １ C ７－９ The compound according to any one of claims 51 to 56, which is an alkylene.

58. Y １ and Y ２ However, each is independent of C ８－１０ The compound according to any one of claims 51 to 57, wherein it is alkyl.

59. R １ However, C ３－５ Alkyl or C ７－９ The compound according to any one of claims 51 to 58, wherein it is alkyl.

60. R ２ and R ３ However, each is independent of C １－３ The compound according to any one of claims 51 to 59, wherein it is alkyl.

61. R ２ However, R ３ The compound according to any one of claims 51 to 59, wherein the nitrogen atoms to which they are bonded together form a 5, 6, or 7-membered ring.

62. The compound according to claim 51, wherein the compound is represented by one of the following structural formulas. 【Chemistry 20-1】 【Chemistry 20-2】 or its salt.

63. A compound represented by one of the following structural formulas 【Chemistry 21-1】 【Chemistry 21-2】 【Chemistry 21-3】 【Chemistry 21-4】 【Chemistry 21-5】 or its salt.

64. The compound according to any one of claims 1 to 63, wherein the salt is a pharmaceutically acceptable salt.

65. A composition comprising a compound according to any one of claims 1 to 64 in its lipid components.

66. The composition according to claim 65, wherein the lipid component further comprises a helper lipid and a PEG lipid.

67. The composition according to claim 65 or 66, wherein the lipid component further comprises a neutral lipid.

68. The PEG lipids include PEG-dilauroylglycerol, PEG-dimiristoylglycerol (PEG-DMG), PEG-dipalmitoylglycerol, PEG-distearoylglycerol (PEG-DSG), PEG-dilaurylglycamide, PEG-dimiristoylglycamide, PEG-dipalmitoylglycamide, PEG-distearoylglycamide, and 1-[8'-(cholest-5-ene-3[beta]-oxy)carboxamide-3 ',6'-Dioxaoctanyl]carbamoyl-[omega]-methyl-poly(ethylene glycol) (PEG-cholesterol), 3,4-Ditetradecoxylbenzyl-[omega]-methyl-poly(ethylene glycol) ether (PEG-DMB), 1,2-Dimiristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (PEG2K-DMPE), 1,2-Dimiristoyl-ra c-Glycero-3-[methoxy(polyethylene glycol)-2000](PEG2K-DMG), 1,2-Distearoyl-sn-Glycero-3-Phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000](PEG2K-DSPE), 1,2-Distearoyl-sn-Glycerol-[methoxy(polyethylene glycol)-2000](PEG2K-DSG), Poly(ethylene glycol)-2000-Dimethacrylate The composition according to claim 66 or 67, selected from methoxy(PEG2K-DMA), 1,2-distearyloxypropyl-3-amine-N-[methoxy(polyethylene glycol)-2000](PEG2K-DSA), methoxy-PEG2000-carbamoyl-1,2-tetradecyloxypropylamine (C14 ether), and methoxy-PEG2000-carbamoyl-1,2-tridecyloxypropylamine (C13 ether).

69. The composition according to claim 68, wherein the PEG lipid is selected from PEG2K-DMG, C13 ether, and C14 ether.

70. The composition according to any one of claims 66 to 69, wherein the PEG lipid comprises dimyristoyl glycerol (DMG).

71. The aforementioned neutral lipids include dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), dioleoylphosphocholine (DOPC), dimyristoylphosphatidylcholine (DMPC), 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphatidylcholine (PLPC), 1,2-diarachidoyl-sn-glycero-3-phosphocholine (DAPC), phosphatidylethanolamine (PE), egg phosphatidylcholine (EPC), dilauroylphosphatidylcholine (DLPC), dimyristoylphosphatidylcholine (DMPC), 1-myristoyl-2-palmitoylphosphatidylcholine (MPPC), 1-palmitoyl-2-myristoylphosphatidylcholine (PMPC), 1-palmitoyl-2-stearoylphosphatidylcholine (PSPC), and 1,2 A composition according to any one of claims 67 to 70, selected from -dibehenoyl-sn-glycero-3-phosphocholine (DBPC), 1-stearoyl-2-palmitoylphosphatidylcholine (SPPC), 1,2-dieicosenoyl-sn-glycero-3-phosphocholine (DEPC), palmitoyloleylphosphatidylcholine (POPC), lysophosphatidylcholine, dioleoylphosphatidylethanolamine (DOPE), dilinoleoylphosphatidylcholine distearoylphosphatidylethanolamine (DSPE), dimyristoylphosphatidylethanolamine (DMPE), dipalmitoylphosphatidylethanolamine (DPPE), palmitoyloleoylphosphatidylethanolamine (POPE), and lysophosphatidylethanolamine, or a combination thereof.

72. The composition according to claim 70, wherein the neutral lipid is DSPC or DMPE.

73. The composition according to claim 72, wherein the neutral lipid is DSPC.

74. The composition according to any one of claims 66 to 73, wherein the helper lipid is selected from cholesterol, 5-heptadecylresorcinol, and cholesterol hemisuccinate.

75. The composition according to claim 74, wherein the helper lipid is cholesterol.

76. The composition according to any one of claims 67 to 75, wherein the lipid component comprises 45 to 55 mol% of the compound according to any one of claims 1 to 64, 35 to 40 mol% of the helper lipid, 7 to 10 mol% of the neutral lipid, and 1 to 5 mol% of the PEG lipid.

77. The composition according to claim 76, wherein the lipid component comprises about 50 mol% of the compound according to any one of claims 1 to 64, about 38 mol% of the helper lipid, about 9 mol% of the neutral lipid, and about 3 mol% of the PEG lipid.

78. The composition according to any one of claims 65 to 77, further comprising a cryoprotectant.

79. The composition according to any one of claims 65 to 78, further comprising a buffer.

80. The composition according to any one of claims 65 to 79, further comprising an aqueous component containing a biologically active agent.

81. The composition according to claim 80, wherein the biologically active agent comprises a polypeptide.

82. The composition according to claim 80 or 81, wherein the biologically active agent comprises or encodes a therapeutically active protein.

83. The composition according to any one of claims 80 to 82, wherein the biologically active agent comprises or encodes a genome editing tool.

84. The composition according to any one of claims 80 to 83, wherein the biologically active agent comprises or encodes one or more nucleases capable of single-strand or double-strand breaks of DNA or RNA.

85. The composition according to claim 84, wherein the biologically active agent comprises nucleic acid.

86. The composition according to claim 85, wherein the nucleic acid comprises RNA.

87. The composition according to claim 85 or 86, wherein the composition has an N / P ratio of about 5 to about 7.

88. The composition according to claim 87, wherein the N / P ratio is approximately 6 ± 1.

89. The composition according to claim 87, wherein the N / P ratio is approximately 6 ± 0.

5.

90. The composition according to claim 87, wherein the N / P ratio is approximately 6.

91. The composition according to any one of claims 65 to 90, comprising an RNA component, wherein the RNA component comprises mRNA.

92. The composition according to claim 91, wherein the RNA component comprises a sequence encoding an RNA guide DNA binder such as Cas nuclease mRNA.

93. The composition according to claim 91 or 92, wherein the RNA component comprises class 2 Cas nuclease mRNA.

94. The composition according to any one of claims 91 to 93, wherein the RNA component comprises Cas9 nuclease mRNA.

95. The composition according to any one of claims 91 to 94, wherein the RNA component comprises modified RNA.

96. The composition according to any one of claims 91 to 95, wherein the RNA component comprises gRNA nucleic acid.

97. The composition according to claim 96, wherein the gRNA nucleic acid is gRNA.

98. The composition according to any one of claims 91 to 97, wherein the RNA component comprises class 2 Cas nuclease mRNA and gRNA.

99. The composition according to any one of claims 96 to 98, wherein the gRNA nucleic acid is a dual guide RNA (dgRNA) or encodes one.

100. The composition according to any one of claims 96 to 99, wherein the gRNA nucleic acid is a single guide RNA (sgRNA) or encodes one.

101. The composition according to any one of claims 96 to 100, wherein the gRNA is a modified gRNA.

102. The composition according to claim 101, wherein the modified gRNA includes a modification to one or more of the first five nucleotides at the 5' end.

103. The composition according to claim 101 or 102, wherein the modified gRNA includes a modification to one or more of the last five nucleotides at the 3' end.

104. The composition according to any one of claims 91 to 103, wherein the composition comprises a guide RNA nucleic acid, the mRNA is a class 2 Cas nuclease mRNA, and the weight ratio of the mRNA to the guide RNA nucleic acid is about 2:1 to 1:

4.

105. The composition according to any one of claims 65 to 104, further comprising at least one template nucleic acid.

106. A method for cutting DNA, comprising contacting a cell with a composition according to any one of claims 83 to 105.

107. A method for gene editing, comprising contacting a cell with a composition according to any one of claims 83 to 105.

108. The method according to claim 106 or 107, wherein the contact step results in a single-stranded DNA nick.

109. The method according to claim 106 or 107, wherein the contact step results in a double-strand DNA break.

110. The method according to any one of claims 106 to 109, wherein the composition comprises class 2 Cas mRNA and gRNA nucleic acids.

111. The method according to any one of claims 106 to 110, further comprising introducing at least one template nucleic acid into the cells.

112. The method according to claim 111, comprising contacting the cells with a composition containing a template nucleic acid.

113. The method according to any one of claims 106 to 112, wherein the method comprises administering the composition to an animal.

114. The method according to any one of claims 106 to 113, wherein the method comprises administering the composition to a human.

115. The method according to any one of claims 106 to 114, comprising administering the composition to cells.

116. The method according to claim 115, wherein the cells are eukaryotic cells.

117. The method according to claim 115 or 116, wherein the method comprises administering formulated mRNA to a first lipid nanoparticle (LNP) composition comprising one or more of mRNA, gRNA, gRNA nucleic acid, and template nucleic acid, and to a second LNP composition.

118. The method according to claim 117, wherein the first LNP composition and the second LNP composition are administered simultaneously.

119. The method according to claim 117, wherein the first LNP composition and the second LNP composition are administered sequentially.

120. The method according to claim 117, wherein the method comprises administering the mRNA and gRNA nucleic acids formulated in a single LNP composition.

121. The method according to any one of claims 107 to 120, wherein the gene editing results in gene knockout.

122. The method according to any one of claims 107 to 120, wherein the gene editing results in gene modification.

123. The method according to any one of claims 106 to 122, wherein the cells are hematopoietic stem cells (HSCs) or induced pluripotent stem cells (iPSCs).

124. The method according to any one of claims 106 to 122, wherein the cells are immune cells.

125. The method according to claim 124, wherein the immune cells are white blood cells or lymphocytes.

126. The method according to claim 124, wherein the immune cells are lymphocytes.

127. The method according to claim 126, wherein the lymphocytes are T cells, B cells, or NK cells.

128. The method according to claim 126, wherein the lymphocyte is a T cell.

129. The method according to claim 126, wherein the lymphocytes are activated T cells.

130. The method according to claim 126, wherein the lymphocytes are inactivated T cells.

131. The method according to any one of claims 115 to 130, wherein the cells are in contact with the lipid composition in vitro.

132. The method according to any one of claims 115 to 130, wherein the cells are in contact with the lipid composition in ex vivo.

133. The method according to any one of claims 106 to 130, wherein the method comprises contacting animal tissue with the lipid composition.

134. The method according to claim 133, wherein the tissue is liver tissue.

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