IONIZABLE CATIONIC LIPIDS AND LIPID NANOPARTICLES, AS WELL AS METHODS OF THEIR SYNTHESIS AND APPLICATION

Claims

1. Compound of formula (I) (I), or its salt, where each of R 1 , R 2 and R 3 independently represents a bond or C 1-3 alkylene; each of R 1A , R 2A and R 3A independently represents a bond or C 1-10 alkylene; each of R 1A1 , R 1A2 , R 1A3 , R 2A1 , R 2A2 , R 2A3 , R 3A1 , R 3A2 and R 3A3 independently represents H, C 1-20 alkyl, C 1-20 alkenyl, -(CH2) 0-10 C(O)OR a1 or -(CH2) 0-10 OC(O)R a2 ; each of R a1 and R a2 represents C 1-20 alkyl or C 1-20 alkenyl; R 3B represents ; R 3B1 represents C 1-6 alkylene; and each of R 3B2 and R 3B3independently represents H or C 1-6 alkyl.

2. The compound according to claim 1 or a salt thereof, wherein the compound is a compound of formula (Ia), (Ia).

3. The compound according to item 1 or 2 or a salt thereof, where R 3B1 is ethylene or propylene.

4. A compound according to any one of paragraphs 1-3 or a salt thereof, where each of R 3B2 and R 3B3 independently represents H or C 1-6 alkyl optionally substituted with one or more substituents each independently selected from the group consisting of -OH and -O-(C 1-6 alkyl).

5. The compound according to item 4 or a salt thereof, where each of R 3B2 and R 3B3 independently represents methyl or ethyl, each of which is optionally substituted with one or more -OH.

6. The compound according to item 5 or a salt thereof, where each of R 3B2 and R 3B3 is an unsubstituted methyl.

7. The compound according to item 1 or 2 or a salt thereof, wherein represents , , , or .

8. A compound according to any one of paragraphs 1-7 or a salt thereof, wherein each of R 1 , R 2 and R 3 independently represents a bond or methylene.

9. The compound according to item 8 or a salt thereof, wherein each of R 1 and R 2 is methylene, and R 3 represents a connection.

10. The compound according to claim 8 or a salt thereof, wherein each of R 1 , R 2 and R 3 is methylene.

11. The compound according to claim 1 or a salt thereof, wherein the compound is a compound of formula (Ib), (Ib).

12. A compound according to any one of paragraphs 1-11 or a salt thereof, wherein each of R 1A , R 2A and R 3A independently represents a bond or -(CH2) 1-10 -.

13. The compound according to claim 12 or a salt thereof, wherein each of R 1A and R 2A independently represents a bond, -CH2-, -(CH2)2-, -(CH2)3-, -(CH2)4-, -(CH2)5-, -(CH2)6-, -(CH2)7-, or (CH2)8-.

14. The compound according to claim 13 or a salt thereof, wherein each of R 1A and R 2A independently represents a bond, -(CH2)2-, -(CH2)4-, -(CH2)6-, -(CH2)7-, or -(CH2)8-.

15. A compound according to any one of paragraphs 12-14 or a salt thereof, where R 3A is a bond, -CH2-, -(CH2)2- or -(CH 2)7 -.

16. A compound according to any one of paragraphs 1-15 or a salt thereof, wherein each of R 1A1 , R 1A2 , R 1A3 , R 2A1 , R 2A2 and R 2A3 represents H, C 1-15 alkyl, -CH=CH-(C 1-15 alkyl), -CH=CH-CH2-CH=CH-(C 1-10 alkyl), -(CH2) 0-4 C(O)OCH(C 1-10 alkyl)(C 1-15 alkyl), -(CH2) 0-4 OC(O)CH(C 1-10 alkyl)(C 1-15 alkyl), -(CH2) 0-4 C(O)OCH2(C 1-15alkyl) or -(CH2) 0-4 OC(O)CH2(C 1-15 alkyl).

17. The compound according to claim 16 or a salt thereof, wherein each of R 1A1 and R 2A1 independently represents -CH=CH-(C 1-15 alkyl), -CH=CH-CH2-CH=CH-(C 1-10 alkyl), -(CH2) 0-4 C(O)OCH(C 1-10 alkyl)(C 1-15 alkyl) or -(CH2) 0-4 OC(O)CH(C 1-10 alkyl)(C 1-15 alkyl); and each of R 1A2 , R 1A3 , R 2A2 and R 2A3 represents H.

18. The compound according to claim 17 or a salt thereof, wherein each of R 1A1 and R 2A1 represents , , , , , or .

19. The compound according to claim 16 or a salt thereof, wherein each of R 1A1 and R 2A1 represents C 1-15 alkyl; each of R 1A2 and R 2A2 represents C 1-15 alkyl; and each of R 1A3 and R 2A3 represents H.

20. The compound according to claim 19 or a salt thereof, wherein each of R 1A1 and R 2A1 represents ; and each of R 1A2 and R 2A2 represents .

21. The compound according to claim 16 or a salt thereof, wherein each of R 1A1 and R 2A1 is -(CH2) 0-4 OC(O)CH2(C 1-15 alkyl); each of R 2A1 and R 2A2 is -(CH2) 0-4 C(O)OCH2(C 1-15 alkyl); and each of R 1A3 and R 2A3 represents H.

22. The compound according to claim 21 or a salt thereof, where each of R 1A1 and R 2A1 represents ; and each of R 2A1 and R 2A2 represents .

23. The compound according to claim 16 or a salt thereof, wherein each of R 1A1 and R 2A1 is -C(O)OCH2(C 1-15 alkyl); each of R 1A2 and R 2A2 is -(CH2) 0-4 C(O)OCH2(C 1-15 alkyl); and each of R1A3 and R 2A3 represents H.

24. The compound according to item 23 or a salt thereof, where each of R 1A1 and R 2A1 represents ; and each of R 1A2 and R 2A2 represents ; or where each of R 1A1 and R 2A1 represents ; and each of R 2A1 and R 2A2 represents .

25. A compound according to any one of paragraphs 1-24 or a salt thereof, wherein each of R 3A1 , R 3A2 and R 3A3 independently represents H, C 1-15 alkyl, -(CH2) 0-4 C(O)OCH(C 1-5 alkyl)(C 1-10 alkyl), -(CH2) 0-4 OC(O)CH(C 1-5 alkyl)(C 1-10 alkyl), -(CH2) 0-4 C(O)OCH2(C 1-10 alkyl) or -(CH2) 0-4 OC(O)CH2(C 1-10 alkyl).

26. The compound according to claim 25 or a salt thereof, wherein each of R 3A1 and R 3A2 independently represents C 1-15 alkyl; and R3A3 represents H.

27. The compound according to claim 26 or a salt thereof, wherein each of R 3A1 and R 3A2 independently represents ethyl, , , or .

28. The compound according to item 25 or a salt thereof, where R 3A1 represents C 1-15 alkyl; and each of R 3A2 and R 3A3 represents H.

29. The compound according to item 28 or a salt thereof, where R 3A1 represents .

30. The compound according to item 25 or its salt, where R 3A1 is -C(O)OCH(C 1-5 alkyl)(C 1-10 alkyl); and each of R 3A2 and R 3A3 represents H.

31. The compound according to claim 30 or a salt thereof, where R 3A1 represents or .

32. The compound according to item 25 or its salt, where R 3A1 is -(CH2) 0-4 OC(O)CH2(C 1-10 alkyl); R 3A2 is -(CH2) 0-4(O)OCH2(C 1-10 alkyl); and R 3A3 represents H.

33. The compound according to claim 32 or a salt thereof, where R 3A1 represents or ; and R 3A2 represents .

34. The compound according to item 25 or a salt thereof, where R 3A1 is -(CH2) 0-4 C(O)OCH2(C 1-10 alkyl); R 3A2 is -(CH2) 0-4 C(O)OCH2(C 1-10 alkyl); and R 3A3 represents H.

35. The compound according to claim 34 or a salt thereof, where R 3A1 represents ; and R 3A2 represents or .

36. The compound according to claim 25 or a salt thereof, wherein each of R 3A1 , R 3A2 and R 3A3 represents H.

37. A compound according to any one of paragraphs 1-15 or a salt thereof, wherein each of R a1 and R a2 independently represents -(CH2) 0-15 CH3or -CH(C 1-10 alkyl)(C 1-15 alkyl).

38. The compound according to claim 37 or a salt thereof, wherein each of R a1 and R a2 independently represents , , , , , , or .

39. The compound according to claim 1 or a salt thereof, wherein the compound is selected from Table 1.

40. The compound according to claim 1 or a salt thereof, wherein the compound is .

41. The compound according to claim 1 or a salt thereof, wherein the compound is .

42. The compound according to claim 1 or a salt thereof, wherein the compound is .

43. A lipid nanoparticle (LNP) comprising a mixture of lipids for targeted delivery of a nucleic acid to an immune cell, wherein the lipid mixture comprises (a) a lipid-immune cell targeting moiety conjugate comprising a compound of formula II: [lipid]-[optional linker]-[immune cell targeting moiety], and (b) an ionizable cationic lipid comprising any of the compounds of paragraphs 1-42, wherein the LNP further comprises a nucleic acid disposed therein.

44. The LNP of claim 43, wherein the immune cell targeting group comprises an antibody that binds a T cell antigen.

45. The LNP of claim 44, wherein the T cell antigen is CD3, CD4, CD7, CD8, or a combination thereof (e.g., both CD3 and CD8, both CD4 and CD8, or both CD7 and CD8).

46. The LNP of any one of claims 43-45, wherein the immune cell targeting moiety comprises an antibody that binds a natural killer (NK) cell antigen.

47. The LNP of claim 46, wherein the NK cell antigen is CD7, CD8, CD56, or a combination thereof (e.g., both CD7 and CD8).

48. The LNP of any one of claims 43-47, wherein the immune cell targeting moiety is covalently linked to a lipid in a lipid mixture via a linker comprising polyethylene glycol (PEG).

49. The LNP of claim 48, wherein the lipid covalently linked to the immune cell targeting moiety via a PEG-containing linker is distearoylglycerol (DSG), distearoylphosphatidylethanolamine (DSPE), dimyristoylphosphatidylethanolamine (DMPE), distearoylglycerophosphoglycerol (DSPG), dimyristoylglycerol (DMG), dipalmitoylphosphatidylethanolamine (DPPE), dipalmitoylglycerol (DPG), or ceramide.

50. LNP according to claim 48 or 49, wherein PEG is PEG 2000 or PEG 3400.

51. The LNP of any one of claims 43-50, wherein the conjugate of a lipid and an immune cell targeting moiety is present in the lipid mixture in the range of 0.001 to 0.5 mole percent (e.g. 0.002-0.2 mole percent).

52. The LNP of any one of claims 43-51, wherein the lipid mixture further comprises one or more structural lipids (e.g., sterol), a neutral phospholipid, and a free PEG lipid.

53. The LNP of any one of claims 43-52, wherein the ionizable cationic lipid is present in the lipid mixture in the range of 30-70 (e.g. 40-60) mole percent.

54. The LNP of claim 52, wherein the sterol is present in the lipid mixture in the range of 20-70 (e.g., 30-50) mole percent.

55. The LNP of claim 52 or 54, wherein the sterol is cholesterol.

56. The LNP of any one of claims 52-55, wherein the neutral phospholipid is selected from the group consisting of phosphatidylcholine, phosphatidylethanolamine, distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and sphingomyelin.

57. The LNP of any one of claims 52-56, wherein the neutral phospholipid is present in the lipid mixture in the range of 5-15 mole percent.

58. The LNP of any one of claims 52-57, wherein the free PEG lipid is selected from the group consisting of PEG-modified phosphatidylethanolamines, PEG-modified phosphatidic acids, PEG-modified ceramides, PEG-modified dialkylamines, PEG-modified diacylglycerols, and PEG-modified dialkylglycerols. For example, the PEG lipid can be a lipid PEG-dioleoylglycerol (PEG-DOG), PEG-dimyristoylglycerol (PEG-DMG), PEG-dipalmitoylglycerol (PEG-DPG), PEG-dilinoleoylglycerophosphatidylethanolamine (PEG-DLPE), PEG-dimyristoylphosphatidylethanolamine (PEG-DMPE), PEG-dipalmitoylphosphatidylethanolamine (PEG-DPPE), PEG-distearoylglycerol (PEG-DSG), PEG-diacylglycerol (PEG-DAG, such as PEG-DMG, PEG-DPG, and PEG-DSG), PEG-ceramide, PEG-distearoylglycerophosphoglycerol (PEG-DSPG), PEG-dioleoylglycerophosphoethanolamine (PEG-DOPE), 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide or PEG-distearoylphosphatidylethanolamine (PEG-DSPE).

59. The LNP of any one of claims 52-57, wherein the free PEG lipid comprises a diacylphosphatidylethanolamine comprising a dipalmitoyl (C16) chain or a distearoyl (C18) chain, and optionally the free PEG lipid comprises PEG-DPG and PEG-DMG.

60. The LNP of any one of claims 52-59, wherein the free PEG lipid is present in the lipid mixture in the range of 1-4 mole percent.

61. The LNP of any one of claims 52-60, wherein the free PEG lipid comprises the same or a different lipid as the lipid in the conjugate of the lipid and the immune cell targeting moiety.

62. The LNP of any one of claims 43-61, wherein the LNP is characterized by an average diameter in the range of 50-200 nm.

63. The LNP of claim 62, wherein the LNP has an average diameter of approximately 100 nm.

64. LNP according to any one of claims 43-63, wherein the LNP is characterized by a polydispersity index in the range from 0.05 to 1.

65. The LNP of any one of claims 43-64, wherein the LNP has a zeta potential of from about +10 mV to about +30 mV at pH 5.

66. The LNP of any one of claims 43-65, wherein the nucleic acid is DNA or RNA.

67. The LNP of claim 66, wherein the RNA is mRNA.

68. The LNP of claim 67, wherein the mRNA encodes a receptor, growth factor, hormone, cytokine, antibody, antigen, enzyme, or vaccine.

69. The LNP of claim 67, wherein the mRNA encodes a polypeptide capable of regulating an immune response in an immune cell.

70. LNP according to claim 67, wherein the mRNA encodes a polypeptide capable of reprogramming an immune cell.

71. The LNP of claim 69, wherein the mRNA encodes a synthetic T-cell receptor (synTCR) or a chimeric antigen receptor (CAR).

72. The LNP of any one of claims 43-71, wherein the immune cell targeting moiety comprises an antibody, and the antibody is a Fab or a single variable domain of an immunoglobulin (e.g. a nanobody).

73. The LNP of any one of claims 43-71, wherein the immune cell targeting moiety comprises a Fab, F(ab')2, Fab'-SH, Fv or scFv fragment.

74. The LNP of claim 72 or 73, wherein the immune cell targeting moiety comprises a Fab that is engineered to knock out a natural interchain disulfide bond at the C-terminus.

75. LNP according to claim 74, wherein the Fab comprises a heavy chain fragment that contains the C233S substitution and a light chain fragment that contains the C214S substitution, numbering according to Kabat.

76. The LNP of any one of claims 73-75, wherein the immune cell targeting moiety comprises a Fab that comprises a non-natural interchain disulfide bond (e.g., an engineered internal interchain disulfide bond).

77. LNP according to claim 76, wherein the Fab comprises a F174C substitution in the heavy chain fragment and a S176C substitution in the light chain fragment, numbering according to Kabat.

78. The LNP of claims 73-77, wherein the immune cell targeting moiety comprises a Fab that comprises a cysteine at the C-terminus of a heavy or light chain fragment.

79. The LNP of claim 78, wherein the Fab further comprises one or more amino acids between the Fab heavy chain fragment and the C-terminal cysteine.

80. The LNP of claim 72, wherein the immune cell targeting moiety comprises a single variable domain of an immunoglobulin.

81. The LNP of claim 72 or 80, wherein the single variable domain of the immunoglobulin comprises a cysteine at the C-terminus.

82. The LNP of claim 81, wherein the single variable domain of the immunoglobulin comprises a VHH domain and further comprises a spacer comprising one or more amino acids between the VHH domain and the C-terminal cysteine.

83. The LNP of any one of claims 73 and 80-82, wherein the immune cell targeting moiety comprises two or more V domains HH .

84. LNP according to claim 83, wherein two or more V domains HH linked via an amino acid linker.

85. The LNP of claim 83, wherein the immune cell targeting moiety comprises a first V domain HH , associated with the CH1 domain of the antibody, and the second V domain HH , linked to a light chain constant domain of an antibody, and wherein the CH1 domain of the antibody and the light chain constant domain of the antibody are linked via one or more disulfide bonds.

86. The LNP of any one of claims 72 and 80-82, wherein the immune cell targeting moiety comprises a V domain HH , linked to the CH1 domain of an antibody, and wherein the CH1 domain of the antibody is linked to the light chain constant domain of the antibody via one or more disulfide bonds.

87. LNP according to claim 85 or 86, wherein the CH1 domain comprises the substitutions F174C and C233S, and the light chain constant domain comprises the substitutions S176C and C214S, numbering according to Kabat.

88. The LNP of any one of claims 43-69, wherein the immune cell targeting moiety comprises a Fab that comprises (a) a heavy chain fragment comprising the amino acid sequence of SEQ ID NO: 1 and a light chain fragment comprising the amino acid sequence of SEQ ID NO: 2 or 3, or (b) a heavy chain fragment comprising the amino acid sequence of SEQ ID NO: 6 and a light chain fragment comprising the amino acid sequence of SEQ ID NO:

7.

89. The LNP of any one of claims 43-88, wherein the ionizable cationic lipid is .

90. The LNP of any one of claims 43-88, wherein the ionizable cationic lipid is .

91. The LNP of any one of claims 43-88, wherein the ionizable cationic lipid is .

92. The LNP of any one of paragraphs 43-91, wherein the LNP comprises (a) ionizable cationic lipid, (b) a conjugate comprising a compound of the following formula: [lipid] - [optional linker] - [immune cell targeting group]; (c) a sterol or other structural lipid; (d) neutral phospholipid, (e) free lipid polyethylene glycol (PEG) and (f) nucleic acid.

93. The LNP of any one of claims 43-92, wherein the LNP is for delivering a nucleic acid to an immune cell, and wherein the immune cell is an NK cell, and the immune cell targeting moiety comprises an antibody that binds CD56.

94. The LNP of any one of claims 43-92, wherein the LNP is for delivering a nucleic acid to an immune cell, and wherein the immune cell targeting moiety comprises an antibody that binds CD7 or CD8, and the free PEG lipid is DMG-PEG or PEG-DPG.

95. The LNP of any one of claims 43-92, wherein the immune cell targeting moiety comprises an antibody, and the antibody is a Fab or a single variable domain immunoglobulin.

96. The LNP of claim 95, wherein the Fab is engineered to knock out the natural interchain disulfide at the C-terminus.

97. LNP according to claim 96, wherein the Fab comprises a heavy chain fragment that contains C233S substitutions and a light chain fragment that contains C214S substitutions.

98. The LNP of claim 96, wherein the Fab comprises a non-natural interchain disulfide.

99. LNP according to claim 96, wherein the Fab comprises a F174C substitution in the heavy chain fragment and a S176C substitution in the light chain fragment.

100. The LNP of claim 95, wherein the antibody is an immunoglobulin single variable domain (ISV), and the ISV domain is an ISV Nanobody®.

101. The LNP of claim 100, wherein the free PEG lipid comprises PEG with a molecular weight of at least 2000 daltons.

102. The LNP of claim 101, wherein the PEG has a molecular weight of from about 3,000 to 5,000 daltons.

103. The LNP of claim 95, wherein the antibody is a Fab.

104. The LNP of claim 103, wherein the Fab binds CD3 and the free PEG lipid in the LNP comprises PEG with a molecular weight of approximately 2000 daltons.

105. The LNP of claim 103, wherein the Fab is an anti-CD4 antibody and the free PEG lipid in the LNP comprises PEG with a molecular weight of from about 3000 to 3500 daltons.

106. The LNP of claim 95, wherein the immune cell targeting moiety comprises two or more V domains HH .

107. LNP according to claim 106, wherein two or more V domains HH linked via an amino acid linker.

108. The LNP of claim 107, wherein the immune cell targeting moiety comprises a first V domain HH , associated with the CH1 domain of the antibody, and the second V domain HH , linked to the constant domain of the light chain of an antibody.

109. The LNP of any one of claims 43-92, wherein the LNP is for delivering a nucleic acid to an immune cell, and wherein the LNP binds CD3 and further binds CD11a or CD18.

110. The LNP of claim 109, wherein the LNP comprises two conjugates, wherein the first conjugate comprises an antibody that binds CD3 and the second conjugate comprises an antibody that binds CD11a or CD18.

111. The LNP of claim 109, wherein the LNP comprises one conjugate, and the conjugate comprises a bispecific antibody that binds both CD3 and CD11a.

112. The LNP of claim 109, wherein the LNP comprises one conjugate, and the conjugate comprises a bispecific antibody that binds both CD3 and CD18.

113. LNP according to claim 111 or 112, wherein the bispecific antibody is a single variable domain of an immunoglobulin or Fab-ScFv.

114. The LNP of any one of claims 43-92, wherein the LNP is for delivering a nucleic acid to an immune cell, and wherein the LNP binds CD7 and CD8 of the immune cell.

115. The LNP of claim 114, wherein the LNP comprises two conjugates, wherein the first conjugate comprises an antibody that binds CD7 and the second conjugate that binds CD8.

116. The LNP of claim 114, wherein the LNP comprises one conjugate, wherein the conjugate comprises a bispecific antibody that binds CD7 and CD8.

117. The LNP of claim 116, wherein the bispecific antibody is a single variable domain immunoglobulin or Fab-ScFv.

118. The LNP of any one of claims 43-92, wherein the LNP binds to a first antigen on the surface of a first type of immune cell and further binds to a second antigen on the surface of a second type of immune cell.

119. The LNP of claim 118, wherein the two different types of immune cells are CD4+ T cells and a CD8+ T cell.

120. The LNP of claim 118, wherein the LNP comprises two conjugates, and the first conjugate comprises a first antibody that binds to a first antigen of a first type of immune cell, and the second conjugate comprises a second antibody that binds to a second antigen of a second type of immune cell.

121. The LNP of claim 118, wherein the LNP comprises one conjugate, and the conjugate comprises a bispecific antibody, and the bispecific antibody binds to both a first antigen on a first type immune cell and a second antigen on a second type immune cell.

122. The LNP of any one of claims 43-92, wherein the bispecific antibody is a single variable domain immunoglobulin or Fab-ScFv.

123. The LNP of any one of claims 43-92, wherein the LNP is for delivering a nucleic acid to an immune cell, and wherein the immune cell targeting moiety comprises a single antibody that binds to CD3 or CD7.

124. The LNP of any one of claims 43-92, wherein the LNP is for delivering a nucleic acid to an immune cell, and wherein the immune cell targeting moiety binds to CD7, CD8, or both CD7 and CD8.

125. The LNP of any one of claims 43-92, wherein the LNP is designed to deliver nucleic acid to both T cells and NK cells, wherein the immune cell targeting moiety binds (a) with both CD3 and CD56; (b) with both CD8 and CD56 or (c) with both CD7 and CD56.

126. The LNP of any one of claims 93-125, wherein the LNP is characterized by an average diameter in the range of 50-200 nm.

127. The LNP of claim 126, wherein the LNP is characterized by an average diameter of approximately 100 nm.

128. The LNP of any one of paragraphs 93-125, wherein the LNP is characterized by a polydispersity index in the range from 0.05 to 1.

129. The LNP of any one of claims 93-128, wherein the LNP has a zeta potential of from about +10 mV to about +30 mV at pH 5.

130. The LNP of any one of claims 93-129, wherein the nucleic acid is DNA or RNA.

131. The LNP of claim 130, wherein the RNA is mRNA.

132. The LNP of claim 131, wherein the mRNA encodes a receptor, growth factor, hormone, cytokine, antibody, antigen, enzyme, or vaccine.

133. The LNP of claim 131, wherein the mRNA encodes a polypeptide capable of regulating an immune response in an immune cell.

134. The LNP of claim 133, wherein the mRNA encodes a polypeptide capable of reprogramming an immune cell.

135. The LNP of claim 134, wherein the mRNA encodes a synthetic T-cell receptor (synTCR) or a chimeric antigen receptor (CAR).

136. The LNP of any one of claims 43-92, wherein the LNP is for delivering a nucleic acid to an immune cell, and wherein the immune cell targeting moiety comprises a Fab without a native interchain disulfide bond.

137. The LNP of claim 136, wherein the Fab is engineered to replace one or both cysteines in the native light chain constant region and the native heavy chain constant region that form a native interchain disulfide with an amino acid other than cysteine, thereby removing the native interchain disulfide bond in the Fab.

138. A method for targeted delivery of a nucleic acid to an immune cell of a subject, comprising contacting the immune cell with an LNP according to any one of claims 43-137, wherein the LNP comprises a nucleic acid.

139. A method for providing expression of a polypeptide of interest in a target immune cell of a subject, comprising contacting the immune cell with an LNP according to any one of claims 43-137, wherein the LNP comprises a nucleic acid encoding the polypeptide.

140. A method for modulating the cellular function of a target immune cell of a subject, comprising administering to the subject an LNP according to any one of claims 43-137, wherein the LNP comprises a nucleic acid that provides modulation of the cellular function of the immune cell.

141. A method for treating, ameliorating, or preventing a symptom of a disorder or disease in a subject in need thereof, comprising administering to the subject an LNP for delivering a nucleic acid to an immune cell of the subject, wherein the LNP is the LNP of any one of claims 43-137, wherein the LNP comprises a nucleic acid.

142. The method of claim 141, wherein the disorder is an immune disorder, an inflammatory disorder, or cancer.

143. The method of claim 141, wherein the nucleic acid encodes an antigen for use in a therapeutic or prophylactic vaccine for the treatment or prevention of infection caused by a pathogen.

144. The method according to any one of claims 138-143, wherein the ionizable cationic lipid is .

145. The method according to any one of claims 138-143, wherein the ionizable cationic lipid is .

146. The method according to any one of claims 138-143, wherein the ionizable cationic lipid is .

147. The method according to any one of claims 138-146, wherein the immune cell targeting group comprises an antibody that binds a T-cell antigen.

148. The method of claim 147, wherein the T cell antigen is CD3, CD8, or both CD3 and CD8.

149. The method according to any one of claims 138-146, wherein the immune cell targeting moiety comprises an antibody that binds a natural killer (NK) cell antigen.

150. The method according to claim 149, wherein the NK cell antigen is CD7, CD8 or CD56.

151. The method according to any one of claims 138-150, wherein the antibody is a human or humanized antibody.

152. The method according to any one of claims 138-151, wherein the immune cell targeting group is covalently linked to a lipid in a lipid mixture via a linker comprising polyethylene glycol (PEG).

153. The method of claim 152, wherein the lipid covalently linked to the immune cell targeting group via a linker comprising PEG is distearoylglycerol (DSG), distearoylphosphatidylethanolamine (DSPE), dimyristoylphosphatidylethanolamine (DMPE), distearoylglycerophosphoglycerol (DSPG), dimyristoylglycerol (DMG), dipalmitoylphosphatidylethanolamine (DPPE), dipalmitoylglycerol (DPG), or ceramide.

154. The method according to claim 152 or 153, wherein the PEG is PEG 2000.

155. The method according to any one of claims 138-154, wherein the conjugate of a lipid and an immune cell targeting group is present in the lipid mixture in the range of 0.002-0.2 mole percent.

156. The method according to any one of claims 138-155, wherein the ionizable cationic lipid is present in the lipid mixture in the range of 40-60 mole percent.

157. The method according to paragraphs 138-156, wherein the sterol is cholesterol.

158. The method according to any one of claims 49-157, wherein the sterol is present in the lipid mixture in the range of 30-50 mole percent.

159. The method according to claims 138-158, wherein the neutral phospholipid is selected from the group consisting of phosphatidylcholine, phosphatidylethanolamine, distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), sphingomyelin (SM).

160. The method of claims 138-159, wherein the neutral phospholipid is present in the lipid mixture in the range of 5-15 mole percent.

161. The method according to any one of claims 138-160, wherein the free PEG lipid is selected from the group consisting of PEG-modified phosphatidylethanolamines, PEG-modified phosphatidic acids, PEG-modified ceramides, PEG-modified dialkylamines, PEG-modified diacylglycerols, and PEG-modified dialkylglycerols. For example, the PEG lipid can be a lipid PEG-dioleoylglycerol (PEG-DOG), PEG-dimyristoylglycerol (PEG-DMG), PEG-dipalmitoylglycerol (PEG-DPG), PEG-dilinoleoylglycerophosphatidylethanolamine (PEG-DLPE), PEG-dimyristoylphosphatidylethanolamine (PEG-DMPE), PEG-dipalmitoylphosphatidylethanolamine (PEG-DPPE), PEG-distearoylglycerol (PEG-DSG), PEG-diacylglycerol (PEG-DAG, such as PEG-DMG, PEG-DPG, and PEG-DSG), PEG-ceramide, PEG-distearoylglycerophosphoglycerol (PEG-DSPG), PEG-dioleoylglycerophosphoethanolamine (PEG-DOPE), 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide or PEG-distearoylphosphatidylethanolamine (PEG-DSPE).

162. The method according to claims 138-160, wherein the free PEG lipid comprises diacylphosphatidylethanolamines containing a dimyristoyl (C14) chain, a dipalmitoyl (C16) chain, or a distearoyl (C18) chain.

163. The method of any one of claims 138-162, wherein the free PEG lipid is present in the lipid mixture in the range of 0.5-2.5 mole percent.

164. The method of any one of claims 138-163, wherein the free PEG lipid comprises the same or a different lipid as the lipid in the conjugate of the lipid and the immune cell targeting moiety.

165. The method of claims 138-164, wherein the LNP is characterized by an average diameter in the range of 50-200 nm.

166. The method of claim 165, wherein the LNP has an average diameter of approximately 100 nm.

167. The method according to paragraphs 138-166, wherein the LNP is characterized by a polydispersity index in the range from 0.05 to 1.

168. The method of claims 138-167, wherein the LNP has a zeta potential of from about +10 mV to about +30 mV at pH 5.

169. The method according to paragraphs 138-168, wherein the nucleic acid is DNA or RNA.

170. The method according to claim 169, wherein the RNA is mRNA, tRNA, siRNA, gNRA or microRNA.

171. The method according to claim 170, wherein the mRNA encodes a receptor, growth factor, hormone, cytokine, antibody, antigen, enzyme or vaccine.

172. The method according to claim 170, wherein the mRNA encodes a polypeptide capable of regulating an immune response in an immune cell.

173. The method according to claim 170, wherein the mRNA encodes a polypeptide capable of reprogramming an immune cell.

174. The method according to claim 170, wherein the mRNA encodes a synthetic T-cell receptor (synTCR) or a chimeric antigen receptor (CAR).

175. The method according to any one of claims 138-174, wherein the immune cell targeting moiety comprises an antibody, and the antibody is a Fab or a single variable domain of an immunoglobulin.

176. The method according to any one of claims 138-174, wherein the immune cell targeting moiety comprises an antibody fragment selected from the group consisting of a Fab fragment, F(ab')2, Fab'-SH, Fv and scFv.

177. The method of claim 175 or 176, wherein the immune cell targeting group comprises a Fab that contains one or more interchain disulfide bonds.

178. The method according to claim 177, wherein the Fab comprises a heavy chain fragment that contains the substitutions F174C and C233S, and a light chain fragment that contains the substitutions S176C and C214S, numbering according to Kabat.

179. The method according to any one of claims 175-178, wherein the immune cell targeting moiety comprises a Fab that comprises a cysteine at the C-terminus of a heavy or light chain fragment.

180. The method according to claim 175, wherein the Fab further comprises one or more amino acids between the Fab heavy chain fragment and the C-terminal cysteine.

181. The method according to any one of claims 176-180, wherein the Fab comprises a heavy chain variable domain linked to a CH1 domain of an antibody and a light chain variable domain linked to a light chain constant domain of an antibody, wherein the CH1 domain and the light chain constant domain are linked via one or more interchain disulfide bonds, and wherein the immune cell targeting moiety further comprises a single-chain variable fragment (scFv) linked to the C-terminus of the light chain constant domain via an amino acid linker.

182. The method of claim 175, wherein the immune cell targeting moiety comprises a single variable domain of an immunoglobulin.

183. The method according to claim 175 or 182, wherein the single variable domain of the immunoglobulin comprises a cysteine at the C-terminus.

184. The method of claim 183, wherein the single variable domain of the immunoglobulin comprises a V domain HH and further comprises a spacer comprising one or more amino acids between the V domain HH and C-terminal cysteine.

185. The method according to any one of paragraph 175 and paragraphs 182-184, wherein the group targeting the immune cell comprises two or more V domains HH .

186. The method according to claim 185, wherein two or more V domains HH linked via an amino acid linker.

187. The method of claim 185, wherein the immune cell targeting group comprises a first V domain HH , associated with the CH1 domain of the antibody, and the second V domain HH , linked to a light chain constant domain of an antibody, and wherein the CH1 domain of the antibody and the light chain constant domain of the antibody are linked via one or more disulfide bonds.

188. The method according to any one of paragraph 175 and paragraphs 182-184, wherein the group targeting the immune cell comprises domain VHH , linked to the CH1 domain of an antibody, and wherein the CH1 domain of the antibody is linked to the light chain constant domain of the antibody via one or more disulfide bonds.

189. The method according to claim 185 or 186, wherein the CH1 domain comprises the substitutions F174C and C233S, and the light chain constant domain comprises the substitutions S176C and C214S, numbering according to Kabat.

190. The method according to any one of claims 138-174, wherein the immune cell targeting group comprises a Fab that comprises (a) a heavy chain fragment comprising the amino acid sequence of SEQ ID NO: 1 and a light chain fragment comprising the amino acid sequence of SEQ ID NO: 2 or 3; (b) a heavy chain fragment comprising the amino acid sequence of SEQ ID NO: 6 and a light chain fragment comprising the amino acid sequence of SEQ ID NO:

7.

191. The method according to any one of claims 138-190, wherein no more than 5% of the non-immune cells are transfected with LNP.

192. The method according to any one of claims 138-191, wherein the half-life of the nucleic acid delivered by the LNP or the polypeptide encoded by the nucleic acid delivered by the LNP is at least 10% longer than the half-life of the nucleic acid delivered by the reference LNP or the polypeptide encoded by the nucleic acid delivered by the reference LNP.

193. The method according to any one of claims 138-192, wherein at least 10% of the immune cells are transfected with LNP.

194. The method according to any one of claims 138-193, wherein the expression level of the nucleic acid delivered by the LNP is at least 10% higher than the expression level of the nucleic acid delivered by the reference LNP.