Ionizable lipids and lipid carriers containing cleavable linkers for therapeutic compositions - Patent Application 20070122999

Abstract

The present disclosure relates to lipid compounds of formula (Ia) or (AL-GI), having various cleavable linkers defined by the variables Z1 and Z2. The disclosure also relates to lipid carriers or lipid nanoformulations utilizing the lipid compounds, the use of the lipid compounds in pharmaceutical compositions, and methods of delivering therapeutic agents. TIFF2026506611000380.tif33170

Description

[Technical Field]

[0001] Related Applications This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 445,266, filed February 13, 2023, the contents of which are incorporated herein by reference.

[0002] The present disclosure relates generally to novel lipids, lipid-based carriers, pharmaceutical compositions, and methods. [Background technology]

[0003] There is a continuing need in the art for improved lipid vehicles that utilize novel lipids to deliver therapeutic agents such as nucleic acid molecules, proteins, and small molecule drugs. Summary of the Invention [Means for solving the problem]

[0004] In some embodiments, the present disclosure provides a compound of formula (Ia): [ka] or a salt thereof, During the ceremony, R N2 is -(CH2) m (NH) n Q 2 and; Q 2 is —OH, —SONH(alkyl), —SON(alkyl), optionally substituted cycloalkyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted heterocycloalkyl, or optionally substituted heterocycloalkenyl; m is an integer of 2 to 3; n is an integer from 0 to 1; L 1 and L 2 are each independently (C1 to C15 ) alkylene; Z 10 and Z 20 are each independently [ka] and X 1 and X 2 each independently represents O, S, or N(R 21 ) and; R 20 is branched (C1-C 15 ) alkyl or unbranched (C1-C 15 ) alkyl; R 21 is H, (C1-C5) alkyl, or (C3-C8) cycloalkyl; s is an integer from 1 to 4; Z 10 In [ka] L 1 indicates the point of attachment to; Z 20 In [ka] L 2 indicates the point of attachment to; R 22 , R 23 , R 24 , and R 25 Each of the is independently H, branched (C1 to C 15 ) alkyl, or unbranched (C1-C 15 ) alkyl; provided that R 22 and R 23 At least one of them is not H and R 24 and R 25 At least one of them is not H; Here, Q 2 is -OH, the following (i) or (ii): (i)Z 10 and Z20 At least one of [ka] or (ii)L 1 and L 2 are not the same At least one of the following applies: The present invention relates to a compound or a salt thereof.

[0005] In some embodiments, the present disclosure provides a compound of formula (AL-GI): [ka] or a salt thereof, During the ceremony, R N is a substituted or unsubstituted C1-C6 alkyl or C3-C8 cycloalkyl; R1, R 1’ , R2, R 2’ , R3, R 3’ , R4, and R 4’ each independently at each occurrence is H, branched C1-C3 alkyl, unbranched C1-C3 alkyl, branched C2-C3 alkenyl, or unbranched C2-C3 alkenyl; R 10 , R 11 , R 12 , and R 13 each independently represents H or a substituted or unsubstituted branched C1-C 15 Alkyl or C1-C 15 alkyl; provided that R 10 and R 11 At least one of them is not H and R 12 and R 13 At least one of them is not H; Each of Z1 and Z2 independently represents [ka] and; X 1 and X2 each independently represents O, S, or N(R 21 ) and; R 20 is a substituted or unsubstituted branched C1-C 15 Alkyl or unbranched C1-C 15 is alkyl; R 21 is H, substituted or unsubstituted C1-C5 alkyl, or substituted or unsubstituted C3-C8 cycloalkyl; s is an integer from 1 to 4; n1, n2, n3, and n4 are each independently an integer of 0 to 15, where n1+n2 is in the range of 1 to 15, and n3+n4 is in the range of 1 to 15; where R N is a C1-C6 alkyl substituted with hydroxy, the following (i) or (ii): (i) At least one of Z1 and Z2 is [ka] or (ii) The sum of n1 and n2 is not equal to the sum of n3 and n4 At least one of the following applies: The present invention relates to a compound or a salt thereof.

[0006] In some embodiments, the present disclosure provides a compound of formula (Iw): [ka] or a salt thereof, During the ceremony, R N2 teeth, [ka] and; L 1 and L 2 are each independently (C1 to C 15 ) alkylene; Z10 and Z 20 are each independently [ka] and; X 1 and X 2 each independently represents O, S, or N(R 21 ) and; R 20 is branched (C1-C 15 ) alkyl or unbranched (C1-C 15 ) alkyl; R 21 is H, (C1-C5) alkyl, or (C3-C8) cycloalkyl; s is an integer from 1 to 4; Z 10 In [ka] L 1 indicates the point of attachment to; Z 20 In [ka] L 2 indicates the point of attachment to; R 22 , R 23 , R 24 , and R 25 Each of the is independently H, branched (C1 to C 15 ) alkyl, or unbranched (C1-C 15 ) alkyl; provided that R 22 and R 23 At least one of them is not H and R 24 and R 25 At least one of them is not H, The present invention relates to a compound or a salt thereof.

[0007] In some embodiments, the present disclosure provides compounds of formula (Iw-4), (Iw-5), or (Iw-6): [ka] or a salt thereof, During the ceremony, R N2 is -(CH2) m (NH) n Q 2 and; Q 2 is —OH, —SONH(alkyl), —SON(alkyl), optionally substituted cycloalkyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted heterocycloalkyl, or optionally substituted heterocycloalkenyl; m is an integer of 2 to 3; n is an integer from 0 to 1; L 1 and L 2 are each independently (C1 to C 15 ) alkylene; X 1 and X 2 each independently represents O, S, or N(R 21 ) and; R 21 is H, (C1-C5) alkyl, or (C3-C8) cycloalkyl; R 22 , R 23 , R 24 , and R 25 Each of the is independently H, branched (C1 to C 15 ) alkyl, or unbranched (C1-C 15 ) alkyl; provided that R 22 and R 23 At least one of them is not H and R 24 and R 25 At least one of them is not H, The present invention relates to a compound or a salt thereof.

[0008] In some embodiments, the present disclosure provides compounds of formula (Iw-7), (Iw-8), or (Iw-9): [ka] or a salt thereof, During the ceremony, R N2 is -(CH2) m (NH) n Q 2 and; Q 2 is —OH, —SONH(alkyl), —SON(alkyl), optionally substituted cycloalkyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted heterocycloalkyl, or optionally substituted heterocycloalkenyl; m is an integer of 2 to 3; n is an integer from 0 to 1; L 1 and L 2 are each independently (C1 to C 15 ) alkylene; s is an integer from 1 to 4; R 22 , R 23 , R 24 , and R 25 Each of the is independently H, branched (C1 to C 15 ) alkyl, or unbranched (C1-C 15 ) alkyl; provided that R 22 and R 23 At least one of them is not H and R 24 and R 25 At least one of them is not H, The present invention relates to a compound or a salt thereof.

[0009] In certain embodiments, the present disclosure provides compounds of formula (Iw-10), (Iw-11), (Iw-12), (Iw-13), (Iw-14), or (Iw-15): [ka] or a salt thereof, During the ceremony, RN2 is -(CH2) m (NH) n Q 2 and; Q 2 is —OH, —SONH(alkyl), —SON(alkyl), optionally substituted cycloalkyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted heterocycloalkyl, or optionally substituted heterocycloalkenyl; m is an integer of 2 to 3; n is an integer from 0 to 1; L 1 and L 2 are each independently (C1 to C 15 ) alkylene; R 20 is branched (C1-C 15 ) alkyl or unbranched (C1-C 15 ) alkyl; R 21 is H, (C1-C5) alkyl, or (C3-C8) cycloalkyl; R 22 , R 23 , R 24 , and R 25 Each of the is independently H, branched (C1 to C 15 ) alkyl, or unbranched (C1-C 15 ) alkyl; provided that R 22 and R 23 At least one of them is not H and R 24 and R 25 At least one of them is not H, A compound or a salt thereof is provided.

[0010] In some embodiments, the present disclosure provides a compound having the following structure: [ka] [ka] [ka] [ka] [ka] [ka] or a salt thereof, In the formula, R= [ka] and each R 27 are independently H, C1 to C 15 alkyl, C2-C8 alkenyl, or C2-C8 alkynyl; A compound or a salt thereof is provided.

[0011] In some embodiments, the present disclosure relates to a lipid-based carrier comprising a compound of the present disclosure (e.g., a compound of Formula (Ia) or (AL-GI)), wherein the lipid-based carrier is a lipid nanoparticle.

[0012] In some embodiments, the present disclosure relates to a method of delivering an effector (e.g., a therapeutic agent) to a subject, comprising administering to the subject a lipid-based carrier of the present disclosure, wherein the lipid-based carrier comprises the effector.

[0013] In some embodiments, the present disclosure relates to a pharmaceutical composition comprising a lipid-based carrier of the present disclosure and a pharmaceutically acceptable excipient. [Brief explanation of the drawings]

[0014] [Figure 1] 1 is a bar graph showing in vivo hEPO expression in lipids 1-22 compared to control. DETAILED DESCRIPTION OF THE INVENTION

[0015] Disclosed herein are novel amine-containing ionizable lipids with various cleavable linkers useful for forming lipid carriers or lipid nanoformulations (e.g., lipid nanoparticles (LNPs) or liposomes), which can be used in the pharmaceutical compositions or methods described herein. These lipid carriers or lipid nanoformulations utilizing the novel ionizable lipids can have advantageous properties for delivering therapeutic agents (e.g., nucleic acid molecules) to cells. As such, the present disclosure provides lipid carriers or lipid nanoformulations comprising these novel lipids. The present disclosure also provides pharmaceutical compositions comprising these lipid-based carriers or lipid nanoformulations. Additionally, the present disclosure provides methods for delivering an effector (e.g., a therapeutic agent) to a cell or subject by administering a pharmaceutical composition or lipid carrier or lipid nanoformulation containing the effector to the cell or subject.

[0016] In some embodiments, the present disclosure provides a compound of formula (Ia): [ka] or a salt thereof, During the ceremony, R N2 is -(CH2) m (NH) n Q 2 and; Q 2 is —OH, —SONH(alkyl), —SON(alkyl), optionally substituted cycloalkyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted heterocycloalkyl, or optionally substituted heterocycloalkenyl; m is an integer of 2 to 3; n is an integer from 0 to 1; L 1 and L 2 are each independently (C1 to C 15 ) alkylene; Z 10 and Z20 are each independently [ka] and X 1 and X 2 each independently represents O, S, or N(R 21 ) and; R 20 is branched (C1-C 15 ) alkyl or unbranched (C1-C 15 ) alkyl; R 21 is H, (C1-C5) alkyl, or (C3-C8) cycloalkyl; s is an integer from 1 to 4; Z 10 In [ka] L 1 indicates the point of attachment to; Z 20 In [ka] L 2 indicates the point of attachment to; R 22 , R 23 , R 24 , and R 25 Each of the is independently H, branched (C1 to C 15 ) alkyl, or unbranched (C1-C 15 ) alkyl; provided that R 22 and R 23 At least one of them is not H and R 24 and R 25 At least one of them is not H; Here, Q 2 is -OH, the following (i) or (ii): (i)Z 10 and Z 20 At least one of [ka] or (ii)L 1 and L 2 are not the same At least one of the following applies: The present invention relates to a compound or a salt thereof.

[0017] In some embodiments, R N2 is -(CH2) m Q 2 is.

[0018] In some embodiments, Q 2 is -OH.

[0019] In some embodiments, R N2 is -CH2CH2OH.

[0020] In some embodiments, R N2 is -(CH2) m (NH)Q 2 is.

[0021] In some embodiments, Q 2 is -SO2NH(alkyl) or -SO2N(alkyl)2.

[0022] In some embodiments, Q 2 is optionally substituted cycloalkyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted heterocycloalkyl, or optionally substituted heterocycloalkenyl; where, valence permitting, the optional substituents are selected from oxo, amino, alkylamino, and dialkylamino.

[0023] In some embodiments, Q 2 teeth, [ka] where each Rc are independently H or C1-C3 alkyl, and s is an integer of 1 to 4.

[0024] In some embodiments, R N teeth, [ka] is.

[0025] In some embodiments, L 1 and L 2 are each independently (C2~C 10 ) alkylene.

[0026] In some embodiments, L 1 and L 2 are each independently (C2-C8) alkylene.

[0027] In some embodiments, L 1 and L 2 are each independently (C4-C8) alkylene.

[0028] In some embodiments, L 1 and L 2 are not identical.

[0029] In some embodiments, Z 10 and Z 20 are each independently [ka] is.

[0030] In some embodiments, R 22 is ;R, not H 24 is not H; and R 23 and R 25 At least one of them is not H.

[0031] In some embodiments, R 22 , R 23 , R24 , and R 25 independently branched (C1-C 15 ) alkyl or unbranched (C1-C 15 ) alkyl.

[0032] In some embodiments, R 22 and R 23 are identical.

[0033] In some embodiments, R 22 and R 23 are not identical.

[0034] In some embodiments, R 24 and R 25 are identical.

[0035] In some embodiments, R 24 and R 25 are not identical.

[0036] In some embodiments, Z 10 and Z 20 On the other hand, [ka] and Z 10 and Z 20 The other is, [ka] is.

[0037] In some embodiments, Z 10 and Z 20 each of which independently [ka] is.

[0038] In some embodiments, Z 10 but [ka] If R 22 is H; and Z 20 but [ka] If R 24 is H.

[0039] In some embodiments, Z 10 and Z 20 On the other hand, [ka] and Z 10 and Z 20 The other is, [ka] is.

[0040] In some embodiments, X 1 and X 2 are O, respectively.

[0041] In some embodiments, R 22 is ;R, not H 24 is not H; and R 23 and R 25 At least one of them is not H.

[0042] In some embodiments, Z 10 and Z 20 On the other hand, [ka] and Z 10 and Z 20 The other is, [ka] is.

[0043] In some embodiments, s is an integer from 1 to 3.

[0044] In some embodiments, the compound has the formula: [ka] It has.

[0045] In some embodiments, R 22 and R 23 are each independently an unsubstituted C5-C8 alkyl; or R 23 is H and R 22 is the unsubstituted C 10 ~C 14 is alkyl; R 24 and R 25 are each independently an unsubstituted C5-C8 alkyl; or R 25 is H and R 24 is the unsubstituted C 10 ~C 14 is alkyl; where R 23 and R 25 Both are not H.

[0046] In some embodiments, the compound has the formula: [ka] It has.

[0047] In some embodiments, X 1 and X 2 are O, respectively.

[0048] In some embodiments, R 22 is ;R, not H 24 is not H; and R 23 and R 25 At least one of them is not H.

[0049] In some embodiments, the compound has the formula [ka] It has.

[0050] In some embodiments, the compound has the formula: [ka] It has.

[0051] In some embodiments, R 23 is H.

[0052] In some embodiments, the compound has the following structure: [ka] [ka] [ka] [ka] [ka] [ka] [ka] and During the ceremony, R= [ka] and each R 27 are independently H, C1 to C 15 It is alkyl, C2-C8 alkenyl, or C2-C8 alkynyl.

[0053] In some embodiments, the present disclosure provides a compound of formula (AL-GI): [ka] or a salt thereof, During the ceremony, R N is a substituted or unsubstituted C1-C6 alkyl or C3-C8 cycloalkyl; R1, R 1’ , R2, R 2’ , R3, R 3’ , R4, and R 4’ each of which is independently at each occurrence H, branched C1-C3 alkyl, unbranched C1-C3 alkyl, branched C2-C3 alkenyl, or unbranched C2-C3 alkenyl; R 10 , R 11 , R 12 , and R 13 each independently represents H or a substituted or unsubstituted branched C1-C 15 Alkyl or unbranched C1-C 15 alkyl; provided that R 10 and R 11 At least one of them is not H and R 12 and R 13 At least one of them is not H; Each of Z1 and Z2 independently represents [ka] and; X 1 and X 2 each independently represents O, S, or N(R 21 ) and; R 20 is a substituted or unsubstituted branched C1-C 15 Alkyl or unbranched C1-C 15 is alkyl; R 21 is H, substituted or unsubstituted C1-C5 alkyl, or substituted or unsubstituted C3-C8 cycloalkyl; s is an integer from 1 to 4; n1, n2, n3, and n4 are each independently an integer of 0 to 15, where n1+n2 is in the range of 1 to 15, and n3+n4 is in the range of 1 to 15; where R N is a C1-C6 alkyl substituted with hydroxy, the following (i) or (ii): (i) At least one of Z1 and Z2 is [ka] or (ii) The sum of n1 and n2 is not equal to the sum of n3 and n4 At least one of the following applies: The present invention relates to a compound or a salt thereof.

[0054] In some embodiments, R N is C1-C6 alkyl, C3-C8 cycloalkyl, -(CH2) v Q, -(CH2) v N(R")Q, -C(Q)(R), or -(CH) v C(Q)(R)2; Each Q is independently -OR", -SR", C3-C8 cycloalkyl, C3-C8 cycloalkenyl, heterocyclyl, heterocycloalkenyl, aryl, heteroaryl, -O(CH2) v N(R")2, -C(O)OR", -OC(O)R, -C(R')3, -CN, -C(O)N(R”)2, -N(R”)C(O)R, -N(R”)S(O)2R, -N(R”)C(O)N(R”)2, -N(R”)C(S)N(R”)2, -N(R”)R a , -O(CH2) v OR”, -N(R")C(=NR b )N(R”)2, -N(R”=)C(CHR b)N(R”)2, -OC(O)N(R”)2, -N(R”)C(O)OR”, -N(OR”)C(O)R, -N(OR”)S(O)2R, -N(OR”)C(O)OR”, -N(OR”)C(O)N(R”)2, -N(OR”)C(S)N(R”)2, -N(OR”)C(=NR b )N(R")2, -N(OR")C(=CHR b )N(R”)2, -C(=NR b )N(R")2, -C(=NR b )R, or -C(O)N(R")OR"; each R is independently H, C1-C3 alkyl, C2-C3 alkenyl, amino, monoalkylamino, or dialkylamino; each R' is independently H, F, Cl, Br, or I; each R" is independently H, C1-C3 alkyl, or C2-C3 alkenyl; Each R a are independently H or C3-C8 cycloalkyl; Each R b are independently H, CN, NO, C-C alkyl, —OR, —S(O)R, —S(O)N(R”), C-C alkenyl, C-C cycloalkyl, or heterocyclyl; v is an integer from 1 to 6; Here, each of the alkyl group, cycloalkyl group, cycloalkenyl group, heterocyclyl group, heterocycloalkenyl group, aryl group, and heteroaryl group is optionally substituted with one or more substituents selected from the group consisting of oxo (=O), OH, amino, monoalkylamino, dialkylamino, and C1-C3 alkyl.

[0055] In some embodiments, R N is the unsubstituted C 1~4 Alkyl, -(CH2) v N(R")Q, or -(CH2) v Q is; Q is -OH, -SH, -NHC(S)N(R")2, -NHC(O)N(R")2, -N(R")C(O)R, -N(R")S(O)2R; -N(R”)R a , -NHC(=NR b )N(R")2, -NHC(=CHR b )N(R")2, -OC(O)N(R")2, -N(R")C(O)OR", heterocyclyl, or heteroaryl.

[0056] In some embodiments, R N is -(CH2) v OH and v is 2, 3, or 4.

[0057] In some embodiments, R N is -CH2CH2OH.

[0058] In some embodiments, R N is -(CH2) v Q or -(CH2) v N(R")Q, where Q is heterocyclyl, heterocycloalkenyl, aryl, or heteroaryl optionally substituted with one or more substituents.

[0059] In some embodiments, R N is -(CH2) v N(R")S(O)R.

[0060] In some embodiments, R N teeth, [ka] where each R c are independently H or C1-C3 alkyl, and s is an integer of 1 to 4.

[0061] In some embodiments, R N teeth, [ka] is.

[0062] In some embodiments, n1+n2 ranges from 1 to 10, and n3+n4 ranges from 1 to 10.

[0063] In some embodiments, n1+n2 ranges from 2 to 7, and n3+n4 ranges from 2 to 7, and n1+n2 and n3+n4 are the same.

[0064] In some embodiments, n1+n2 ranges from 2 to 7, and n3+n4 ranges from 2 to 7, and n1+n2 and n3+n4 are different.

[0065] In some embodiments, each of Z1 and Z2 is independently: [ka] is.

[0066] In some embodiments, one of Z1 and Z2 is [ka] and the other of Z1 and Z2 is [ka] is.

[0067] In some embodiments, each of Z1 and Z2 is independently: [ka] is.

[0068] In some embodiments, The Z2, [ka] If R 11 is H; and The Z1, [ka] If R 13 is H.

[0069] In some embodiments, one of Z1 and Z2 is [ka] and the other of Z1 and Z2 is [ka] is.

[0070] In some embodiments, each of Z1 and Z2 is independently the same or different. [ka] is.

[0071] In some embodiments, one of Z1 and Z2 is [ka] and the other of Z1 and Z2 is [ka] is.

[0072] In some embodiments, each of Z1 and Z2 is independently the same or different. [ka] is.

[0073] In some embodiments, the compound has the formula: [ka] It has.

[0074] In some embodiments, R 10 and R 11 are each independently an unsubstituted C5-C8 alkyl; or R 11 is H and R 10 is the unsubstituted C 10 ~C 14 is alkyl; R 12 and R 13 are each independently an unsubstituted C5-C8 alkyl; or R 13 is H and R 12 is the unsubstituted C 10 ~C 14 is alkyl; where R 11 and R 13 Both are not H.

[0075] In some embodiments, n1+n2 is an integer from 2 to 4, and n3+n4 is an integer from 5 to 7; or n1+n2 is an integer from 5 to 7, and n3+n4 is an integer from 2 to 4.

[0076] In some embodiments, the compound has the formula: [ka] It has.

[0077] In some embodiments, the compound has the formula: [ka] It has.

[0078] In some embodiments, R 10 and R 11 are each independently an unsubstituted C5-C8 alkyl; or R 11 is H and R 10 is unsubstituted C7~C 11 is alkyl; R 12 and R 13are each independently an unsubstituted C5-C8 alkyl; or R 13 is H and R 12 is unsubstituted C7~C 11 is alkyl; where R 11 and R 13 Both are not H.

[0079] In some embodiments, each of X and X is independently O or N(R 21 ) and R 21 is H or C1-C3 alkyl.

[0080] In some embodiments, n1+n2 is an integer between 4 and 7, and n3+n4 is an integer between 6 and 7; or n1+n2 is an integer between 6 and 7, and n3+n4 is an integer between 4 and 7.

[0081] In some embodiments, the compound has the formula: [ka] It has.

[0082] In some embodiments, R 10 is unsubstituted C7~C 11 is alkyl; R 12 and R 13 are each independently an unsubstituted C5-C8 alkyl; or R 13 is H and R 12 is unsubstituted C7~C 11 is alkyl; Each R 21 is H; Each R 20 is independently unsubstituted C2-C9 alkyl.

[0083] In some embodiments, n1+n2 and n3+n4 are each independently an integer from 5 to 7.

[0084] In some embodiments, the present disclosure relates to a lipid-based carrier comprising a compound of the present disclosure (e.g., a compound of Formula (Ia) or (AL-GI)), wherein the lipid-based carrier is a lipid nanoparticle.

[0085] In some embodiments, the lipid-based carrier further comprises a second lipid.

[0086] In some embodiments, the second lipid is a cationic lipid, an anionic lipid, an ionic lipid, or a zwitterionic lipid.

[0087] In some embodiments, the lipid-based carrier further comprises a PEGylated lipid, a sterol, a phospholipid, and / or a neutral lipid.

[0088] In some embodiments, the lipid component of the lipid-based carrier is Approximately 25 to 100 mol % of this compound, Approximately 0-50 mol% phospholipids, approximately 0-50 mol% sterols, and Approximately 0-10 mol% PEGylated lipid Includes:

[0089] In some embodiments, the lipid component of the lipid-based carrier is Approximately 30 to 60 mol% of this compound, Approximately 0-30 mol% phospholipids, Approximately 15-50 mol% sterols, and Approximately 0-10 mol% PEGylated lipid Includes:

[0090] In some embodiments, the lipid nanoparticle further comprises an effector, such as a therapeutic agent.

[0091] In some embodiments, the therapeutic agent is a nucleic acid molecule.

[0092] In some embodiments, the nucleic acid molecule is a nucleic acid selected from the group consisting of a plasmid, an immunostimulatory oligonucleotide, an antisense oligonucleotide, an antagomir, an aptamer, a deoxyribozyme (DNAzyme), and a ribozyme.

[0093] In some embodiments, the nucleic acid molecule is DNA or RNA.

[0094] In some embodiments, the DNA is linear, circular, single-stranded, or double-stranded.

[0095] In some embodiments, the RNA is selected from the group consisting of mRNA, miRNA, siRNA or siRNA precursor, RNA aptamer, linear RNA, circular RNA, single-stranded RNA, double-stranded RNA, tRNA, microRNA (miRNA) or miRNA precursor, Dicer substrate small interfering RNA (dsiRNA), short hairpin RNA (shRNA), asymmetric interfering RNA (aiRNA), guide RNA (gRNA), lncRNA, ncRNA, sncRNA, rRNA, snRNA, piRNA, snoRNA, snRNA, scaRNA, exRNA, scaRNA, Y RNA, and hnRNA.

[0096] In some embodiments, the RNA is mRNA.

[0097] In some embodiments, the nucleic acid molecule comprises one or more nucleic acid analogs selected from the group consisting of phosphoramide, phosphorothioate, phosphorodithioate, O-methyl phosphoramidate, morpholino, locked nucleic acid (LNA), glycerol nucleic acid (GNA), threose nucleic acid (TNA), and peptide nucleic acid (PNA).

[0098] In some embodiments, the therapeutic agent is a protein or small molecule drug.

[0099] In some embodiments, the lipid nanoparticle comprises an antigen.

[0100] In some embodiments, the antigen is a protein or a nucleic acid.

[0101] In some embodiments, the antigen is a protein.

[0102] In some embodiments, the antigen is a nucleic acid.

[0103] In some embodiments, the lipid nanoparticle comprises an mRNA molecule comprising a nucleotide sequence encoding an antigen.

[0104] In some embodiments, the present disclosure relates to a method of delivering an effector, such as a therapeutic agent, to a subject, comprising administering to the subject a lipid-based carrier of the present disclosure, wherein the lipid-based carrier comprises the effector.

[0105] In some embodiments, the present disclosure relates to a method of vaccinating a subject in need thereof, comprising administering to the subject an effective amount of a lipid-based carrier of the present disclosure, wherein the lipid-based carrier comprises an antigen.

[0106] In some embodiments, the present disclosure relates to a pharmaceutical composition comprising a lipid-based carrier of the present disclosure and a pharmaceutically acceptable excipient.

[0107] Novel ionizable lipids One aspect of the present invention is a compound of formula (AL-GI): [ka] A compound of the formula: During the ceremony, R N is a substituted or unsubstituted C1-C6 alkyl or C3-C8 cycloalkyl; R1, R 1’ , R2, R 2’ , R3, R 3’ , R4, and R 4’each of which is independently at each occurrence H, branched or unbranched C1-C3 alkyl, or branched or unbranched C2-C3 alkenyl; R 10 , R 11 , R 12 , and R 13 each independently represents H or a branched or unbranched, substituted or unsubstituted C1-C 15 alkyl; provided that R 10 and R 11 At least one of them is not H and R 12 and R 13 At least one of them is not H; Each of Z1 and Z2 independently represents [ka] and; Each of X1 and X2 independently represents O, S, or N(R 21 ) and; R 20 is a branched or unbranched, substituted or unsubstituted C1-C 15 is alkyl; R 21 is H, substituted or unsubstituted C1-C5 alkyl, or substituted or unsubstituted C3-C8 cycloalkyl; s is an integer from 1 to 4; n1, n2, n3, and n4 are each independently an integer of 0 to 15, where n1+n2 is in the range of 1 to 15, and n3+n4 is in the range of 1 to 15. Concerning compounds.

[0108] In any of the formulas described herein, R N can be a C1-C6 alkyl or a C3-C8 cycloalkyl. Each of these alkyl and cycloalkyl groups can be unsubstituted or substituted with one or more substituents.

[0109] In some embodiments, in any of the formulas described herein, R N is C1-C6 alkyl, C3-C8 cycloalkyl, -(CH2) v Q, -(CH2) v N(R)Q, -C(Q)(R)2, or -(CH2) v C(Q)(R)2.

[0110] Each Q is independently -OR, -SR, C3-C8 cycloalkyl, heterocyclyl, heteroaryl, -O(CH2) v N(R)2, -C(O)OR, -OC(O)R, -C(R')3, -CN, -C(O)N(R)2, -N(R)C(O)R, -N(R)S(O)2R, -N(R)C(O)N(R)2, -N(R)C(S)N(R)2, -N(R)R a , -O(CH2) v OR, -N(R)C(=NR b )N(R)2, -N(R)C(=CHR b )N(R)2, -OC(O)N(R)2, -N(R)C(O)OR, -N(OR)C(O)R, -N(OR)S(O)2R, -N(OR)C(O)OR, -N(OR)C(O)N(R)2, -N(OR)C(S)N(R)2, -N(OR)C(=NR b )N(R)2, -N(OR)C(=CHR b )N(R)2, -C(=NR b )N(R)2, -C(=NR b )R, or -C(O)N(R)OR.

[0111] Each of the alkyl, cycloalkyl, heterocyclyl, and heteroaryl groups can be optionally substituted with one or more substituents selected from the group consisting of oxo (=O), OH, amino, mono- or di-alkylamino, and C1-C3 alkyl. Each R is independently H, C1-C3 alkyl, C2-C3 alkenyl, amino, or mono- or di-alkylamino. Each R' is independently H, F, Cl, Br, or I. Each R a are independently H or C3-C8 cycloalkyl. b is independently H, CN, NO, C-C alkyl, —OR, —S(O)R, —S(O)N(R), C-C alkenyl, C-C cycloalkyl, or heterocyclyl. Each v is independently an integer from 1 to 6.

[0112] In some embodiments, in any of the formulas described herein, R N is unsubstituted C1-C6 alkyl, for example, unsubstituted C1-C4 alkyl.

[0113] In some embodiments, in any of the formulas described herein, R N is a C1-C6 alkyl (e.g., a C1-C4 alkyl) substituted with one or more substituents. In some embodiments, the substituent is OH, oxo (=O), amino, mono- or di-alkylamino, or a C1-C3 alkyl.

[0114] In some embodiments, in any of the formulas described herein, R N is unsubstituted C3-C8 cycloalkyl, for example, C3-C6 cycloalkyl (eg, C5-C6 cycloalkyl).

[0115] In some embodiments, in any of the formulas described herein, R Nis a C3-C8 cycloalkyl (e.g., a C3-C6 cycloalkyl (e.g., a C5-C6 cycloalkyl) substituted with one or more substituents. In some embodiments, the substituent is OH, oxo (=O), amino, mono- or di-alkylamino, or C1-C3 alkyl.

[0116] In some embodiments, in any of the formulas described herein, R N is -(CH2) v Q or -(CH2) v N(R)Q, where Q is OH, SH, -NHC(S)N(R), -NHC(O)N(R), -N(R)C(O)R, -N(R)S(O)R, -N(R)R a , NHC(=NR b )N(R)2, -NHC(=CHR b )N(R)2, —OC(O)N(R)2, —N(R)C(O)OR, heterocyclyl, or heteroaryl. Each heterocyclyl and heteroaryl group may be substituted with one or more substituents. Each v is independently 2, 3, 4, or 5. In some embodiments, Q is OH, -NHC(S)N(R)2, -NHC(O)N(R)2, -N(R)C(O)R, or -N(R)S(O)2R.

[0117] In some embodiments, in any of the formulas described herein, R N is -(CH2) v OH, and v is an integer of 1 to 6, for example, v is 2, 3, or 4.

[0118] In some embodiments, in any of the formulas described herein, R N is -(CH2) v Q or -(CH2) vN(R)Q. Q is -N(R)S(O)R, heterocyclyl, or heteroaryl. Each heterocyclyl and heteroaryl group can be substituted with one or more substituents. Each v is independently 2, 3, 4, or 5.

[0119] In some embodiments, in any of the formulas described herein, R N is -(CH2) v Q is heterocyclyl, which may be substituted with one or more substituents. In some embodiments, the substituents may be oxo (=O), OH, amino, mono- or di-alkylamino, or C1-C3 alkyl.

[0120] In some embodiments, in any of the formulas described herein, R N is -(CH2) v Q is heteroaryl, which may be substituted with one or more substituents. In some embodiments, the substituents may be oxo (=O), OH, amino, mono- or di-alkylamino, or C1-C3 alkyl.

[0121] In some embodiments, in any of the formulas described herein, R N is -(CH2) v N(R)Q. Q is heterocyclyl, which may be substituted with one or more substituents. In some embodiments, the substituents may be oxo (=O), OH, amino, mono- or di-alkylamino, or C1-C3 alkyl. Each R is independently H, C1-C3 alkyl, C2-C3 alkenyl, amino, or mono- or di-alkylamino.

[0122] In some embodiments, in any of the formulas described herein, R N is -(CH2) vN(R)Q. Q is heteroaryl, which may be substituted with one or more substituents. In some embodiments, the substituents may be oxo (=O), OH, amino, mono- or di-alkylamino, or C1-C3 alkyl. Each R is independently H, C1-C3 alkyl, C2-C3 alkenyl, amino, or mono- or di-alkylamino.

[0123] In some embodiments, in any of the formulas described herein, R N is -(CH2) v N(R)S(O)R, where each R is independently H, C1-C3 alkyl, C2-C3 alkenyl, amino, or mono- or di-alkylamino.

[0124] In some embodiments, in any of the formulas described herein, R N has the following structure: [ka] Each R c are independently H or C1-C3 alkyl. v is an integer of 1 to 6, for example, v is 2, 3, or 4. s is an integer of 1 to 4.

[0125] In some embodiments, in any of the formulas described herein, R N is as follows: [ka] It has the following structure.

[0126] In any of the formulas described herein, n1, n2, n3, and n4 are each independently an integer from 0 to 15, e.g., n1, n2, n3, and n4 are each independently an integer from 0 to 10, from 0 to 7, or from 0 to 4. In some embodiments, n1 + n2 ranges from 1 to 15, and n3 + n4 ranges from 1 to 15. For example, n1 + n2 can range from 1 to 10, and n3 + n4 can range from 1 to 10.

[0127] In some embodiments, in any of the formulas described herein, n1+n2 ranges from 2 to 7, and n3+n4 ranges from 2 to 7, and n1+n2 and n3+n4 are the same.

[0128] In some embodiments, in any of the formulas described herein, n1+n2 ranges from 2 to 7, and n3+n4 ranges from 2 to 7, and n1+n2 and n3+n4 are different.

[0129] In any of the formulas described herein, R, R 1’ , R2, R 2’ , R3, R 3’ , R4, and R 4’ are each independently H, a branched or unbranched C1-C3 alkyl, or a branched or unbranched C2-C3 alkenyl. Each of the alkyl group and the C2-C3 alkenyl group can be unsubstituted or substituted with one or more substituents. In some embodiments, each of the alkyl group and the C2-C3 alkenyl group is unsubstituted. In some embodiments, the alkyl group or the C2-C3 alkenyl group is substituted with one or more substituents. In some embodiments, R1, R 1’ , R2, R 2’ , R3, R 3’ , R4, and R 4’ Each of R, R is independently H or unsubstituted C1-C3 alkyl. 1’ , R2, R 2’ , R3, R 3’ , R4, and R4’ Each of R, R is independently H or methyl. 1’ , R2, R 2’ , R3, R 3’ , R4, and R 4’ Each of is H.

[0130] In some embodiments, n1 is at least 1, and R1 and R 1’ In some embodiments, n1 is at least 1 and R1 and R2 are different for at least one occurrence. 1’ are identical for at least one occurrence. In some embodiments, n1 is at least 2, and R1 and R2 are 1’ In some embodiments, n1 is at least 2 and R1 and R2 are different for at least two occurrences. 1’ In some embodiments, n1 is at least 3 and R1 and R2 are identical for at least two occurrences. 1’ In some embodiments, n1 is at least 3 and R1 and R2 are different for at least three occurrences. 1’ In some embodiments, n1 is at least 4 and R1 and R2 are identical for at least 3 occurrences. 1’ In some embodiments, n1 is at least 4 and R1 and R2 are different for at least 4 occurrences. 1’ In some embodiments, n1 is at least 5 and R1 and R2 are identical for at least 4 occurrences. 1’ In some embodiments, n1 is at least 5 and R1 and R2 are different for at least 5 occurrences. 1’ are identical for at least 5 occurrences. In some embodiments, R and R 1’ In some embodiments, R and R 1’ is identical for each occurrence.

[0131] In some embodiments, n2 is at least 1, and R2 and R 2’In some embodiments, n2 is at least 1 and R2 and R 2’ are identical for at least one occurrence. In some embodiments, n2 is at least 2 and R and R 2’ In some embodiments, n2 is at least 2 and R and R 2’ In some embodiments, n2 is at least 3 and R2 and R 2’ In some embodiments, n2 is at least 3 and R2 and R 2’ In some embodiments, n2 is at least 4 and R and R 2’ In some embodiments, n2 is at least 4 and R2 and R 2’ In some embodiments, n2 is at least 5 and R2 and R 2’ In some embodiments, n2 is at least 5 and R2 and R 2’ are identical for at least 5 occurrences. In some embodiments, R and R 2’ is different for each occurrence. In some embodiments, R and R 2’ is identical for each occurrence.

[0132] In some embodiments, n3 is at least 1, and R3 and R 3’ In some embodiments, n3 is at least 1 and R and R 3’ are identical for at least one occurrence. In some embodiments, n3 is at least 2, and R and R 3’ are different for at least two occurrences. In some embodiments, n3 is at least 2 and R and R 3’In some embodiments, n3 is at least 3 and R and R are identical for at least two occurrences. 3’ In some embodiments, n3 is at least 3 and R and R 3’ In some embodiments, n3 is at least 4 and R and R 3’ In some embodiments, n3 is at least 4 and R and R 3’ In some embodiments, n3 is at least 5 and R and R are identical for at least 4 occurrences. 3’ In some embodiments, n3 is at least 5 and R and R 3’ are identical for at least 5 occurrences. In some embodiments, R and R 3’ is different for each occurrence. In some embodiments, R and R 3’ is identical for each occurrence.

[0133] In some embodiments, n4 is at least 1, and R4 and R 4’ In some embodiments, n4 is at least 1 and R4 and R 4’ are identical for at least one occurrence. In some embodiments, n4 is at least 2, and R4 and R 4’ In some embodiments, n4 is at least 2 and R4 and R 4’ In some embodiments, n4 is at least 3 and R4 and R 4’ In some embodiments, n4 is at least 3 and R4 and R 4’ In some embodiments, n4 is at least 4 and R4 and R 4’In some embodiments, n4 is at least 4 and R4 and R 4’ In some embodiments, n4 is at least 5 and R4 and R 4’ In some embodiments, n4 is at least 5 and R4 and R 4’ are identical for at least 5 occurrences. In some embodiments, R and R 4’ is different for each occurrence. In some embodiments, R and R 4’ is identical for each occurrence.

[0134] In any of the formulas described herein, R 10 , R 11 , R 12 , and R 13 each independently represents H or a branched or unbranched, substituted or unsubstituted C1-C 15 In some embodiments, R 10 , R 11 , R 12 , and R 13 each independently represents H or a C1-C substituted with one or more substituents. 15 In some embodiments, R 10 , R 11 , R 12 , and R 13 each independently represents H or a branched C1-C 15 In some embodiments, R 10 , R 11 , R 12 , and R 13 each independently represents H, or oxo (=O), OH, amino, mono- or di-alkylamino, C1-C 15 C1-C optionally substituted with one or more substituents selected from the group consisting of alkyl, C2-C8 alkenyl, and C2-C8 alkynyl 15 In some embodiments, R 10 , R11 , R 12 , and R 13 each independently represents H or an unbranched, unsubstituted C1-C 15 It is alkyl.

[0135] In some embodiments, R 10 and R 11 At least one of them is not H and R 12 and R 13 At least one of R is not H. In some embodiments, 11 is H and R 10 , R 12 , and R 13 is not H. In some embodiments, R 13 is H and R 10 , R 11 , and R 12 is not H. In some embodiments, R 11 and R 13 is H and R 10 and R 12 is not H. In some embodiments, R 10 and R 11 are identical and are not H. In some embodiments, R 12 and R 13 are identical and not H.

[0136] In some embodiments, R 10 and R 11 are each independently C5-C8 alkyl (e.g., unsubstituted and / or unbranched); or R 11 is H, and R 10 is C 10 ~C 14 alkyl (e.g., unsubstituted and / or unbranched); and R 12 and R 13 are each independently C5-C8 alkyl (e.g., unsubstituted and / or unbranched); or R 13 is H, and R 12 is C 10 ~C 14alkyl (e.g., unsubstituted and / or unbranched); where R 11 and R 13 Both are not H.

[0137] In some embodiments, R 10 and R 11 are each independently C5-C8 alkyl (e.g., unsubstituted and / or unbranched); or R 11 is H, and R 10 is C7~C 11 alkyl (e.g., unsubstituted and / or unbranched); and R 12 and R 13 are each independently C5-C8 alkyl (e.g., unsubstituted and / or unbranched); or R 13 is H, and R 12 is C7~C 11 alkyl (e.g., unsubstituted and / or unbranched), where R 11 and R 13 Both are not H.

[0138] In any of the formulas described herein, each of Z1 and Z2 is independently a cleavable linker, [ka] is. [ka] indicates the attachment of Z1 or Z2 to this formula and does not imply directionality. For example, [ka] is in both orientations, [ka] Similarly, [ka] is in both orientations, [ka] and [ka] This indicates that it is possible.

[0139] In some embodiments, in any of the formulas described herein, each of Z and Z is independently: [ka] Therefore, the lipid compound contains at least two ester linker groups.

[0140] For example, the lipid compound: [ka] The variable R N , R 10 , R 11 , R 12 , R 13 , n 1、 n2, n3, and n4 are defined in the various embodiments above.

[0141] In formula (AL-Ia), (AL-Ib), or (AL-Ic), in some embodiments, R 10 and R 11 are each independently C5-C8 alkyl (e.g., unsubstituted and / or unbranched); or R 11 is H, and R 10 is C 10 ~C 14 alkyl (e.g., unsubstituted and / or unbranched); and R 12 and R 13 are each independently C5-C8 alkyl (e.g., unsubstituted and / or unbranched); or R 13 is H, and R 12 is C 10 ~C14 alkyl (e.g., unsubstituted and / or unbranched); where R 11 and R 13 are not both H. In some embodiments, each alkyl group is selected from the group consisting of oxo (=O), OH, amino, mono- or di-alkylamino, C1-C 15 It may be optionally substituted with one or more substituents selected from the group consisting of alkyl, C2-C8 alkenyl, and C2-C8 alkynyl. In some embodiments, n1 + n2 are integers from 2 to 4, and n3 + n4 are integers from 5 to 7; or n1 + n2 are integers from 5 to 7, and n3 + n4 are integers from 2 to 4.

[0142] In some embodiments, in any of the formulas described herein, at least one of Z1 and Z2 is [ka] Each of X1 and X2 independently represents O, S, or N(R 21 For example, this lactide or derivative is [ka] R 21 is H, substituted or unsubstituted C1-C5 alkyl, or substituted or unsubstituted C3-C8 cycloalkyl. In some embodiments, R 21 is H. In some embodiments, R 21 is C1-C5 alkyl, for example, C1-C3 alkyl (eg, unsubstituted and / or unbranched).

[0143] In some embodiments, one of Z1 and Z2 is [ka] and the other of Z1 and Z2 is [ka] Thus, the lipid contains at least one ester linker group and one lactide (or derivative thereof) linker group.

[0144] In some embodiments, each of Z1 and Z2 is independently: [ka] Thus, the lipid contains at least two lactide (or derivatives thereof) linker groups. In some embodiments, Z1 and Z2 each have the same [ka] wherein the variables X1 and X2 for Z1 are the same as the variables X1 and X2 for Z2. In some embodiments, Z1 and Z2 are each different [ka] wherein the variables X1 and X2 for Z1 are different from the variables X1 and X2 for Z2.

[0145] For example, the lipid compound: [ka] The variable R N , R 10 , R 11 , R 12 , R 13 , X1, X2, n1, n2, n3, and n4 are defined in the various embodiments above.

[0146] In Formula (AL-IIa), (AL-IIb), or (AL-IIc), in some embodiments, R 10 and R 11 are each independently C5-C8 alkyl (e.g., unsubstituted and / or unbranched); or R 11 is H, and R 10 is C7~C 11alkyl (e.g., unsubstituted and / or unbranched); and R 12 and R 13 are each independently C5-C8 alkyl (e.g., unsubstituted and / or unbranched); or R 13 is H, and R 12 is C7~C 11 alkyl (e.g., unsubstituted and / or unbranched); where R 11 and R 13 are not both H. In some embodiments, each alkyl group is selected from the group consisting of oxo (=O), OH, amino, mono- or di-alkylamino, C1-C 15 It may be optionally substituted with one or more substituents selected from the group consisting of alkyl, C2-C8 alkenyl, and C2-C8 alkynyl. In some embodiments, n1 + n2 are integers from 4 to 7, and n3 + n4 are integers from 6 to 7; or n1 + n2 are integers from 6 to 7, and n3 + n4 are integers from 4 to 7.

[0147] In some embodiments, each of X and X is independently O or N(R 21 In some embodiments, R 21 is H or C1-C3 alkyl.

[0148] In some embodiments, in any of the formulas described herein, at least one of Z1 and Z2 is [ka] R 20 is a branched or unbranched, substituted or unsubstituted C1-C 15 In some embodiments, R 20 is C2-C9 alkyl (e.g., unsubstituted and / or unbranched). 21 is H, substituted or unsubstituted C1-C5 alkyl, or substituted or unsubstituted C3-C8 cycloalkyl. In some embodiments, R 21 is H. In some embodiments, R21 is C1-C5 alkyl, for example, C1-C3 alkyl (eg, unsubstituted and / or unbranched).

[0149] In some embodiments, one of Z1 and Z2 is [ka] and the other of Z1 and Z2 is [ka] Thus, the lipid compound contains one ester linker group and one phosphoramidate linker group.

[0150] In some embodiments, each of Z1 and Z2 is independently: [ka] Thus, the lipid compound contains at least two phosphoramidate linker groups. In some embodiments, Z1 and Z2 each represent the same [ka] wherein the variable R for Z1 20 and R 21 is the variable R with respect to Z2 20 and R 21 is the same as

[0151] In some embodiments, Z1 and Z2 are each different [ka] wherein the variable R for Z1 20 and R 21 is the variable R with respect to Z2 20 and R 21 In some embodiments, Z1 and Z2 are each different from [ka] wherein one of Z1 and Z2 is [ka] and the other of Z1 and Z2 represents [ka] where the variable R 20 and R 21 is the variable R with respect to Z2 20 and R 21 may be the same as or different from.

[0152] In some embodiments, Z2 is [ka] If R 11 is H and Z1 is [ka] If R 13 is H.

[0153] For example, the lipid compound: [ka] The variable R N , R 10 , R 11 , R 12 , R 13 , R 20 , R 21 , n1, n2, n3, and n4 are defined in the various embodiments above.

[0154] In Formulas (AL-IIIa), (AL-IIIb), or (AL-IIIc), (AL-IIId), (AL-IIIe), (AL-IIIf), and (AL-IIIg), in some embodiments, R 10 is C7~C 11alkyl (e.g., unsubstituted and / or unbranched); R 12 and R 13 are each independently C5-C8 alkyl (e.g., unsubstituted and / or unbranched); or R 13 is H, and R 12 is C7~C 11 alkyl (e.g., unsubstituted and / or unbranched); each R 21 is H; and each R 20 is independently C2-C9 alkyl (e.g., unsubstituted and / or unbranched). In some embodiments, each alkyl group is selected from the group consisting of oxo (=O), OH, amino, mono- or di-alkylamino, C1-C 15 It may be optionally substituted with one or more substituents selected from the group consisting of alkyl, C2-C8 alkenyl, and C2-C8 alkynyl. In some embodiments, n1+n2 and n3+n4 are each independently an integer from 5 to 7.

[0155] In some embodiments, in any of the formulas described herein, at least one of Z1 and Z2 is [ka] where s is an integer of 1 to 4. For example, the lactone linker group is [ka] It may have the formula:

[0156] In some embodiments, one of Z1 and Z2 is [ka] and the other of Z1 and Z2 is [ka] Thus, the lipid contains at least one ester linker group and one lactone linker group.

[0157] In some embodiments, each of Z1 and Z2 is independently the same or different. [ka] Thus, the lipid contains at least two lactone linker groups. In some embodiments, Z and Z are each the same [ka] wherein the variable and position of s on the lactone ring connected to the formula for Z1 is the same as the variable and position of s on the lactone ring connected to the formula for Z2.

[0158] In some embodiments, Z1 and Z2 are each different [ka] wherein the variable s for Z1 is different from the variable s for Z2. In some embodiments, Z1 and Z2 are each different [ka] wherein the variable s for Z1 is the same as the variable s for Z2, but the position on the lactone ring linked to the formula for Z1 is different from the position on the lactone ring linked to the formula for Z2.

[0159] For example, the lipid compound: [ka] The variable R N , R 10 , R 11 , R 12 , R 13 , X1, X2, n1, n2, n3, and n4 are defined in the various embodiments above.

[0160] In formula (AL-IVa), (AL-IVb), or (AL-IVc), in some embodiments, R 10 and R 11 are each independently C5-C8 alkyl (e.g., unsubstituted and / or unbranched); or R 11 is H, and R 10 is C7~C 11 alkyl (e.g., unsubstituted and / or unbranched); and R 12 and R 13 are each independently C5-C8 alkyl (e.g., unsubstituted and / or unbranched); or R 13 is H, and R 12 is C7~C 11 alkyl (e.g., unsubstituted and / or unbranched); where R 11 and R 13 are not both H. In some embodiments, each alkyl group is selected from the group consisting of oxo (=O), OH, amino, mono- or di-alkylamino, C1-C 15 It may be optionally substituted with one or more substituents selected from the group consisting of alkyl, C2-C8 alkenyl, and C2-C8 alkynyl. In some embodiments, n1 + n2 are integers from 4 to 7, and n3 + n4 are integers from 6 to 7; or n1 + n2 are integers from 6 to 7, and n3 + n4 are integers from 4 to 7.

[0161] Further exemplary formulas for the present lipid compounds include, but are not limited to, the following:

[0162] a. A lipid having at least two ester groups: [ka] In the above formula, A=H, OH, Q, or NHQ Q = heterocycle, heteroaryl, or S(O)NR 21 ; R 21 =H or methyl.

[0163] b. Lipids containing a lactide (or its derivative) group: [ka] [ka] [ka] In all the above formulas, A=H, OH, Q, or NHQ; Q = heterocycle, heteroaryl, or S(O)NR 21 ; R 21 =H or methyl; X1, X2 = O or NH.

[0164] c. Lipids containing lactone groups: [ka] [ka] In all the above formulas, A=H, OH, Q, or NHQ; Q = heterocycle, heteroaryl, or S(O)NR 21 ; R 21 =H or methyl.

[0165] d. Lipids containing phosphoramidate groups: [ka] [ka] In all the above formulas, A=H, OH, Q, or NHQ; Q = heterocycle, heteroaryl, or S(O)NR 21 ; R 20 = C2-C9 alkyl; R 21 =H or methyl.

[0166] Non-limiting examples of lipid compounds disclosed herein are listed below. [ka] [ka] [ka] [ka] [ka]

[0167] In a first embodiment, the present invention provides a compound of formula (AL-GI): [ka] A compound of the formula: During the ceremony, R N is a substituted or unsubstituted C1-C6 alkyl or C3-C8 cycloalkyl; R1, R 1’ , R2, R 2’ , R3, R 3’ , R4, and R 4’ each of which is independently at each occurrence H, branched or unbranched C1-C3 alkyl, or branched or unbranched C2-C3 alkenyl; R 10 , R 11 , R 12 , and R 13 each independently represents H or a branched or unbranched, substituted or unsubstituted C1-C 15 alkyl; provided that R 10 and R 11 At least one of them is not H and R 12 and R 13 At least one of them is not H; Each of Z1 and Z2 independently represents [ka] and; Each of X1 and X2 independently represents O, S, or N(R 21 ) and; R 20 is a branched or unbranched, substituted or unsubstituted C1-C 15 is alkyl; R 21 is H, substituted or unsubstituted C1-C5 alkyl, or substituted or unsubstituted C3-C8 cycloalkyl; s is an integer from 1; n1, n2, n3, and n4 are each independently an integer of 0 to 15, where n1+n2 is in the range of 1 to 15, and n3+n4 is in the range of 1 to 15. Concerning compounds.

[0168] In a first aspect of the first embodiment, R N is C1-C6 alkyl, C3-C8 cycloalkyl, -(CH2) v Q, -(CH2) v N(R)Q, -C(Q)(R)2, or -(CH2) v C(Q)(R)2; each Q is independently -OR, -SR, C3-C8 cycloalkyl, heterocyclyl, heteroaryl, -O(CH2) v N(R)2, -C(O)OR, -OC(O)R, -C(R')3, -CN, -C(O)N(R)2, -N(R)C(O)R, -N(R)S(O)2R, -N(R)C(O)N(R)2, -N(R)C(S)N(R)2, -N(R)R a , -O(CH2) v OR, -N(R)C(=NR b )N(R)2, -N(R)C(=CHR b )N(R)2, -OC(O)N(R)2, -N(R)C(O)OR, -N(OR)C(O)R, -N(OR)S(O)2R, -N(OR)C(O)OR, N(OR)C(O)N(R)2, -N(OR)C(S)N(R)2, -N(OR)C(=NRb )N(R)2, -N(OR)C(=CHR b )N(R)2, -C(=NR b )N(R)2, -C(=NR b )R, or —C(O)N(R)OR, wherein each of the alkyl, cycloalkyl, heterocyclyl, and heteroaryl groups is optionally substituted with one or more substituents selected from the group consisting of oxo (═O), OH, amino, mono- or di-alkylamino, and C1-C3 alkyl; each R is independently H, C1-C3 alkyl, C2-C3 alkenyl, amino, or mono- or di-alkylamino; each R′ is independently H, F, Cl, Br, or I, and each R a are independently H or C3-C8 cycloalkyl, and each R b is independently H, CN, NO, C-C alkyl, —OR, —S(O)R, —S(O)N(R), C-C alkenyl, C-C cycloalkyl, or heterocyclyl; and each v is independently an integer from 1 to 6.

[0169] In a second aspect of the first embodiment, R N is the unsubstituted C 1~4 Alkyl, -(CH2) v N(R)Q or -(CH2) v Q is OH, SH, -NHC(S)N(R)2, -NHC(O)N(R)2, -N(R)C(O)R, -N(R)S(O)2R, -N(R)R a , -NHC(=NR b )N(R)2, -NHC(=CHR b )N(R), —OC(O)N(R), —N(R)C(O)OR, heterocyclyl, or heteroaryl; and each v is independently 2, 3, 4, or 5. The remainder of the features and exemplary features of the second aspect are as described above for the first aspect of the first embodiment.

[0170] In a third aspect of the first embodiment, R N is -(CH2) vOH and v is independently 2, 3, or 4. The remainder of the features and exemplary features of the third aspect are as described above for the first and second aspects of the first embodiment.

[0171] In a fourth aspect of the first embodiment, R N is -(CH2) v Q or -(CH2) v N(R)Q, where Q is heterocyclyl or heteroaryl optionally substituted with one or more substituents. The remainder of the features and exemplary features of the fourth aspect are as described above for the first through third aspects of the first embodiment.

[0172] In a fifth aspect of the first embodiment, R N is -(CH2) v Q or -(CH2) v N(R)Q, where Q is heterocyclyl or heteroaryl optionally substituted with one or more substituents. The remainder of the features and exemplary features of the fifth aspect are as described above for the first through fourth aspects of the first embodiment.

[0173] In a sixth aspect of the first embodiment, R N is -(CH2) v N(R)S(O)R. The remainder of the features and exemplary features of the sixth aspect are as described above with respect to the first through fifth aspects of the first embodiment.

[0174] In a seventh aspect of the first embodiment, R N has the following structure: [ka] wherein each R c are independently H or C1-C3 alkyl, and s is an integer from 1 to 4. The remainder of the features and exemplary features of the seventh aspect are as described above for the first through sixth aspects of the first embodiment.

[0175] In an eighth aspect of the first embodiment, R N teeth, [ka] The remainder of the features and exemplary features of the eighth aspect are as described above with respect to the first to seventh aspects of the first embodiment.

[0176] In a ninth aspect of the first embodiment, n1 + n2 ranges from 1 to 10, and n3 + n4 ranges from 1 to 10. For example, n1 + n2 ranges from 2 to 7, and n3 + n4 ranges from 2 to 7, and n1 + n2 and n3 + n4 are the same. In certain embodiments, n1 + n2 ranges from 2 to 7, and n3 + n4 ranges from 2 to 7, and n1 + n2 and n3 + n4 are different. The remainder of the features and exemplary features of the ninth aspect are as described above with respect to the first to eighth aspects of the first embodiment.

[0177] In a tenth aspect of the first embodiment, each of Z1 and Z2 independently represents [ka] The remainder of the features and exemplary features of the tenth aspect are as described above with respect to the first to ninth aspects of the first embodiment.

[0178] In an eleventh aspect of the first embodiment, one of Z1 and Z2 is [ka] and the other of Z1 and Z2 is [ka] The remainder of the features and exemplary features of the eleventh aspect are as described above with respect to the first to tenth aspects of the first embodiment.

[0179] In a twelfth aspect of the first embodiment, each of Z1 and Z2 is the same or different. [ka] The remainder of the features and exemplary features of the twelfth aspect are as described above with respect to the first through eleventh aspects of the first embodiment.

[0180] In a thirteenth aspect of the first embodiment, Z2 is [ka] If R 11 is H and Z1 is [ka] If R 13 is H. The remainder of the features and exemplary features of the thirteenth aspect are as described above with respect to the first through twelfth aspects of the first embodiment.

[0181] In a fourteenth aspect of the first embodiment, one of Z1 and Z2 is [ka] and the other of Z1 and Z2 is [ka] The remainder of the features and exemplary features of the fourteenth aspect are as described above with respect to the first to thirteenth aspects of the first embodiment.

[0182] In a fifteenth aspect of the first embodiment, each of Z1 and Z2 is independently the same or different. [ka] The remainder of the features and exemplary features of the fifteenth aspect are as described above with respect to the first to fourteenth aspects of the first embodiment.

[0183] In a sixteenth aspect of the first embodiment, one of Z1 and Z2 is [ka] and the other of Z1 and Z2 is [ka] The remainder of the features and exemplary features of the sixteenth aspect are as described above with respect to the first to fifteenth aspects of the first embodiment.

[0184] In a seventeenth aspect of the first embodiment, each of Z1 and Z2 is independently the same or different. [ka] The remainder of the features and exemplary features of the seventeenth aspect are as described above with respect to the first to sixteenth aspects of the first embodiment.

[0185] In an eighteenth aspect of the first embodiment, the compound has the formula: [ka] The remainder of the features and exemplary features of the eighteenth aspect are as described above with respect to the first to seventeenth aspects of the first embodiment. The remainder of the features and exemplary features of the nineteenth aspect are as described above with respect to the first to eighteenth aspects of the first embodiment.

[0186] In a nineteenth aspect of the first embodiment, R 10 and R 11 are each independently an unsubstituted C5-C8 alkyl; or R 11 is H, and R 10 is the unsubstituted C 10 ~C 14 alkyl; and R 12 and R 13 are each independently an unsubstituted C5-C8 alkyl; or R 13 is H, and R 12 is the unsubstituted C 10 ~C 14 alkyl; where R 11 and R 13and are not both H. The remainder of the features and exemplary features of the nineteenth aspect are as described above with respect to the first through eighteenth aspects of the first embodiment.

[0187] In a twenty-first aspect of the first embodiment, n1 + n2 are integers from 2 to 4, and n3 + n4 are integers from 5 to 7, or n1 + n2 are integers from 5 to 7, and n3 + n4 are integers from 2 to 4. The remainder of the features and exemplary features of the twenty-first aspect are as described above with respect to the first to twentieth aspects of the first embodiment.

[0188] In a twenty-second aspect of the first embodiment, the compound has the formula: [ka] The remainder of the features and exemplary features of the twenty-second aspect are as described above with respect to the first to twenty-first aspects of the first embodiment.

[0189] In a twenty-third aspect of the first embodiment, R 10 and R 11 are each independently an unsubstituted C5-C8 alkyl; or R 11 is H, and R 10 is unsubstituted C7~C 11 alkyl; and R 12 and R 13 are each independently an unsubstituted C5-C8 alkyl; or R 13 is H, and R 12 is unsubstituted C7~C 11 alkyl, where R 11 and R 13 and are not both H. The remainder of the features and exemplary features of the twenty-third aspect are as described above with respect to the first through twenty-second aspects of the first embodiment.

[0190] In a twenty-fourth aspect of the first embodiment, each of X1 and X2 is independently O or N(R 21 ) and R 21is H or C1-C3 alkyl. In some embodiments, n1 + n2 are integers from 4 to 7, and n3 + n4 are integers from 6 to 7; or n1 + n2 are integers from 6 to 7, and n3 + n4 are integers from 4 to 7. The remainder of the features and exemplary features of the twenty-fourth aspect are as described above for the first through twenty-third aspects of the first embodiment.

[0191] In a twenty-fifth aspect of the first embodiment, the compound has the formula: [ka] In some embodiments, R 10 and R 11 are each independently an unsubstituted C5-C8 alkyl; or R 11 is H, and R 10 is unsubstituted C7~C 11 alkyl; and R 12 and R 13 are each independently an unsubstituted C5-C8 alkyl; or R 13 is H, and R 12 is unsubstituted C7~C 11 alkyl, where R 11 and R 13 are not both H. The remainder of the features and exemplary features of the twenty-fifth aspect are as described above with respect to the first through twenty-fourth aspects of the first embodiment.

[0192] In a 26th aspect of the first embodiment, n1 + n2 are integers from 4 to 7, and n3 + n4 are integers from 6 to 7; or n1 + n2 are integers from 6 to 7, and n3 + n4 are integers from 4 to 7. The remainder of the features and exemplary features of the 26th aspect are as described above with respect to the first to 25th aspects of the first embodiment.

[0193] In a twenty-seventh aspect of the first embodiment, the compound has the formula: [ka] The remainder of the features and exemplary features of the 27th aspect are as described above with respect to the first to 26th aspects of the first embodiment.

[0194] In a twenty-eighth aspect of the first embodiment, R 10 is unsubstituted C7~C 11 alkyl; R 12 and R 13 are each independently an unsubstituted C5-C8 alkyl; or R 13 is H and R 12 is unsubstituted C7~C 11 alkyl; each R 21 is H; each R 20 is independently unsubstituted C2-C9 alkyl. The remainder of the features and exemplary features of the twenty-eighth aspect are as described above for the first through twenty-seventh aspects of the first embodiment.

[0195] In a 29th aspect of the first embodiment, n1 + n2 and n3 + n4 are each independently an integer from 5 to 7. The remainder of the features and exemplary features of the 29th aspect are as described above with respect to the first through 28th aspects of the first embodiment.

[0196] In a thirtieth aspect of the first embodiment, the compound has the following structure: [ka] [ka] [ka] [ka] and During the ceremony, R= [ka] and each R 22 are independently H, C1 to C 15It is alkyl, C2-C8 alkenyl, or C2-C8 alkynyl.

[0197] In a second embodiment, the present invention relates to a lipid-based carrier comprising a compound of formula (AL-GI) as described herein, wherein the lipid-based carrier is a lipid nanoparticle.

[0198] In a first aspect of the second embodiment, the lipid-based carrier further comprises a second lipid.

[0199] In a second aspect of the second embodiment, the second lipid is a cationic lipid, an anionic lipid, an ionic lipid, or a zwitterionic lipid. The remainder of the features and exemplary features of the second aspect are as described above with respect to the first aspect of the second embodiment.

[0200] In a third aspect of the second embodiment, the lipid-based carrier further comprises a PEGylated lipid, a sterol, a phospholipid, and / or a neutral lipid. The remainder of the features and exemplary features of the third aspect are as described above with respect to the first and second aspects of the second embodiment.

[0201] In a fourth aspect of the second embodiment, the lipid component of the lipid-based carrier is Approximately 25 to 100 mol % of this compound, Approximately 0-50 mol% phospholipids, approximately 0-50 mol% sterols, and Approximately 0-10 mol% PEGylated lipid Includes: The remainder of the features and exemplary features of the fourth aspect are as described above with respect to the first to third aspects of the second embodiment.

[0202] In a fifth aspect of the second embodiment, the lipid component of the lipid-based carrier is Approximately 30 to 60 mol% of this compound, Approximately 0-30 mol% phospholipids, Approximately 15-50 mol% sterols, and Approximately 0-10 mol% PEGylated lipid Includes: The remainder of the features and exemplary features of the fifth aspect are as described above with respect to the first through fourth aspects of the second embodiment.

[0203] In a third embodiment, the present invention relates to a pharmaceutical composition comprising a lipid-based carrier as described herein and a pharmaceutically acceptable excipient, which may further comprise a therapeutic agent.

[0204] In a first aspect of the third embodiment, the pharmaceutical composition further comprises a therapeutic agent.

[0205] In a second aspect of the third embodiment, the therapeutic agent is a nucleic acid molecule. For example, the nucleic acid molecule is a nucleic acid selected from the group consisting of a plasmid, an immunostimulatory oligonucleotide, an antisense oligonucleotide, an antagomir, an aptamer, a deoxyribozyme (DNAzyme), and a ribozyme. The remainder of the features and exemplary features of the second aspect are as described above with respect to the first aspect of the third embodiment.

[0206] In a third aspect of the third embodiment, the nucleic acid molecule is DNA or RNA. The remainder of the features and exemplary features of the third aspect are as described above with respect to the first and second aspects of the third embodiment.

[0207] In a fourth aspect of the third embodiment, the nucleic acid molecule is DNA. In some embodiments, the DNA is linear, circular, single-stranded, or double-stranded. The remainder of the features and exemplary features of the fourth aspect are as described above with respect to the first through third aspects of the third embodiment.

[0208] In a fifth aspect of the third embodiment, the nucleic acid molecule is RNA. In some embodiments, the RNA is selected from the group consisting of mRNA, miRNA, siRNA or siRNA precursor, RNA aptamer, linear RNA, circular RNA, single-stranded RNA, double-stranded RNA, tRNA, microRNA (miRNA) or miRNA precursor, Dicer substrate small interfering RNA (dsiRNA), short hairpin RNA (shRNA), asymmetric interfering RNA (aiRNA), guide RNA (gRNA), lncRNA, ncRNA, sncRNA, rRNA, snRNA, piRNA, snoRNA, snRNA, scaRNA, exRNA, scaRNA, Y RNA, and hnRNA. In one embodiment, the RNA is mRNA. The remainder of the features and exemplary features of the fifth aspect are as described above with respect to the first through fourth aspects of the third embodiment.

[0209] In a sixth aspect of the third embodiment, the nucleic acid molecule comprises one or more nucleic acid analogs selected from the group consisting of phosphoramide, phosphorothioate, phosphorodithioate, O-methyl phosphoramidate, morpholino, locked nucleic acid (LNA), glycerol nucleic acid (GNA), threose nucleic acid (TNA), and peptide nucleic acid (PNA). The remainder of the features and exemplary features of the sixth aspect are as described above with respect to the first through fifth aspects of the third embodiment.

[0210] In a seventh aspect of the third embodiment, the therapeutic agent is a protein or small molecule drug. The remainder of the features and exemplary features of the seventh aspect are as described above with respect to the first through sixth aspects of the third embodiment.

[0211] In an eighth aspect of the third embodiment, the pharmaceutical composition is a vaccine. The remainder of the features and exemplary features of the eighth aspect are as described above for the first to seventh aspects of the third embodiment.

[0212] In a fourth embodiment, the present invention relates to a method of delivering a therapeutic agent to a subject.

[0213] In a first aspect of the fourth embodiment, the method comprises administering to the subject a pharmaceutical composition described herein.

[0214] Additional aspects, advantages, and features of the present invention are described herein, and in part will become apparent to those skilled in the art upon examination of the following or may be learned by practice of the invention. The inventions disclosed in this application are not limited to any particular set or combination of aspects, advantages, and features. Various combinations of the described aspects, advantages, and features are contemplated to comprise the inventions disclosed in this application.

[0215] definition As used in this specification and claims, unless the context dictates otherwise, the singular forms "a," "an," and "the" include plural references and a reference to a particular numerical value includes at least that particular value. For example, a reference to a "substance" is a reference to at least one of such substance and equivalents thereof known to those of ordinary skill in the art, and so forth.

[0216] When values ​​are expressed as approximations by use of the descriptor "about," it will be understood that the particular value forms another embodiment. In general, use of the term "about" indicates an approximation that may vary depending on the desired properties sought to be obtained by the disclosed subject matter and may be interpreted in the particular context in which it is used based on its function. Those skilled in the art will be able to interpret this as a matter of routine. In some cases, the number of significant figures used for a particular value may be one non-limiting method of determining the scope of the term "about." In other cases, the step numbers used in a series of values ​​may be used to determine the intended range that can be used for the term "about" for each value. Where present, all ranges are inclusive and combinable. That is, reference to values ​​specified in a range includes all values ​​within that range.

[0217] When lists are presented, it is to be understood that each individual element and every combination is to be construed as a separate embodiment unless otherwise stated. For example, a list of embodiments presented as "A, B, or C" should be construed to include the embodiments "A," "B," "C," "A or B," "A or C," "B or C," or "A, B, or C."

[0218] It should be understood that certain features of the invention, which are described herein in the context of separate embodiments for clarity, may also be provided in combination in a single embodiment. That is, unless clearly incompatible or excluded, each individual embodiment is deemed combinable with any other embodiment, and such combinations are deemed to be separate embodiments. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any subcombination. Finally, while an embodiment may be described as part of a series of steps or as part of a more general structure, each step may also be considered an independent embodiment in itself.

[0219] It should be noted that the claims may be drafted to exclude optional elements, and thus, this statement is intended to presuppose the use of exclusive language such as "only," "only," and the like in connection with the recitation of claimed elements, or the use of "negative" limitations.

[0220] As used herein, the term "compound" is intended to include all isomers and isotopes of the depicted structure, all pharmaceutically acceptable salts, solvates, or hydrates thereof, and all crystalline forms (e.g., crystalline polymorphs), mixtures of crystalline forms, or anhydrates or hydrates thereof.

[0221] "Isotopes" refer to atoms with the same atomic number but different mass numbers due to different numbers of neutrons in the nucleus. For example, isotopes of hydrogen include tritium ( 3 H) and deuterium ( 2H) are listed.

[0222] "Isomers." The compounds described herein, or pharmaceutically acceptable salts thereof, can include all isomers, such as geometric isomers, optical isomers based on asymmetric carbons, stereoisomers, tautomers, and the like. For example, the compounds can contain one or more stereocenters and thus give rise to geometric isomers (e.g., double bonds giving rise to geometric E / Z isomers), enantiomers, diastereomers (e.g., enantiomers (i.e., (+) or (-)) or cis / trans isomers), and other stereoisomeric configurations that can be defined in terms of absolute stereochemistry as (R)- or (S)- (e.g., in the case of sugar anomers) or (D)- or (L)- (e.g., in the case of amino acids). The present disclosure is intended to include all such possible isomers, as well as their racemic and optically pure forms. Optically active (+) and (-), (R)- and (S)-, or (D)- and (L)-isomers can be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques (e.g., chromatography and fractional crystallization). Conventional techniques for the preparation / isolation of individual enantiomers include chiral synthesis from suitable optically pure precursors, or resolution of the racemate (or racemate of a salt or derivative) using, for example, chiral high-pressure liquid chromatography (HPLC). Enantiomeric and stereoisomeric mixtures of compounds, as well as means for resolving them into their component enantiomers or stereoisomers, are known. When a compound described herein contains an olefinic double bond or other center of geometric asymmetry, unless otherwise specified, the compound is intended to include both the E and Z geometric isomers. Likewise, all tautomeric forms are intended to be included.

[0223] The terms "crystalline polymorph," "polymorph," or "crystalline form" refer to crystalline structures in which a compound (or a salt or solvate thereof) can crystallize in different crystal packing arrangements, all of which have the same elemental composition. Different crystalline forms typically differ in X-ray diffraction patterns, infrared spectra, melting points, density hardness, crystal shape, optical and electrical properties, stability, and solubility. Recrystallization solvent, crystallization rate, storage temperature, and other factors can cause one crystalline form to dominate. Crystalline polymorphs of a compound can be prepared by crystallization under various conditions.

[0224] The crystallization of the compound disclosed herein can produce solvate.As used herein, the term " solvate " refers to the aggregate that comprises one or more molecules of the ionized lipid of the present disclosure together with one or more solvent molecules.This solvent can be water, and in this case, solvate can be hydrate such as monohydrate, dihydrate, hemihydrate, sesquihydrate, trihydrate, tetrahydrate and the like.Alternatively, this solvent can be organic solvent.

[0225] As used herein, "pharmaceutically acceptable salts" refers to derivatives of the compounds of the present disclosure, in which the parent compound has been modified by converting an existing acid or base moiety into its salt form (e.g., by reacting a free base group with a suitable organic acid). The salts retain the biological effectiveness and properties of the free acids, which are not biologically or otherwise undesirable. Examples of pharmaceutically acceptable salts include, but are not limited to, inorganic or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as carboxylic acids; and the like. Representative acid addition salts include acetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptonate, hexanoate, hydrobromide, hydrochloride, hydroiodide, 2-hydroxybenzoate, benzoic acid salts, benzoates ... ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, toluenesulfonate, undecanoate, valerate, and the like. Representative alkali or alkaline earth metal salts include: sodium, lithium, potassium, calcium, magnesium, and the like, as well as non-toxic ammonium, quaternary ammonium, and amine cations, such as, but not limited to, ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, and the like. The pharmaceutically acceptable salts of the present disclosure include the conventional non-toxic salts of the parent compound formed, for example, from non-toxic inorganic or organic acids.Non-limiting examples of inorganic salts are ammonium, sodium, potassium, calcium, and magnesium salts. Salts derived from organic bases include, but are not limited to, salts of primary, secondary, and tertiary amines, substituted amines such as naturally occurring substituted amines, cyclic amines, and basic ion exchange resins such as ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, diethanolamine, ethanolamine, deanol, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, benethamine, benzathine, ethylenediamine, glucosamine, methylglucamine, theobromine, triethanolamine, tromethamine, purines, piperazine, piperidine, N-ethylpiperidine, polyamine resins, and the like. Non-limiting examples of organic bases are isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline, and caffeine. Pharmaceutically acceptable salts of compounds can be synthesized from parent compounds containing basic or acidic moieties by conventional chemical methods. Generally, such salts can be prepared by reacting the free acid form or free base form of these compounds with a stoichiometric amount of an appropriate base or acid in water or an organic solvent, or a mixture of the two, and generally, non-aqueous media such as ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred.Lists of suitable salts can be found in Remington's Pharmaceutical Sciences (17th ed., Mack Publishing Company, Easton, Pa., 1985, p. 1418); Pharmaceutical Salts: Properties, Selection, and Use (P.H. Stahl and C.G. Wermuth (eds.), Wiley-VCH, 2008); and Berge et al., Journal of Pharmaceutical Science, 66:1-19 (1977), each of which is incorporated herein by reference in its entirety.

[0226] "Pharmaceutically acceptable excipients," as used herein, refer to any component other than the compounds described herein that is substantially non-toxic and non-inflammatory in patients. Excipients may include, for example, adjuvants, anti-adherents, antioxidants, binders, coatings, compression aids, disintegrants, dyes (colorants), emollients, emulsifiers, fillers, solvents, diluents, film-forming agents or coatings, flavorings, flavor enhancers, flavorings, glidants (flow enhancers), surfactants, wetting agents, lubricants, preservatives, stabilizers, printing inks, adsorbents, suspending or dispersing agents, sweeteners, isotonicity agents, and hydration water, which are approved by the United States Food and Drug Administration as acceptable for use in humans or veterinary medicine. Exemplary excipients include, but are not limited to, butylated hydroxytoluene (BHT), calcium carbonate, calcium phosphate (dibasic), calcium stearate, croscarmellose, cross-linked polyvinylpyrrolidone, citric acid, crospovidone, cysteine, ethylcellulose, gelatin, hydroxypropyl cellulose, hydroxypropylmethylcellulose, lactose, magnesium stearate, maltitol, mannitol, methionine, methylcellulose, methylparaben, microcrystalline cellulose, polyethylene glycol, polyvinylpyrrolidone, povidone, pregelatinized starch, propylparaben, retinyl palmitate, shellac, silicon dioxide, sodium carboxymethylcellulose, sodium citrate, sodium starch glycolate, sorbitol, starch (corn), stearic acid, sucrose, talc, titanium dioxide, vitamin A, vitamin E (α-tocopherol), vitamin C, xylitol, and other species disclosed herein.

[0227] The term "halo" or "halogen" refers to any radical of fluorine, chlorine, bromine, or iodine.

[0228] The term "alkyl" refers to a hydrocarbon chain, which may be straight or branched, containing the indicated number of carbon atoms. Unless otherwise indicated, "alkyl" generally refers to a hydrocarbon chain having a C1-C 24 Alkyl (e.g., C1-C 15 Alkyl, C1-C 12 alkyl, C1-C8 alkyl, C1-C6 alkyl, C1-C4 alkyl, or C1-C3 alkyl). Unless stated otherwise specifically in the specification, an alkyl group is optionally substituted.

[0229] The term "alkylene" refers to a divalent alkyl group. An "alkylene chain" is a polymethylene group, i.e., -(CH2) n In the formula, n is a positive integer, for example, an integer of 1 to 6, 1 to 4, 1 to 3, 1 to 2, or 2 to 3.

[0230] The term "haloalkyl" refers to an alkyl in which one or more hydrogen atoms are replaced by halo, and includes alkyl moieties in which all hydrogens are replaced by halo (e.g., perfluoroalkyl). Alkyl and haloalkyl groups can be optionally interrupted by O, N, or S.

[0231] The term "aralkyl" refers to an alkyl moiety in which an alkyl hydrogen atom is replaced by an aryl group. Aralkyls include groups in which multiple hydrogen atoms are replaced by aryl groups. Examples of "aralkyls" include benzyl, 9-fluorenyl, benzhydryl, and trityl groups.

[0232] The term "alkenyl" refers to a straight or branched hydrocarbon chain, characterized by having one or more double bonds. Unless otherwise indicated, "alkenyl" generally refers to a C2-C8 alkenyl (e.g., a C2-C6 alkenyl, a C2-C4 alkenyl, or a C2-C3 alkenyl). Examples of typical alkenyl groups are allyl, propenyl, 2-butenyl, 3-hexenyl, and 3-octenyl groups.

[0233] The term "alkynyl" refers to a straight or branched hydrocarbon chain, characterized by having one or more triple bonds. Unless otherwise indicated, "alkynyl" generally refers to a C2-C8 alkynyl (e.g., a C2-C6 alkynyl, a C2-C4 alkynyl, or a C2-C3 alkynyl). Some examples of typical alkynyl groups are ethynyl, 2-propynyl, and 3-methylbutynyl, and propargyl. Sp 2 Carbon and Sp 3 The carbons can optionally serve as points of attachment for alkenyl and alkynyl groups, respectively.

[0234] The term "cycloalkyl" includes saturated and partially unsaturated, but not aromatic, cyclic hydrocarbon groups having 3 to 12 carbons (e.g., 3 to 8 carbons, 3 to 7 carbons, 3 to 6 carbons, or 3 to 5 carbons), where the cycloalkyl group can be further optionally substituted. Some examples of typical cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, and cyclooctyl.

[0235] The terms "heterocyclyl," "heterocycle," or "heterocyclic ring" refer to a non-aromatic 5- to 8-membered monocyclic, 8- to 12-membered bicyclic, or 11- to 14-membered tricyclic ring system having 1 to 3 heteroatoms in the monocyclic ring, 1 to 6 heteroatoms in the bicyclic ring, or 1 to 9 heteroatoms in the tricyclic ring. The heteroatoms may be selected from O, N, or S (e.g., carbon atoms and 1 to 3, 1 to 6, or 1 to 9 heteroatoms N, O, or S in the monocyclic, bicyclic, or tricyclic ring, respectively). For each ring of a heterocycle, 0, 1, 2, or 3 atoms may be substituted with substituents. When used with reference to a ring atom of a heterocycle, the term "nitrogen" includes a substituted nitrogen. As an example, in a saturated or partially unsaturated ring having 0-3 heteroatoms selected from oxygen, sulfur, or nitrogen, the nitrogen can be N (as in 3,4-dihydro-2H-pyrrolyl), NH (as in pyrrolidinyl), or +NR (as in N-substituted pyrrolidinyl). Examples of heterocyclyl groups include trizolyl, tetrazolyl, piperazinyl, pyrrolidinyl, dioxanyl, dioxolanyl, diazepinyl, oxazepinyl, thiazepinyl, morpholinyl, tetrahydrofuranyl, tetrahydrothiophenylpyrrolidinyl, piperidinyl, pyrrolinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl, oxazolidinyl, quinuclidinyl, and the like.

[0236] The term "heterocyclylalkyl" refers to an alkyl group substituted with a heterocyclyl, where the alkyl and heterocyclyl portions independently are optionally substituted.

[0237] The term "aryl" refers to a 6-carbon monocyclic aromatic ring system or a 10-carbon bicyclic aromatic ring system in which 0, 1, 2, 3, or 4 atoms of each ring may be substituted with a substituent. The term "aryl" may be used interchangeably with the term "aryl ring." Examples of aryl groups include phenyl, biphenyl, naphthyl, anthracyl, and the like, which may bear one or more substituents. Also included within the scope of the term "aryl," as used herein, are groups in which an aromatic ring is fused to one or more non-aromatic rings, such as indanyl, phthalimidyl, naphthymidyl, phenanthridinyl, or tetrahydronaphthyl, and the like. The term "arylalkyl" or "aralkyl" refers to an alkyl substituted with an aryl. The term "arylalkoxy" refers to an alkoxy substituted with an aryl.

[0238] The terms "heteroaryl" or "heteroar-" refer to aromatic 5- to 8-membered monocyclic, 8- to 12-membered bicyclic, or 11- to 14-membered tricyclic ring systems having 1 to 3 heteroatoms in the monocyclic ring, 1 to 6 heteroatoms in the bicyclic ring, and 1 to 9 heteroatoms in the tricyclic ring, selected from O, N, or S (e.g., carbon atoms and 1 to 3, 1 to 6, or 1 to 9 heteroatoms N, O, or S in the monocyclic, bicyclic, or tricyclic ring, respectively), where 0, 1, 2, 3, or 4 atoms of each ring can be substituted by a substituent. The terms also include groups in which a heteroaromatic ring is fused to one or more aryl, cycloalkyl, or heterocyclyl rings, where the radical or point of attachment is on the heteroaromatic ring. Examples of heteroaryl groups include pyrrolyl, pyridyl, pyridazinyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, oxadiazolyl, furanyl, imidazolyl, benzimidazolyl, pyrimidinyl, pyrazinyl, indolizinyl, thiophenyl or thienyl, quinolinyl, indolyl, thiazolyl, isothiazolyl, thiadiazolyl, purinyl, naphthyridinyl, pteridinyl, isoindo allyl, benzothienyl, benzofuranyl, dibenzofuranyl, indazolyl, benzothiazolyl, quinolyl, isoquinolyl, cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, 4H-quinolidinyl, carbazolyl, acridinyl, phenazinyl, phenothiazinyl, phenoxazinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, and pyrido[2,3-b]-1,4-oxazin-3(4H)-one, and the like.

[0239] The term "heteroarylalkyl" or "heteroaralkyl" refers to an alkyl substituted with a heteroaryl. The term "heteroarylalkoxy" refers to an alkoxy substituted with a heteroaryl.

[0240] Divalent alkyl, alkenyl, aryl, heteroaryl, cycloalkyl, and heterocyclyl radicals are formed by the removal of a hydrogen atom from an alkyl, alkenyl, aryl, heteroaryl, cycloalkyl, and heterocyclyl radical, respectively (or by the removal of two hydrogen atoms from an alkane, alkene, arene, heteroarene, cycloalkane, or heterocycle, respectively).

[0241] The term "alkoxy" refers to an -O-alkyl radical. The term "aminoalkyl" refers to an alkyl substituted with an amino. The term "mercapto" refers to an -SH radical. The term "thioalkoxy" refers to an -S-alkyl radical.

[0242] The term "oxo" refers to an oxygen atom which forms a carbonyl when attached to carbon, an N-oxide when attached to nitrogen, and a sulfoxide or sulfone when attached to sulfur.

[0243] The term "acyl" refers to an alkylcarbonyl, cycloalkylcarbonyl, arylcarbonyl, heterocyclylcarbonyl, or heteroarylcarbonyl substituent, any of which can be further substituted by substituents.

[0244] The term "substituent," when applied to any of the above groups (e.g., alkyl, cycloalkyl, heterocyclyl, alkenyl, alkynyl, aryl, heteroaryl, etc.), means that one or more hydrogen radicals in the group are replaced with the radical of the specified substituent, including, but not limited to, halo (e.g., F, CI, Br, or I), oxo (=O), hydroxyl (-OH), alkoxy, alkoxyalkyl, aralkoxy, alkyl, cycloalkyl, alkenyl, alkynyl, aryl, aralkylheterocyclyl, heterocyclyl, heteroaryl, thiol, alkylthio, arylthio, alkylthioalkyl, aryl ...

[0033] Examples of substituents include alkyl, alkylsulfonyl, alkylsulfonylalkyl, arylsulfonylalkyl, aryloxy, carboxyalkyl, alkoxycarbonylalkyl, aminocarbonylalkyl, acyl, aminocarbonyl, alkylaminocarbonyl, arylaminocarbonyl, alkoxycarbonyl, aryloxycarbonyl, haloalkyl, amino, trifluoromethyl, cyano, nitro, alkylamino, arylamino, alkylaminoalkyl, arylaminoalkyl, aminoalkylamino, aralkoxycarbonyl, sulfonylalkylaminolactam, alkylaminoheteroaryl, alkylaminoheterocyclosilyl, and aminosulfonamido. Exemplary substituents also include: -(C=O)OR s , -O(C=O)R s , -C(=O)R s , -OR s , -S(O) k R s , -S-SR s , -C(=O)SR s , -SC(=O)R s , -NR s R s’ , -R l C(=O)R s , -C(=O)R s R s’ , -R l C(=O)R s R s’ ;-OC(=O)R s R s’ , -Rl C(=O)OR s , -R l S(O) k R s R s’ , -R l S(O) k R s , and -S(O) k R s R s’ (In the formula, R s and R s’ are each independently H, C1 to C 15 alkyl, or cycloalkyl, and each R l is C1~C 15 alkylene, and k is 0, 1, or 2. In some embodiments, the substituents are C1-C 12 In some embodiments, the substituent is a C3-C8 cycloalkyl group. In some embodiments, the substituent is a C2-C3 alkenyl group. In some embodiments, the substituent is a halo group such as F or Br. In some embodiments, the substituent is an oxo group. In some embodiments, the substituent is a hydroxyl group. In some embodiments, the substituent is a hydroxyalkylene group (-R l In some embodiments, the substituent is an alkoxy group (-OR s In some embodiments, the substituent is a carboxyl group. In some embodiments, the substituent is an amino group (—NR s R s’ Suitable substituents also include divalent substituents on saturated carbon atoms, including, but not limited to: =O, =S, =NNR*2, =NNHC(O)R*, =NNHC(O)OR*, =NNHS(O)2R*, =NR*, =NOR*, -O(C(R*2)) 2~3 O- or -S(C(R*2)) 2~3 S—(wherein each independent occurrence of R* is hydrogen, substituted or unsubstituted C 1~6alkyl, or an unsubstituted 5-6 membered saturated or partially unsaturated ring, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur).

[0245] As used herein, "expression" of a nucleic acid sequence refers to translation of mRNA into a polypeptide or protein and / or post-translational modification of a polypeptide or protein.

[0246] As used herein, "encapsulation efficiency" refers to the percentage of encapsulated cargo (e.g., therapeutic and / or prophylactic agent) successfully incorporated (e.g., encapsulated or otherwise associated) into a lipid-based carrier or lipid nanoformulation relative to the initial total amount of therapeutic and / or prophylactic agent provided. For example, if 97 mg of therapeutic and / or prophylactic agent is encapsulated in a lipid-based carrier or lipid nanoformulation out of a total of 100 mg of therapeutic and / or prophylactic agent initially provided, the encapsulation efficiency may be expressed as 97%. Encapsulation efficiency may be used to indicate the efficiency of loading encapsulated cargo (e.g., nucleic acid molecules) into a lipid-based carrier or lipid nanoformulation using a particular formulation method and recipe.

[0247] As used herein, the term "lipid component" refers to a component in a lipid carrier or lipid nanoformulation, such as one or more lipids. For example, the lipid component may include one or more of cationic / anionic / ionic / zwitterionic lipids, neutral lipids, PEGylated lipids, or other lipids such as phospholipids.

[0248] The term "lipid nanoformulation" generally refers to lipid vesicles that carry a cargo (e.g., an encapsulated therapeutic agent such as a nucleic acid) at least partially within a protective layer of lipid and can deliver this cargo to a desired target site. Exemplary lipid nanoformulations described herein include liposomes and lipid nanoparticles (LNPs).

[0249] As used herein, the terms "lipid carrier" and "lipid nanoformulation" may be used interchangeably to refer to compositions comprising one or more lipids, and include lipid nanoparticles (LNPs), liposomes (e.g., lipid vesicles), and lipoplexes. These compositions are typically on the order of micrometers or less in size and may include a lipid bilayer.

[0250] The term "formulation recipe," as used herein, is meant to define the molar ratio of components in a lipid nanoformulation that are mixed with an encapsulated molecule (e.g., a nucleic acid molecule) and formulated together to produce a lipid nanoformulation (e.g., an LNP composition).

[0251] The term "liposome," as used herein, refers to a composition comprising an outer lipid membrane (e.g., a single lipid bilayer, known as a unilamellar liposome, or multiple lipid bilayers, known as a multilamellar liposome) surrounding an internal aqueous space that may contain cargo. See, e.g., Cullis et al., Biochim. Biophys Acta, 559:399-420 (1987), incorporated herein by reference in its entirety. Unilamellar liposomes generally range in diameter from about 20 to about 400 nanometers (nm), from about 50 to about 300 nm, from about 100 to about 200 nm, or from about 300 to about 400 nm. Multilamellar liposomes typically range in diameter from about 1 to about 10 μm and may comprise two to several hundred concentric lipid bilayers alternating with layers of aqueous phase.

[0252] The term "lipid nanoparticle" or "LNP" refers to a composition comprising lipids (e.g., ionic (e.g., cationic or anionic), zwitterionic, or ionizable lipids) for encapsulation of cargo. LNPs may also comprise neutral lipids, such as phospholipid molecules belonging to the phosphatidylcholine (PC) class; sterols, such as cholesterol; and polyethylene glycol (PEG). LNPs can be taken up into cells via endocytosis, and the ionizable ability of lipids at low pH allows for endosomal escape, thereby allowing release of cargo into the cytoplasm. LNPs are liposome-like structures. However, LNPs may not have a continuous bilayer; some LNPs have micelle-like structures (e.g., [ka] ) enclosing an interior, which may have a non-aqueous core. Exemplary lipid nanoparticle compositions are formulations of ionizable lipids, sterols (or hydrophobic molecules), structural lipids, e.g., phospholipids, polyethylene glycol (PEG) lipids, and potentially additional components (see Nature Nanotechnology 15:313-320 (2020), incorporated herein by reference in its entirety), or single molecules containing a combination of ionizable lipids, sterols, structural phospholipids, and shielding groups (see Nature Materials 20:701-710 (2021), incorporated herein by reference in its entirety). These components can be mixed with cargo molecules (e.g., nucleic acid molecules such as mRNA) to be formulated into the LNP composition.

[0253] The term "ionizable lipid" refers to a molecule having both an ionizable component and a lipophilic component. "Ionizable" means that a group (e.g., a head group) contained in the lipid can be ionized under given conditions (e.g., pH), e.g., can dissociate to generate one or more charged species. For example, an ionizable lipid can carry a net positive charge at a selected pH, such as physiological pH (e.g., a pH of about 7.0). In some embodiments, the hydrophilic component contains an ionizable amine. In some embodiments, the hydrophobic component contains one or more linear or branched lipids.

[0254] As used herein, the terms "PEG-lipid" and "PEGylated lipid" are synonymous and refer to a lipid that includes a polyethylene glycol moiety.

[0255] As used herein, "phospholipid" refers to a lipid comprising a phosphate moiety and one or more carbon chains, such as unsaturated fatty acid chains. Phospholipids may comprise one or more multiple (e.g., double or triple) bonds (e.g., one or more unsaturations). Certain phospholipids may facilitate fusion with membranes. For example, cationic phospholipids may interact with one or more negatively charged phospholipids in membranes (e.g., cell membranes or intracellular membranes). The fusion of phospholipids with membranes may allow one or more components of lipid-containing compositions to pass through the membrane, for example, allowing one or more components to be delivered to cells.

[0256] As used herein, the term "size" refers to the hydrodynamic diameter of a lipid nanoparticle population. Measurement of the size of a lipid nanoformulation can be used to indicate the size and population distribution (polydispersity index, PDI) of the composition.

[0257] As used herein, "polydispersity index" refers to the ratio of the weight-average molar mass to the number-average molar mass, Mn, which describes the uniformity of the particle size distribution of a system. A small value (e.g., less than 0.3) indicates a narrow particle size distribution.

[0258] As used herein, the term "apparent pKa" refers to the pH at which 50% of a lipid nanoformulation (e.g., LNP) is protonated. This can be used as an indicator of the pH range at which a lipid nanoformulation (e.g., LNP) becomes protonated and thus initiates the endosomal escape process in nucleotide delivery.

[0259] As used herein, the term "zeta potential" refers to the electrokinetic potential of lipids, for example, in a lipid nanoformulation (e.g., an LNP composition). Zeta potential can describe the surface charge of an LNP composition. Zeta potential is useful for predicting organ tropism and potential interactions with serum proteins.

[0260] As used herein, "administration method" can include both systemic delivery and local delivery. "Systemic delivery" means that a useful amount of a drug, such as a therapeutic agent, is delivered to most parts of the body. Systemic delivery of liposomes or LNPs can be performed by any means known in the art, such as intravenous, intraarterial, intramuscular, intradermal, subcutaneous, and intraperitoneal delivery. In some embodiments, systemic delivery of lipid nanoparticles is by intravenous delivery. "Local delivery," as used herein, refers to the direct delivery of a drug to a target site within an organism. For example, a drug can be delivered locally by direct injection into a disease site such as a tumor, another target site such as a site of inflammation, or a target organ such as the liver, heart, pancreas, kidney, and the like. Local delivery can also include topical application or local injection techniques, such as intramuscular, subcutaneous, or intradermal injection. Local delivery does not exclude systemic pharmacological effects.

[0261] As used herein, the term "polypeptide" or "polypeptide of interest" refers to a polymer of amino acid residues, typically joined by peptide bonds, that may be naturally produced (e.g., isolated or purified) or synthetically produced.

[0262] "Nucleic acid" is meant to define an oligonucleotide or polynucleotide sequence. Non-limiting examples of oligonucleotides or polynucleotides are DNA, plasmid DNA, self-amplifying RNA, mRNA, siRNA, and tRNA. The term also encompasses RNA / DNA hybrids. Nucleotides are typically linked in nucleic acids by phosphodiester bonds, but the term "nucleic acid" also encompasses nucleic acid analogs with other types of linkages or backbones, such as phosphoamide, phosphorothioate, phosphorodithioate, O-methylphosphoramidate, morpholino, locked nucleic acid (LNA), glycerol nucleic acid (GNA), threose nucleic acid (TNA), and peptide nucleic acid (PNA) linkages or backbones, among others. Nucleic acids can be single-stranded, double-stranded, or contain portions of both single-stranded and double-stranded sequences. Nucleic acids can contain any combination of deoxyribonucleotides and ribonucleotides, and any combination of bases (e.g., adenine, thymine, cytosine, guanine, uracil, and modified or non-standard bases (e.g., hypoxanthine, xanthine, 7-methylguanine, 5,6-dihydrouracil, 5-methylcytosine, and 5-hydroxymethylcytosine).

[0263] As used herein, "RNA" refers to a ribonucleic acid, which may or may not occur naturally. For example, RNA may contain modified and / or non-naturally occurring components, such as one or more nucleobases, nucleosides, nucleotides, or linkers. RNA may contain a cap structure, a chain-terminating nucleoside, a stem-loop, a polyA sequence, and / or a polyadenylation signal. RNA may have a nucleotide sequence encoding a polypeptide of interest. For example, RNA may be messenger RNA (mRNA). Translation of an mRNA encoding a specific polypeptide (e.g., in vivo translation of an mRNA in a mammalian cell) may produce the encoded polypeptide. RNA may be selected from the non-limiting group consisting of small interfering RNA (siRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), dicer substrate RNA (dsRNA), short hairpin RNA (shRNA), mRNA, and mixtures thereof.

[0264] As used herein, the term "subject" or "patient" refers to any organism to which a composition according to the present disclosure can be administered, for example, for experimental, diagnostic, prophylactic, and / or therapeutic purposes. Typical subjects include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and humans) and / or plants. In some embodiments, the subject is a mammal, such as a human. In some embodiments, the subject is a veterinary or farm animal, a domestic animal or pet, or an animal used in clinical research. In some embodiments, the subject is an adult, i.e., 18 years of age or older. In some embodiments, the subject is a pediatric subject, i.e., under 18 years of age.

[0265] The term "therapeutic agent" or "prophylactic agent" refers to any agent that has a therapeutic, diagnostic, and / or prophylactic effect and / or induces a desired biological and / or pharmacological effect when administered to a subject. Therapeutic agents are also referred to as "active agents." Such agents include, but are not limited to, cytotoxins, radioactive ions, chemotherapeutic agents, small molecule drugs, proteins, and nucleic acids.

[0266] As used herein, the term "effective amount" or "therapeutically effective amount" refers to an amount of an active agent or therapeutic agent (e.g., a nucleic acid, small molecule drug, therapeutic peptide or protein composition, diagnostic agent, prophylactic agent, etc.) delivered that is sufficient to treat, ameliorate the symptoms of, diagnose, prevent, and / or delay the onset of an infection, disease, disorder, and / or condition when administered to a subject suffering from or susceptible to the infection, disease, disorder, and / or condition. Effective amounts will vary depending on the route of administration, excipient use, and co-use with other active agents, as will be recognized by those of skill in the art. Such amounts will, of course, depend on the particular condition being treated, the severity of the condition, individual patient parameters (e.g., age, physical condition, size, sex, and weight), duration of treatment, the nature of concurrent therapy (if any), the specific route of administration, and similar factors within the knowledge and expertise of a medical practitioner. These factors are known to those of skill in the art and can be addressed with no more than routine experimentation. Generally, it is preferred to use the maximum dose of each component or combination thereof, i.e., the safest dose according to sound medical judgment, however, it will be understood by those skilled in the art that a patient may insist on a lower or tolerated dose for medical, psychological, or virtually any other reason.

[0267] Lipid nanoformulations / lipid-based carriers In some embodiments, the compounds described herein are formulated in a lipid-based carrier (or lipid nanoformulation). In some embodiments, the lipid-based carrier (or lipid nanoformulation) is a liposome or a lipid nanoparticle (LNP). In one embodiment, the lipid-based carrier is an LNP.

[0268] In some embodiments, the lipid-based carrier (or lipid nanoformulation) comprises a cationic lipid (e.g., an ionizable lipid), a non-cationic lipid (e.g., a phospholipid), a structured lipid (e.g., cholesterol), and a PEG-modified lipid. In some embodiments, the lipid-based carrier (or lipid nanoformulation) contains one or more compounds described herein, or pharmaceutically acceptable salts thereof.

[0269] All of the above statements relating to aspects of lipid compounds such as those encompassed by formula (IA) or (AL-GI) and all of the embodiments discussed in the above aspects are all applicable to these aspects of the invention relating to lipid-based carriers (or lipid nanoformulations).

[0270] As described herein, suitable compounds for use in lipid-based carriers (or lipid nanoformulations) include all isomers and isotopes of the compounds described above, as well as all pharmaceutically acceptable salts, solvates, or hydrates thereof, and all crystalline forms, mixtures of crystalline forms, and anhydrates or hydrates.

[0271] In addition to one or more compounds described herein, the lipid-based carrier (or lipid nanoformulation) may further comprise a second lipid. In some embodiments, the second lipid is a cationic lipid, a non-cationic (e.g., neutral, anionic, or zwitterionic) lipid, or an ionizable lipid.

[0272] One or more naturally occurring and / or synthetic lipid compounds may be used in the preparation of the lipid-based carrier (or lipid nanoformulation).

[0273] Lipid-based carriers (or lipid nanoformulations) can contain positively charged (cationic) lipids, neutral lipids, negatively charged (anionic) lipids, or combinations thereof.

[0274] In some embodiments, the lipid nanoparticles of the present invention may be conjugated to a targeting moiety (e.g., an antibody or antigen-binding fragment thereof) via a linking group. The lipid nanoparticles of the present invention may use a variety of linking groups known in the art, which may include one or more of the following: optionally substituted alkylene, optionally substituted heteroalkylene, optionally substituted alkenylene, optionally substituted heteroalkenylene, optionally substituted alkynylene, optionally substituted heteroalkynylene, optionally substituted cycloalkylene, optionally substituted heterocycloalkylene, optionally substituted arylene, optionally substituted heteroarylene, peptide moiety, dipeptide moiety, -(C=O)-, disulfide, hydrazone, thioester, sulfone, sulfoxide, thiosulfinate, thiosulfonate, sulfate, sulfonate, sulfonylurea, ether, thioether, ester, amide, carbonate, carbamate, urea, sulfamide, succinimide, maleimide, phosphate, diphosphate, triazole, or saccharide, or a combination thereof. Suitable linking groups are described, for example, in WO 2024 / 015229, WO 2024 / 006272, and WO 2023 / 225359.

[0275] Cationic lipids (positively charged) and ionizable lipids In some embodiments, the lipid-based carrier (or lipid nanoformulation) comprises one or more cationic lipids (e.g., cationic lipids that can exist in a positively charged or neutral form depending on the pH) or amine-containing lipids that can be readily protonated. In some embodiments, the cationic lipids are, for example, lipids that can be positively charged under physiological conditions.

[0276] Exemplary cationic lipids include one or more amine groups with a positive charge. Examples of positively charged (cationic) lipids include, but are not limited to, N,N'-dimethyl-N,N'-dioctadecylammonium bromide (DDAB) and chloride (DDAC), N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), 3β-[N',N'-dimethylaminoethyl)carbamoyl)cholesterol (DC-chol), 1,2-dioleoyloxy-3-[trimethylammonio]-propane (DOTAP), 1,2-dioctadecyloxy-3-[trimethylammonio]-propane (DSTAP), and 1,2-dioleoyloxypropyl-3-dimethyl-hydroxyethylammonium chloride (DORI), N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N -dimethyl-2,3-dioleyloxy)propylamine (DODMA), 1,2-dioleoyl-3-dimethylammonium-propane (DODAP), 1,2-dioleoylcarbamyl-3-dimethylammonium-propane (DOCDAP), 1,2-dilineoyl-3-dimethylammonium-propane (DLINDAP), 3-dimethylamino-2-(cholest-5-ene-3-beta-oxybutan-4-oxy)-1-(cis,cis-9,12-octadecadienoxy)propane (CLinDMA), 2-[5'-(cholest-5-ene-3-beta-oxy)-3'-oxapentoxy)-3-dimethyl-1-(cis,cis-9',12'-octadecadienoxy)propane (CpLinDMA), N,N-dimethyl-3,4-dioleyloxybenzylamine (DMOBA), as well as compounds such as those manufactured by Martin et al. et al., Current Pharmaceutical Design, pages 1-394, which is incorporated herein by reference in its entirety. In some embodiments, the lipid-based carrier (or lipid nanoformulation) comprises multiple types of cationic lipids.

[0277] In some embodiments, the lipid-based carrier (or lipid nanoformulation) comprises a cationic lipid having an effective pKa greater than 6.0, hi some embodiments, the lipid-based carrier (or lipid nanoformulation) further comprises a second cationic lipid having an effective pKa different from (e.g., greater than) the first effective pKa of the first cationic lipid.

[0278] In some embodiments, cationic lipids that may be used in lipid-based carriers (or lipid nanoformulations) include, for example, those described in Table 4 of WO 2019 / 217941, which is incorporated by reference.

[0279] In some embodiments, the cationic lipid is an ionizable lipid (e.g., a lipid that is protonated at low pH but remains neutral at physiological pH). In some embodiments, the lipid-based carrier (or lipid nanoformulation) may include one or more additional ionizable lipids that are different from the ionizable lipids described herein. Exemplary ionizable lipids include, but are not limited to: [ka] (See WO 2017 / 004143 A1, which is incorporated by reference in its entirety.)

[0280] In some embodiments, the lipid-based carrier (or lipid nanoformulation) further comprises one or more compounds described in WO 2021 / 113777, the entirety of which is incorporated herein by reference (e.g., a lipid of Formula (3), such as the lipids in Table 3 of WO 2021 / 113777).

[0281] In one embodiment, the ionizable lipid is a lipid disclosed in Hou, X., et al. Nat Rev Mater 6, 1078-1094 (2021). https: / / doi.org / 10.1038 / s41578-021-00358-0 (e.g., L319, C12-200, and DLin-MC3-DMA), which is incorporated herein by reference in its entirety.

[0282] Examples of other ionizable lipids that may be used in lipid-based carriers (or lipid nanoformulations) include, but are not limited to, one or more of the following formulas: X of U.S. Patent Application Publication No. 2016 / 0311759; I of U.S. Patent Application Publication No. 20150376115 or U.S. Patent Application Publication No. 2016 / 0376224; Compound 5 or Compound 6 of U.S. Patent Application Publication No. 2016 / 0376224; I, IA, or II of U.S. Patent Application Publication No. 9,867,888; I, II, or III of U.S. Patent Application Publication No. 2016 / 0151284; or U.S. Patent Application Publication No. 2017 / 021096 No. 7, I, IA, II, or IIA; No. 2015 / 0140070, Ic; No. 2013 / 0178541, A; No. 2013 / 0303587 or No. 2013 / 0123338, I; No. 2015 / 0141678, I; No. 2015 / 0239926, II, III, IV, or V; No. 2017 / 0119904, I; No. 2017 / 117528, I or II; U.S. Patent Application Publication No. 2012 / 0149894, A; U.S. Patent Application Publication No. 2015 / 005 7373 A; WO 2013 / 116126 A; U.S. Patent Application Publication No. 2013 / 0090372 A; WO 2013 / 0274523 A; WO 2013 / 0274504 A; WO 2013 / 0053572 A; WO 2013 / 016058 A; WO 2012 / 162210 A; U.S. Patent Application Publication No. 2008 / 042973 I; U.S. Patent Application Publication No. 2012 / 01287670 I, II, III, or IV; U.S. Patent Application Publication No. 2014 / 02 No. 00257, I or II; No. 2015 / 0203446, I, II, or III; No. 2015 / 0005363, I or III; No. 2014 / 0308304, I, IA, IB, IC, ID, II, IIA, IIB, IIC, IID, or III-XXIV; No. 2013 / 0338210; No. 2009 / 132131, I, II, III, or IV; U.S. Patent Application Publication No. 2012 / 01011478, A; U.S. Patent Application Publication No. 2012 / 0027796, I or XXXV;No. 2012 / 0058144, No. XIV or XVII; No. 2013 / 0323269; No. 2011 / 0117125, No. I; No. 2011 / 0256175, No. I, II, or III; No. 2012 / 0202871, No. I, II, III, IV, V, VI, VII, VIII, IX, X, XI, or XII; No. 2011 / 0076335, No. I, II, III, IV, V, VI, VII, VIII, X, XII, XIII, XIV, XV, or XVI; No. 2006 / 008378, No. I or or II; I (e.g., ATX-002) of WO 2015 / 074085; I of WO 2013 / 0123338; ​​I or XAYZ of WO 2015 / 0064242; XVI, XVII, or XVIII of WO 2013 / 0022649; I, II, or III of WO 2013 / 0116307; I, II, or III of WO 2013 / 0116307; I or II of WO 2010 / 0062967; I to X of WO 2013 / 0189351; WO 2014 / 003 No. 9032, I; No. 2018 / 0028664, V; No. 2016 / 0317458, I; No. 2013 / 0195920, I; U.S. Pat. No. 10,221,127, 5, 6, or 10; No. WO 2018 / 081480, III-3; No. 2020 / 081938, I-5 or I-8; No. 2015 / 199952, I (e.g., compound 6 or 22) and Table 1; No. 9,867,888, 18 or 25; U.S. Patent Application Publication No. 2019 / 0 No. 136231 A; WO 2020 / 219876 II; U.S. Patent Application Publication No. 2012 / 0027803 I; U.S. Patent Application Publication No. 2019 / 0240349 OF-02; U.S. Patent Application No. 10,086,013 23; Miao et al. (2020) cKK-E12 / A6; WO 2010 / 053572 C12-200; Dahlman et al. (2017) 7C1; Whitehead et al. 304-O13 or 503-O13; U.S. Patent Application No. 9,708,628 TS-P4C2;I of WO 2020 / 106946; I of WO 2020 / 106946; (1), (2), (3), or (4) of WO 2021 / 113777; and any one of Tables 1-16 of WO 2021 / 113777, all of which are incorporated by reference in their entirety.

[0283] In some embodiments, the lipid-based carrier (or lipid nanoformulation) further includes a biodegradable ionizable lipid, such as (9Z,12Z)-3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl octadeca-9,12-dienoate (also referred to as 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate)). See, for example, WO 2019 / 067992, WO 2017 / 173054, WO 2015 / 095340, and WO 2014 / 136086, which are incorporated by reference in their entireties.

[0284] Non-cationic lipids (e.g., phospholipids) In some embodiments, the lipid-based carrier (or lipid nanoformulation) further comprises one or more non-cationic lipids. In some embodiments, the non-cationic lipid is a phospholipid. In some embodiments, the non-cationic lipid is a substitution or replacement for a phospholipid. In some embodiments, the non-cationic lipid is a negatively charged (anionic) lipid.

[0285] Exemplary non-cationic lipids include, but are not limited to, distearoyl-sn-glycero-phosphoethanolamine, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidylethanolamine amine (DSPE), monomethyl-phosphatidylethanolamine (e.g., 16-O-monomethyl PE), dimethyl-phosphatidylethanolamine (e.g., 16-O-dimethyl PE), 18-1-trans PE, 1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE), hydrogenated soy phosphatidylcholine (HSPC), egg phosphatidylcholine (EPC), dioleoylphosphatidylserine (DOPS), sphingomyelin Sodium acetylcholine (SM), dimyristoyl phosphatidylcholine (DMPC), dimyristoyl phosphatidylglycerol (DMPG), distearoyl phosphatidylglycerol (DSPG), dierucoyl phosphatidylcholine (DEPC), palmitoyl oleyl phosphatidylglycerol (POPG), dielaidoyl phosphatidylethanolamine (DEPE), 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC), sodium 1,2-ditetradecanoyl-sn-glycero-3-phosphate (DMPA), phosphatidylcholine (lecithin), phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM), phosphatidylethanolamine (cephalin), cardiolipin, phosphatidic acid, cerebroside, dicetyl phosphate, lysophosphatidylcholine, dilinoleoylphosphatidylcholine, or mixtures thereof. It is understood that other diacylphosphatidylcholine and diacylphosphatidylethanolamine phospholipids can also be used. The acyl group in these lipids is preferably C, 10 ~C 24 The acyl group is derived from a fatty acid having a carbon chain, such as lauroyl, myristoyl, paimitoyl, stearoyl, or oleoyl. Additional exemplary lipids include, but are not limited to, those described in Kim et al. (2020) dx.doi.org / 10.1021 / acs.nanolett.0c01386, which is incorporated herein by reference in certain embodiments. In some embodiments, such lipids include plant lipids (e.g., DGTS), which have been found to improve liver transfection with mRNA. In some embodiments, saturated long-chain phosphatidylcholines are less permeable and more stable in vivo than their unsaturated counterparts.

[0286] In some embodiments, the lipid-based carrier (or lipid nanoformulation) may include a combination of distearoylphosphatidylcholine / cholesterol, dipalmitoylphosphatidylcholine / cholesterol, dimyristoylphosphatidylcholine / cholesterol, 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) / cholesterol, or egg sphingomyelin / cholesterol.

[0287] Other examples of suitable non-cationic lipids include, but are not limited to, non-phospholipids such as stearylamine, dodecylamine, hexadecylamine, acetyl palmitate, glycerol ricinoleate, hexadecyl stearate, isopropyl myristate, amphoteric acrylic polymers, triethanolamine-lauryl sulfate, alkyl-aryl sulfate polyethyloxylated fatty acid amides, dioctadecyldimethylammonium bromide, ceramide, sphingomyelin, and the like. Other non-cationic lipids are described in WO 2017 / 099823 or U.S. Patent Application Publication No. 2018 / 0028664, which are incorporated herein by reference in their entireties.

[0288] In some embodiments, the lipid-based carrier (or lipid nanoformulation) further comprises one or more non-cationic lipids that are oleic acid or compounds of Formula I, II, or IV of U.S. Patent Application Publication No. 2018 / 0028664, the entirety of which is incorporated herein by reference.

[0289] The amount of non-cationic lipid present can be, for example, 0-30% (mol) of the total lipid components present, hi some embodiments, the amount of non-cationic lipid present is 5-20% (mol) or 10-15% (mol) of the total lipid components present.

[0290] In some embodiments, the lipid-based carrier (or lipid nanoformulation) further comprises a neutral lipid, wherein the molar ratio of ionizable lipid to neutral lipid ranges from about 2:1 to about 8:1 (e.g., about 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, or 8:1).

[0291] In some embodiments, the lipid-based carrier (or lipid nanoformulation) does not include any phospholipids.

[0292] In some embodiments, the lipid-based carrier (or lipid nanoformulation) may further comprise one or more phospholipids and, optionally, one or more additional molecules of similar molecular shape and size having both hydrophobic and hydrophilic portions (e.g., cholesterol).

[0293] structured lipids The lipid-based carriers (or lipid nanoformulations) described herein may further comprise one or more structured lipids. As used herein, the term "structured lipid" refers to a sterol (e.g., cholesterol), and also refers to lipids containing a sterol moiety.

[0294] The incorporation of structural lipids into lipid nanoparticles can help alleviate the aggregation of other lipids in the particles. The structural lipids can be selected from the group including, but not limited to, cholesterol or cholesterol derivatives, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, tomatine, ursolic acid, alpha-tocopherol, hopanoids, plant sterols, steroids, and mixtures thereof. In some embodiments, the structural lipid is a sterol. In certain embodiments, the structural lipid is a steroid. In certain embodiments, the structural lipid is cholesterol. In certain embodiments, the structural lipid is a cholesterol analog. In certain embodiments, the structural lipid is alpha-tocopherol.

[0295] In some embodiments, structured lipids may be incorporated into lipid-based carriers at molar ratios ranging from about 0.1 to 1.0 (cholesterol phospholipids).

[0296] In some embodiments, the sterol, if present, may include one or more of cholesterol or cholesterol derivatives, such as those described in WO 2009 / 127060 or U.S. Patent Application Publication No. 2010 / 0130588, which are incorporated by reference in their entireties. Additional exemplary sterols include plant sterols, such as those described in Eygeris et al. (2020), Nano Lett. 2020;20(6):4543-4549, which are incorporated by reference in their entireties.

[0297] In some embodiments, the structured lipid is a cholesterol derivative.Non-limiting examples of cholesterol derivatives include: polar analogs, such as 5a-cholestanol, 53-coprostanol, cholesteryl-(2'-hydroxy)-ethyl ether, cholesteryl-(4'-hydroxy)-butyl ether, and 6-ketocholestanol; non-polar analogs, such as 5a-cholestan, cholestenone, 5a-cholestanone, 5p-cholestanone, and cholesteryl decanoate; and mixtures thereof.In some embodiments, the cholesterol derivative is a polar analog, such as cholesteryl-(4'-hydroxy)-butyl ether.Exemplary cholesterol derivatives are described in International Publication No. 2009 / 127060 and US Patent Publication No. 2010 / 0130588, each of which is incorporated herein by reference in its entirety.

[0298] In some embodiments, the lipid-based carrier (or lipid nanoformulation) further comprises a sterol in an amount of 0-50 mol% (e.g., 0-10 mol%, 10-20 mol%, 20-50 mol%, 20-30 mol%, 30-40 mol%, or 40-50 mol%) of the total lipid component.

[0299] Polymers and Polyethylene Glycol (PEG)-Lipids In some embodiments, the lipid-based carrier (or lipid nanoformulation) may comprise one or more polymers or copolymers, for example, poly(lactic-co-glycolic acid) (PFAG) nanoparticles.

[0300] In some embodiments, the lipid-based carrier (or lipid nanoformulation) may include one or more polyethylene glycol (PEG) lipids. Examples of useful PEG lipids include, but are not limited to, 1,2-diacyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-350] (mPEG 350 PE); 1,2-diacyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-550] (mPEG 550 PE); 1,2-diacyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-750] (mPEG 750 PE); 1,2-diacyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-1000] (mPEG 1000 PE); 1,2-diacyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (mPEG 2000 PE); 1,2-diacyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-3000] (mPEG 3000 PE); 1,2-diacyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-5000] (mPEG 5000 PE); N-acyl-sphingosine-1-[succinyl(methoxypolyethylene glycol)750] (mPEG 750 ceramide); N-acyl-sphingosine-1-[succinyl(methoxypolyethylene glycol)2000] (mPEG 2000 ceramide); and N-acyl-sphingosine-1-[succinyl(methoxypolyethylene glycol)5000] (mPEG 5000 ceramide). In some embodiments, the PEG lipid is a polyethylene glycol-diacylglycerol (i.e., polyethylene glycol diacylglycerol (PEG-DAG), PEG-cholesterol, or PEG-DMB) conjugate.

[0301] In some embodiments, the lipid-based carrier (or nanoformulation) includes one or more conjugated lipids (e.g., PEG-conjugated lipids or lipids conjugated to polymers as described in Table 5 of WO 2019 / 217941, the entirety of which is incorporated herein by reference). In some embodiments, the one or more conjugated lipids are formulated with one or more ionic lipids (e.g., non-cationic lipids such as neutral, anionic, or zwitterionic lipids); and one or more sterols (e.g., cholesterol). In some embodiments, the conjugated lipid molecules may be used to inhibit aggregation and / or provide steric stabilization of the lipid nanoparticles.

[0302] PEG conjugates can include PEG-dilaurylglycerol (C12), PEG-dimyristylglycerol (C14), PEG-dipalmitoylglycerol (C16), PEG-disterylglycerol (C18), PEG-dilaurylglycamide (C12), PEG-dimyristylglycamide (C14), PEG-dipalmitoylglycamide (C16), and PEG-disterylglycamide (C18). PEG conjugates can also include PEG-cholesterol (1-[8'-(cholest-5-ene-3[beta]-oxy)carboxamido-3',6'-dioxaotanyl]carbamoyl-[omega]-methyl-poly(ethylene glycol), PEG-DMB (3,4-ditetradecoxylbenzyl-[omega]-methyl-poly(ethylene glycol) ether), and 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000]. In some embodiments, the PEG-lipid comprises PEG-DMG, 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000].

[0303] In some embodiments, the conjugated lipid, if present, may comprise one or more of the following: PEG-diacylglycerol (DAG) (e.g., 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG-DMG)), PEG-dialkyloxypropyl (DAA), PEG-phospholipid, PEG-ceramide (Cer), PEGylated phosphatidylethanolamine (PEG-PE), PEG succinate diacylglycerol (PEGS-DAG) (e.g., 4 -O-(2',3'-di(tetradecanoyloxy)propyl-1-O-(w-methoxy(polyethoxy)ethyl)butanedioate (PEG-S-DMG)), PEG dialkoxypropylcarbam, N-(carbonyl-methoxypolyethylene glycol 2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine sodium salt, and those set forth in Table 2 of WO 2019 / 051289, which is incorporated herein by reference in its entirety, as well as combinations of the foregoing.

[0304] Further exemplary PEG-lipid conjugates are described, for example, in U.S. Pat. Nos. 5,885,613, 6,287,591, U.S. Patent Application Publication Nos. 2003 / 0077829, 2003 / 0077829, 2005 / 0175682, 2008 / 0020058, 2011 / 0117125, 2010 / 0130588, 2016 / 0376224, 2017 / 0119904, 2018 / 0028664, and WO 2017 / 099823, all of which are incorporated herein by reference in their entireties.

[0305] In some embodiments, the PEG-lipid is a compound of Formula III, III-aI, III-a-2, III-b-1, III-b-2, or V of U.S. Patent Application Publication No. 2018 / 0028664, the entire contents of which are incorporated herein by reference. In some implementations, the PEG-lipid is of Formula II of U.S. Patent Application Publication No. 2015 / 0376115 or U.S. Patent Application Publication No. 2016 / 0376224, both of which are incorporated herein by reference in their entireties. In some embodiments, the PEG-DAA conjugate can be, for example, PEG-dilauryloxypropyl, PEG-dimyristyloxypropyl, PEG-dipalmityloxypropyl, or PEG-distearyloxypropyl. In some embodiments, the PEG-lipid is [ka] Contains one of the following:

[0306] In some embodiments, lipids conjugated with molecules other than PEG can also be used in place of PEG-lipids, for example, polyoxazoline (POZ)-lipid conjugates, polyamide-lipid conjugates (e.g., ATTA-lipid conjugates), and cationic-polymer lipid (CPL) conjugates can be used in place of or in addition to PEG-lipids.

[0307] Exemplary conjugated lipids (e.g., PEG-lipids, (POZ)-lipid conjugates, ATTA-lipid conjugates, and cationic polymer-lipids) include those described in Table 2 of WO 2019 / 051289 A9, the entirety of which is incorporated herein by reference.

[0308] In some embodiments, the conjugated lipid (e.g., PEGylated lipid) may be present in an amount of 0-20 mol% of the total lipid components present in the lipid-based carrier (or lipid nanoformulation). In some embodiments, the content of the conjugated lipid (e.g., PEGylated lipid) is 0.5-10 mol% or 2-5 mol% of the total lipid components.

[0309] Optionally, the lipid-based carriers (or lipid nanoformulations) described herein can be coated with a polymer layer to enhance in vivo stability (e.g., sterically stabilized LNPs).

[0310] Examples of suitable polymers include, but are not limited to, poly(ethylene glycol), which can form a hydrophilic surface layer that improves the circulation half-life of liposomes and enhances the amount of lipid nanoformulations (e.g., liposomes or LNPs) that reach therapeutic targets. See, for example, Working et al., J Pharmacol Exp Ther, 289: 1128-1133 (1999); Gabizon et al., J Controlled Release 53: 275-279 (1998); Adlakha Hutcheon et al., Nat Biotechnol 17: 775-779 (1999); and Koning et al., Biochim Biophys Acta 1420: 153-167 (1999) (which are incorporated herein by reference in their entirety).

[0311] Percentage of lipid nanoformulation ingredients In some embodiments, the lipid-based carrier (or lipid nanoformulation) comprises one or more of the compounds described herein, optionally a non-cationic lipid (e.g., a phospholipid), a sterol, a neutral lipid, and optionally a conjugated lipid (e.g., a PEGylated lipid) that inhibits particle aggregation. The amounts of these components can be varied independently and to achieve desired properties. For example, in some embodiments, ionizable lipids, including lipid compounds described herein, are present in an amount of about 20 mol% to about 100 mol% (e.g., 20-90 mol%, 20-80 mol%, 20-70 mol%, 25-100 mol%, 30-70 mol%, 30-60 mol%, 30-40 mol%, 40-50 mol%, or 50-90 mol%) of the total lipid components; and non-cationic lipids (e.g., phospholipids) are present in an amount of about 0 mol% to about 50 mol% (e.g., 0-40 mol%, 0-30 mol%, 5-50 mol%, 5-40 mol%) of the total lipid components. mol%, 5-30 mol%, or 5-10 mol%) of the total lipid components; the conjugated lipid (e.g., PEGylated lipid) is present in an amount of about 0.5 mol% to about 20 mol% (e.g., 1-10 mol% or 5-10 mol%) of the total lipid components; and the sterol is present in an amount of about 0 mol% to about 60 mol% (e.g., 0-50 mol%, 10-60 mol%, 10-50 mol%, 15-60 mol%, 15-50 mol%, 20-50 mol%, 20-40 mol%) of the total lipid components; provided that the total mol% of the lipid components does not exceed 100%.

[0312] In some embodiments, the lipid-based carrier (or lipid nanoformulation) comprises about 25-100 mol % of an ionizable lipid comprising a lipid compound described herein, about 0-50 mol % of a phospholipid, about 0-50 mol % of a sterol, and about 0-10 mol % of a PEGylated lipid.

[0313] In one embodiment, the lipid-based carrier (or lipid nanoformulation) comprises about 25-100 mol % of an ionizable lipid comprising a lipid compound described herein; about 0-40 mol % of a phospholipid (e.g., DSPC), about 0-50 mol % of a sterol (e.g., cholesterol), and about 0-10 mol % of a PEGylated lipid.

[0314] In some embodiments, the lipid-based carrier (or lipid nanoformulation) comprises about 30-60 mol % (e.g., about 35-55 mol % or about 40-50 mol %) of an ionizable lipid comprising a lipid compound described herein, about 0-30 mol % (e.g., 5-25 mol % or 10-20 mol %) of a phospholipid, about 15-50 mol % (e.g., 18.5-48.5% or 30-40 mol %) of a sterol, and about 0-10 mol % (e.g., 1-5 mol % or 1.5-2.5 mol %) of a PEGylated lipid.

[0315] In some embodiments, the molar ratio of ionizable lipid / sterol / phospholipid (or other structured lipid) / PEG-lipid / additional component varies between the following ranges: ionizable lipid (25-100%); phospholipid (DSPC) (0-40%); sterol (0-50%); and PEG-lipid (0-5%).

[0316] In some embodiments, the lipid-based carrier (or lipid nanoformulation) comprises, by mol% or wt% of the total lipid components, 50-75% ionizable lipid (including lipid compounds described herein), 20-40% sterol (e.g., cholesterol or derivatives), 0-10% non-cationic lipid, and 1-10% conjugated lipid (e.g., PEGylated lipid).

[0317] The molar ratios of ionizable lipids, non-cationic lipids, sterols, and conjugated lipids (e.g., PEGylated lipids) can be varied as needed. For example, a lipid-based carrier (or lipid nanoformulation) can contain, in mol% or wt% of the total lipid components, 30-70% ionizable lipids (including lipid compounds described herein), 0-60% sterols (e.g., cholesterol or derivatives), 0-30% non-cationic lipids, and 1-10% conjugated lipids (e.g., PEGylated lipids). For example, a lipid-based carrier (or lipid nanoformulation) can contain, in mol% or wt% of the total lipid components, 30-40% ionizable lipids (including lipid compounds described herein), 40-50% sterols (e.g., cholesterol or derivatives), and 10-20% non-cationic lipids. In some embodiments, a lipid-based carrier (or lipid nanoformulation) may comprise, by mol % or wt % of the total lipid components, 50-75% ionizable lipids (including lipid compounds described herein), 20-40% sterol (e.g., cholesterol or derivatives), 5-10% non-cationic lipids, and 1-10% conjugated lipids (e.g., PEGylated lipids). This composition may contain 60-70% ionizable lipids by molar or total weight, 25-35% cholesterol by molar or total weight, and 5-10% non-cationic lipids by molar or total weight. In some embodiments, a lipid-based carrier (or lipid nanoformulation) may comprise, by mol % or wt % of the total lipid components, up to 90% ionizable lipids (including lipid compounds described herein) and 2-15% non-cationic lipids.

[0318] In some embodiments, the lipid-based carrier (or lipid nanoformulation) may comprise, by mol% or wt% of the total lipid components, 8-30% ionizable lipids (including the lipid compounds described herein), 5-30% non-cationic lipids, and 0-20% sterol (e.g., cholesterol or derivatives).

[0319] In some embodiments, the lipid-based carrier (or lipid nanoformulation) may comprise, by mol% or wt% of the total lipid components, 4-25% ionizable lipids (including lipid compounds described herein), 4-25% non-cationic lipids, 2-25% sterols (e.g., cholesterol or derivatives), and 10-35% conjugated lipids (e.g., PEGylated lipids).

[0320] In some embodiments, the lipid-based carrier (or lipid nanoformulation) may comprise, by mol% or wt% of the total lipid components, 2-30% ionizable lipids (including lipid compounds described herein), 2-30% non-cationic lipids, 1-15% sterol (e.g., cholesterol or derivatives), and 2-35% conjugated lipids (e.g., PEGylated lipids).

[0321] In some embodiments, the lipid-based carrier (or lipid nanoformulation) may comprise, by mol% or wt% of the total lipid components, up to 90% ionizable lipids (including the lipid compounds described herein) and 2-10% non-cationic lipids.

[0322] In some embodiments, the lipid compounds described herein are components of a lipid-based carrier (or lipid nanoformulation) and constitute 10 mol% to 95 mol%, 10 mol% to 90 mol%, 10 mol% to 80 mol%, 10 mol% to 70 mol%, 10 mol% to 60 mol%, 20 mol% to 55 mol%, 20 mol% to 45 mol%, 20 mol% to 40 mol%, 25 mol% to 50 mol%, 25 mol% to 45 mol%, 30 mol% to 50 mol%, 30 mol% to 45 mol%, 30 mol% to 40 mol%, 35 mol% to 45 mol%, or 37 mol% to 42 mol% (or any portion of these ranges) of the total lipid component.

[0323] In some embodiments, when the lipid-based carrier (or lipid nanoformulation) contains a mixture of phospholipids and a sterol (e.g., cholesterol or a derivative), this mixture may be present at up to 40 mol%, 45 mol%, 50 mol%, 55 mol%, or 60 mol% of the total lipid component.

[0324] In some embodiments, the phospholipid component in this mixture can be present at 2 mol% to 20 mol%, 2 mol% to 15 mol%, 2 mol% to 12 mol%, 4 mol% to 15 mol%, 4 mol% to 10 mol%, 5 mol% to 10 mol% (or any portion of these ranges) of the total lipid component. In some embodiments, the lipid-based carrier (or lipid nanoformulation) does not comprise a phospholipid.

[0325] In some embodiments, the sterol component (e.g., cholesterol or derivatives) in this mixture can comprise 25 mol% to 45 mol%, 25 mol% to 40 mol%, 25 mol% to 35 mol%, 25 mol% to 30 mol%, 30 mol% to 45 mol%, 30 mol% to 40 mol%, 30 mol% to 35 mol%, 35 mol% to 40 mol%, 27 mol% to 37 mol%, or 27 mol% to 35 mol% (or any portion of these ranges) of the total lipid component.

[0326] In some embodiments, when the lipid-based carrier (or lipid nanoformulation) does not include phospholipids, the sterol component (e.g., cholesterol or a derivative) may be present at up to 25 mol%, 30 mol%, 35 mol%, 40 mol%, 45 mol%, 50 mol%, 55 mol%, or 60 mol% of the total lipid component. For example, the sterol component (e.g., cholesterol or a derivative) may be present at 25 mol% to 65 mol%, 25 mol% to 60 mol%, 25 mol% to 55 mol%, 25 mol% to 50 mol%, 25 mol% to 45 mol%, 25 mol% to 40 mol%, 30 mol% to 45 mol%, 30 mol% to 40 mol%, 35 mol% to 45 mol%, 30 mol% to 35 mol%, or 35 mol% to 40 mol% (or any fraction thereof or range therein) of the total lipid component.

[0327] In some embodiments, the non-ionized lipid component in the lipid-based carrier (or lipid nanoformulation) may be present at 5 mol% to 90 mol%, 10 mol% to 85 mol%, or 20 mol% to 80 mol% (or any portion of these ranges) of the total lipid component.

[0328] The ratio of total lipid components to cargo (e.g., encapsulated therapeutic agent such as a nucleic acid) can be varied as needed. For example, the ratio of total lipid components to cargo (mass or weight) can be from about 10:1 to about 30:1. In some embodiments, the ratio of total lipid components to cargo (mass / mass ratio; w / w ratio) can be in the range of about 1:1 to about 25:1, about 10:1 to about 14:1, about 3:1 to about 15:1, about 4:1 to about 10:1, about 5:1 to about 9:1, or about 6:1 to about 9:1. The total lipid components and cargo amounts can be adjusted to obtain a desired N / P ratio (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or higher). Typically, the overall lipid content of a lipid-based carrier (or lipid nanoformulation) can range from about 5 mg / mL to about 30 mg / mL. The nitrogen:phosphate ratio (N:P ratio) is evaluated at a value between 0.1 and 100.

[0329] In some embodiments, the lipid-based carrier (or lipid nanoformulation) comprises an ionizable lipid compound described herein, a phospholipid, cholesterol, and a PEGylated lipid in a molar ratio of 50:10:38.5:1.5. In some embodiments, the lipid-based carrier (or lipid nanoformulation) comprises an ionizable lipid compound described herein, cholesterol, and a PEGylated lipid in a molar ratio of 60:38.5:1.5.

[0330] In some embodiments of any of the aspects or embodiments herein, the lipid-based carrier (or lipid nanoformulation) further comprises a tissue-targeting moiety. This tissue-targeting moiety can be a peptide, oligosaccharide, or the like, which can be used to deliver the lipid-based carrier (or lipid nanoformulation) to one or more specific tissues, such as the liver. In some embodiments, the tissue-targeting moiety is a ligand for a liver-specific receptor. In one embodiment, the liver-specific receptor ligand used for liver targeting is an oligosaccharide (e.g., N-acetylgalactosamine (GalNAc)) covalently attached to a component of the lipid-based carrier (or lipid nanoformulation) (e.g., a PEG-lipid conjugate or the like). In some embodiments, the GalNAc is covalently attached to, for example, a PEG-lipid conjugate. In some embodiments, the GalNAc is conjugated to DSPE-PEG2000. In some embodiments, the GalNAc-PEG-lipid conjugate is present in the lipid-based carrier (or lipid nanoformulation) at a molar percentage of 1.5%, 1.4%, 1.3%, 1.2%, 1.1%, 1.0%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1% of the total lipid. In some embodiments, the GalNAc-PEG-lipid conjugate is present in the lipid-based carrier (or lipid nanoformulation) at a molar percentage of 0.2% of the total lipid. In some embodiments, the GalNAc-PEG-lipid conjugate is present in the lipid-based carrier (or lipid nanoformulation) at a molar percentage of 0.3% of the total lipid. In some embodiments, the GalNAc-PEG-lipid conjugate is present in the lipid-based carrier (or lipid nanoformulation) at a molar percentage of 0.4% of the total lipid. In some embodiments, the GalNAc-PEG-lipid conjugate is present in the lipid-based carrier (or lipid nanoformulation) at a molar percentage of 0.5% of the total lipids. In some embodiments, the GalNAc-PEG-lipid conjugate is present in the lipid-based carrier (or lipid nanoformulation) at a molar percentage of 0.6% of the total lipids.In some embodiments, the GalNAc-PEG-lipid conjugate is present in the lipid-based carrier (or lipid nanoformulation) at a molar percentage of 0.7% of the total lipid. In some embodiments, the GalNAc-PEG-lipid conjugate is present in the lipid-based carrier (or lipid nanoformulation) at a molar percentage of 0.8% of the total lipid. In some embodiments, the GalNAc-PEG-lipid conjugate is present in the lipid-based carrier (or lipid nanoformulation) at a molar percentage of 0.9% of the total lipid. In some embodiments, the GalNAc-PEG-lipid conjugate is present in the lipid-based carrier (or lipid nanoformulation) at a molar percentage of 1.0% of the total lipid. In some embodiments, the GalNAc-PEG-lipid conjugate is present in the lipid-based carrier (or lipid nanoformulation) at a molar percentage of about 1.5% of the total lipid. In some embodiments, the GalNAc-PEG-lipid conjugate is present in the lipid-based carrier (or lipid nanoformulation) at a molar percentage of 2.0% of the total lipid.

[0331] Properties of lipid nanoformulations In some embodiments, the average particle size of the lipid-based carrier (or lipid nanoformulation) can be from 10 nm to 100 nm, for example, as measured by dynamic light scattering (DLS). In some embodiments, the average particle size of the lipid-based carrier (or lipid nanoformulation) can be from about 1 mm to about 500 mm, from about 5 mm to about 200 mm, from about 10 mm to about 100 mm, from about 20 mm to about 80 mm, from about 25 mm to about 60 mm, from about 30 mm to about 55 mm, from about 35 mm to about 50 mm, from about 38 mm to about 42 mm, from about 40 nm to about 150 nm (e.g., about 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 130 nm, 135 nm, 140 nm, 145 nm, 150 nm, 160 nm, 165 nm, 170 nm, 175 nm, 180 nm, 185 nm, 190 nm, 200 nm, 210 nm, 220 nm, 230 nm, 240 nm, 250 nm, 260 nm, 270 nm, 280 nm, 290 nm, 300 nm, 310 nm, 320 nm, 330 nm, 340 nm, 350 nm, 360 nm, 370 nm, 380 nm, 390 nm, 400 nm, 410 nm, 420 nm, 430 nm, 440 nm, 45 50 nm to about 100 nm, about 50 nm to about 90 nm, about 50 nm to about 80 nm, about 50 nm to about 70 nm, about 50 nm to about 60 nm, about 60 nm to about 100 nm, about 60 nm to about 90 nm, about 60 nm to about 80 nm, about 60 nm to about 70 nm, about 70 nm to about 100 nm, about 70 nm to about 90 nm, about 70 nm to about 80 nm, about 80 nm to about 100 nm, about 80 nm to about 90 nm, or about 90 nm to about 100 nm.

[0332] Lipid-based carriers or lipid nanoformulations (e.g., liposomes or LNPs) can be relatively homogeneous in some cases. The polydispersity index can be used to indicate the homogeneity of lipid nanoformulations (e.g., liposomes or LNPs) (e.g., the particle size distribution of liposomes or LNPs). A small polydispersity index (e.g., less than 0.3) generally indicates a narrow particle size distribution. The lipid-based carrier or lipid nanoformulation (e.g., liposome or LNP) can have a polydispersity index of about 0 to about 0.25, such as 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, or 0.25. In some embodiments, the polydispersity index of the lipid-based carrier or lipid nanoformulation (e.g., liposome or LNP) can be about 0.10 to 0.20.

[0333] The zeta potential of a lipid-based carrier or lipid nanoformulation (e.g., liposome or LNP) can be used to indicate the electrokinetic potential of the composition. In some embodiments, the zeta potential can describe the surface charge of the liposome or LNP. Lipid nanoformulations (e.g., liposome or LNP) with a relatively low positive or negative charge are generally desirable because more highly charged species may undesirably interact with cells, tissues, and other elements in the body. In some embodiments, the zeta potential of the liposome or LNP can be about -10 mV to about +20 mV, about -10 mV to about +15 mV, about 10 mV to about +10 mV, about -10 mV to about +5 mV, about -10 mV to about 0 mV, about -10 mV to about -5 mV, about -5 mV to about +20 mV, about -5 mV to about +15 mV, about -5 mV to about +10 mV, about -5 mV to about +5 mV, about -5 mV to about 0 mV, about 0 mV to about +20 mV, about 0 mV to about +15 mV, about 0 mV to about +10 mV, about 0 mV to about +5 mV, about +5 mV to about +20 mV, about +5 mV to about +15 mV, or about +5 mV to about +10 mV.

[0334] The efficiency of encapsulation of cargo, such as proteins and / or nucleic acids, describes the amount of protein and / or nucleic acid encapsulated or otherwise associated with a lipid nanoformulation (e.g., liposome or LNP) after preparation relative to the initial amount provided. The encapsulation efficiency is desirably high (e.g., at least 70%, 80%, 90%, 95%, or close to 100%). The encapsulation efficiency can be measured, for example, by comparing the amount of protein or nucleic acid in a solution containing liposomes or LNPs before and after liposome or LNP collapse and one or more organic solvents or surfactants. Anion exchange resins can be used to measure the amount of free protein or nucleic acid (e.g., RNA) in solution. Fluorescence can be used to measure the amount of free protein and / or nucleic acid (e.g., RNA) in solution. For liposomes or LNPs described herein, the encapsulation efficiency of proteins and / or nucleic acids may be at least 50%, e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the encapsulation efficiency may be at least 80%. In some embodiments, the encapsulation efficiency may be at least 90%. In some embodiments, the encapsulation efficiency may be at least 95%.

[0335] The lipid carrier or lipid nanoformulation may optionally include one or more coatings. In some embodiments, the lipid carrier or lipid nanoformulation (e.g., liposome or LNP) may be formulated into a capsule, film, or tablet having a coating. The capsule, film, or tablet containing the composition described herein may have any useful size, tensile strength, hardness, or density.

[0336] Further exemplary lipids, formulations, methods, and characterization of lipid carriers or lipid nanoformulations (e.g., liposomes or LNPs) are taught by WO 2020 / 061457 and WO 2021 / 113777, which are incorporated by reference in their entireties. Further exemplary lipids, formulations, methods, and characterization of LNPs are taught by Hou et al. Lipid nanoparticles for mRNA delivery. Nat Rev Mater (2021). doi.org / 10.1038 / s41578-021-00358-0, which is incorporated by reference in its entirety (see, e.g., exemplary lipids and lipid derivatives in Figure 2 of Hou et al.).

[0337] In some embodiments, in vitro or ex vivo cell lipofection is performed using Lipofectamine MessengerMax (Thermo Fisher) or TransIT-mRNA Transfection Reagent (Mirus Bio). In certain embodiments, LNPs are formulated using GenVoy_ILM Ionized Lipid Mix (Precision NanoSystems). In certain embodiments, LNPs are formulated using 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA) or Dilinoleylmethyl-4-dimethylaminobutyrate (DLin-MC3-DMA or MC3), the formulations and their in vivo use taught in Jayaraman et al. Angew Chem Int Ed Engl 51(34):8529-8533 (2012), the entire contents of which are incorporated herein by reference.

[0338] Lipid nanoformulations (e.g., liposomes or LNPs) optimized for delivery of CRISPR-Cas systems (e.g., Cas9-gRNP RNP, gRNA, Cas9 mRNA) are described in WO 2019067992 and WO 2019067910, which are incorporated by reference in their entireties.

[0339] Further specific lipid nanoformulations (e.g., liposomes or LNPs) useful for delivery of nucleic acid effector molecules are described in U.S. Pat. Nos. 8,158,601 and 8,168,775, the disclosures of which are incorporated by reference in their entireties.

[0340] A variety of methods can be used to prepare the lipid carriers or lipid nanoformulations (e.g., liposomes or LNPs) described herein. Such methods are known in the art or are disclosed herein, for example, the method described by Lichtenberg and Barenholz in Methods of Biochemical Analysis, 33:337-462 (1988), which is incorporated herein by reference in its entirety. See also Szoka et al., Ann. Rev. Biophys. Bioeng. 9:467 (1980); U.S. Patent Nos. 4,235,871; 4,501,728; and 4,837,028; Liposomes, Marc J. Ostro, ed., Marcel Dekker, Inc., New York, 1983, Chapter 1; and Hope et al., Chem. Phys. Lip. 40:89 (1986), which are incorporated herein by reference in their entirety. Small unilamellar vesicles (SUVs, less than 100 nm in size) can be prepared by a combination of standard methods of thin film hydration and repeated extrusion.

[0341] Techniques for sizing lipid carriers or lipid nanoformulations (e.g., liposomes or LNPs) to a desired size are known to those skilled in the art. See, for example, U.S. Pat. No. 4,737,323 and Hope et al., Biochim. Biophys. Acta, 812:55-65, which are incorporated by reference in their entirety. Sonicating a lipid nanoformulation (e.g., liposomes or LNPs) suspension by either bath or probe sonication results in a gradual size reduction to small unilamellar vesicles less than about 50 nm in size. Homogenization or microfluidization are other methods that rely on shear energy to fragment large lipid nanoformulations (e.g., liposomes or LNPs) into smaller ones. In a typical homogenization procedure, multilamellar vesicles are recirculated through a standard emulsion homogenizer until a selected lipid nanoformulation (e.g., liposome or LNP) size, typically about 100-500 nm, is observed. In both methods, the particle size distribution can be monitored by conventional laser beam particle size discrimination.

[0342] Extrusion of lipid nanoformulations (e.g., liposomes or LNPs) through small-pore polycarbonate or asymmetric ceramic membranes is a highly effective method for reducing the size of liposomes or LNPs to a relatively well-defined size distribution. Typically, the suspension is cycled through the membrane one or more times until the desired liposome or LNP size distribution is achieved. The lipid-based carrier or lipid nanoformulation can be extruded through successively smaller-pore membranes to achieve a gradual reduction in the size of the liposomes or LNPs.

[0343] Any of the lipid-based carriers or lipid nanoformulations described herein can be analyzed to determine their physical and / or chemical characteristics using methods known to those skilled in the art. For example, a phosphate assay can be used to determine the concentration of a lipid nanoformulation. One phosphate assay is based on the interaction of molybdate with malachite green dye. The main principle involves reacting inorganic phosphate with molybdate to form a colorless, non-reduced phosphomolybdate complex, which is converted to a blue-colored complex upon reduction under acidic conditions. When complexed with malachite green, phosphomolybdate becomes 20- or 30-fold more color-enhancing. The final product, a reduced, green, soluble complex, is measured by its absorbance at 620 nm, which is a direct measure of the inorganic phosphate in solution.

[0344] In some embodiments, the lipid-based carriers or lipid nanoformulations disclosed herein are tested for particle size, lipid concentration, and active agent encapsulation.

[0345] More ionizable lipids Some non-limiting examples of additional lipid compounds that can be used (e.g., in combination with the lipid compounds and other lipid components described herein) to form lipid-based carriers (or lipid nanoformulations) include: [ka] [ka]

[0346] In some embodiments, the lipid-based carrier (or lipid nanoformulation) further comprises a lipid of formula (i), (ii), (iii), (iv), (v), (vi), (vii), (viii), or (ix).

[0347] In some embodiments, the lipid-based carrier (or lipid nanoformulation) has the following structure: [ka] A compound having (In the formula, X 1 , O, NR 1 , or a direct bond, and X 2 is a C2-5 alkylene, and X 3 is C(=O) or a direct bond; R 1 is H or Me, and R 3 is C1-3 alkyl, and R 2 is C1-3 alkyl, or R 2 is the nitrogen atom to which it is attached and X 2 together with 1 to 3 carbon atoms of X 1 is NR 1 and R 1 and R 2 together with the nitrogen atom to which they are attached form a 5- or 6-membered ring, or R 2 is R 3 and together with the nitrogen atom to which they are attached form a 5-, 6-, or 7-membered ring; Y 1 is C2~ 12 is alkylene, and Y 2 teeth, [ka] is selected from n is 0 to 3; R 4 is C 1~15 is alkyl; Z 1 is C 1~6 alkylene or a direct bond, and Z 2 teeth, [ka] or does not exist, except that Z 1 If is a direct bond, then Z 2 does not exist; R 5 is C 5~9Alkyl or C 6~10 is alkoxy, and R 6 is C 5~9 Alkyl or C 6~10 is alkoxy; W is methylene or a direct bond; R 7 is H or Me) or a salt thereof, However, R 3 and R 2 is a C2 alkyl, and X 1 is O and X 2 is a linear C3 alkylene, and X 3 is C(=O) and Y 1 is a linear C5 alkylene, and (Y 2 )nR 4 but [ka] and R 4 is a linear C5 alkyl, and Z 1 is C2 alkylene, and Z 2 is absent, W is methylene, and R 7 If is H, then R 5 and R 6 is not a C2 alkoxy.

[0348] In some embodiments, the lipid-based carrier (or lipid nanoformulation) further comprises one or more compounds of formula (x).

[0349] Further non-limiting examples of lipid compounds that may be further included in the lipid-based carrier (or lipid nanoformulation) include: [ka] [ka] [ka] (e.g., in combination with the lipid compounds described herein, and other lipid components).

[0350] In some embodiments, the lipid-based carrier (or lipid nanoformulation) further comprises one or more compounds of formula (xi), (xii), (xiii), (xiv), (xv), (xvi), (xvii), (xviii) (e.g., (xviii)a, (xviii)b), or (xix).

[0351] In some embodiments, the lipid-based carrier (or lipid nanoformulation) can be prepared by the following reaction: [ka] The lipid further comprises a lipid formed by one of:

[0352] In some embodiments, the lipid-based carrier (or lipid nanoformulation) has the formula (xxi): [ka] and further comprising a lipid (e.g., in combination with the lipid compounds described herein, and other lipid components) having the formula: During the ceremony: each n is independently an integer from 2 to 15; L1 and L3 are each independently -OC(O)-* or -C(O)O-*, where "*" indicates the point of attachment to R1 or R3; R1 and R3 are each independently oxo, halo, hydroxy, cyano, alkyl, alkenyl, aldehyde, heterocyclylalkyl, hydroxyalkyl, dihydroxyalkyl, hydroxyalkylaminoalkyl, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, (heterocyclyl)(alkyl)aminoalkyl, heterocyclyl, heteroaryl, alkylheteroaryl, alkynyl, alkoxy, amino, dialkylamino, aminoalkylcarbonylamino, aminocarbonylalkylamino, (aminocarbonylalkyl)(alkyl)amino, alkenylcarbonylamino, hydroxycarbonyl, alkyl straight or branched chain C9-C alkyl groups optionally substituted with one or more substituents selected from the group consisting of alkyloxycarbonyl, aminocarbonyl, aminoalkylaminocarbonyl, alkylaminoalkylaminocarbonyl, dialkylaminoalkylaminocarbonyl, heterocyclylalkylaminocarbonyl, (alkylaminoalkyl)(alkyl)aminocarbonyl, alkylaminoalkylcarbonyl, dialkylaminoalkylcarbonyl, heterocyclylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, alkylsulfoxide, alkylsulfoxidealkyl, alkylsulfonyl, and alkylsulfonealkyl; 20 Alkyl or C9-C 20 is alkenyl; R2 is [ka] is selected from the group consisting of:

[0353] In some embodiments, the lipid-based carrier (or lipid nanoformulation) further comprises one or more compounds of formula (xxi). In some embodiments, the compounds of formula (xxi) include those described in WO 2021 / 113777 (e.g., lipids of formula (1), such as the lipids in Table 1 of WO 2021 / 113777), which is incorporated herein by reference in its entirety.

[0354] In some embodiments, the lipid-based carrier (or lipid nanoformulation) has the formula (xxii): [ka] and further comprising a lipid (e.g., in combination with the lipid compounds described herein, and other lipid components) having the formula: During the ceremony, each n is independently an integer from 1 to 15; R1 and R2 are each independently [ka] [ka] is selected from the group consisting of R3 is [ka] is selected from the group consisting of:

[0355] In some embodiments, the lipid-based carrier (or lipid formulation) further comprises one or more compounds of formula (xxii). In some embodiments, the compounds of formula (xxii) include those described in WO 2021 / 113777 (e.g., lipids of formula (2), such as the lipids in Table 2 of WO 2021 / 113777), which is incorporated herein by reference in its entirety.

[0356] In some embodiments, the lipid-based carrier (or lipid nanoformulation) has the formula (xxiii): [ka] and further comprising a lipid (e.g., in combination with the lipid compounds described herein, and other lipid components) having the formula: During the ceremony, X is selected from -O-, -S-, or -OC(O)-*, where * indicates the point of attachment to R1; R1 is [ka] is selected from the group consisting of R2 is [ka] is selected from the group consisting of:

[0357] In some embodiments, the lipid-based carrier (or lipid nanoformulation) further comprises one or more compounds of formula (xxiii). In some embodiments, the compounds of formula (xxiii) include those described in WO 2021 / 113777 (e.g., lipids of formula (3), such as the lipids in Table 3 of WO 2021 / 113777), which is incorporated herein by reference in its entirety.

[0358] In some embodiments, the lipid-based carrier (or lipid nanoformulation) further comprises one or more additional ionizable lipids.

[0359] In one embodiment, the additional ionizable lipid is heptadecan-9-yl 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate (SM-102), as described, for example, in Example 1 of U.S. Pat. No. 9,867,888, which is incorporated herein by reference in its entirety.

[0360] In one embodiment, the additional ionizable lipid is (9Z,12Z)-3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl octadeca-9,12-dienoate (LP01), as synthesized, for example, in Example 13 of WO 2015 / 095340, which is incorporated herein by reference in its entirety.

[0361] In one embodiment, the additional ionizable lipid is di((Z)-non-2-en-1-yl)9-((4-dimethylamino)butanoyl)oxy)heptadecanedioate (L319), for example, as synthesized in Example 7, 8, or 9 of U.S. Patent Application Publication No. 2012 / 0027803, which is incorporated herein by reference in its entirety.

[0362] In one embodiment, the additional ionizable lipid is 1,1'-((2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethyl)azanediyl)bis(dodecan-2-ol) (C12-200), for example, as synthesized in Examples 14 and 16 of WO 2010 / 053572, which is incorporated herein by reference in its entirety.

[0363] In one embodiment, the additional ionizable lipid is the imidazole cholesterol ester (ICE) lipid (3S,10R,13R,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl 3-(1H-imidazol-4-yl)propanoate, e.g., structure (I) from WO 2020 / 106946, which is incorporated herein by reference in its entirety.

[0364] In one embodiment, the additional ionizable lipid is MC3(6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butanoate (DLin-MC3-DMA or MC3), as described, for example, in Example 9 of WO 2019 / 051289 A9, which is incorporated herein by reference in its entirety.

[0365] In one embodiment, the additional ionizable lipid is the lipid ATX-002, for example, as described in Example 10 of WO 2019 / 051289 A9, which is incorporated herein by reference in its entirety.

[0366] In one embodiment, the additional ionizable lipid is (13Z,16Z)-A,A-dimethyl-3-nonyldocosa-13,16-dien-1-amine (compound 32), as described, for example, in Example 11 of WO 2019 / 051289 A9, which is incorporated herein by reference in its entirety.

[0367] In one embodiment, the additional ionizable lipid is, for example, compound 6 or compound 22, as described in Example 12 of WO 2019 / 051289 A9, which is incorporated herein by reference in its entirety.

[0368] Examples of additional ionizable lipids that may be used in lipid-based carriers (or lipid nanoformulations) include, but are not limited to, those listed in Table 1 of WO 2019 / 051289, which is incorporated herein by reference.

[0369] Pharmaceutical Composition The present disclosure also provides a pharmaceutical composition comprising a lipid-based carrier described herein and a pharmaceutically acceptable excipient, which may further comprise a therapeutic agent.

[0370] All of the above descriptions and all of the embodiments discussed in the above aspects of the lipid compounds, such as compounds encompassed by formulae (AL-GI), (AL-Ia) to (AL-Ic), (AL-IIa) to (AL-IIc), (AL-IIIa) to (AL-IIIg), (AL-IVa) to (AL-IVc), and exemplary formulae of lipids having at least two ester groups, lipids containing lactide (or derivatives thereof) groups, and lipids containing phosphoramidate groups, are all applicable to these aspects of the invention relating to this pharmaceutical composition.

[0371] All of the above statements regarding the lipid-based carrier (or lipid nanoformulation) aspects including various other lipid components and all of the embodiments discussed in the above aspects are applicable to these aspects of the invention regarding this pharmaceutical composition.

[0372] Treatment drugs In some embodiments, the present invention provides methods of delivering an effector, comprising administering a lipid nanoparticle of the present invention, e.g., to a patient, wherein the lipid nanoparticle comprises an effector. In certain embodiments, the effector comprises a therapeutic agent.

[0373] nucleic acid molecule In some embodiments, the therapeutic agent is a nucleic acid molecule. The nucleic acid molecule can be any nucleic acid molecule that can function as a therapeutic or diagnostic agent. For example, the nucleic acid molecule can be DNA or RNA.

[0374] In some embodiments, the nucleic acid molecule is a nucleic acid selected from the group consisting of a plasmid, an immunostimulatory oligonucleotide, an antisense oligonucleotide, an antagomir, an aptamer, a deoxyribozyme (DNAzyme), and a ribozyme.

[0375] In some embodiments, the therapeutic agent is DNA. The DNA can be selected by one of skill in the art. In some embodiments, the DNA is linear, circular, single-stranded, or double-stranded.

[0376] In one embodiment, the therapeutic agent is linear DNA.

[0377] In one embodiment, the therapeutic agent is circular DNA.

[0378] In one embodiment, the therapeutic agent is single-stranded DNA.

[0379] In one embodiment, the therapeutic agent is double-stranded DNA.

[0380] In some embodiments, the therapeutic agent is RNA. The RNA can be selected by one skilled in the art. In certain embodiments, the RNA is mRNA, miRNA, siRNA or siRNA precursor, RNA aptamer, linear RNA, circular RNA, single-stranded RNA, double-stranded RNA, tRNA, microRNA (miRNA) or miRNA precursor, Dicer substrate small interfering RNA (dsiRNA), short hairpin RNA (shRNA), asymmetric interfering RNA (aiRNA), guide RNA (gRNA), lncRNA, ncRNA, sncRNA, rRNA, snRNA, piRNA, snoRNA, snRNA, scaRNA, exRNA, scaRNA, Y RNA, or hnRNA.

[0381] In some embodiments, the nucleic acid molecule comprises one or more nucleic acid analogs selected from the group consisting of phosphoramide, phosphorothioate, phosphorodithioate, O-methyl phosphoramidate, morpholino, locked nucleic acid (LNA), glycerol nucleic acid (GNA), threose nucleic acid (TNA), and peptide nucleic acid (PNA).

[0382] In one embodiment, the therapeutic agent is mRNA (messenger RNA).

[0383] In one embodiment, the therapeutic agent is a miRNA (microRNA) or a miRNA precursor.

[0384] In one embodiment, the therapeutic agent is a siRNA (small interfering RNA) or an siRNA precursor.

[0385] In one embodiment, the therapeutic agent is a Dicer substrate small interfering RNA (dsiRNA).

[0386] In one embodiment, the therapeutic agent is a short hairpin RNA (shRNA).

[0387] In one embodiment, the therapeutic agent is an asymmetric interfering RNA (aiRNA).

[0388] In one embodiment, the therapeutic agent is a guide RNA (gRNA).

[0389] In one embodiment, the therapeutic agent is an RNA aptamer.

[0390] In one embodiment, the therapeutic agent is a circular RNA, eg, a circular RNA that encodes a therapeutic polypeptide or a non-coding circular RNA.

[0391] In one embodiment, the therapeutic agent is a tRNA (transfer RNA).

[0392] In one embodiment, the therapeutic agent is rRNA (ribosomal RNA).

[0393] In one embodiment, the therapeutic agent is a lncRNA (long non-coding RNA).

[0394] In one embodiment, the therapeutic agent is a snRNA (small nuclear RNA).

[0395] In one embodiment, the therapeutic agent is a ncRNA (non-coding RNA).

[0396] In one embodiment, the therapeutic agent is a sncRNA (small non-coding RNA).

[0397] In one embodiment, the therapeutic agent is a snoRNA (small nuclear RNA).

[0398] In one embodiment, the therapeutic agent is a piRNA (piwi-interacting RNA).

[0399] In one embodiment, the therapeutic agent is a scaRNA (small Cajal body-specific RNA).

[0400] In one embodiment, the therapeutic agent is exRNA (extracellular RNA).

[0401] In one embodiment, the therapeutic agent is Y RNA, a small non-coding RNA that is a component of the Ro60 ribonucleoprotein particle.

[0402] In one embodiment, the therapeutic agent is hnRNA (heterogeneous nuclear RNA).

[0403] In one embodiment, the therapeutic agent is an shRNA (short hairpin RNA).

[0404] In some embodiments, the therapeutic agent is an enzymatic nucleic acid molecule. The term "enzymatic nucleic acid molecule" refers to a nucleic acid molecule that has complementarity to a specific gene target in its substrate-binding region and also has enzymatic activity active in specifically cleaving the target RNA. That is, the enzymatic nucleic acid molecule is capable of intermolecularly cleaving RNA, thereby inactivating the target RNA molecule. These complementary regions allow sufficient hybridization of the enzymatic nucleic acid molecule to the target RNA, thereby enabling cleavage. While 100 percent complementarity is preferred, complementarity as low as 50-75% can also be useful in the present invention (see, e.g., Werner et al., Nucleic Acids Research 23:2092-2096 (1995); Hammann et al., Antisense and Nucleic Acid Drug Dev. 9:25-31 (1999), both of which are incorporated herein by reference in their entireties).

[0405] The term enzymatic nucleic acid is used interchangeably with phrases such as ribozyme, catalytic RNA, enzymatic RNA, catalytic DNA, aptazyme or aptamer-binding ribozyme, regulatable ribozyme, catalytic oligonucleotide, nucleozyme, DNAzyme, RNA enzyme, endoribonuclease, endonuclease, minizyme, leadzyme, oligozyme, or DNA enzyme, all of which describe nucleic acid molecules with enzymatic activity.

[0406] In some embodiments, the therapeutic agent is an antisense nucleic acid. The term "antisense nucleic acid" refers to a non-enzymatic nucleic acid molecule that binds to a target RNA through RNA-RNA, RNA-DNA, or RNA-PNA (protein nucleic acid) interactions and alters the activity of the target RNA. Typically, an antisense molecule is complementary to a target sequence along a single, continuous sequence of the antisense molecule. However, in certain embodiments, an antisense molecule may bind to a substrate molecule such that the substrate molecule forms a loop, and / or an antisense molecule may bind to a substrate molecule such that the antisense molecule forms a loop. Thus, an antisense molecule may be complementary to two (or more) discontinuous substrate sequences, or two (or more) discontinuous sequence portions of an antisense molecule may be complementary to a target sequence, or both. In addition, antisense DNA may be used to target RNA through DNA-RNA interactions, thereby activating RNase H, which digests the double-stranded target RNA.

[0407] In some embodiments, the nucleic acid molecule can be a 2-5A antisense chimera. The term "2-5A antisense chimera" refers to an antisense oligonucleotide containing a 5'-phosphorylated 2',5'-linked adenylate residue. This chimera binds to a target RNA in a sequence-specific manner, activates cellular 2-5A-dependent ribonuclease, and subsequently cleaves the target RNA.

[0408] In some embodiments, the nucleic acid molecule can be a triplex-forming oligonucleotide. The term "triplex-forming oligonucleotide" refers to an oligonucleotide that can bind to double-stranded DNA in a sequence-specific manner to form a triple-stranded helix.

[0409] In some embodiments, the nucleic acid molecule can be a decoy RNA. The term "decoy RNA" refers to an RNA molecule or aptamer that is designed to preferentially bind to a predetermined ligand. Such binding can result in the inhibition or activation of the target molecule.

[0410] In some embodiments, the nucleic acid molecule (e.g., RNA or DNA) encodes a therapeutic peptide or polypeptide. In the case of DNA, the nucleic acid comprises a promoter operably linked to a sequence encoding the therapeutic peptide or polypeptide. The therapeutic peptide or therapeutic polypeptide can be, for example, a transcription factor; a chromatin remodeling factor; an antigen; a hormone; an enzyme (e.g., a nuclease, e.g., an endonuclease, e.g., a nuclease component of a CRISPR system, e.g., Cas9, dCas9, aCas9-nickase, Cpf / Cas12a); a Crispr-binding enzyme, e.g., a base editor or a prime editor; a mobile genetic element protein (e.g., a transposase, retrotransposase, recombinase, integrase); a gene writer; a polymerase; a methylase; a demethylase; an acetylase; a deacetylase; a kinase; a phosphatase; a ligase; a deubiquitinase; an integrase, a recombinase; a topoisomerase; a gyrase; a helicase; a lysosomal acid hydrolase; an antibody; a receptor ligand; a receptor; a coagulation factor; a membrane protein, a mitochondrial protein, a nuclear protein, an antibody or other protein scaffold binder, e.g., a centrin, a darpin, or an adnectin.

[0411] In some embodiments, the nucleic acid molecule (e.g., DNA or RNA) encodes (in the case of DNA) or is (in the case of RNA) one or more of the following: a non-coding region, such as an siRNA, miRNA, long non-coding RNA, piRNA, snoRNA, scaRNA, tRNA, rRNA, therapeutic RNA aptamer, and snRNA.

[0412] In some embodiments, the therapeutic nucleic acid molecule targets a host gene, e.g., the nucleic acid effector hybridizes to an endogenous gene.

[0413] In some embodiments, the nucleic acid molecule is an antisense RNA; a guide RNA; a nucleic acid that hybridizes to an exogenous nucleic acid such as viral DNA or RNA, a nucleic acid that hybridizes to an RNA; a nucleic acid that interferes with gene transcription; a nucleic acid that interferes with RNA translation; a nucleic acid that stabilizes RNA or destabilizes RNA, such as by targeting it for degradation; or a nucleic acid that modulates a DNA- or RNA-binding factor.

[0414] In some embodiments, the nucleic acid molecule targets the sense strand of the host gene. In some embodiments, the nucleic acid molecule targets the antisense strand of the host gene.

[0415] In some embodiments, the nucleic acid molecule is a guide RNA or encodes a guide RNA. Guide RNA sequences are generally 15-30 nucleotides in length (e.g., 17, 19, 20, 21, or 24 nucleotides) and are designed to be complementary to the targeted nucleic acid sequence. Custom gRNA generators and algorithms are commercially available for use in designing effective guide RNAs. Gene editing has also been achieved using chimeric "single guide RNAs" ("sgRNAs"), which are engineered (synthetic) single RNA molecules that mimic the naturally occurring crRNA-tracrRNA complex and contain both a tracrRNA (to bind nucleases) and at least one crRNA (to guide nucleases to the sequence targeted for editing). Chemically modified sgRNAs have also been demonstrated to be effective for genome editing; see, e.g., Hendel et al. (2015) Nature Biotechnol., 985-991. The gRNA can recognize a specific DNA sequence (e.g., a sequence adjacent to or within a gene promoter, enhancer, silencer, or repressor). In one embodiment, the gRNA is used as part of a CRISPR system for gene editing. For the purpose of gene editing, the ssDNA constructs or sequences disclosed herein can be designed to include one or more sequences encoding guide RNA sequences corresponding to the desired target DNA sequence; see, e.g., Cong et al. (2013) Science, 339:819-823; Ran et al. (2013) Nature Protocols, 8:2281-2308.

[0416] In some embodiments, a nucleic acid molecule can include multiple sequences, which can be of the same or different types, and which can be the same sequence or different sequences of the same type.

[0417] All of the nucleic acid molecules described herein can be chemically modified. Various modification strategies for nucleic acid molecules are known to those skilled in the art. In some embodiments, the nucleic acid molecule contains one or more modifications selected from the group consisting of pseudouridine, 5-bromouracil, 5-methylcytosine, peptide nucleic acid, xeno nucleic acid, morpholino, locked nucleic acid, glycol nucleic acid, threose nucleic acid, dideoxynucleotide, cordycepin, 7-deaza-GTP, fluorophore (e.g., rhodamine or fluorescein linked to a sugar), thiol-containing nucleotide, biotin-linked nucleotide, fluorescent base analog, CpG island, methyl-7-guanosine, methylated nucleotide, inosine, thiouridine, pseudouridine, dihydrouridine, queusine, and wyosine. In some embodiments, the antisense oligonucleotide can be a locked nucleic acid oligonucleotide (LNA). The term "locked nucleic acid (LNA)" refers to an oligonucleotide containing one or more nucleotide components in which an extra methylene bridge locks the ribose moiety in either a C3'-endo (beta-D-LNA) or C2'-endo (alpha-L-LNA) conformation (Grunweller A, Hartmann RK, BioDrugs, 21(4):235-243(2007)).

[0418] Further examples of nucleic acid molecules (e.g., tumor suppressor genes, antisense oligonucleotides, siRNA, miRNA, or shRNA) can be found in U.S. Patent Application Publication No. 2007 / 0065499 and U.S. Patent No. 7,780,882, which are incorporated by reference in their entireties.

[0419] In some embodiments, the pharmaceutical composition may comprise multiple nucleic acid molecules, which may be of the same or different types.

[0420] In some embodiments, the N:P ratio of the lipid-based carrier or lipid nanoformulation in which the nucleic acid molecule is encapsulated ranges from 1:1 to 30:1, such as 3:1 to 20:1, 3:1 to 15:1, 3:1 to 10:1, or 3:1 to 6:1. The N:P ratio refers to the molar ratio of amines (e.g., amines in ionizable lipids) present in the lipid-based carrier or lipid nanoformulation to phosphates present in the nucleic acid molecule. This is a factor in efficient packaging and efficacy. In one embodiment, the N:P ratio of the lipid-based carrier or lipid nanoformulation in which the nucleic acid molecule is encapsulated ranges from 3:1 to 15:1.

[0421] Other medications The therapeutic agent can be a nucleic acid, peptide or protein, or a small molecule drug encapsulated in a lipid-based carrier or lipid nanoformulation. The pharmaceutical composition can contain two or more therapeutic agents different from the nucleic acid molecule, peptide or protein, and the small molecule drug.

[0422] In some embodiments, the protein effector can be any peptide or protein molecule that can function as a therapeutic or diagnostic agent.

[0423] In some embodiments, the protein can be a peptide or polypeptide, such as a transcription factor; a chromatin remodeling factor; an antigen; a hormone; an enzyme (e.g., a nuclease, e.g., an endonuclease, e.g., a nuclease element of a CRISPR system, e.g., Cas9, dCas9, aCas9-nickase, Cpf / Cas12a); a Crispr-binding enzyme, e.g., a base editor or a prime editor; a mobile genetic element protein (e.g., a transposase, retrotransposase, recombinase, integrase); a gene writer; a polymerase; a methylase; a demethylase; an acetylase; a deacetylase; a kinase; a phosphatase; a ligase; a deubiquitinase; an integrase; a recombinase; a topoisomerase; a gyrase; a helicase; a lysosomal acid hydrolase); an antibody; a receptor ligand; a receptor; a coagulation factor; a membrane protein; a mitochondrial protein; a nuclear protein, an antibody, or other protein scaffold binder, such as a centrin, a darpin, or an adnectin.

[0424] In one embodiment, the protein is a ribonucleoprotein (RNP), which is a complex of ribonucleic acid and an RNA-binding protein.

[0425] In one embodiment, the protein is a recombinant cytokine.

[0426] In some embodiments, the pharmaceutical composition may comprise multiple protein molecules, which may be of the same or different types.

[0427] In some embodiments, the therapeutic agent is a small molecule drug, eg, a small molecule drug that has been approved for use in humans by an appropriate regulatory agency.

[0428] In some embodiments, the small molecule drug is an HDAC inhibitor, a kinase inhibitor, a cytotoxic molecule, a chromatin modulating agent, an RNAi modulating agent, a transcription factor, an adjuvant, or a combination of two or more.

[0429] In some embodiments, the small molecule drug can be a small molecule that lacks cell-penetrating properties.

[0430] In some embodiments, the pharmaceutical composition may include multiple small molecule drugs that may be of the same or different types.

[0431] In some embodiments, the therapeutic agent is a vaccine, hi some embodiments, the vaccine is an RNA vaccine, such as an RNA cancer vaccine or an RNA vaccine for an infectious disease (e.g., a virus, such as an influenza virus vaccine or a coronavirus vaccine (e.g., a COVID-19 vaccine)).

[0432] Other ingredients The pharmaceutical composition may contain one or more pharmaceutically acceptable excipients. The pharmaceutically acceptable excipients are selected based on the mode and route of administration. Suitable pharmaceutical carriers and excipients for use in pharmaceutical formulations are described in Remington: The Science and Practice of Pharmacy, 21 st Ed.,Gennaro,Ed.,Lippencott Williams & Wilkins(2005);Handbook of Pharmaceutical Excipients,6 th Edition, Rowe et al., Eds., Pharmaceutical Press (2009); and USP / NF (United States Pharmacopeia and the National Formulary), which are incorporated herein by reference in their entireties.

[0433] In some embodiments, the pharmaceutically acceptable excipient comprises one or more of an antioxidant, binder, antiadherent, buffer, colorant, diluent (e.g., solid or liquid), disintegrant (e.g., coating disintegrant), dispersant, dye, filler, emulsifier, flavoring agent, lubricant, pH adjuster, pigment, preservative, stabilizer, solubilizer, solvent, suspending agent, sweetener, or wetting agent, or combinations thereof.

[0434] Examples of suitable excipients include, but are not limited to, acacia, alginate, calcium phosphate, calcium carbonate, calcium silicate, carbopol gel, carboxymethylcellulose, carnauba wax, cellulose, crospovidone, dextrose, diacetylated monoglyceride, ethylcellulose, gelatin, glyceryl monostearate 40-50, acacia gum, gum arabic, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, hypromellose phthalate, hypromellose, lactose, lecithin, magnesium stearate, kaolin, methacrylic acid copolymer type C, mannitol, methylcellulose, methylhydroxybenzoate, microcrystalline cellulose, povidone, polyethylene glycol, polysorbate 80, polyvinylpyrrolidone, propylhydroxybenzoate, sodium carboxymethylcellulose, sodium hydroxide, sodium stearyl fumarate, sodium starch glycolate, starch, sorbitan monooleate. Sorbitol, sorbic acid, sucrose, talc, tragacanth, talc, triethyl citrate, titanium dioxide, yellow iron oxide, talc, an oily vehicle (e.g., peanut oil, liquid paraffin, mineral oil, olive oil, almond oil, glycerin, propylene glycol), or water.

[0435] When the excipient serves as a diluent, it can be a solid, semi-solid, or liquid material (e.g., saline) that acts as a vehicle, carrier, or medium for the active ingredient. As is known in the art, the type of diluent can vary depending on the intended route of administration.

[0436] The pharmaceutical compositions may contain pharmaceutically acceptable carriers, excipients, or stabilizers in the form of lyophilized formulations or aqueous solutions. Acceptable carriers, excipients, or stabilizers are non-toxic to recipients at the dosages and concentrations employed and include buffers such as phosphates, citrates, and other organic acids; antioxidants such as ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzyl chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl, or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins such as serum alcohols. amino acids, such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, such as glucose, mannose, or dextran; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions, such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants, such as TWEEN®, PLURONICS®, or polyethylene glycol (PEG).

[0437] Suitable carriers or excipients for the pharmaceutical composition may also include substances that enhance the ability of an individual's body to absorb LNPs or liposomes. Suitable carriers and / or excipients may also include any substance that can be used to bulk the formulation with LNPs or liposomes to allow for convenient and accurate dosing. In addition, carriers and / or excipients may be used in the manufacturing process to aid in the handling of LNPs or liposomes. Depending on the route of administration and the form of the drug, various carriers and / or excipients may be used.

[0438] Carriers and / or excipients can also include vehicles and / or diluents. "Vehicle" generally refers to any of a variety of media that act as a solvent or carrier; "diluent" refers to a diluent added to dilute the active ingredients of the composition; suitable diluents include any substance that can reduce the viscosity of the drug. The type and amount of carriers and / or excipients are selected according to the selected pharmaceutical form; suitable pharmaceutical forms include liquid systems such as solutions, infusions, and suspensions; semi-solid systems such as colloids, gels, pastes, or creams; solid systems such as powders, granules, tablets, capsules, pellets, microgranules, minitablets, microcapsules, micropellets, and suppositories; etc.

[0439] Each of the above systems may be suitably formulated for normal, delayed, or accelerated release using techniques known in the art.

[0440] Formulation, Dosage, and Route of Administration The pharmaceutical compositions described herein can be prepared according to standard techniques and those techniques described herein. For example, the pharmaceutical compositions can be prepared by conventional methods, for example, by conventional mixing, dissolving, granulating, dragee-making, pulverizing, emulsifying, encapsulating, encapsulating, or lyophilizing processes. Methods for preparing formulations known in the art are known in the art. For example, see Remington: The Science and Practice of Pharmacy, 21 st See Ed., Gennaro, Ed., Lippencott Williams & Wilkins (2005), and Encyclopedia of Pharmaceutical Technology, eds. J. Swarbrick and J.C. Boylan, 1988-1999, Marcel Dekker, New York.

[0441] A therapeutic agent can be encapsulated in a lipid-based carrier (or lipid nanoformulation); for example, the therapeutic agent can be located completely or partially within the interior space of the LNP, within the lipid layer / membrane, or associated with the outer surface of the lipid layer / membrane. One purpose of incorporating a therapeutic agent into an LNP is to protect the therapeutic agent from the environment, which may contain enzymes, chemicals, or conditions that degrade the therapeutic agent and / or systems or receptors, causing rapid excretion of the therapeutic agent. Furthermore, incorporating a therapeutic agent into an LNP can facilitate uptake of the therapeutic agent, thus enhancing therapeutic efficacy.

[0442] In some embodiments, the ratio of lipid component to therapeutic agent (mass / mass ratio; w / w ratio) in the pharmaceutical composition can range from about 1:1 to about 25:1, about 10:1 to about 14:1, about 3:1 to about 15:1, about 4:1 to about 10:1, about 5:1 to about 9:1, or about 6:1 to about 9:1.

[0443] The lipid-based carrier or pharmaceutical composition may contain from about 5 to about 95% by weight of the therapeutic agent, based on the weight of the lipid-based carrier or pharmaceutical composition. In some embodiments, the lipid-based carrier or pharmaceutical composition contains about 5, about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, or about 95% by weight of the therapeutic agent, based on the weight of the LNP or pharmaceutical composition. In some embodiments, the lipid-based carrier or pharmaceutical composition may comprise, by weight of the lipid-based carrier or pharmaceutical composition, about 5 to 95%, about 5 to 90%, about 5 to 80%, about 5 to 70%, about 5 to 60%, about 5 to 50%, about 5 to 40%, about 5 to 30%, about 5 to 20%, about 5 to 10%, about 10 to 95%, about 10 to 90%, about 10 to 80%, about 10 to 70%, about 10 to 60%, about 10 to 50%, about 10 to 40%, about 10 to 30%, about 10 to 20%, about 20 to 95%, about 20 to 90%, about 20 to 80%, about 20 to 70%, about 20 to 60%, about 20 to 50%, about 20 to 50%, about 20 to 60%, about 20 to 50%, about 20 to 70%, about 20 to 80%, about 20 to 5 ... about 40%, about 20-30%, about 30-95%, about 30-90%, about 30-80%, about 30-70%, about 30-60%, about 30-50%, about 30-40%, about 40-95%, about 40-90%, about 40-80%, about 40-70%, about 40-60%, about 40-50%, about 50-95%, about 50-90%, about 50-80%, about 50-70%, about 50-60%, about 60-95%, about 60-90%, about 60-80%, about 60-70%, about 70-95%, about 70-90%, about 70-80%, about 80-95%, about 80-90%, or about 90-95% of the therapeutic agent.

[0444] The lipid-based carrier (or lipid nanoformulation) or pharmaceutical composition may contain total lipids in an amount of about 5 to about 95% by weight, based on the weight of the lipid-based carrier (or lipid nanoformulation) or pharmaceutical composition. In some embodiments, the lipid-based carrier (or lipid nanoformulation) or pharmaceutical composition may comprise, by weight of the lipid-based carrier or pharmaceutical composition, about 5-95%, about 5-90%, about 5-80%, about 5-70%, about 5-60%, about 5-50%, about 5-40%, about 5-30%, about 5-20%, about 5-10%, about 10-95%, about 10-90%, about 10-80%, about 10-70%, about 10-60%, about 10-50%, about 10-40%, about 10-30%, about 10-20%, about 20-95%, about 20-90%, about 20-80%, about 20-70%, about 20-60%, about 20-50%, about 20-60%, about 20-50%, about 20-60%, about 20-70%, about 20-80%, about 20-90%, about 20-90%, about 20-80%, about 20-70%, about 20-50%, about 20-50%, about 20-60%, about 20-50%, about 20-95%, about 20-90%, about 20-80%, about 20-70%, about 20-60%, about 20-5 ...50%, about 20-60%, about 20-50%, about 20-50%, about 20-95 0%, approx. 20-40%, approx. 20-30%, approx. 30-95%, approx. 30-90%, approx. 30-80%, approx. 30-70%, approx. 30-60%, approx. 30-50%, approx. 30-40%, approx. 40-95%, approx. 40-90%, approx. 40-80%, approx. 40-70%, approx. 40-60%, approx. 40-50%, approx. 50-9 The total lipid content is 5%, about 50-90%, about 50-80%, about 50-70%, about 50-60%, about 60-95%, about 60-90%, about 60-80%, about 60-70%, about 70-95%, about 70-90%, about 70-80%, about 80-95%, about 80-90%, or about 90-95%.

[0445] In some embodiments, the pharmaceutical compositions may be formulated for parenteral administration, for example, for intracanalicular, intravenous, subcutaneous, or intramuscular administration.

[0446] As used herein, the term "parenteral" refers to a route of administration other than enteral administration. Examples of parenteral administration include, but are not limited to, buccal, epicutaneous, epidural, extra-amniotic, intra-arterial, intra-articular, intracardiac, intracavernosal, intracerebral, intraventricular, intradermal, intralesional, intramuscular, intraocular, intraosseous injection, intraperitoneal, intrapulmonary, intrathecal, intrauterine, intravaginal, intravenous, intravesical, intravitreal, nasal, perivascular, subcutaneous, sublingual, transdermal, topical, transepithelial, or transmucosal. Parenteral administration may be performed by continuous infusion over a selected period of time.

[0447] In some embodiments, the pharmaceutical composition is administered intravenously by bolus injection or infusion. Suitable formulations for use can be found in Remington's Pharmaceutical Sciences, Mack Publishing Company, Philadelphia, Pa., 17th ed. (1985), which is incorporated herein by reference in its entirety.

[0448] In some embodiments, the pharmaceutical composition is formulated for injection, such as intravenous infusion. Sterile injectable compositions (e.g., sterile injectable aqueous or oleaginous suspensions) can be formulated according to techniques known in the art using suitable dispersing or wetting agents (e.g., Tween 80) or suspending agents. The sterile injectable preparation can also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, such as a sterile injectable solution or suspension in 1,3-butanediol. Among the acceptable vehicles and solvents that can be used are mannitol, water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile fixed oils are conventionally used as solvents or suspending media (e.g., synthetic monoglycerides or diglycerides). Fatty acids (e.g., oleic acid and its glyceride derivatives) are useful in the preparation of injectables, and natural pharmaceutically acceptable oils are available, such as olive oil or castor oil, especially their polyoxyethylated versions. These oil solutions or suspensions may also contain long-chain alcohol diluents or dispersants, or carboxymethylcellulose or similar dispersing agents. Other commonly used surfactants, such as Tweens or Spans, or other similar emulsifiers or bioavailability enhancers commonly used in pharmaceutical manufacturing are commonly used in pharmaceutical manufacturing.

[0449] Any of the pharmaceutical compositions described herein can be used to deliver an active molecule or therapeutic agent (e.g., a nucleic acid molecule) encapsulated in a lipid-based carrier (or lipid nanoformulation) to a desired target. To practice this use, an effective amount of a pharmaceutical composition described herein can be administered to a subject (e.g., a human subject) in need of treatment via a suitable route, such as those described herein.

[0450] The present disclosure also provides dosage units containing the lipid-based carrier or pharmaceutical composition disclosed herein.Those skilled in the art can select dosage forms for use herein.For example, dosage units can be solid dosage forms, liquid dosage forms, or solid / liquid dosage forms.In some embodiments, dosage units are solid dosage forms.In some embodiments, dosage forms are liquid dosage forms.

[0451] The dosage unit can be formulated for delivery most useful to the subject. In some embodiments, the dosage unit is for enteral or parenteral administration. Examples of enteral administration include, but are not limited to, oral, rectal, sublingual, or buccal.

[0452] In some embodiments, the dosage unit is administered intravenously, intraperitoneally, intramuscularly, or subcutaneously. In some embodiments, the dosage unit is administered orally, intravenously, intraperitoneally, intramuscularly, or subcutaneously. In some embodiments, the dosage unit is administered orally.

[0453] In some embodiments, the dosage unit is for parenteral administration, i.e., a parenteral dosage unit. Parenteral dosage units are known in the art and include, but are not limited to, injections, inhalants, infusions, patches, and suppositories. In certain aspects, the parenteral dosage unit is an injection solution. In other embodiments, the dosage unit is formulated for oral delivery, i.e., an oral dosage unit. In certain aspects, the oral dosage unit is a pill (e.g., a tablet, a caplet, a capsule (e.g., soft gelatin, hard gelatin, gel capsule)), an effervescent dosage form, an elixir, a film, a liquid / solution (e.g., a suspension, an emulsion), a candy, a lozenge, a paste, a powder, a sachet, or a syrup. In some embodiments, the oral dosage unit is a pill, a tablet, a capsule, a syrup, a solution, a powder, a paste, a patch, a pump, or a film. In some embodiments, the oral dosage unit is a dry product for reconstitution with water or other suitable vehicle before use.

[0454] If the dosage form is a solid dosage form, an enteric coating may be applied or the solid dosage form may be scored. The enteric coating may be stable at low pH (e.g., in the stomach) and dissolve at high pH (e.g., in the small intestine).

[0455] Regardless of the type of dosage unit, it contains a therapeutically effective amount of one or more lipid compounds or lipid nanoformulations described herein. One skilled in the art can determine the amount of lipid-based carrier, or lipid nanoformulation, or compound suitable for incorporation into a pharmaceutical composition in a dosage unit.

[0456] In some embodiments, the lipid-based carrier, pharmaceutical composition, or dosage unit contains about 0.01 to about 1000 mg of one or more lipid compounds described herein. In some embodiments, the lipid-based carrier, pharmaceutical composition, or dosage unit contains about 0.01, about 0.1, about 0.5, about 1, about 5, about 10, about 25, about 50, about 75, about 100, about 125, about 150, about 175, about 200, about 225, 250, about 275, about 300, about 350, about 400, about 450, about 500, about 550, about 600, about 650, about 700, about 750, about 800, about 850, about 900, about 950, or about 1000 mg of one or more lipid compounds described herein.In some embodiments, the lipid-based carrier, pharmaceutical composition, or dosage unit contains from about 0.01 to about 750 mg, from about 0.01 to about 500 mg, from about 0.01 to about 250 mg, from about 0.01 to about 100 mg, from about 0.01 to about 50 mg, from about 0.01 to about 25 mg, from about 0.01 to about 10 mg, from about 0.01 to about 5 mg, from about 0.01 to about 0.1 mg, from about 0.1 to about 1000 mg, from about 0.1 to about 750 mg, from about 0.1 to about 500 mg, from about 0.1 to about 250 mg, from about 0.1 to about 100 mg, from about 0.1 to about 5 mg, Approximately 50 mg, approximately 0.1 to approximately 25, approximately 0.1 to approximately 10 mg, approximately 0.1 to approximately 5 mg, approximately 0.1 to approximately 1 mg, approximately 1 to approximately 1000 mg, approximately 1 to approximately 750 mg, approximately 1 to approximately 500 mg, approximately 1 to approximately 250 mg, approximately 1 ~100mg, 1~50mg, 1~25mg, 1~10mg, 1~5mg, 5~1000mg, 5~750mg, 5~500mg, 5~250mg, 5~10 0 mg, about 5 to about 50 mg, about 5 to about 25 mg, about 5 to about 10 mg, about 10 to about 1000 mg, about 10 to about 750 mg, about 10 to about 500, about 10 to about 250 mg, about 10 to about 100 mg, about 10 to about About 50 mg, about 10 to about 25 mg, about 25 to about 1000 mg, about 25 to about 750 mg, about 25 to about 500 mg, about 25 to about 250 mg, about 25 to about 100 mg, about 25 to about 50 mg, about 50 to about 100 0 mg, about 50 to about 750 mg, about 50 to about 500 mg, about 50 to about 250 mg, about 50 to about 100 mg, about 100 to about 1000 mg, about 100 to about 750 mg, about 100 to about 500 mg, about 100 to about 250 mg, about 250 to about 1000 mg, about 250 to about 750 mg, about 250 to about 500 mg, about 500 to about 1000 mg, about 500 to about 750 mg, or about 750 to about 1000 mg.

[0457] Methods of Using Pharmaceutical Compositions Certain aspects of the present invention also relate to various methods of using the pharmaceutical compositions described herein.

[0458] All of the above descriptions of aspects of lipid compounds, such as compounds encompassed by formulas (Ia), (AL-GI), (AL-Ia)-(AL-Ic), (AL-IIa)-(AL-IIc), (AL-IIIa)-(AL-IIIg), (AL-IVa)-(AL-IVc), and exemplary formulas of lipids having at least two ester groups, lipids containing lactide (or derivatives thereof) groups, and lipids containing phosphoramidate groups, and all of the embodiments discussed in the above aspects, are all applicable to these aspects of the invention relating to various methods of using the pharmaceutical compositions described herein.

[0459] All of the above statements regarding aspects of lipid-based carriers (or lipid nanoformulations) that include various other lipid components and all of the embodiments discussed in those aspects are applicable to these aspects of the invention regarding the various methods of using the pharmaceutical compositions described herein.

[0460] All of the above descriptions of aspects of pharmaceutical compositions, including various aspects of therapeutic agents and other ingredients, and all of the embodiments discussed in the above aspects, are applicable to these aspects of the invention relating to the various methods of using the pharmaceutical compositions described herein.

[0461] Some embodiments relate to methods of delivering a therapeutic agent (encapsulated in a lipid-based carrier or pharmaceutical composition described herein) to one or more cells of a subject or organism, comprising administering a pharmaceutical composition described herein (containing a lipid-based carrier that contains a lipid compound described herein) under conditions suitable for delivery of the pharmaceutical composition described herein to one or more cells of the subject or organism.

[0462] Some embodiments relate to a method of modulating expression of a target gene in a cell, comprising introducing into the cell a pharmaceutical composition described herein (containing a lipid-based carrier containing a lipid compound described herein and a therapeutic agent such as a nucleic acid (e.g., mRNA)) under conditions suitable for modulating expression of the target gene in the cell. In one embodiment, the cell is a liver cell (e.g., a hepatocyte).

[0463] Some embodiments relate to a method of modulating expression of multiple target genes in a cell, comprising introducing into the cell a pharmaceutical composition described herein (comprising a lipid-based carrier containing a lipid compound described herein and a therapeutic agent, such as a nucleic acid (e.g., mRNA)) under conditions suitable for modulating expression of the target genes in the cell. In one embodiment, the cell is a liver cell (e.g., a hepatocyte).

[0464] Some embodiments relate to methods for expressing an RNA or polypeptide in a subject or organism in need thereof, comprising contacting the subject or organism with a pharmaceutical composition described herein (comprising a lipid-based carrier containing a lipid compound described herein and a therapeutic agent, such as a nucleic acid (e.g., RNA)) under conditions suitable for expressing the RNA or polypeptide in the subject or organism.

[0465] Some embodiments relate to methods of preventing or treating a disease, disorder, and / or condition in a subject in need thereof, which disease, disorder, and / or condition may be characterized by deficient or abnormal protein or polypeptide activity. The method includes administering a pharmaceutical composition described herein (containing a lipid-based carrier containing a lipid compound described herein and a therapeutic agent, such as a nucleic acid (e.g., RNA)), where the RNA may be mRNA encoding a polypeptide that antagonizes or otherwise overcomes the activity of an abnormal protein or polypeptide present in a cell of the subject, thereby preventing or treating the disease, disorder, and / or condition. In one embodiment, the cell is a liver cell (e.g., a hepatocyte).

[0466] "Treatment" or variations thereof refers to ameliorating or alleviating the onset of a disease or disorder, i.e., delaying the onset of the disease. In certain embodiments, "treatment" refers to improving or reducing at least one physical parameter of the disease or disorder. In other embodiments, "treatment" is directed to ameliorating the disease or disorder. In further embodiments, "treatment" is directed to the cause of the disease or disorder. In yet other embodiments, "treatment" is directed to alleviating the symptoms of the disease or disorder. In yet further embodiments, "treatment" is directed to treating a disease or disorder as a complement to another therapy.

[0467] In one embodiment, in any of the above methods, the method comprises contacting the subject or organism with a pharmaceutical composition described herein via local administration to the relevant tissue or cells.

[0468] In one embodiment, in any of the above methods, the method comprises contacting a subject or organism with a pharmaceutical composition described herein via systemic administration to the relevant tissue or cells (such as intravenous or subcutaneous administration of the formulation or composition). The formulations or compositions of the invention may be formulated or conjugated to target the appropriate tissue or cells of the subject or organism, as described herein or otherwise known in the art.

[0469] In any of the above methods, the pharmaceutical compositions described herein may be administered at various time intervals, for example, once daily, once every two days, once every three days, once every four days, once every five days, once every six days, once every week, once every two weeks, once every month, etc. In one embodiment, administration is once every 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 weeks.

[0470] In any of the above methods, the pharmaceutical compositions described herein may be administered to the subject systemically, as described herein or otherwise known in the art. Systemic administration may include, for example, intravenous, subcutaneous, intramuscular, catheterized, nasopharyngeal, transdermal, or gastrointestinal administration, as generally known in the art.

[0471] In one embodiment, in any of the above methods of treatment or prevention, the pharmaceutical compositions described herein may be administered locally to a subject or administered to a local tissue, as described herein or otherwise known in the art. Local administration may include, for example, catheter insertion, implantation, osmotic pumping, direct injection, intrathecal, ventricular, dermal / transdermal application, stent placement, ear / eye drops, or portal vein administration to the relevant tissue, or any other local administration technique, method, or procedure, as generally known in the art.

[0472] kit The present disclosure also provides kits for use in delivering pharmaceutical compositions to a target site or for diagnostic or therapeutic purposes. Such kits may include one or more containers containing any of the pharmaceutical compositions described herein and a pharmaceutically acceptable carrier / excipient.

[0473] In some embodiments, the kit may include instructions for use according to any of the methods described herein. The included instructions may include instructions for administering the pharmaceutical composition according to any of the methods described herein. The kit may further include instructions for selecting individuals suitable for diagnosis or treatment.

[0474] Instructions for use of the pharmaceutical compositions described herein generally include information regarding dosages, administration schedules, and routes of administration for the intended treatment. Containers may be unit doses, bulk packages (e.g., multi-dose packages), or sub-unit doses. Instructions provided in the kit are typically written instructions on a label or package insert (e.g., a paper sheet included in the kit), although machine-readable instructions (e.g., instructions written on a magnetic or optical storage disk) are also acceptable.

[0475] The kits described herein are in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags), and the like. Also contemplated are packaging for use in combination with a specific device, such as an inhaler, an intranasal administration device (e.g., an atomizer), or an infusion device such as a minipump. The kit may have a sterile access port (e.g., the container may be an intravenous solution bag or vial with a stopper pierceable by a hypodermic needle). The container may also have a sterile access port (e.g., the container may be an intravenous solution bag or vial with a stopper pierceable by a hypodermic needle). The kits described herein may optionally provide additional components, such as buffers and interpretive information. Typically, the kit includes a container and a label or package insert on or associated with the container. In some embodiments, the present disclosure provides an article of manufacture containing the contents of the kit described above. [Example]

[0476] The following examples are for illustrative purposes only and are not intended to limit the scope of the present invention in any way. To the extent that specific materials are mentioned, this is for illustrative purposes only and is not intended to limit the present invention. Those skilled in the art may develop equivalent methods or reactants without the exercise of inventive capacity and without departing from the scope of the present invention.

[0477] Example 1 - General Reaction for the Synthesis of Ionizable Lipid Compounds General reaction schemes for the synthesis of exemplary ionizable lipid compounds containing various cleavable linkers are shown in Schemes 1-3.

[0478] Scheme 1 [ka] As shown in Scheme 1, the synthesis begins with coupling an acrylated lipid with ethanolamine, which is then alkylated with a brominated lipid to produce a final product containing an ester-cleavable linker in the lipid tail. Both cis and trans stereoisomers can be synthesized.

[0479] Scheme 2 [ka] As shown in Scheme 2, the synthesis begins with functionalizing POCl with alkenyl alcohols, alkyl alcohols, and amines to generate alkenyl phosphoramidates. This intermediate compound is then hydrogenated to form a brominated phosphoramidate lipid, which is then reacted with ethanolamine and another brominated lipid to form the final product. Both cis and trans stereoisomers can be synthesized.

[0480] Scheme 3 [ka] As shown in Scheme 3, the synthesis is based on coupling two lipid building blocks, each bearing a lactic acid-like unit, via a coupling agent (e.g., N,N'-diisopropylcarbodiimide) and cyclizing these two lipid building blocks to form the final product. Both cis and trans stereoisomers can be synthesized.

[0481] Example 2 - Preparation of an exemplary lipid nanoparticle composition Lipid nanoparticle compositions, such as therapeutic compositions, can be prepared as described herein by the selection of lipid compounds described herein (e.g., those encompassed by the exemplary formulas (AL-GI), (AL-Ia)-(AL-Ic), (AL-IIa)-(AL-IIc), (AL-IIIa)-(AL-IIIg), (AL-IVa)-(AL-IVc), and lipids having at least two ester groups, lipids containing lactide (or derivatives thereof) groups, and lipids containing phosphoramidate groups), the selection of additional lipids, the amount of each lipid in the lipid component, and the wt:wt ratio of the lipid components.

[0482] Preparation of lipid nanoparticles: Specific lipid nanoparticle (LNP) components and ratios are selected to achieve various lipid nanoparticle properties. Lipid nanoparticle formulation recipes for LNP compositions include various molar ratios of ionizable lipids, cholesterol, phospholipids (or other structured lipids), PEG-lipids, and / or additional components (e.g., RNA cargo). LNP composition formulations are evaluated to determine properties such as encapsulation, size, zeta potential, apparent pKa, and in vitro / in vivo protein expression levels.

[0483] In one example, the molar ratio of ionizable lipid / sterol / phospholipid (or other structured lipid) / PEG-lipid / additional component is varied within the following ranges: ionizable lipid (25-100%), sterol (0-50%), phospholipid (DSPC) (0-40%), and PEG-lipid (0-5%). The nitrogen:phosphate ratio (N:P ratio) is evaluated on a scale of 0.1 to 100.

[0484] Lipid nanoparticle formulation procedure: In one example, ionizable lipids, structural lipids (e.g., phospholipids such as DSPC), sterols (e.g., cholesterol), and PEG lipids (e.g., DSPE-PEG 2k) are individually dissolved in ethanol. The separate lipid solutions are combined by pipetting in a molar ratio of ionizable lipid / cholesterol / DSPC / DSPE-PEG 2k of 50 / 38.5 / 10 / 1.5 to produce a lipid stock solution. Nucleic acid (e.g., mRNA) is diluted in a buffer (e.g., 10 mM citrate) and mixed with this ethanolic lipid solution using a syringe pump or microfluidic mixing. The resulting LNP composition is collected for further processing.

[0485] In another example, the NanoAssemblr® Ignite™ (Precision Nanosystems) system is used. A lipid solution containing lipids at the molar ratio described above is loaded into a syringe. An mRNA solution (0.25 mg / mL) is prepared using citrate buffer (10 mM, pH 4) and loaded into a syringe. A NanoAssembler® microfluidic chip is used to mix the lipid and RNA solutions at 2.5 and 7.5 mL / min. The resulting nanoparticles, with a buffer to ethanol ratio of 3:1, are ready for further downstream processing and purification.

[0486] Processing / Refining Processing of LNP compositions is important to maintain the size and physical properties of formulated lipid nanoparticles. Storage buffers and residual ethanol can affect the stability of lipid nanoparticles. Processing can remove ethanol present during formulation and allow for buffer exchange.

[0487] After formulation, the LNP composition is left for 30 minutes, and then diluted 1:1 with deionized water.To purify and concentrate the lipid nanoparticle composition, the solution is loaded onto a desalting column (e.g., PD-10 Sepharose desalting column) to exchange the buffer solution with 1xPBS, and then concentrated to about 0.5mg / ml (nucleotide cargo) with an Amicon centrifugal spin filter.Finally, the LNP composition is filtered through a 0.2 μm filter.

[0488] Example 3 - Characterization of lipid nanoparticle compositions To determine the safety and efficacy of lipid nanoparticle compositions for use in the delivery of therapeutic molecules to cells, various lipid nanoparticle formulations are tested and characterized.

[0489] Various lipid nanoparticle compositions containing nucleic acid payloads are characterized by various methods. For example, dynamic light scattering (DLS) is used to determine particle size (Wyatt Dynapro Platereader III). In addition, instruments such as the Zetasizer Nano ZS (Malvern Instruments Ltd) are used to measure multiple physical properties of lipid nanoparticle compositions, such as particle size, polydispersity index (PDI), and zeta potential. A fluorescence-based assay is used to determine the efficiency of nucleic acid encapsulation in lipid nanoparticles. A fluorescence-based assay is used to determine the "apparent pKa" of lipid nanoparticles. Both fluorescence assays are evaluated on a Varioskanlux plate reader (ThermoScientific).

[0490] Nanoparticle size After formulation and processing of the lipid nanoparticle composition, the Z-average size of the lipid nanoparticle composition is assayed by dynamic light scattering (DLS) (e.g., using a Wyatt Dynapro Platereader III or a Malvern Zetasizer ZS). Briefly, a 2 μL aliquot of the processed nanoparticle composition mixture (approximately 0.5 mg / mL RNA concentration) is diluted with 50 μL of phosphate-buffered saline (0.1×PBS) and loaded into a 384-well plate. The sample is then analyzed to determine the nanoparticle size.

[0491] Encapsulation efficiency assay / Ribogreen assay For lipid nanoparticle formulations containing RNA, the QUANTIT™ RIBOGREEN® RNA assay (Invitrogen Corporation) is used to assess encapsulation of RNA within the lipid nanoparticle formulation.

[0492] The sample is diluted to approximately 10 μg / ml with TE buffer. In a 96-well plate, 50 μl of the diluted sample is added twice to 50 μL of TE buffer, and 20 μL of the diluted sample is added twice to 80 μL of Triton X buffer (2% Triton in TE buffer). The plate is incubated at room temperature for 10 minutes. Next, RIBOGREEN® is diluted 1:200 with TE buffer, and 100 μL is added to each sample well. Fluorescence intensity is measured using a fluorescent plate reader (e.g., Varioskanlux, ThermoScientific) at an excitation wavelength of approximately 480 nm and an emission wavelength of approximately 520 nm. Encapsulation efficiency can be determined by using the percentage ratio of non-encapsulated RNA (TE sample) to total RNA (TX sample) and then subtracting it from 100 to determine the percentage of encapsulated RNA.

[0493] TNS Assay / Protocol for Determining "Apparent pKa" The apparent pKa of lipid nanoparticle compositions is measured using a 6-(p-toluidino)-2-naphthalenesulfonic acid (TNS) assay. Briefly, lipid nanoparticles (10 uL of approximately 0.5 mg mRNA / mL) and TNS probe (40 uL of 1 mM stock) are added to 750 uL of TNS buffer (25 mM citric acid, 20 mM sodium phosphate, 150 mM NaCl, and 20 mM ammonium acetate) at pHs ranging from 2 to 10. 20 uL of each sample is added to 80 uL of buffer at each pH in a 96-well plate. The plate is incubated for 10 minutes, and the λ ex = 321 nm and λ em The fluorescence signal is measured using a spectrofluorometer (Varioskanlux, ThermoScientific) with excitation / emission settings of 445 nm. The apparent pKa of the nanoparticle composition is determined by deriving the pH at which 50% of the pH2 fluorescence signal is present. All data points are normalized to the fluorescence signal at pH 2.

[0494] Zeta potential assay The zeta potential of the nanoparticle composition is determined using a Zetasizer Nano ZS (Malvern Instruments Ltd). Briefly, a 5 μL aliquot of approximately 0.5 mg mRNA / mL sample is diluted with 800 μL of 0.1×PBS. The sample is then transferred to a disposable conductive cuvette and the zeta potential is measured.

[0495] Example 4 - In vitro and in vivo testing of lipid nanoparticle compositions Lipid nanoparticle compositions containing polynucleotides such as mRNA are useful in assessing the efficacy and biological activity of various lipid nanoparticle formulations, both in in vitro and in vivo settings. Higher levels of protein expression resulting from administration of a formulation containing mRNA would indicate higher mRNA translation and / or lipid nanoparticle mRNA delivery efficiency.

[0496] After administration of the lipid nanoparticle composition to mice or cells, the dose delivery profile, dose response, and toxicity of a particular formulation, as well as the dose used, are measured by enzyme-linked immunosorbent assay (ELISA), bioluminescence imaging, fluorescence-assisted cell sorting (FACS), or other methods. For lipid nanoparticle compositions containing mRNA, the time course of protein expression may also be evaluated. Samples used for analysis may include cell suspensions, supernatants, or adherent cells. For in vivo studies, samples collected from rodents for evaluation may include blood, serum, and tissues (e.g., muscle tissue from the intramuscular injection site or harvested organs); sample collection may involve sacrificing the animal.

[0497] In vitro hEPO assay Briefly, 10,000 cells from selected cell lines (e.g., immortalized cells such as HeLas and primary cells such as PBMCs) are plated in a 96-well plate and maintained overnight in an incubator. The medium is replaced with Opti-MEM, and for dose-response evaluation, the cells are treated in triplicate with hEPO-mRNA-containing lipid nanoparticles at doses ranging from 500 ng / well to 50 pg / well. After 6 hours of incubation, 20 μL of supernatant is collected from each well and quantified using an EPO Human ProQuantum Immunoassay Kit (ThermoFisher Scientific) according to the manufacturer's protocol. After 24 hours, cytotoxicity is assessed by a fluorescence-based assay (e.g., CellTiter-Fluor™ Cell Viability Assay, Promega) using a spectrofluorometer (Varioskanlux, ThermoScientific). EPO expression levels indicate protein expression resulting from administration of a particular lipid nanoparticle formulation. Cytotoxicity also provides information about the therapeutic potential of a particular lipid nanoparticle formulation.

[0498] In vivo EPO assay Mice are administered a single dose of a lipid nanoparticle composition with a formulation such as that provided in Example 2 intravenously, intramuscularly, intraarterially, or intratumorally. Doses can range from 0.001 mg / kg to 10 mg / kg, where 10 mg / kg describes a dose containing 10 mg of polynucleotide in the lipid nanoparticle composition for every kg of mouse body weight. A control composition containing PBS is used.

[0499] In one example, a group of mice (e.g., n=5) is injected via tail vein (IV) or intramuscularly (IM) and imaged by IVIS (luciferase mRNA) at 6, 24, and 48 hours post-injection. In another example, secreted protein levels of human erythropoietin (EPO mRNA) are measured from blood drawn at 6, 24, and 48 hours post-injection and assayed for EPO concentration by ELISA. Blood / serum samples allow assessment of liver enzyme levels such as ALT / AST, which are measured by ELISA. Blood / serum samples are also used to assay cytokine production, which is measured by Luminex (TRONSITE-LX200, Millipore Sigma).

[0500] Example 5: Synthesis of general intermediates: Compounds A-G were synthesized in bulk and then used as intermediates throughout these experimental procedures. For the synthesis of each lipid (Example 6), compound numbering begins with compound 1.

[0501] Compound A: [ka]

[0502] Synthesis of Compound A: [ka]

[0503] Preparation of Compound 3: [ka] To a solution of compound 1 (9 g, 35.09 mmol, 1 equiv.) in DCM (90 mL) was added compound 2 (11.74 g, 52.64 mmol, 1.5 equiv.), DMAP (857.43 mg, 7.02 mmol, 0.2 equiv.), EDCI (8.07 g, 42.11 mmol, 1.2 equiv.), and DIEA (9.07 g, 70.18 mmol, 12.22 mL, 2 equiv.). The resulting mixture was stirred at 20 °C for 16 h. TLC showed that compound 1 was consumed and one major new spot was detected. The reaction mixture was diluted with DCM (100 mL), washed with NH Cl (2 x 30 mL), dried over Na SO , filtered, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, petroleum ether) to give compound 3 (6.25 g, 13.54 mmol, 38.59% yield, 100% purity) as a white solid. 1 H NMR (400 MHz, chloroform-d) δ = 4.92-4.83 (m, 1H), 3.56-3.50 (m, 1H), 3.43-3.36 (m, 1H), 2.31-2.26 (m, 2H), 1.90-1.72 (m, 2H), 1.70-1.59 (m, 2H), 1.55-1.39 (m, 6H), 1.39-1.05 (m, 28H), 0.91-0.85 (m, 6H).

[0504] Preparation of Compound A: [ka] To a solution of compound 3 (3 g, 6.50 mmol, 1 equiv.) in ACN (30 mL) was added compound 3A (2.78 g, 45.50 mmol, 2.75 mL, 7 equiv.), K2CO3 (1.80 g, 13.00 mmol, 2 equiv.), and KI (1.19 g, 7.15 mmol, 1.1 equiv.). The resulting mixture was heated to 80 °C for 16 h. LCMS showed that compound 3 was consumed and the desired MS was detected. The reaction mixture was filtered, and the filtrate was concentrated to give a residue. The residue was purified by column chromatography (SiO2, dichloromethane:methanol = 40:1 to 4:1) to give compound A (1.7 g, 3.85 mmol, 59.21% yield, 100% purity) as a yellow oil. LCMS [M+1] + =442.5 1 H NMR(400MHz,chloroform-d)δ=4.91-4.83(m,1H),3.71-3.65(m,2H),3.03-2.91(m,2H),2.85-2.80(m,2H),2.70- 2.63(m,2H),2.32-2.25(m,2H),1.68-1.58(m,2H),1.55-1.50(m,4H),1.37-1.22(m,32H),0.92-0.85(m,6H)

[0505] Compound B: [ka]

[0506] Synthesis of Compound B: [ka]

[0507] Preparation of Compound 3: [ka] To a solution of compound 2 (2 g, 11.61 mmol, 1 equiv.) in toluene (20 mL), compound 1 (2.72 g, 13.93 mmol, 1.2 equiv.) and 4-methylbenzenesulfonic acid (999.39 mg, 5.80 mmol, 0.5 equiv.) were added, and the mixture was heated to 50 °C under a N atmosphere for 16 h. LCMS showed that compound 2 was completely consumed and one major peak with the desired MS was detected. The residue was diluted with HO (50 mL) and extracted with EA (50 mL x 3). The combined organic layers were dried over NaSO, filtered, and concentrated under reduced pressure to give the residue. The residue was purified by column chromatography (SiO, petroleum ether:ethyl acetate = 99 / 1 to 95 / 5) to give compound 3 (4.25 g, crude) as a colorless oil. LCMS [M+1] + =348.2 1H NMR(400MHz,chloroform-d)δ=4.07(t,J=6.8Hz,2H),3.42(t,J=6.8Hz,2H),2.33(t,J=7.2Hz,2H), 1.94-1.84(m,2H),1.71-1.58(m,4H),1.55-1.43(m,2H),1.36-1.23(m,16H),0.93-0.85(m,3H)

[0508] Preparation of Compound B: [ka] A mixture of compound 3 (5 g, 14.31 mmol, 1 equiv.), compound 3A (4.37 g, 71.56 mmol, 4.32 mL, 5 equiv.), K2CO3 (3.96 g, 28.62 mmol, 2 equiv.), and KI (237.59 mg, 1.43 mmol, 0.1 equiv.) in ACN (20 mL) was degassed and purged with N2 three times, and then the mixture was stirred at 80 °C under a N2 atmosphere for 16 h. LCMS showed that compound 3 was completely consumed and one major peak with the desired MS was detected. The residue was diluted with H2O (50 mL) and extracted with DCM (100 mL x 3). The combined organic layers were dried over Na2SO4, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, DCM:MeOH = 50:1 to 4:1) to give compound B (3.13 g, 9.50 mmol, yield 66.37%) as a white solid. LCMS [M+1] + =330.4 1H NMR (400MHz, chloroform-d) δ=4.06(t,J=6.8Hz,2H),3.69-3.60(m,2H),2.82-2.76(m,2H),2.65(t,J=7.2Hz,2H ),2.31(t,J=7.6Hz,2H),1.70-1.58(m,4H),1.53(quin,J=7.2Hz,2H),1.44-1.20(m,18H),0.94-0.85(m,3H)

[0509] Compound C: [ka]

[0510] Compound D: [ka]

[0511] Synthesis of Compound D: [ka]

[0512] Preparation of Compound 2: [ka] POCl (10.63 g, 69.32 mmol, 6.46 mL, 2 equiv.) was added dropwise to compound 1 (5 g, 34.66 mmol, 1 equiv.) at 20 °C. After the addition, the mixture was stirred at this temperature for 16 h. TLC showed that compound 1 was consumed and one major new spot was detected. The reaction mixture was concentrated under reduced pressure to give compound 2 (7 g, 26.81 mmol, 77.34% yield, 100% purity) as a yellow oil, which was used without further purification. 1 H NMR (400 MHz, chloroform-d) δ = 4.39-4.30 (m, 2H), 1.86-1.76 (m, 2H), 1.48-1.21 (m, 12H), 0.93-0.85 (m, 3H)

[0513] Preparation of Compound 4: [ka] To a solution of compound 2 (5 g, 19.15 mmol, 1 equiv.) in toluene (100 mL) was added compound 3 (2.19 g, 15.32 mmol, 0.8 equiv.) and TEA (1.55 g, 15.32 mmol, 2.13 mL, 0.8 equiv.). The resulting mixture was stirred at 20 °C for 2 h. TLC showed that compound 2 was consumed and one major new spot was detected. The reaction mixture was diluted with saturated NH4Cl (50 mL), extracted with ethyl acetate (2 x 50 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, petroleum ether:ethyl acetate = 100 / 1 to 10 / 1) to give compound 4 (2.5 g, 6.79 mmol, 35.49% yield, 100% purity) as a white solid. LCMS[M+1] + =368.3 1H NMR (400 MHz, chloroform-d) δ = 4.06 (br s, 2H), 3.29-3.09 (m, 1H), 3.07-2.96 (m, 2H), 1.79-1.68 (m, 2H), 1.59-1.50 (m, 2H), 1.46-1.17 (m, 24H), 0.99-0.81 (m, 6H)

[0514] Preparation of Compound C: [ka] To a solution of compound 4 (2.3 g, 6.25 mmol, 1 equiv.) in DCM (14 mL) was added compound 5 (1.34 g, 6.88 mmol, 1.1 equiv.) and TEA (1.27 g, 12.50 mmol, 1.74 mL, 2 equiv.). The resulting mixture was stirred at 30° C. for 16 h. LCMS showed that more than 90% of compound 4 was consumed, and several new peaks were observed by LCMS. The reaction mixture was diluted with saturated NH4Cl (20 mL), extracted with DCM (2×10 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by reverse-phase HPLC (column: Welch Xtimate C1 100 × 30 mm × 5 μm; mobile phase: [HO (10 mM NHHCO)-THF:ACN = 1:3]; gradient: 50% to 80% B over 20.0 min) to give compound C (0.52 g, 986.04 μmol, 15.77% yield, 99.85% purity) as a yellow oil. LCMS [M+1] + =527.3,LCMS [2M+1] + =1053.5 1 H NMR(400MHz,chloroform-d)δ=4.06-3.91(m,4H),3.41(t,J=6.8Hz,2H),2.94-2.83(m,2H),2.51-2.40(m,1 H),1.91-1.77(m,2H),1.73-1.64(m,4H),1.54-1.39(m,6H),1.38-1.21(m,26H),0.89(t,J=6.8Hz,6H)

[0515] Preparation of Compound D: [ka] To a solution of compound C (2 g, 3.80 mmol, 1 equiv.) in EtOH (20 mL) was added compound C1 (6.96 g, 113.95 mmol, 6.88 mL, 30 equiv.). The mixture was heated to 80 °C for 16 h. LCMS showed that compound C was completely consumed and one major peak with the desired MS was detected. The residue was diluted with HO (15 mL), extracted with ethyl acetate (10 mL x 3), dried over NaSO, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO, DCM:MeOH = 50:1 to 3:1) to give compound D (1.34 g, 2.64 mmol, 69.62% yield) as a yellow oil. LCMS[M+1] + =507.5 1 H NMR(400MHz,chloroform-d)δ=4.04-3.89(m,4H),3.72-3.57(m,2H),2.93-2.76(m,4H),2.71-2 .53(m,6H),1.72-1.62(m,4H),1.58-1.44(m,4H),1.42-1.21(m,30H),0.88(t,J=6.8Hz,6H)

[0516] Compound E: [ka]

[0517] Preparation of Compound E: [ka] To a solution of compound 1 (10 g, 44.82 mmol, 1 eq.) and compound 2 (9.27 g, 53.79 mmol, 1.2 eq.) in DCM (100 mL) was added EDCI (10.31 g, 53.79 mmol, 1.2 eq.), DMAP (1.10 g, 8.96 mmol, 0.2 eq.), and DIEA (11.59 g, 89.64 mmol, 15.61 mL, 2 eq.). The mixture was stirred at 20° C. for 16 hours. TLC (petroleum ether:ethyl acetate=10:1, R f =0.5), indicating the complete consumption of compound 1 and the appearance of one new spot. The reaction mixture was diluted with DCM (100 mL) and washed with HO (200 mL). The combined organic layers were dried over NaSO, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO, petroleum ether:ethyl acetate = 1:0 to 300:1) to give compound E (8.7 g, 22.82 mmol, 50.92% yield, 99% purity) as a colorless oil. 1 H NMR(400MHz,chloroform-d)δ=4.83-4.67(m,1H),3.37-3.30(m,2H),2.25-2.18(m,2H),1.78(quin,J=7.2Hz,2 H),1.60-1.52(m,2H),1.46-1.40(m,3H),1.39(s,2H),1.30(s,5H),1.23-1.14(m,12H),0.84-0.77(m,6H)

[0518] Compound F: [ka]

[0519] Synthesis of compound F: [ka]

[0520] Preparation of Compound 4: [ka] A mixture of compound E (8.7 g, 23.05 mmol, 1 equiv.) and compound 3 (20.08 g, 115.26 mmol, 20.12 mL, 5 equiv.) in EtOH (70 mL) was degassed and purged with N2 three times, and then the mixture was stirred at 80 °C under N2 atmosphere for 16 h. LCMS showed that compound E was completely consumed and one major peak with the desired MS was detected. The reaction mixture was partitioned between DCM (60 mL) and HCl (0.5 mol, 60 mL). The organic phase was separated, dried over Na2SO4, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, dichloromethane:methanol = 50:1 to 8:1) to give compound 4 (7.2 g, 15.14 mmol, 65.69% yield, 99% purity) as a yellow solid. LCMS[M+1] + =471.5 1 H NMR(400MHz,chloroform-d)δ=4.86-4.73(m,1H),3.40-3.22(m,2H),3.08-2.82(m,4H),2.33-2.22(m,2H),2.16-1.98(m,2H),1.91-1.83(m, 2H),1.82-1.68(m,3H),1.66-1.57(m,3H),1.57-1.47(m,4H),1.47(d,J=4.4Hz,9H),1.31(s,6H),1.30-1.24(m,11H),0.92-0.84(m,6H)

[0521] Preparation of Compound 6: [ka] To a solution of compound 4 (6.2 g, 13.17 mmol, 1 equiv.) and compound A (6.08 g, 13.17 mmol, 1 equiv.) in ACN (130 mL) was added K2CO3 (5.46 g, 39.51 mmol, 3 equiv.) and KI (2.19 g, 13.17 mmol, 1 equiv.). The mixture was heated to 90 °C for 16 h. LCMS showed that compound 4 was completely consumed and one major peak with the desired MS was detected. The reaction mixture was concentrated to give a residue. The residue was diluted with DCM (20 mL) and washed with H2O (30 mL). The combined organic layers were dried over Na2SO4, filtered, and concentrated under reduced pressure to give the crude product. The crude product was purified by column chromatography (SiO2, dichloromethane:methanol = 1:0 to 10:1) to give compound 6 (10 g, 11.28 mmol, yield 85.61%, purity 96%) as a yellow oil. LCMS[M+1] + =851.7 1 H NMR (400MHz, chloroform-d) δ=5.70-5.48(m,1H),4.91-4.75(m,2H),3.27-3.09(m,2H),2.57-2.46(m,2H),2.44-2.35(m ,3H),2.31(s,4H),1.70-1.57(m,7H),1.57-1.47(m,9H),1.47-1.41(m,12H),1.35-1.23(m,48H),0.93-0.82(m,12H)

[0522] Preparation of Compound F: [ka] A mixture of compound 6 (10 g, 11.75 mmol, 1 equiv.) in HCl / dioxane (4 M, 50 mL) was degassed and purged with N three times, and then the mixture was stirred at 20 °C under N atmosphere for 3 h. LCMS showed that compound 6 was completely consumed and one major peak with the desired MS was detected. The mixture was concentrated to give the crude product. The crude product was dissolved in DCM (100 mL), and the mixture was washed with saturated NaHCO (30 mL). The combined organic layers were dried over NaSO, filtered, and concentrated under reduced pressure to give compound F (8.6 g, 11.45 mmol, 90.17% yield, 100% purity) as a colorless oil. LCMS [M+1] + =751.8 1 H NMR(400MHz,chloroform-d)δ=4.97-4.79(m,2H),2.80-2.68(m,2H),2.51-2.43(m,2H),2.41-2.35(m,4H),2.3 3-2.23(m,4H),1.56(d,10H),1.54-1.47(m,6H),1.45-1.39(m,4H),1.35-1.23(m,48H),0.93-0.80(m,12H)

[0523] Compound G: [ka]

[0524] Synthesis of compound G: [ka]

[0525] Preparation of Compound 2: [ka] A mixture of compound E (2.5 g, 6.62 mmol, 1 equiv.) and compound 1 (9.95 g, 132.49 mmol, 10.22 mL, 20 equiv.) in EtOH (50 mL) was degassed and purged with N2 three times, and then the mixture was stirred at 60 °C under N2 atmosphere for 16 h. LCMS showed that compound E was completely consumed and one major peak with the desired MS was detected. The residue was diluted with HCl (0.5 M, 50 mL) and extracted with DCM (60 mL x 2). The combined organic layers were dried over Na2SO4, filtered, and concentrated under reduced pressure to give the residue. The residue was purified by column chromatography (SiO2, DCM:MeOH = 99:1 to MeOH) to give compound 2 (6.85 g, 18.43 mmol, 68.50% yield) as a pale yellow oil. LCMS [M+1] + =372.4 1 H NMR(400MHz,chloroform-d)δ=4.89-4.77(m,1H),3.88-3.75(m,2H),2.94-2.85(m,2H),2.62(t,J=7.2Hz,2H),2.29(t,J=7.6Hz,2H ),1.71(td,J=5.6,10.8Hz,2H),1.65-1.58(m,2H),1.57-1.44(m,5H),1.66-1.43(m,1H),1.41-1.14(m,18H),0.98-0.82(m,6H)

[0526] Preparation of Compound 3: [ka] A mixture of compound 2 (1 g, 2.69 mmol, 1 equiv.), compound A (1.24 g, 2.69 mmol, 1 equiv.), K2CO3 (1.49 g, 10.76 mmol, 4 equiv.), and KI (446.73 mg, 2.69 mmol, 1 equiv.) in ACN (20 mL) was degassed and purged with N2 three times, and then the mixture was stirred at 90 °C under a N2 atmosphere for 16 h. LCMS showed that compound A was completely consumed and one major peak with the desired MS was detected. The reaction mixture was diluted with H2O (30 mL) and extracted with DCM (2 x 40 mL). The combined organic layers were dried over Na2SO4, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, DCM:MeOH = 98:2 to 96:4) to give compound 3 (1.3 g, 1.73 mmol, yield 64.22%) as a yellow oil. LCMS [M+1] + =752.7 1 H NMR(400MHz,chloroform-d)δ=4.91-4.76(m,2H),3.81(t,J=5.2Hz,2H),2.72(br s,2H),2.50(br s,4H),2.29(dt,J=4.0,7.6Hz,4H),1.73(br s,2H),1.67-1.58(m,5H),1.58-1.50(m,10H),1.40-1.16(m,50H),0.95-0.83(m,12H)

[0527] Preparation of Compound G: [ka] A mixture of compound 3 (1 g, 1.33 mmol, 1 equiv.) and PBr (737.67 mg, 2.73 mmol, 2.05 equiv.) in MeCN (10 mL) was degassed and purged with N three times, and then the mixture was stirred at 75 °C under a N atmosphere for 2 h. LCMS showed that compound 3 was completely consumed and one major peak with the desired MS was detected. The residue was diluted with HO (80 mL) and extracted with DCM (2 x 100 mL). The combined organic layers were dried over NaSO, filtered, and concentrated under reduced pressure to give compound G (1.56 g, crude) as a yellow oil. LCMS [M+1] + =814.6 1 H NMR(400MHz,chloroform-d)δ=11.36(br d,J=2.0Hz,1H),4.84(td,J=6.0,20.0Hz,2H),3.52(t,J=5.6Hz,2H),3.24-3.16(m,2H),3.07-2.97(m ,4H),2.59-2.48(m,2H),2.30(dt,J=4.4,7.6Hz,4H),1.91-1.78(m,7H),1.69-1.46(m,13H),1.38(br s,12H),1.27(s,40H),0.97-0.79(m,12H)

[0528] Example 6: Synthesis of lipids 1-22: Fat 1: [ka]

[0529] Lipid 1 synthesis: [ka]

[0530] Preparation of Compound 3: [ka] To a solution of compound 1 (2.00 g, 13.06 mmol, 1.35 mL, 1.4 equiv) in toluene (20 mL) at 20 °C, compound 2 (2 g, 9.33 mmol, 1 equiv) and TsOH (803.23 mg, 4.66 mmol, 0.5 equiv) were added. The resulting mixture was stirred at 50 °C for 16 h. LCMS showed that compound 1 was consumed and the desired MS was detected. The reaction mixture was diluted with HO (20 mL), extracted with DCM (70 mL), dried over NaSO, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO, petroleum ether:ethyl acetate = 30:1 to 10:1) to give compound 3 (2.6 g, 7.44 mmol, 79.78% yield, 100% purity) as a white solid. 1 H NMR(400MHz,DMSO-d6)δ=4.08-4.02(m,2H),3.67-3.61(m,2H),2.97-2.91(m,2H),1.61-1.52(m,2H),1.35-1.17(m,22H),0.88-0.83(m,3H)

[0531] Preparation of lipid 1: [ka] To a solution of compound A (1.82 g, 5.21 mmol, 2.3 equiv.) in ACN (10 mL) at 20 °C, compound 3 (1 g, 2.26 mmol, 1 equiv.), K2CO3 (625.77 mg, 4.53 mmol, 2 equiv.), and NaI (67.87 mg, 452.77 μmol, 0.2 equiv.) were added. The resulting mixture was stirred at 80 °C for 16 h. LCMS indicated that compound A was consumed and the desired MS was detected. The mixture was diluted with ACN (10 mL) and filtered. The filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, dichloromethane:methanol = 1:0 to 20:1) to give lipid 1 (0.475 g, 668.86 μmol, 29.55% yield, 100% purity) as a yellow oil. LCMS [M+1] + =710.7 1 H NMR(400MHz,chloroform-d)δ=4.91-4.83(m,1H),4.11-4.05(m,2H),3.60-3.54(m,2H),2.83(br t,J=6.8Hz,2H),2.61(br t,J=5.2Hz,2H),2.47(br t,J=6.8Hz,4H),2.31-2.25(m,2H),1.66-1.59(m,4H),1.53-1.43(m,6H),1.34-1.24(m,54H),0.88(t,J=6.8Hz,9H)

[0532] Lipid 2: [ka]

[0533] Lipid 2 synthesis: [ka]

[0534] Preparation of Compound 6: [ka] To a solution of compound 1 (916.20 mg, 5.99 mmol, 619.05 μL, 1.2 equiv.) in toluene (20 mL) at 20 °C, compound 2 (1 g, 4.99 mmol, 1 equiv.) and TsOH (429.73 mg, 2.50 mmol, 0.5 equiv.) were added. The mixture was stirred at 50 °C for 16 h. LCMS showed that compound 2 was completely consumed and one major peak with the desired MS was detected. The mixture was diluted with DCM (30 mL) and washed with NH Cl (3 x 10 mL). The combined organic layers were washed with brine (3 x 10 mL), dried over Na SO , filtered, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, petroleum ether:ethyl acetate=100:1 to 80:1) to give compound 3 (1.36 g, 4.06 mmol, yield 81.26%) as a yellow oil. 1H NMR(400MHz,DMSO-d6)δ=4.09-4.01(m,2H),3.68-3.60(m,2H),2.99-2.90(m,2H),1.61-1.51(m,2H),1.34-1.19(m,20H),0.88-0.82(m,3H)

[0535] Preparation of lipid 2: [ka] A mixture of compound A (569.33 mg, 1.70 mmol, 1.5 equiv.), compound 3 (0.5 g, 1.13 mmol, 1 equiv.), K2CO3 (312.88 mg, 2.26 mmol, 2 equiv.), and NaI (16.97 mg, 113.19 μmol, 0.1 equiv.) in ACN (5 mL) was degassed and purged with N2 three times, and then the mixture was stirred at 80 °C under a N2 atmosphere for 16 h. LCMS showed that compound 3 was completely consumed and one major peak with the desired MS was detected. The reaction was filtered and concentrated under reduced pressure to give a residue. The residue was purified by preparative TLC (SiO2, DCM:MeOH = 20:1) to give lipid 2 (102.57 mg, 147.34 μmol, 13.02% yield, 100% purity) as a yellow oil. LCMS [M+1] + =696.7 1 H NMR(400MHz,chloroform-d)δ=4.91-4.83(m,1H),4.16(t,J=6.8Hz,1H),4.11-4.05(m,2H),3.66-3.53(m,2H),2.91-2.77(m,2H),2.68- 2.56(m,2H),2.56-2.37(m,4H),2.32-2.24(m,2H),1.66-1.59(m,4H),1.54-1.42(m,6H),1.34-1.24(m,50H),0.89(t,J=6.8Hz,9H)

[0536] Fat 3: [ka]

[0537] Lipid 3 synthesis: [ka]

[0538] Preparation of Compound 3: [ka] To a solution of compound 1 (985.17 mg, 6.44 mmol, 665.65 μL, 1.2 equiv.) and compound 2 (1 g, 5.37 mmol, 1 equiv.) in toluene (20 mL) was added TsOH (462.08 mg, 2.68 mmol, 0.5 equiv.). The mixture was stirred at 50° C. for 16 hours. TLC (petroleum ether:ethyl acetate=10:1, R1:R f =0.2, P1:R f =0.6) indicated the complete consumption of compound 2 and the appearance of one new spot. The reaction was clean by TLC. The mixture was concentrated to give the crude product. The residue was diluted with HO (20 mL) and extracted with EA (60 mL). The combined organic layers were dried over NaSO, filtered, and concentrated under reduced pressure to give a residue that gave compound 3 (850 mg, 2.38 mmol, 44.37% yield, 90% purity) as a colorless oil. 1 H NMR(400MHz,DMSO-d6)δ=4.11-4.00(m,2H),3.68-3.58(m,2H),2.99-2.88(m,2H),1.63-1.48(m,2H),1.36-1.19(m,18H),0.93-0.78(m,3H)

[0539] Preparation of lipid 3: [ka] To a solution of compound 3 (240.03 mg, 747.07 μmol, 1.5 equiv.) and compound A (220 mg, 498.04 μmol, 1 equiv.) in ACN (3 mL) was added KCO (103.25 mg, 747.07 μmol, 1.5 equiv.) and NaI (7.47 mg, 49.80 μmol, 0.1 equiv.). The mixture was stirred at 80° C. for 16 hours. LCMS showed that compound A was completely consumed and one major peak with the desired MS was detected. The mixture was filtered through celatom, and the filtrate was concentrated to give a residue. The residue was purified by preparative HPLC (HO (0.04% HCl)-THF:ACN=1:3) to give lipid 3 (117.95 mg, 73.30 μmol, 14.72% yield, 100% purity, HCl) as a yellow oil. LCMS [M+1] + =682.7 1 H NMR(400MHz,DMSO-d6)δ=9.32-9.12(m,1H),5.39-5.26(m,1H),4.83-4.70(m,1H),4.15-3.97(m,2H),3.79-3.64(m,2H),3.46-3.33(m,2H) ),3.22-3.15(m,2H),3.12-3.03(m,2H),2.88-2.78(m,2H),2.29-2.23(m,2H),1.67-1.41(m,10H),1.32-1.20(m,48H),0.91-0.78(m,9H)

[0540] Fat 4: [ka]

[0541] Synthesis of lipid 4: [ka]

[0542] Preparation of Compound 3: [ka] To a solution of compound 1 (2.13 g, 13.93 mmol, 1.44 mL, 1.2 equiv.) and compound 2 (2 g, 11.61 mmol, 1 equiv.) in toluene (40 mL) was added TsOH (999.39 mg, 5.80 mmol, 0.5 equiv.). The mixture was stirred at 50° C. for 16 hours. TLC (petroleum ether:ethyl acetate=10:1, R1:R f =0.2, P1:R f =0.6), which indicated the complete consumption of compound 2 and the appearance of one new spot. The mixture was concentrated to give a residue. The residue was diluted with HO (30 mL) and extracted with EA (60 mL). The combined organic layers were dried over NaSO, filtered, and concentrated under reduced pressure to give a residue that gave compound 3 (2.65 g, 7.76 mmol, 66.87% yield, 90% purity) as a colorless oil. 1 H NMR(400MHz,DMSO-d6)δ=4.11-4.00(m,2H),3.66-3.60(m,2H),2.99-2.92(m,2H),1.63-1.50(m,2H),1.35-1.21(m,16H),0.89-0.82(m,3H)

[0543] Preparation of lipid 4: [ka] To a solution of compound 3 (250.42 mg, 814.98 μmol, 1.2 equiv.) and compound A (300 mg, 679.15 μmol, 1 equiv.) in ACN (3 mL) was added K2CO3 (112.63 mg, 814.98 μmol, 1.2 equiv.) and NaI (10.18 mg, 67.92 μmol, 0.1 equiv.). The mixture was stirred at 80 °C for 16 h. LCMS showed that compound A was completely consumed and one major peak with the desired MS was detected. The mixture was filtered through Ceratom, and the filtrate was concentrated to give the crude product. The crude product was purified by preparative TLC (SiO2, DCM:MeOH = 8:1) to give lipid 4 (101 mg, 151.18 μmol, 22.26% yield, 100% purity) as a colorless oil. LCMS [M+1]+ =668.6 1 H NMR(400MHz,DMSO-d6)δ=4.85-4.69(m,1H),4.25-4.18(m,1H),4.01-3.94(m,2H),3.42-3.35(m,2H),2.72-2.64(m,2 H),2.46-2.41(m,2H),2.38-2.32(m,4H),2.27-2.21(m,2H),1.60-1.41(m,8H),1.36-1.16(m,48H),0.91-0.78(m,9H)

[0544] Fat 5: [ka]

[0545] Synthesis of lipid 5: [ka]

[0546] Preparation of Compound 2: [ka] A mixture of compound 1 (1 g, 6.93 mmol, 1 equiv.) was added dropwise to POCl (2.13 g, 13.86 mmol, 1.29 mL, 2 equiv.) at 20 °C, and then the mixture was stirred at 20 °C under N atmosphere for 3 h. TLC (petroleum ether: ethyl acetate = 5:1, R f =0.56), indicating the complete consumption of compound 1 and the appearance of one new spot. The reaction mixture was concentrated under reduced pressure to give compound 2 (1.8 g, 6.89 mmol, 99.44% yield) as a deep yellow oil, which was used without further purification.

[0547] Preparation of Compound 4: [ka] To a solution of compound 3 (977.80 mg, 6.78 mmol, 1 equiv.) and TEA (685.90 mg, 6.78 mmol, 943.46 μL, 1 equiv.) in toluene (10 mL) was added compound 2 (1.77 g, 6.78 mmol, 1 equiv.) in toluene (30 mL) at 20° C. The mixture was stirred at 20° C. for 16 hours. TLC (petroleum ether:ethyl acetate=5:1, R f =0.56), indicating the complete consumption of compound 2 and the appearance of one new spot. The residue was diluted with HO (10 mL) and extracted with DCM (10 mL x 2). The combined organic layers were dried over NaSO, filtered, and concentrated under reduced pressure to give a residue. This residue was purified by column chromatography (SiO, petroleum ether:ethyl acetate = 99:1 to 96:4) to give compound 4 (2.8 g, 7.59 mmol, 55.99% yield) as a colorless oil. 1 H NMR(400MHz,chloroform-d)δ=4.27-4.11(m,4H),1.74(quin,J=6.8Hz,4H),1.47-1.36(m,4H),1.35-1.21(m,20H),0.95-0.83(m,6H)

[0548] Preparation of Compound 6: [ka] To a solution of compound 4 (606.55 mg, 1.65 mmol, 1 equiv.) in DCM (9 mL), TEA (550.48 mg, 5.44 mmol, 757.20 μL, 3.3 equiv.) and compound 5 (320.00 mg, 1.65 mmol, 1 equiv.) were added, degassed, and purged with N2 three times. The mixture was then stirred under N2 atmosphere for 16 h at 20 °C. LCMS showed that compound 4 was completely consumed and one major peak with the desired MS was detected. The reaction mixture was diluted with HO (10 mL) and extracted with DCM (10 mL x 3). The combined organic layers were dried. The residue was purified by column chromatography (SiO2, petroleum ether:ethyl acetate = 10:1 to 4:1) to give compound 6 (618 mg, 1.18 mmol, 71.33% yield) as a colorless oil. LCMS [M+1] + =526.3 1H NMR(400MHz,chloroform-d)δ=4.09-3.84(m,4H),3.41(t,J=6.8Hz,2H),2.90(q,J=7.7Hz,2H),1.86 (quintet, J=7.1Hz,2H),1.73-1.63(m,4H),1.54-1.41(m,4H),1.41-1.20(m,27H),0.92-0.85(m,6H)

[0549] Preparation of lipid 5: [ka] A mixture of compound B (612.49 mg, 1.17 mmol, 1.2 equiv.), compound 6 (320 mg, 971.12 μmol, 1 equiv.), K2CO3 (268.43 mg, 1.94 mmol, 2 equiv.), and NaI (14.56 mg, 97.11 μmol, 0.1 equiv.) in ACN (8 mL) was degassed and purged with N2 three times, and then the mixture was stirred at 80 °C under a N2 atmosphere for 16 h. LCMS showed that compound 6 was completely consumed and one major peak with the desired MS was detected. The residue was diluted with H2O (10 mL) and extracted with DCM (2 x 10 mL). The combined organic layers were dried over Na2SO4, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO, dichloromethane:methanol = 80:1 to 3:1) to give lipid 5 (400 mg, 516.01 μmol, 53.14% yield) as a pale red oil. LCMS [M+1] + =775.7 1H NMR(400MHz,DMSO-d6)δ=4.84-4.72(m,1H),4.23(m,1H),3.99(t,J=6.4Hz,2H),3.8 0(dq,J=3.2,6.4Hz,4H),3.52-3.34(m,4H),2.75-2.68(m,2H),2.34(m,2H),2.27(br t,J=7.2Hz,2H),1.61-1.48(m,9H),1.43-1.19(m,55H),0.86(br t,J=6.8Hz,9H)

[0550] Fat 6: [ka]

[0551] Synthesis of lipid 6: [ka]

[0552] Preparation of Compound 2: [ka] POCl3 (11.77 g, 76.79 mmol, 7.16 mL, 2 equiv.) in DCM (2.5 mL) was added dropwise to compound 1 (5 g, 38.39 mmol, 6.07 mL, 1 equiv.) at 20 °C. After addition, the mixture was stirred at this temperature for 3 h. TLC (petroleum ether:ethyl acetate = 5:1, P:R f =0.72), indicated that a trace of compound 1 remained and a new spot appeared. The reaction mixture was concentrated under reduced pressure to remove POCl. The crude product was then evaporated with toluene three times (3×2 mL) to give compound 2 (9.49 g, crude) as a colorless oil. This product was used directly in the next step. 1 H NMR (400 MHz, chloroform-d) δ = 4.34 (td, J = 6.4, 10.0 Hz, 2H), 1.93-1.66 (m, 2H), 1.50-1.37 (m, 2H), 1.37-1.21 (m, 8H), 1.00-0.75 (m, 3H)

[0553] Preparation of Compound 4: [ka] Compound 2 (4.75 g, 36.49 mmol, 5.77 mL, 0.95 equiv.) and TEA (3.89 g, 38.41 mmol, 5.35 mL, 1 equiv.) in toluene (50 mL) were added dropwise to a solution of compound 3 (9.49 g, 38.41 mmol, 1 equiv.) in toluene (150 mL). The mixture was stirred at 20° C. for 16 hours. TLC (petroleum ether:ethyl acetate=5:1, P:R f =0.66), indicating that compound 2 had been consumed and a new spot had appeared. The reaction mixture was diluted with HO (40 mL) and extracted with DCM (200 mL). The combined organic layers were washed with brine (50 mL), dried over NaSO, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO, petroleum ether:ethyl acetate = 200:1 to 100:1) to give compound 4 (7.3 g, 21.42 mmol, 55.76% yield) as a pale yellow oil. 1 H NMR(400MHz,chloroform-d)δ=4.36-4.04(m,4H),1.74(quin,J=6.8Hz,4H),1.46-1.36(m,4H),1.36-1.22(m,16H),1.02-0.77(m,6H)

[0554] Preparation of Compound 6: [ka] To a solution of compound 4 (1.2 g, 3.52 mmol, 1 equiv.) in DCM (18 mL) at 0° C., TEA (783.71 mg, 7.75 mmol, 1.08 mL, 2.2 equiv.) and compound 5 (683.36 mg, 3.52 mmol, 1 equiv.) were added. The mixture was stirred at 20° C. for 16 hours. LCMS showed that the desired product was detected. TLC (petroleum ether:ethyl acetate=1:1, P:R f =0.40), indicating that compound 4 had been consumed and a new spot had appeared. The reaction mixture was diluted with HO (30 mL) and extracted with DCM (80 mL). The combined organic layers were washed with brine (30 mL), dried over NaSO, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO, petroleum ether:ethyl acetate = 10:1 to 5:1) to give compound 6 (1.5 g, 3.00 mmol, 85.21% yield, 99.7% purity) as a pale yellow oil. LCMS [M+1] +=498.5 1 H NMR(400MHz,chloroform-d)δ=4.08-3.87(m,4H),3.41(t,J=6.8Hz,2H),2.99-2.80(m,2H),2.46(br d,J=5.6Hz,1H),1.86(quin,J=7.1Hz,2H),1.67(quin,J=7.2Hz,4H),1.55-1.40(m,4H),1.39-1.15(m,24H),0.96-0.79(m,6H)

[0555] Preparation of lipid 6: [ka] A mixture of compound 6 (726.18 mg, 1.46 mmol, 1.2 equiv.), K2CO3 (335.54 mg, 2.43 mmol, 2 equiv.), NaI (18.20 mg, 121.39 μmol, 0.1 equiv.), and compound B (400 mg, 1.21 mmol, 1 equiv.) in ACN (6 mL) was degassed and purged with N2 three times, and then the mixture was stirred at 80 °C under a N2 atmosphere for 16 h. LCMS showed that compound 6 was consumed and the desired MS was detected. The reaction mixture was concentrated under reduced pressure to give a residue. The residue was diluted with H2O (20 mL) and extracted with DCM (60 mL). The combined organic layers were washed with brine (20 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO, DCM:MeOH = 80:1 to 50:1) to give lipid 6 (200 mg, 267.69 μmol, yield 22.05%, purity 100%) as a colorless oil. LCMS [M+1] + =747.8 1 H NMR(400MHz,chloroform-d)δ=4.06(t,J=6.8Hz,2H),4.03-3.90(m,4H),3.58(br s,2H),2.89(qd,J=7.2,9.6Hz,2H),2.63(br s,2H),2.58-2.40(m,5H),2.31(t,J=7.6Hz,2H),1.75-1.57(m,9H),1.55-1.42(m,6H),1.40-1.21(m,44H),1.02-0.72(m,9H)

[0556] Fat 7: [ka]

[0557] Preparation of lipid 7: [ka] To a solution of compound C (300 mg, 569.73 μmol, 1 equiv.) in ACN (6 mL) was added compound B (187.73 mg, 569.73 μmol, 1 equiv.), KCO (157.48 mg, 1.14 mmol, 2 equiv.), and NaI (17.08 mg, 113.95 μmol, 0.2 equiv.). The resulting mixture was heated to 80° C. for 16 hours. LCMS indicated that compound C was consumed and the desired MS was detected. The reaction mixture was filtered, and the filtrate was diluted with HO (10 mL), extracted with DCM (2×10 mL), dried over NaSO, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO, dichloromethane:methanol = 40:1 to 35:1) to give lipid 7 (100 mg, 129.00 μmol, yield 22.64%, purity 100%) as a brown oil. LCMS [M+1] + =775.7 1 H NMR(400MHz,DMSO-d6)δ=4.84-4.71(m,1H),4.05-3.95(m,2H),3.87-3.70(m,4H),3.61-3.41(m,2H),2.53(br s,6H),2.49-2.38(m,2H),2.30-2.23(m,2H),1.61-1.50(m,8H),1.46-1.18(m,54H),0.89-0.81(m,9H)

[0558] Fat 8: [ka]

[0559] Synthesis of lipid 8: [ka]

[0560] Preparation of Compound 2: [ka] Phosphoryl trichloride (18.84 g, 122.86 mmol, 11.45 mL, 2 equiv.) was added dropwise to compound 1 (8 g, 61.43 mmol, 9.71 mL, 1 equiv.) at 0° C., and the mixture was then warmed to 20° C. over 2 h. TLC showed that compound 1 was completely consumed and one new spot appeared. The mixture was concentrated to give compound 2 (14 g, 56.66 mmol, 92.23% yield) as a colorless oil, which was used without further purification. 1 H NMR(400MHz,chloroform-d)δ=4.34(td,J=6.4,9.6Hz,2H),1.81(q,J=6.8Hz,2H),1.50-1.17(m,10H),0.97-0.85(m,3H)

[0561] Preparation of Compound 4: [ka] A mixture of compound 3 (9 g, 36.42 mmol, 1 equiv.), compound 2 (3.77 g, 29.14 mmol, 4.82 mL, 0.8 equiv.), and TEA (2.95 g, 29.14 mmol, 4.06 mL, 0.8 equiv.) in toluene (135 mL) was stirred at 20 °C for 2 h. LCMS showed that compound 2 was completely consumed and one major peak with the desired MS was detected. The mixture was quenched with HO (100 mL), extracted with EtOAc (2 x 200 mL), dried over NaSO, filtered, and the filtrate was concentrated to give a residue. The residue was purified by column chromatography (SiO, petroleum ether:ethyl acetate = 10:1 to 3:1) to give compound 4 (10 g, 29.42 mmol, 80.78% yield) as a yellow oil. LCMS [M+1] + =340.4 1 H NMR (400 MHz, chloroform-d) δ = 4.28-4.03 (m, 2H), 3.23-3.09 (m, 1H), 3.09-2.96 (m, 2H), 1.79-1.49 (m, 5H), 1.47-1.18 (m, 20H), 1.02-0.77 (m, 6H)

[0562] Preparation of Compound 6: [ka] A mixture of compound 4 (1 g, 2.94 mmol, 1 equiv.), compound 5 (631.42 mg, 3.24 mmol, 1.1 equiv.), and TEA (595.45 mg, 5.88 mmol, 819.04 μL, 2 equiv.) in DCM (10 mL) was stirred at 30 °C for 12 h. LCMS showed that compound 4 was completely consumed and one major peak with the desired MS was detected. The mixture was quenched with HO (20 mL), extracted with ethyl acetate (2 x 20 mL), dried over NaSO, filtered, and the filtrate was concentrated to give a residue. The residue was purified by column chromatography (SiO, petroleum ether:ethyl acetate = 10:1 to 3:1) to give compound 6 (0.48 g, 962.86 μmol, 32.73% yield) as a yellow oil. LCMS [M+1] + =498 1 H NMR(400MHz,chloroform-d)δ=4.09-3.89(m,4H),3.41(t,J=6.8Hz,2H),2.97-2.84( m,2H),1.87(m,2H),1.73-1.58(m,5H),1.56-1.21(m,29H),0.89(t,J=6.8Hz,6H)

[0563] Preparation of lipid 8: [ka] A mixture of compound 6 (480 mg, 962.86 μmol, 1 equiv.), compound B (395.18 mg, 962.86 μmol, 1 equiv.), KCO (266.15 mg, 1.93 mmol, 2 equiv.), and NaI (72.16 mg, 481.43 μmol, 0.5 equiv.) in ACN (5 mL) was stirred at 80° C. for 12 h. LCMS showed that compound 6 was completely consumed and one major peak with the desired MS was detected. The mixture was quenched with HO (10 mL), extracted with ethyl acetate (2×20 mL), dried over NaSO, filtered, and the filtrate was concentrated to give a residue. The residue was purified by column chromatography (SiO2, DCM:MeOH = 50:1 to 10:1) to give lipid 8 (0.2 g, 267.69 μmol, 27.80% yield) as a yellow oil. LCMS [M+1] + =747.7 1 H NMR (400 MHz, chloroform-d) δ = 4.08-3.95 (m, 6H), 3.62 (m, 2H), 2.93-2.85 (m, 2H), 2.57-2.47 (m, 7H), 2.33-2.29 (t, J = 8.0 Hz, 2H), 1.69-1.59 (m, 8H), 1.57-1.45 (m, 6H), 1.45-1.17 (m, 44H), 0.89 (t, J = 6.4 Hz, 9H).

[0564] Fat 9: [ka]

[0565] Synthesis of lipid 9: [ka]

[0566] Preparation of Compound 3: [ka] A solution of compound 1 (8 g, 49.10 mmol, 5.83 mL, 1 equiv.), compound 2 (7.19 g, 41.73 mmol, 0.85 equiv.), and TEA (7.45 g, 73.65 mmol, 10.25 mL, 1.5 equiv.) in DCM (80 mL) was degassed and purged with N three times, and the mixture was then stirred under N atmosphere for 2 h at 20 °C. TLC (dichloromethane:methanol = 25:1, P1:R f =0.6), which indicated that compound 1 was consumed and one new spot appeared. The mixture was concentrated to give a residue, which was purified by column chromatography (SiO, petroleum ether:ethyl acetate = 100:1 to 50:1) to give compound 3 (2.2 g, 6.63 mmol, 13.50% yield, 90% purity) as a colorless oil. 1 H NMR (400 MHz, chloroform-d) δ = 4.35-4.05 (m, 4H), 1.80-1.67 (m, 2H), 1.37 (s, 5H), 1.35-1.21 (m, 14H), 0.90-0.84 (m, 3H)

[0567] Preparation of Compound 5: [ka] To a solution of compound 3 (1.48 g, 4.94 mmol, 1 equiv.) in DCM (5 mL) was added compound 4 (1 g, 4.94 mmol, 1 equiv., HCl) and TEA (2.00 g, 19.75 mmol, 2.75 mL, 4 equiv.), and the mixture was stirred at 30 °C under a N atmosphere for 16 h. LCMS showed that compound 3 was completely consumed and one major peak with the desired MS was detected. The reaction mixture was diluted with saturated NH4Cl (30 mL) and extracted with DCM (10 mL). The combined organic layers were dried over Na2SO4, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, dichloromethane:methanol = 100:1 to 80:1) to give compound 5 (1.1 g, 2.31 mmol, 46.80% yield, 90% purity) as a yellow oil. 1H NMR(400MHz,chloroform-d)δ=4.15-3.89(m,4H),3.47-3.35(m,2H),3.12-3.06(m,1H),2.98-2.85(m,2H),2.57- 2.39(m,1H),1.91-1.85(m,1H),1.72-1.61(m,2H),1.57-1.45(m,4H),1.40-1.20(m,19H),0.94-0.80(m,3H)

[0568] Preparation of lipid 9 [ka] A solution of compound 5 (1.11 g, 2.58 mmol, 1 equiv.), compound A (1.14 g, 2.58 mmol, 1 equiv.), K2CO3 (713.35 mg, 5.16 mmol, 2 equiv.), and NaI (77.37 mg, 516.15 μmol, 0.2 equiv.) in ACN (20 mL) was degassed and purged with N2 three times, and the mixture was then stirred at 80 °C under a N2 atmosphere for 16 h. LCMS showed that compound 5 was consumed and one peak with the desired MS was detected. The residue was diluted with H2O (20 mL) and extracted with DCM (2 x 20 mL). The combined organic layers were dried over Na2SO4, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, dichloromethane:methanol=40:1) to give lipid 9 (153.53 mg, 194.54 μmol, 7.54% yield, 100% purity) as a yellow oil. LCMS [M+1] + =789.7 1 H NMR (400MHz, chloroform-d) δ=4.87-4.68(m,1H),4.06-3.83(m,4H),3.59-3.47(m,2H),2.88-2.76(m,2H),2.64-2.59(m ,2H),2.55-2.41(m,5H),2.30-2.14(m,3H),1.66-1.52(m,4H),1.50(s,10H),1.33-1.12(m,51H),0.86-0.73(m,9H)

[0569] Fat 10: [ka]

[0570] Synthesis of lipid 10: [ka]

[0571] Preparation of Compound 3: [ka] To a solution of compound 2 (5 g, 30.69 mmol, 3.64 mL, 1 equiv.) in DCM (50 mL), TEA (4.66 g, 46.03 mmol, 6.41 mL, 1.5 equiv.) and compound 1 (4.13 g, 26.08 mmol, 4.98 mL, 0.85 equiv.) were added. The mixture was stirred at 20 °C for 2 h. LCMS showed that compound 1 was completely consumed and one major peak with the desired MS was detected. The residue was diluted with DCM (30 mL), washed with saturated NH4Cl (3 x 10 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure to give a residue, which was then purified by column chromatography (SiO2, petroleum ether:ethyl acetate = 100:1 to 40:1) to give compound 3 (3.29 g, 11.55 mmol, 37.65% yield) as a yellow oil. 1 H NMR (400 MHz, chloroform-d) δ = 4.42-4.09 (m, 4H), 1.79-1.70 (m, 2H), 1.45-1.36 (m, 5H), 1.35-1.23 (m, 12H), 0.92-0.87 (m, 3H)

[0572] Preparation of Compound 5: [ka] To a solution of compound 3 (1.5 g, 5.27 mmol, 1 equiv.) in DCM (15 mL) was added TEA (1.17 g, 11.59 mmol, 1.61 mL, 2.2 equiv.) and compound 4 (1.07 g, 5.27 mmol, 1.10 mL, 1 equiv.). The mixture was stirred at 30 °C for 16 h. LCMS showed that compound 3 was completely consumed and one major peak with the desired MS was detected. The reaction mixture was diluted with DCM (30 mL), washed with saturated NH4Cl (3 x 10 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, petroleum ether:ethyl acetate = 15:1 to 1:1) to give compound 5 (2 g, 4.44 mmol, 84.26% yield) as a yellow oil. LCMS [M+1-100] + =351.3 1 H NMR (400MHz, chloroform-d) δ=4.60-4.46(m,1H),4.14-3.91(m,4H),3.17-3.05(m,2H),2.96-2.86( m,2H),1.72-1.62(m,2H),1.54-1.48(m,4H),1.45(s,9H),1.40-1.23(m,20H),0.92-0.86(m,3H)

[0573] Preparation of Compound 6: [ka] To a solution of compound 5 (1.5 g, 3.33 mmol, 1 equiv.) in DCM (15 mL) was added TFA (7.59 g, 66.58 mmol, 4.95 mL, 20 equiv.). The mixture was stirred at 20 °C for 1 h. LCMS showed that compound 5 was completely consumed and one major peak with the desired MS was detected. The reaction mixture was diluted with DCM (40 mL), washed with saturated NaHCO3, dried over Na2SO4, filtered, and concentrated under reduced pressure to give compound 6 (1 g, 2.85 mmol, 85.71% yield) as a yellow oil. 1H NMR (400 MHz, chloroform-d) δ = 8.76-8.48 (m, 2H), 4.84-4.51 (m, 1H), 4.11-3.86 (m, 3H), 3.86-3.77 (m, 1H), 3.00-2.83 (m, 2H), 1.79-1.68 (m, 1H), 1.67-1.55 (m, 3H), 1.53-1.44 (m, 1H), 1.37-1.23 (m, 18H), 0.91-0.86 (m, 3H).

[0574] Preparation of Compound 7: [ka] A mixture of compound 7A (10.44 g, 46.79 mmol, 1.2 equiv.), compound 7B (10 g, 38.99 mmol, 1 equiv.), DMAP (952.70 mg, 7.80 mmol, 0.2 equiv.), EDCI (8.97 g, 46.79 mmol, 1.2 equiv.), and DIEA (10.08 g, 77.98 mmol, 13.58 mL, 2 equiv.) in DCM (100 mL) was degassed and purged with N three times, and then the mixture was stirred under N atmosphere for 16 h at 20 °C. TLC (petroleum ether:ethyl acetate = 10:1, R f =0.67), indicating the complete consumption of compound 7B and the appearance of many new spots. The reaction mixture (two batches) was partitioned between DCM (200 mL) and saturated NH4Cl (100 mL). The organic phase was dried over Na2SO4, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, petroleum ether:ethyl acetate = 100:1 to 20:1) to give compound 7 (29 g, 62.83 mmol, 63.24% yield) as a colorless oil. LCMS [M+23] + =483.43 1H NMR(400MHz,chloroform-d)δ=4.87(quin,J=6.4Hz,1H),3.40(t,J=6.8Hz,2H),2.29(t,J=7.6Hz,2H),1.92-1.73(m,2H),1.68-1.58(m,2H),1.51(br d,J=5.6Hz,4H),1.47-1.40(m,2H),1.39-1.30(m,6H),1.32-1.18(m,21H),0.88(t,J=6.8Hz,6H)

[0575] Preparation of Compound 8: [ka] To a solution of compound 6 (700 mg, 2.00 mmol, 1 equiv.) in DMF (14 mL) were added CsCO (452.92 mg, 1.39 mmol, 0.696 equiv.) and compound 7 (921.86 mg, 2.00 mmol, 1 equiv.). The mixture was stirred at 50 °C for 16 h. LCMS showed that compound 7 was completely consumed and one major peak with the desired MS was detected. The residue was diluted with ethyl acetate (50 mL), washed with HO (20 mL), brine (20 mL), dried over NaSO, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO, DCM:MeOH = 30:1 to 15:1) to give compound 8 (0.6 g, 820.66 μmol, 41.09% yield) as a yellow oil. LCMS [M+1] + =731.7 1 H NMR(400MHz,DMSO-d6)δ=4.85-4.73(m,2H),3.93-3.72(m,4H),3.70-3.55(m,1H),2.76(br s,6H),2.28-2.20(m,2H),1.58-1.36(m,14H),1.30-1.18(m,48H),1.14-1.06(m,2H),0.90-0.80(m,9H)

[0576] Preparation of lipid 10: [ka] To a solution of compound 8 (200 mg, 273.55 μmol, 1 equiv.) in dioxane (10 mL) was added K2CO3 (56.71 mg, 410.33 μmol, 1.5 equiv.), NaI (20.50 mg, 136.78 μmol, 0.5 equiv.), and 2-bromoethanol (170.92 mg, 1.37 mmol, 96.95 μL, 5 equiv.). The mixture was stirred at 110 °C for 16 h. LCMS showed that compound 8 was completely consumed and one major peak with the desired MS was detected. The reaction mixture was concentrated under reduced pressure to give a residue. The residue was diluted with DCM (30 mL), washed with saturated NH4Cl (3 x 10 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO, DCM:MeOH = 20:1 to 15:1) to give lipid 10 (59 mg, 76.11 μmol, 27.82% yield, 100% purity) as a yellow oil. LCMS [M+1] + =775.7 1 H NMR(400MHz,chloroform-d)δ=4.91-4.82(m,1H),4.12-4.02(m,2H),4.01-3.91(m,2H),3.83-3.62(m,2H),3.05-2.61(m,8 H),2.60-2.52(m,1H),2.32-2.25(m,2H),1.71-1.59(m,7H),1.56-1.47(m,7H),1.37-1.22(m,50H),0.93-0.85(m,9H)

[0577] Lipid 11: [ka]

[0578] Synthesis of lipid 11: [ka]

[0579] Preparation of Compound 3 [ka] To a solution of compound 1 (8 g, 49.10 mmol, 5.83 mL, 1 eq.) and compound 2 (6.02 g, 41.73 mmol, 0.85 eq.) in DCM (80 mL) was added TEA (7.45 g, 73.65 mmol, 10.25 mL, 1.5 eq.). The mixture was stirred at 20° C. for 16 hours. TLC (dichloromethane:methanol=25:1, P1:R f =0.6), indicating the complete consumption of compound 2 and the appearance of one new spot. The residue was diluted with saturated NH4Cl (10 mL) and extracted with DCM (20 mL). The combined organic layers were dried over Na2SO4, filtered, and concentrated under reduced pressure to give a residue. This residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 100:1 to 50:1) to give compound 3 (1.8 g, 6.65 mmol, 13.54% yield, 100% purity) as a colorless oil. 1 H NMR (400 MHz, chloroform-d) δ = 4.29-4.03 (m, 4H), 1.73-1.59 (m, 2H), 1.38-1.29 (m, 5H), 1.28-1.16 (m, 10H), 0.86-0.74 (m, 3H)

[0580] Preparation of Compound 5 [ka] To a solution of compound 3 (996.54 mg, 3.68 mmol, 1 equiv.) in DCM (8.2 mL) was added TEA (1.49 g, 14.72 mmol, 2.05 mL, 4 equiv.) and compound 4 (0.82 g, 4.05 mmol, 1.1 equiv.). The mixture was stirred at 30 °C for 4 h. LCMS showed that compound 3 was completely consumed and one major peak with the desired MS was detected. The residue was diluted with DCM (30 mL), washed with saturated NH4Cl (3 x 10 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure to give the residue. The residue was purified by column chromatography (SiO2, DCM:MEOH = 100:1 to 1:1) to give compound 5 (1.07 g, 2.67 mmol, 72.61% yield) as a yellow oil. LCMS [M+1] + =400.2 1 H NMR(400MHz,chloroform-d)δ=4.12-3.89(m,4H),3.45-3.37(m,1H),3.14-3.06(m,1H),2.98-2.88(m,2H),2.53- 2.40(m,1H),1.92-1.75(m,2H),1.71-1.58(m,4H),1.57-1.46(m,4H),1.41-1.24(m,14H),0.92-0.84(m,3H)

[0581] Preparation of lipid 11: [ka] To a solution of compound 5 (0.3 g, 749.38 μmol, 1 equiv.) in ACN (6 mL) was added K2CO3 (207.14 mg, 1.50 mmol, 2 equiv.), NaI (22.46 mg, 149.88 μmol, 0.2 equiv.), and compound A (331.02 mg, 749.38 μmol, 1 equiv.). The mixture was stirred at 80 °C for 16 h. LCMS showed that compound 5 was completely consumed and one major peak with the desired MS was detected. The residue was diluted with DCM (30 mL), washed with saturated NH4Cl (3 x 10 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure to give the residue. The residue was purified by column chromatography (SiO, DCM:MEOH = 50:1 to 40:1) to give lipid 11 (109 mg, 141.77 μmol, 18.92% yield, 99% purity) as a yellow oil. LCMS [M+1] + =761.7 1 H NMR(400MHz,DMSO-d6)δ=9.37-9.24(m,1H),5.36-5.25(m,1H),4.87-4.73(m,2H),3.94-3.76(m,4H),3.75-3.68(m,2H) ),3.19-2.99(m,6H),2.79-2.69(m,2H),2.29-2.23(m,2H),1.65-1.38(m,14H),1.30-1.17(m,47H),0.89-0.81(m,9H).

[0582] Fat 12: [ka]

[0583] Synthesis of lipid 12: [ka]

[0584] Preparation of Compound 3: [ka] To a solution of compound 1 (9.04 g, 55.45 mmol, 6.58 mL, 1 equiv.) and TEA (5.89 g, 58.22 mmol, 8.10 mL, 1.05 equiv.) in DCM (90 mL) at 0 °C, compound 2 (9.5 g, 55.45 mmol, 1 equiv.) was added dropwise. The resulting mixture was stirred at 20 °C for 2 h. LCMS showed that compound 2 was consumed and the desired MS was detected. The reaction mixture was concentrated to give a residue. The residue was purified by column chromatography (SiO, petroleum ether:ethyl acetate = 10:1 to 5:1) to give compound 3 (4.1 g, 6.79 mmol, 13.22% yield, 100% purity) as a yellow oil. LCMS [M+1] + =298.2 1 H NMR (400 MHz, chloroform-d) δ = 4.32-4.13 (m, 2H), 3.56-3.44 (m, 1H), 3.08-2.92 (m, 2H), 1.59-1.49 (m, 2H), 1.43-1.21 (m, 19H), 0.89-0.85 (m, 3H)

[0585] Preparation of Compound 5: [ka] To a solution of compound 3 (2 g, 6.72 mmol, 1 equiv.) in DCM (20 mL) was added TEA (1.36 g, 13.43 mmol, 1.87 mL, 2 equiv.) and compound 4 (1.23 g, 7.39 mmol, 1.1 equiv.). The resulting mixture was stirred at 20° C. for 16 h. LCMS showed that no compound 3 remained and the desired MS was detected. The reaction mixture was diluted with DCM (20 mL), washed with saturated NH4Cl (3 x 10 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by reverse-phase HPLC (column: Welch Xtimate C1 250 × 50 mm × 10 μm; mobile phase: [HO (10 mM NHHCO)-ACN:THF = 1:1]; gradient: 40–80% B over 20.0 min) to give compound 5 (0.55 g, 1.28 mmol, 19.12% yield, 100% purity) as a white solid. LCMS [M+1] + =428.3 1 H NMR(400MHz,DMSO-d6)δ=4.83-4.73(m,1H),3.93-3.78(m,4H),3.59-3.48(m,2H),2 .76-2.66(m,2H),1.87-1.77(m,2H),1.64-1.55(m,2H),1.49-1.42(m,2H),1.37(br d,J=6.4Hz,1H),1.30-1.17(m,20H),0.89-0.82(m,3H)

[0586] Preparation of lipid 12: [ka] To a solution of compound C (659.39 mg, 1.54 mmol, 1.3 equiv) in ACN (12 mL) was added compound 5 (0.6 g, 1.18 mmol, 1 equiv), KCO (327.28 mg, 2.37 mmol, 2 equiv), and NaI (35.50 mg, 236.81 μmol, 0.2 equiv). The resulting mixture was stirred at 80° C. for 16 hours. LCMS indicated that compound C was consumed and the desired MS was detected. The reaction mixture was diluted with DCM (20 mL), washed with HO (6 mL), dried over NaSO, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO, dichloromethane:methanol = 40:1 to 4:1) to give lipid 12 (102 mg, 119.41 μmol, yield 51.00%, purity 100%) as a yellow oil. LCMS [M+1] + =854.7 1 H NMR(400MHz,chloroform-d)δ=4.10-3.92(m,8H),3.81-3.60(m,2H),2.95-2.56(m,12H),1.70-1.63(m,6H),1.56-1.19(m,59H),0.89(t,J=6.8Hz,9H)

[0587] Lipid 13: [ka]

[0588] Synthesis of lipid 13: [ka]

[0589] Preparation of Compound 2: [ka] To a solution of compound 1 (10 g, 69.32 mmol, 1 equiv.) in DCM (2 mL) was added POCl (21.26 g, 138.64 mmol, 12.92 mL, 2 equiv.). The mixture was stirred at 20° C. for 3 h. TLC (petroleum ether:ethyl acetate=3:1, R f =0.76) indicated that compound 1 was consumed and one major new spot was detected. The reaction mixture was evaporated three times with toluene (5 mL x 3) to give compound 2 (21.42 g, crude) as a brown oil. 1 H NMR (400 MHz, chloroform-d) δ = 4.42-4.29 (m, 2H), 1.88-1.71 (m, 2H), 1.49-1.24 (m, 10H), 0.95-0.87 (m, 3H)

[0590] Preparation of Compound 4: [ka] To a solution of compound 2 (21.42 g, 82.01 mmol, 1 equiv.) in toluene (460 mL) were added TEA (6.64 g, 65.61 mmol, 9.13 mL, 0.8 equiv.) and compound 3 (9.4 g, 65.61 mmol, 0.8 equiv.). The mixture was stirred at 20 °C for 3 h. LCMS showed that compound 2 was completely consumed and one major peak with the desired MS was detected. The reaction mixture was diluted with saturated NH4Cl (20 mL) and extracted with DCM (25 mL). The combined organic layers were washed with brine (20 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, petroleum ether:ethyl acetate = 100:1 to 10:1) to give compound 4 (14 g, 38.05 mmol, 46.39% yield) as a yellow oil. LCMS [M+1] + =368.3 1H NMR (400 MHz, chloroform-d) δ = 4.25-4.03 (m, 2H), 3.22-3.11 (m, 1H), 3.24-3.10 (m, 1H), 3.08-2.93 (m, 2H), 1.78-1.68 (m, 2H), 1.60-1.50 (m, 2H), 1.43-1.22 (m, 24H), 0.89 (br t, J = 6.8 Hz, 6H)

[0591] Preparation of Compound 6: [ka] To a solution of compound 4 (3 g, 8.15 mmol, 1 equiv.) in toluene (30 mL), TEA (1.65 g, 16.31 mmol, 2.27 mL, 2 equiv.) and compound 5 (1.36 g, 8.15 mmol, 1 equiv.) were added. The mixture was stirred at 50 °C for 16 h. LCMS showed that compound 4 was completely consumed and one major peak with the desired MS was detected. The reaction mixture was concentrated under reduced pressure to give a residue. The residue was purified by preparative HPLC (column: Welch Xtimate C1 100 × 30 mm × 5 μm; mobile phase: [HO (10 mM NHHCO)-THF:ACN = 1:3]; gradient: 48% to 88% B over 20.0 min) to give compound 6 (1.7 g, 3.41 mmol, 28.33% yield) as a yellow oil. LCMS[M+1] + =498.3 1H NMR(400MHz,chloroform-d)δ=4.06-3.92(m,4H),3.57-3.52(m,1H),3.42(t,J=6.8Hz,2H),2.92-2.83(m,2H),2.53- 2.36(m,1H),1.96-1.78(m,2H),1.76-1.62(m,5H),1.61-1.53(m,4H),1.40-1.21(m,24H),0.89(t,J=6.8Hz,6H)

[0592] Preparation of lipid 13: [ka] To a solution of compound 6 (511.56 mg, 1.03 mmol, 1.3 equiv) in ACN (8 mL) was added NaI (23.66 mg, 157.87 μmol, 0.2 equiv), KCO (218.19 mg, 1.58 mmol, 2 equiv), and compound D (0.4 g, 789.36 μmol, 1 equiv). The mixture was stirred at 80° C. for 16 hours. LCMS showed that compound D was completely consumed and one major peak with the desired MS was detected. The reaction mixture was poured into HO (10 mL), extracted with DCM (3×10 mL), dried over NaSO, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO, DCM:MeOH = 50:1 to 10:1) to give lipid 13 (109.17 mg, 118.11 μmol, 15.60% yield) as a white solid. LCMS [M+1] + =924.7 1 H NMR(400MHz,DMSO-d6)δ=4.83-4.72(m,2H),3.89-3.71(m,8H),3.45(br s,2H),2.79-2.54(m,6H),2.49-2.25(m,4H),1.62-1.49(m,8H),1.45-1.15(m,64H),0.85(br t,J=6.8Hz,12H)

[0593] Fat 14: [ka]

[0594] Synthesis of lipid 14: [ka]

[0595] Preparation of Compound 3 [ka] To a solution of compound 1 (5 g, 30.69 mmol, 3.64 mL, 1 equiv.) and TEA (3.26 g, 32.22 mmol, 4.48 mL, 1 equiv.) in DCM (50 mL) at 0 °C, compound 2 (4.47 g, 26.08 mmol, 0.85 equiv.) was added dropwise. The resulting mixture was stirred at 20 °C for 2 h. TLC showed that compound 2 was consumed and one major new spot was formed. The reaction mixture was concentrated to give a residue. The residue was purified by column chromatography (SiO, petroleum ether:ethyl acetate = 10:1 to 10:1) to give compound 3 (3.5 g, 11.75 mmol, 38.30% yield, 100% purity) as a yellow oil. LCMS [M+1] + =298.2 1 H NMR (400 MHz, chloroform-d) δ = 4.34-4.14 (m, 2H), 3.28-3.14 (m, 1H), 3.07-2.95 (m, 2H), 1.60-1.51 (m, 2H), 1.40-1.25 (m, 19H), 0.92-0.86 (m, 3H)

[0596] Preparation of Compound 5 [ka] To a solution of compound 3 (1.18 g, 3.96 mmol, 1 equiv.) and TEA (801.90 mg, 7.92 mmol, 1.10 mL, 2 equiv.) in DCM (20 mL) was added compound 4 (728.08 mg, 4.36 mmol, 1.1 equiv.). The resulting mixture was stirred at 20 °C for 16 h. LCMS showed that compound 3 was consumed and the desired MS was detected. The reaction mixture was diluted with NH4Cl (10 mL), extracted with DCM (3 x 10 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by reverse-phase HPLC (column: Welch Xtimate C1 250 × 50 mm × 10 μm; mobile phase: [HO (10 mM NHHCO)-ACN:THF = 1:1]; gradient: 40% to 50% B over 20.0 min) to give compound 5 (0.812 g, 1.90 mmol, 47.84% yield, 100% purity) as a yellow oil. LCMS [M+1] + =429.2 1 H NMR (400MHz, chloroform-d) δ=4.13-3.95(m,4H),3.58-3.52(m,1H),3.55(t,J=6.4Hz,1H),2.94-2.84(m,2H),2.59- 2.43(m,1H),1.94-1.80(m,2H),1.75-1.67(m,2H),1.59-1.52(m,2H),1.37-1.23(m,20H),0.89(t,J=6.8Hz,3H)

[0597] Preparation of lipid 14: [ka] To a solution of compound A (290.94 mg, 679.15 μmol, 1 equiv.) in ACN (6 mL) was added compound 5 (0.3 g, 679.15 μmol, 1 equiv.), KCO (187.72 mg, 1.36 mmol, 2 equiv.), and NaI (20.36 mg, 135.83 μmol, 0.2 equiv.). The resulting mixture was heated to 80° C. for 5 h. TLC showed that compound A was consumed and one major new spot appeared. The reaction mixture was diluted with DCM (20 mL), washed with HO (2×10 mL), dried over NaSO, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO, dichloromethane:methanol = 100:1 to 20:1) to give lipid 14 (0.101 g, 3.85 mmol, 32.24% yield, 98.02% purity) as a brown oil. LCMS [M+1] + =789.7 1 H NMR(400MHz,chloroform-d)δ=4.90-4.82(m,1H),4.20-3.83(m,1H),3.00(br d,J=6.0Hz,3H),2.93-2.84(m,2H),2.79(br s,1H),2.31-2.26(m,2H),1.80-1.70(m,4H),1.69-1.56(m,4H),1.54-1.46(m,8H),1.41-1.20(m,52H),0.94-0.83(m,9H)

[0598] Fat 15: [ka]

[0599] Synthesis of lipid 15: [ka]

[0600] Preparation of Compound 3: [ka] To a solution of compound 1 (2 g, 12.27 mmol, 1.46 mL, 1 equiv.) and TEA (1.30 g, 12.89 mmol, 1.79 mL, 1.05 equiv.) in DCM (20 mL) was added compound 2 (1.93 g, 12.27 mmol, 1 equiv.) in DCM (10 mL) at 20° C. The mixture was stirred for 2 hours at 20° C. TLC (dichloromethane:methanol=10:1, R f =0.8), which indicated that compound 1 was completely consumed and many new spots were generated. The reaction mixture was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO, petroleum ether:ethyl acetate = 96:4 to 88:12) to give compound 3 (2 g, 7.05 mmol, 57.42% yield, 100% purity) as a white solid. LCMS [M+1] + =284.2 1 H NMR (400 MHz, chloroform-d) δ 4.33-4.12 (m, 2H), 3.29-3.15 (m, 1H), 3.06-2.97 (m, 2H), 1.59-1.53 ​​(m, 2H), 1.44-1.40 (m, 3H), 1.34-1.24 (m, 14H), 0.91-0.88 (m, 3H)

[0601] Preparation of Compound 5: [ka] To a solution of compound 3 (2 g, 7.05 mmol, 1.0 equiv.) and TEA (1.43 g, 14.10 mmol, 1.96 mL, 2 equiv.) in DCM (20 mL) at 20 °C, compound 4 (1.30 g, 7.75 mmol, 1.1 equiv.) was added. The mixture was stirred at 20 °C for 16 h. LCMS showed that compound 3 was completely consumed and the desired MS was detected. The reaction mixture was concentrated under reduced pressure to give a residue. The residue was purified by preparative HPLC (column: Welch Xtimate C1 250 × 50 mm × 10 μm; mobile phase: [HO (10 mM NHHCO)-ACN:THF = 1:1]; gradient: 40% to 80% B over 20.0 min) to give compound 5 (460 mg, 1.11 mmol, 15.75% yield) as a white solid. LCMS [M+1] + =414.2 1 H NMR(400MHz,chloroform-d)δ=4.15-3.91(m,4H),3.42(t,J=6.8Hz,2H),2.89(qd,J=7.2,9.2Hz ,2H),2.59-2.45(m,1H),1.97-1.85(m,2H),1.77-1.66(m,2H),1.61-1.52(m,2H),1.48(br t,J=6.8Hz,2H),1.35-1.24(m,19H),0.92-0.85(m,1H)

[0602] Preparation of lipid 15: [ka] A mixture of compound A (460 mg, 1.11 mmol, 1.05 equiv.), compound 5 (457.69 mg, 1.04 mmol, 1.0 equiv.), K2CO3 (204.57 mg, 1.48 mmol, 2 equiv.), and NaI (11.09 mg, 74.01 μmol, 0.1 equiv.) in ACN (5 mL) was degassed and purged with N2 three times, and then the mixture was stirred at 80 °C under a N2 atmosphere for 16 h. LCMS showed complete consumption of the starting material. The reaction mixture was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, DCM:MeOH = 100:1 to 90:10) to give lipid 15 (120 mg, 154.80 μmol, 20.92% yield, 100% purity) as a white solid. LCMS [M+1] + =775.7 1 H NMR(400MHz,chloroform-d)δ 4.86(t,J=6.4Hz,1H),4.12-3.94(m,4H),3.94-3.79(m,2H),3.02(br s,2H),3.11-2.81(m,7H),2.75-2.58(m,1H),2.28(t,J=7.2Hz,2H),1.73(td,J=6.8,13. 6Hz,4H),1.69-1.55(m,3H),1.55-1.42(m,8H),1.40-1.18(m,49H),0.88(t,J=6.8Hz,9H)

[0603] Fat 16: [ka]

[0604] Synthesis of lipid 16: [ka]

[0605] Preparation of Compound 3: [ka] A solution of compound 2 (2.11 g, 14.73 mmol, 0.8 equiv.) and TEA (1.96 g, 19.33 mmol, 2.69 mL, 1.05 equiv.) in toluene (10 mL) was added dropwise to compound 1 (3 g, 18.41 mmol, 2.18 mL, 1 equiv.) in toluene (20 mL). The mixture was stirred at 20° C. under a N atmosphere for 16 hours. TLC (dichloromethane:methanol=10:1, R f =0.5), indicating the complete consumption of compound 2 and the appearance of one new spot. The mixture was filtered through a Celite pad, and the filtrate was concentrated to give the crude product. The crude product was diluted with HO (20 mL) and extracted with DCM (2 x 20 mL). The combined organic layers were dried over NaSO, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO, petroleum ether:ethyl acetate = 96:4 to 90:10) to give compound 3 (2.96 g, 10.97 mmol, 59.60% yield) as a pale yellow oil. 1 H NMR(400MHz,chloroform-d)δ=4.36-4.15(m,2H),3.13(br s,1H),3.08-2.96(m,2H),1.60-1.50(m,2H),1.40(dt,J=0.8,7.2Hz,3H),1.37-1.18(m,12H),0.89(br t,J=6.8Hz,3H)

[0606] Preparation of Compound 5: [ka] A mixture of compound 3 (2.94 g, 10.90 mmol, 1 equiv.), compound 4 (2.37 g, 14.17 mmol, 1.3 equiv.), and TEA (2.21 g, 21.79 mmol, 3.03 mL, 2 equiv.) in DCM (30 mL) was degassed and purged with N three times, and then the mixture was stirred under a N atmosphere at 20° C. for 16 hours. LCMS showed that compound 3 was completely consumed and one major peak with the desired MS was detected. The reaction mixture was concentrated under reduced pressure to give a residue. The residue was purified by preparative HPLC (column: Welch Xtimate C1 100 × 30 mm × 5 μm, mobile phase: A: HO (0.05% HCl); B: THF:ACN = 1:3, gradient: B from 40.00% to 80.00% in 20.00 min, flow rate: 100.00 ml / min; monitor wavelength: 220-254 nm) to give compound 5 (530 mg, 1.32 mmol, 12.15% yield) as a white solid. LCMS [M+1] + =400.4 1 H NMR (400MHz, chloroform-d) δ=8.13(m,1H),4.16-3.93(m,4H),3.55(t,J=6.8Hz,1H),3.42(t ,J=6.8Hz,2H),3.07-2.95(m,1H),2.93-2.83(m,2H),1.91(quin,J=7.2Hz,2H),1.81(br dd,J=7.2,14.4Hz,1H),1.85-1.76(m,1H),1.76-1.66(m,2H),1.60-1.53(m,2H),1.52-1.44(m,2H),1.44-1.14(m,16H),0.89(t,J=6.8Hz,3H)

[0607] Preparation of lipid 16: [ka] A mixture of compound 5 (498.46 mg, 1.25 mmol, 1.1 equiv.), K2CO3 (312.87 mg, 2.26 mmol, 2 equiv.), NaI (16.97 mg, 113.19 μmol, 0.1 equiv.), and compound A (500 mg, 1.13 mmol, 1 equiv.) in ACN (10 mL) was degassed and purged with N2 three times, and then the mixture was stirred at 80 °C under a N2 atmosphere for 16 h. LCMS showed that compound A was completely consumed and one major peak with the desired MS was detected. The reaction mixture was quenched by the addition of saturated NH4Cl (5 mL) at 20 °C, then diluted with HO (5 mL), and extracted with DCM (3 x 10 mL). The combined organic layers were washed with brine (5 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO, DCM:MeOH = 100:1 to 10:1) to give lipid 16 (133.9 mg, 175.92 μmol, 14.88% yield, 100% purity) as a colorless oil. LCMS [M+1] + =761.7 1 H NMR(400MHz,chloroform-d)δ=4.86(t,J=6.0Hz,1H),4.14-3.84(m,6H),3.26-2.80(m,7H),2.69 -2.59(m,1H),2.29(t,J=7.6Hz,2H),1.78-1.70(m,3H),1.67-1.57(m,4H),1.50(br d,J=4.0Hz,9H),1.41-1.16(m,46H),0.89(t,J=6.8Hz,9H).

[0608] Lipid 17: [ka]

[0609] Synthesis of lipid 17: [ka]

[0610] Preparation of Compound 2: [ka] To a solution of compound 1 (2 g, 10.36 mmol, 1 equiv.) in THF (20 mL) at 0° C. was added NaH (497.47 mg, 12.44 mmol, 60% purity, 1.2 equiv.), and the mixture was stirred at 0° C. for 1 h under a N atmosphere. SEM-Cl (2.25 g, 13.47 mmol, 2.38 mL, 1.3 equiv.) was then added, and the mixture was stirred at 20° C. for 2 h under a N atmosphere. TLC (petroleum ether:ethyl acetate=3:1, R f =0.42), compound 1 was completely consumed and one new spot appeared. The reaction mixture was quenched with saturated NH4Cl (10 mL) and extracted with ethyl acetate (10 mL x 3). The combined organic layer was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, petroleum ether:ethyl acetate = 100:1 to 3:1) to give compound 2 (5.9 g, 18.25 mmol, 88.06% yield) as a colorless oil. 1 H NMR(400MHz,DMSO-d6)δ=5.64(s,2H),3.66(t,J=8.0Hz,2H),0.95-0.74(m,2H),0.03(s,9H)

[0611] Preparation of Compound 3: [ka] A mixture of compound 2 (51.63 mg, 159.73 μmol, 1.2 eq.), compound F (100 mg, 133.11 μmol, 1 eq.) in dioxane (1 mL), tBuXPhos Pd-G3 (31.72 mg, 39.93 μmol, 0.3 eq.), NaOt-Bu (2 M, 133.11 μL, 2 eq.) was degassed and purged with N2 three times, and then the mixture was stirred at 110 °C under a N2 atmosphere for 16 h. LCMS showed that compound F was completely consumed and one major peak with the desired MS was detected. The reaction mixture (45 batches) was diluted with HO (80 mL) and extracted with DCM (2 x 80 mL). The combined organic layers were dried over Na2SO4, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, dichloromethane:methanol=100:1 to 20:1) to give compound 3 (3 g, crude) as a yellow oil. LCMS [M+1] + =993.8 1 H NMR(400MHz,chloroform-d)δ=5.42-5.22(m,2H),4.93-4.66(m,2H),3.70-3.42(m,4H),3.40-2.85(m,2H),2.71-2.56(m,1 H),2.52-2.38(m,2H),2.29(dt,J=4.0,7.6Hz,4H),1.90-1.68(m,2H),1.67-1.46(m,18H),1.36-1.21(m,48H),0.94(br d,J=8.8Hz,2H),0.92-0.84(m,13H),0.05-0.03(m,9H)

[0612] Preparation of Compound 4: [ka] To a solution of compound 3 (101 mg, 101.65 μmol, 1 equiv.) in THF (0.5 mL) at 20° C., TBAF (1 M, 1.02 mL, 10 equiv.) was added, and the mixture was heated to 80° C. under a N atmosphere for 3 h. LCMS showed that compound 3 was completely consumed, and one major peak with the desired MS was detected. The reaction mixture was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO, DCM:MeOH=96:4 to 9:1) followed by preparative TLC to give compound 4 (180 mg, 208.50 μmol, 41.02% yield) as a yellow oil. LCMS [M+1] + =863.7 1 H NMR (400 MHz, chloroform-d) δ = 4.93-4.76 (m, 2H), 3.48 (br s, 1H), 2.96-2.72 (m, 5H), 2.29 (m, 11H), 2.06-1.93 (m, 2H), 1.72-1.45 (m, 16H), 1.38-1.21 (m, 48H), 0.97-0.80 (m, 12H)

[0613] Preparation of lipid 17: [ka] To a solution of compound 4 (100 mg, 115.83 μmol, 1 equiv.) in TFE (2 mL) was added Pd / C (29.89 mg, 56.18 μmol, 20% purity) under a N atmosphere. The suspension was degassed and purged with H three times. The mixture was stirred at 30 °C under H (15 psi) for 2 h. LCMS showed that compound 4 was completely consumed and one major peak with the desired MS was detected. The reaction mixture was filtered, and the filter cake was washed with DCM:IPA = 1:1 (40 mL x 3). The filtrate was concentrated under reduced pressure to give lipid 17 (44.67 mg, 53.60 μmol, 46.28% yield) as a yellow oil. LCMS [M+1] + =833.5 1H NMR(400MHz,DMSO-d6)δ=5.70-5.36(m,1H),5.32-5.00(m,1H),4.89-4.53(m,2H),3.51(m,2H),2.98(br s,2H),2.71(br t,J=5.6Hz,1H),2.41-2.19(m,8H),2.19-2.03(m,2H),1.64-1.42(m,11H),1.34(br s,6H),1.29-1.01(m,46H),0.91-0.66(m,12H)

[0614] Fat 18: [ka]

[0615] Synthesis of lipid 18: [ka] [ka]

[0616] Preparation of Compound 3: [ka] To a solution of compound 1 (300 mg, 1.90 mmol, 1 equiv.) in acetone (5 mL) was added KCO (1.31 g, 9.49 mmol, 5 equiv.) and compound 2 (1.15 g, 5.69 mmol, 580.54 μL, 3 equiv.). The mixture was heated to 60° C. for 2 h. TLC (ethyl acetate, R f =0.24) indicated that approximately 5% of compound 1 remained and one major new spot was formed. The reaction mixture (three batches) was filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO, petroleum ether:ethyl acetate = 25:1 to 0:1) to give compound 3 (640 mg, 2.29 mmol, 40.40% yield) as a yellow oil. 1H NMR (400 MHz, chloroform-d) δ = 7.65 (s, 1H), 4.91 (t, J = 7.2 Hz, 2H), 3.48 (t, J = 6.4 Hz, 2H), 2.62-2.51 (m, 2H)

[0617] Preparation of Compound 4: [ka] A mixture of compound 4A (10.44 g, 46.79 mmol, 1.2 equiv.), compound 4B (10 g, 38.99 mmol, 1 equiv.), DMAP (952.70 mg, 7.80 mmol, 0.2 equiv.), EDCI (8.97 g, 46.79 mmol, 1.2 equiv.), and DIEA (10.08 g, 77.98 mmol, 13.58 mL, 2 equiv.) in DCM (100 mL) was degassed and purged with N three times, and then the mixture was stirred under N atmosphere for 16 h at 20 °C. TLC (petroleum ether:ethyl acetate = 10:1, R f =0.67), indicating the complete consumption of compound 4B and the appearance of many new spots. The reaction mixture (two batches) was partitioned between DCM (200 mL) and saturated NH4Cl (100 mL). The organic phase was dried over Na2SO4, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, petroleum ether:ethyl acetate = 100:1 to 20:1) to give compound 4 (29 g, 62.83 mmol, 63.24% yield) as a colorless oil. LCMS [M+23] + =483.43 1 H NMR(400MHz,chloroform-d)δ=4.87(quin,J=6.4Hz,1H),3.40(t,J=6.8Hz,2H),2.29(t,J=7.6Hz,2H),1.92-1.73(m,2H),1.68-1.58(m,2H),1.51(br d,J=5.6Hz,4H),1.47-1.40(m,2H),1.39-1.30(m,6H),1.32-1.18(m,21H),0.88(t,J=6.8Hz,6H)

[0618] Preparation of compound 5C: [ka] A mixture of compound 5A (13.36 g, 51.51 mmol, 1 equiv.), compound 5B (7.1 g, 41.21 mmol, 0.8 equiv.), DMAP (1.26 g, 10.30 mmol, 0.2 equiv.), EDCI (11.85 g, 61.81 mmol, 1.2 equiv.), and DIEA (13.31 g, 103.01 mmol, 17.94 mL, 2 equiv.) in DCM (71 mL) was degassed and purged with N three times, and then the mixture was stirred at 20 °C under a N atmosphere for 16 h. LCMS indicated that compound 5B had been consumed and the desired MS was detected. The reaction mixture was diluted with DCM (71 mL), washed with saturated NH4Cl (3 x 50 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, petroleum ether:ethyl acetate=1:0 to 9:1) to give compound 5C (15 g, 36.26 mmol, yield 70.41%, purity 100%) as a yellow oil. 1 H NMR (400 MHz, chloroform-d) δ = 4.89-4.70 (m, 1H), 4.51 (br s, 1H), 3.18-2.98 (m, 2H), 2.28 (t, J = 7.6 Hz, 2H), 1.69-1.40 (m, 17H), 1.38-1.18 (m, 18H), 0.93-0.83 (m, 6H)

[0619] Preparation of Compound 5: [ka] A mixture of compound 5C (5 g, 12.09 mmol, 1 equiv.) in HCl / dioxane (250 mL) was degassed and purged with N three times, and then the mixture was stirred under a N atmosphere for 2 hours at 20 °C. LCMS showed that compound 5C was consumed and the desired MS was detected. The reaction solution was concentrated to give compound 5 (8.1 g, 23.14 mmol, 95.73% yield, HCl) as a yellow oil. LCMS [M+1]+ =314.4 1 H NMR(400MHz,chloroform-d)δ=8.29(br s,3H),4.81(quin,J=6.4Hz,1H),2.99(br d,J=5.6Hz,2H),2.28(t,J=7.6Hz,2H),1.90(br s,1H),1.77(br t,J=7.2Hz,2H),1.70-1.47(m,6H),1.46-1.14(m,18H),0.94-0.80(m,6H)

[0620] Preparation of Compound 6: [ka] A mixture of compound 5 (6.1 g, 17.43 mmol, 1 equiv., HCl), compound 4 (7.24 g, 15.69 mmol, 0.9 equiv.), and CsCO (3.95 g, 12.13 mmol, 0.9 equiv.) in DMF (61 mL) was degassed and purged with N three times, and then the mixture was stirred at 20 °C under N atmosphere for 2 h. LCMS showed that compound 4 was consumed and the desired MS was detected. The reaction mixture was diluted with ethyl acetate (60 mL), washed with H0 (30 mL x 3), dried over NaSO, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO, petroleum ether:ethyl acetate = 80:1 to 8:1) to give compound 6 (1.6 g, 2.30 mmol, 16.00% yield, 98.33% purity) as a white solid. LCMS [M+1] + =694.7 1 H NMR (400MHz, chloroform-d)δ=4.96-4.71(m,2H),2.61(t,J=7.6Hz,4H),2.28(dt,J=3.6,7. 6Hz,4H),1.67-1.59(m,4H),1.55-1.43(m,11H),1.40-1.09(m,50H),0.95-0.78(m,12H)

[0621] Preparation of Compound 7: [ka] To a solution of compound 3 (96.48 mg, 345.74 μmol, 1.2 equiv.) in ACN (4 mL) was added KCO (79.64 mg, 576.23 μmol, 2 equiv.), NaI (8.64 mg, 57.62 μmol, 0.2 equiv.), and compound 6 (0.2 g, 288.12 μmol, 1 equiv.). The mixture was stirred at 80° C. for 16 h. LCMS showed that compound 6 was completely consumed and one major peak with the desired MS was detected. The reaction mixture (two batches) was concentrated under reduced pressure to give a residue. The residue was purified by preparative TLC (SiO, DCM:MeOH=15:1) to give compound 7 (100 mg, 112.07 μmol, 19.23% yield) as a yellow solid. LCMS [M+1] + =892.8 1 H NMR (400MHz, chloroform-d) δ=7.69-7.55(m,1H),4.97-4.68(m,4H),2.54-2.47(m,2H),2.39-2.24(m,7 H),2.12-2.02(m,2H),1.67-1.60(m,5H),1.55-1.47(m,8H),1.41-1.16(m,52H),0.92-0.84(m,12H)

[0622] Preparation of lipid 18: [ka] To a solution of compound 7 (140 mg, 156.90 μmol, 1 equiv.) in TFE (15 mL) was added Pd / C (312.24 mg, 293.40 μmol, 10% purity) under a N atmosphere. The suspension was degassed and purged with H three times. The mixture was stirred at 30 °C under H (15 psi) for 2 h. LCMS showed that compound 7 was completely consumed and one major peak with the desired MS was detected. The reaction mixture was filtered, and the filter cake was washed with DCM:IPA = 1:1 (40 mL x 3). The filtrate was concentrated under reduced pressure to give lipid 18 (113.69 mg, 136.59 μmol, 87.06% yield) as a yellow oil. LCMS [M+1] + =832.8 1H NMR(400MHz,DMSO-d6)δ=4.88-4.66(m,4H),4.55(s,1H),4.14-4.06(m,2H),3.58-3.48(m,2H),2 .34-2.18(m,11H),1.72-1.59(m,2H),1.55-1.40(m,12H),1.36-1.13(m,54H),0.90-0.77(m,12H)

[0623] Lipid 19: [ka]

[0624] Preparation of lipid 19: [ka] To a solution of compound F (400 mg, 485.33 μmol, 1 eq., 2HCl) and TEA (392.89 mg, 3.88 mmol, 540.42 μL, 8 eq.) in DCM (8 mL) at 0° C., compound 1 (150.92 mg, 1.16 mmol, 2.4 eq.) was added. The reaction mixture was stirred at 20° C. for 2 h. LCMS showed that compound F was consumed and one major peak with the desired MS was detected. The reaction mixture was partitioned between saturated NaHCO (15 mL) and DCM (30 mL). The organic phase was separated, dried over NaSO, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by preparative HPLC (column: X-Select CSH Phenyl-Hexyl 100 × 30 5 μl; mobile phase: [HO (0.04% HCl)-THF:ACN = 1:3]; gradient: 40% to 80% B over 8.0 min) to give lipid 19 (185 mg, 219.10 μmol, 45.14% yield) as a colorless oil. LCMS [M+1] + =844.8 1 H NMR(400MHz,chloroform-d)δ=11.27(br s,1H),4.90-4.75(m,2H),3.32(br s,2H),3.20(br d,J=4.8Hz,2H),3.12-2.92(m,5H),2.74(s,3H),2.29(dt,J=4.4,7.6Hz,4H),2.11(br s,2H),1.89-1.69(m,4H),1.67-1.45(m,13H),1.37(br s,12H),1.32-1.18(m,36H),0.92-0.83(m,12H)

[0625] Fat 20: [ka]

[0626] Preparation of lipid 20: [ka] To a solution of compound F (300 mg, 399.33 μmol, 1 equiv.) in DCM (2 mL) at 0° C., TEA (121.22 mg, 1.20 mmol, 166.75 μL, 3 equiv.) and compound 1 (137.62 mg, 958.39 μmol, 102.93 μL, 2.4 equiv.) were added. The mixture was stirred at 25° C. for 2 hours. LCMS showed that compound F was completely consumed and one major peak with the desired MS was detected. The reaction mixture was concentrated under reduced pressure to give a residue. The residue was purified by preparative HPLC (column: X-Select CSH Phenyl-Hexyl 100 × 305 μl; mobile phase: [HO (0.04% HCl)-THF:ACN = 1:3]; gradient: 30% to 80% B over 10.0 min) to give lipid 20 (120 mg, 139.80 μmol, 35.01% yield, HCl) as a white solid. LCMS [M+1] + =858.7 1 H NMR(DMSO-d6,400MHz)δ =10.01(br s,1H),7.30(br t,J=5.6Hz,1H),4.7-4.8(m,2H),2.9-3.1(m,8H),2.67(s,6H),2.2-2.3(m,4H),1.7-1.9(m,2H),1.62(br s,4H),1.4-1.6(m,13H),1.2-1.3(m,50H),0.8-0.9(m,12H)

[0627] Lipids 21: [ka]

[0628] Preparation of lipid 21: [ka] To a solution of compound G (200 mg, 245.36 μmol, 1 equiv.) in DMF (6 mL) was added compound 1 (24.31 mg, 245.36 μmol, 1 equiv.), NaI (7.36 mg, 49.07 μmol, 0.2 equiv.), and K2CO3 (67.82 mg, 490.71 μmol, 2 equiv.), and the mixture was stirred at 80 °C under a N2 atmosphere for 3 h. LCMS showed that compound G was completely consumed and one major peak with the desired MS was detected. The residue was diluted with HO (50 mL) and extracted with DCM (2 x 60 mL). The combined organic layers were dried over Na2SO4, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by preparative HPLC (column: X-Select CSH Phenyl-Hexyl 100 × 30 5 μl; mobile phase: [HO (0.04% HCl)-THF:ACN = 1:3]; gradient: 45% to 85% B over 8.0 min) to give lipid 21 (120 mg, 144.00 μmol, 14.67% yield, HCl) as a yellow oil. LCMS [M+1] + =833.7 1 H NMR(400MHz,DMSO-d6)δ=13.26-11.41(m,1H),10.71(br s,1H),8.21(br s,2H),6.77-6.11(m,1H),4.86-4.65(m,2H),3.95(br t,J=6.4Hz,2H),3.08(br s,2H),2.98(br s,4H),2.35-2.16(m,4H),2.07(br s,2H),1.65(br s,5H),1.57-1.35(m,13H),1.36-1.09(m,48H),0.97-0.73(m,12H)

[0629] Lipids 22: [ka]

[0630] Synthesis of lipid 22: [ka]

[0631] Preparation of Compound 2: [ka] To a solution of compound 1 (4.5 g, 23.44 mmol, 1 equiv.) in THF (45 mL) was added NaH (1.88 g, 46.88 mmol, 60% purity, 2 equiv.) and SEM-Cl (5.08 g, 30.47 mmol, 5.39 mL, 1.3 equiv.) at 0° C. The mixture was stirred at 20° C. for 16 h. TLC (petroleum ether:ethyl acetate=3:1, R f =0.5), indicating the complete consumption of compound 1 and the appearance of one new spot. The residue was quenched with saturated NH4Cl (50 mL) and extracted with EtOAc (50 mL x 3). The combined organic layers were concentrated under reduced pressure to give a residue. This residue was purified by column chromatography (SiO2, petroleum ether:ethyl acetate = 200:1 to 10:1) to give compound 2 (4.2 g, 11.73 mmol, 50.04% yield, 90% purity) as a colorless oil. 1 H NMR (400 MHz, chloroform-d) δ = 7.06-6.96 (m, 1H), 5.67-5.54 (m, 2H), 3.75-3.58 (m, 2H), 1.62-1.49 (m, 1H), 1.01-0.88 (m, 2H), 0.07-0.06 (m, 9H)

[0632] Preparation of Compound 3: [ka] To a solution of compound 2 (3.08 g, 9.583 mmol, 1.2 equiv.) and compound F (6 g, 7.986 mmol, 1 equiv.) in dioxane (60 mL) was added tBuXPhos Pd-G3 (1.2 g, 1.597 mmol, 0.2 equiv.) and NaOtBu (2 M, 7.98 mL, 2 equiv.). The mixture was stirred at 110 °C for 16 h. LCMS showed that compound 2 was completely consumed and one major peak with the desired MS was detected. The residue was diluted with DCM (50 mL) and extracted with H2O (25 mL). The combined organic layers were dried over Na2SO4, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, dichloromethane:methanol = 500:1 to 125:1) to give compound 3 (1.6 g, 1.60 mmol, yield 92.22%, purity 99%) as a yellow oil. LCMS [M+1] + =993.0 1 H NMR(400MHz,chloroform-d)δ=5.97- 5.89(m,2H),5.42-5.31(m,2H),4.90-4.77(m,2H),3.68-3.53(m,2H),3.25-3.14(m,2H),2.97-2.56(m,5H),2.34-2.21(m ,5H),1.89-1.71(m,2H),1.66-1.55(m,8H),1.54-1.46(m,8H),1.35-1.23(m,52H),0.95-0.84(m,15H),0.06--0.07(m,9H)

[0633] Preparation of Compound 4: [ka] To a solution of compound 3 (700 mg, 705.46 μmol, 1 eq.) in THF (3.5 mL), TBAF (1 M, 3.5 mL, 5 eq.) was added. The mixture was stirred at 80° C. for 3 h. LCMS showed that compound 10 was completely consumed and one major peak with the desired MS was detected. The mixture was concentrated to give a residue. The residue was purified by preparative TLC (SiO, DCM:MeOH=10:1) to give compound 4 (100 mg, 104.37 μmol, 14.80% yield, 90% purity) as a yellow oil. LCMS [M+1] + =862.8 1 H NMR(400MHz,chloroform-d)δ=5.91-5.80(m,1H),4.85-4.60(m,2H),3.31-3.14(m,2H),2.89-2.76(m,2H),2.72-2 .57(m,3H),2.28-2.13(m,4H),1.94-1.77(m,2H),1.63-1.36(m,16H),1.32-1.10(m,51H),0.85-0.70(m,12H)

[0634] Preparation of lipid 22: [ka] To a solution of compound 4 (100 mg, 120.14 μmol, 1 equiv.) in 2,2,2-trifluoroethanol (1 mL) was added Pd / C (63.93 mg, 10% purity) under a N atmosphere. The suspension was degassed and purged with H three times. The mixture was stirred at 20 °C under H (15 psi) for 2 h. LCMS showed that compound 4 was consumed and one major peak with the desired MS was detected. The reaction mixture was filtered, and the filter cake was washed with DCM:IPA = 1:1 (40 mL x 3). The filtrate was concentrated under reduced pressure to give lipid 22 (62.75 mg, 21.87 μmol, 43.41% yield, 94.62% purity) as a brown oil. LCMS [M+1] + =832.9 1H NMR (400MHz, chloroform-d) δ=4.84-4.69(m,2H),3.12(m,3H),2.85-2.42(m,8H),2.21(m ,4H),1.81-1.67(m,2H),1.62-1.37(m,16H),1.31-1.10(m,50H),0.87-0.75(m,12H)

[0635] Example 7. Lipid screening by in vivo expression assessment in lipid nanoparticles Formulation In this example, messenger RNA molecules encoding hEPO protein were formulated in lipid nanoparticles for in vivo delivery. The lipid nanoparticle (LNP) formulation consisted of a lipid composition of ionizable lipid:helper lipi...

Claims

1. The following structure: 【Chemistry 1】 【Chemistry 2】 【Transformation 3】 【Chemistry 4】 【Transformation 5】 【Transformation 6】 or a salt thereof, During the ceremony, R = 【Transformation 7】 and each R ２７ are independently H, C １ ~C １５ Alkyl, C ２ ~C ８ Alkenyl, or C ２ ~C ８ is alkynyl, A compound or a salt thereof.

2. Formula (Iw): 【Transformation 8】 or a salt thereof, During the ceremony, R Ｎ２ teeth, 【Chemistry 9】 and L １ and L ２ are each independently １ ~C １５ ) alkylene; Z １０ and Z ２０ are each independently 【Chemistry 10】 and X １ and X ２ Each of is independently O, S, or N(R ２１ ) and R ２０ is branched (C １ ~C １５ ) alkyl or unbranched (C １ ~C １５ ) alkyl; R ２１ is H, (C １ ~C ５ ) alkyl, or (C ３ ~C ８ ) cycloalkyl; s is an integer from 1 to 4; Z １０ In 【Chemistry 11】 Is, L １ indicates the point of attachment to; Z ２０ In 【Chemistry 12】 Is, L ２ indicates the point of attachment to; R ２２ , R ２３ , R ２４ , and R ２５ each independently selected from H, branched (C １ ~C １５ ) alkyl, or unbranched (C １ ~C １５ ) alkyl; provided that R ２２ and R ２３ At least one of is not H and R ２４ and R ２５ At least one of them is not H, A compound or a salt thereof.

3. Formula (I-w-4), (I-w-5), or (I-w-6): 【Chemistry 13】 A compound having the structure or a salt thereof, During the ceremony, R Ｎ２ is -(CH ２ ) ｍ (NH) ｎ Q ２ and Q ２ is -OH, -SO ２ NH(alkyl), -SO ２ N(alkyl) ２ , optionally substituted cycloalkyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted heterocycloalkyl, or optionally substituted heterocycloalkenyl; m is an integer from 2 to 3; n is an integer from 0 to 1; L １ and L ２ are each independently １ ~C １５ ) alkylene; X １ and X ２ Each of is independently O, S, or N(R ２１ ) and R ２１ is H, (C １ ~C ５ ) alkyl, or (C ３ ~C ８ ) cycloalkyl; R ２２ , R ２３ , R ２４ , and R ２５ each independently selected from H, branched (C １ ~C １５ ) alkyl, or unbranched (C １ ~C １５ ) alkyl; provided that R ２２ and R ２３ At least one of is not H and R ２４ and R ２５ At least one of them is not H, A compound or a salt thereof.

4. Formula (I-w-7), (I-w-8), or (I-w-9): 【Chemistry 14】 A compound having the structure or a salt thereof, During the ceremony: R Ｎ２ is -(CH ２ ) ｍ (NH) ｎ Q ２ and Q ２ is -OH, -SO ２ NH(alkyl), -SO ２ N(alkyl) ２ , optionally substituted cycloalkyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted heterocycloalkyl, or optionally substituted heterocycloalkenyl; m is an integer from 2 to 3; n is an integer from 0 to 1; L １ and L ２ are each independently １ ~C １５ ) alkylene; s is an integer from 1 to 4; R ２２ , R ２３ , R ２４ , and R ２５ each independently selected from H, branched (C １ ~C １５ ) alkyl, or unbranched (C １ ~C １５ ) alkyl; provided that R ２２ and R ２３ At least one of is not H and R ２４ and R ２５ At least one of them is not H, A compound or a salt thereof.

5. Formula (I-w-10), (I-w-11), (I-w-12), (I-w-13), (I-w-14), or (I-w-15): 【Chemistry 15】 A compound having the structure or a salt thereof, During the ceremony: R Ｎ２ is -(CH ２ ) ｍ (NH) ｎ Q ２ and Q ２ is -OH, -SO ２ NH(alkyl), -SO ２ N(alkyl) ２ , optionally substituted cycloalkyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted heterocycloalkyl, or optionally substituted heterocycloalkenyl; m is an integer from 2 to 3; n is an integer from 0 to 1; L １ and L ２ are each independently １ ~C １５ ) alkylene; R ２０ is branched (C １ ~C １５ ) alkyl or unbranched (C １ ~C １５ ) alkyl; R ２１ is H, (C １ ~C ５ ) alkyl, or (C ３ ~C ８ ) cycloalkyl; R ２２ , R ２３ , R ２４ , and R ２５ each independently selected from H, branched (C １ ~C １５ ) alkyl, or unbranched (C １ ~C １５ ) alkyl; provided that R ２２ and R ２３ At least one of is not H and R ２４ and R ２５ At least one of them is not H, A compound or a salt thereof.

6. Formula (Ia): 【Chemistry 16】 A compound having the structure or a salt thereof, During the ceremony: R Ｎ２ is -(CH ２ ) ｍ (NH) ｎ Q ２ and Q ２ is -OH, -SO ２ NH(alkyl), -SO ２ N(alkyl) ２ , optionally substituted cycloalkyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted heterocycloalkyl, or optionally substituted heterocycloalkenyl; m is an integer from 2 to 3; n is an integer from 0 to 1; L １ and L ２ are each independently １ ~C １５ ) alkylene; Z １０ and Z ２０ are each independently 【Chemistry 17】 and X １ and X ２ Each of is independently O, S, or N(R ２１ ) and R ２０ is branched (C １ ~C １５ ) alkyl or unbranched (C １ ~C １５ ) alkyl; R ２１ is H, (C １ ~C ５ ) alkyl, or (C ３ ~C ８ ) cycloalkyl; s is an integer from 1 to 4; Z １０ In [Chemistry 18] Is, L １ indicates the point of attachment to; Z ２０ In 【Chemistry 19】 Is, L ２ indicates the point of attachment to; R ２２ , R ２３ , R ２４ , and R ２５ each independently selected from H, branched (C １ ~C １５ ) alkyl, or unbranched (C １ ~C １５ ) alkyl; provided that R ２２ and R ２３ At least one of is not H and R ２４ and R ２５ at least one of is not H; Here, Q ２ is —OH, the following (i) or (ii): (i) Z １０ and Z ２０ At least one of 【Chemistry 20】 or (ii) L １ and L ２ are not the same At least one of the following applies: A compound or a salt thereof.

7. R Ｎ２ is -(CH ２ ) ｍ Q ２ 7. The compound of claim 6, wherein:

8. Q ２ The compound according to claim 6 or 7, wherein is —OH.

9. R Ｎ２ is -CH ２ CH ２ The compound according to any one of claims 6 to 8, wherein the aryl group is OH.

10. R Ｎ２ is -(CH ２ ) ｍ (NH)Q ２ 7. The compound of claim 6, wherein:

11. Q ２ is -SO ２ NH(alkyl) or -SO ２ N(alkyl) ２ 11. The compound of claim 10, wherein:

12. Q ２ is an optionally substituted cycloalkyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted heterocycloalkyl, or optionally substituted heterocycloalkenyl; where, valence permitting, the optional substituents are selected from oxo, amino, alkylamino, and dialkylamino.

13. Q ２ teeth, 【Chemistry 21】 wherein each R ｃ are independently H or C １ ~C ３ The compound of claim 10, wherein s is alkyl and s is an integer from 1 to 4.

14. R Ｎ teeth, 【Chemistry 22】 7. The compound of claim 6, wherein:

15. L １ and L ２ are each independently ２ ~C １０ 15. The compound according to any one of claims 6 to 14, wherein:

16. L １ and L ２ are each independently ２ ~C ８ 15. The compound according to any one of claims 6 to 14, wherein:

17. L １ and L ２ are each independently ４ ~C ８ 15. The compound according to any one of claims 6 to 14, wherein:

18. L １ and L ２ The compound according to any one of claims 1 to 17, wherein:

19. Z １０ and Z ２０ are each independently 【Chemistry 23】 The compound according to any one of claims 1 to 18, wherein

20. R ２２ is not H;R ２４ is not H; and R ２３ and R ２５ 20. The compound of claim 19, wherein at least one of is not H.

21. R ２２ , R ２３ , R ２４ , and R ２５ are independently branched (C １ ~C １５ ) alkyl or unbranched (C １ ~C １５ 20. The compound of claim 19, wherein:

22. R ２２ and R ２３ and are the same.

23. R ２２ and R ２３ and n is 0 or 1. The compound of claim 21 , wherein

24. R ２４ and R ２５ The compound according to any one of claims 21 to 23, wherein are identical.

25. R ２４ and R ２５ The compound according to any one of claims 21 to 23, wherein:

26. Z １０ and Z ２０ On the other hand, 【Chemistry 24】 and Z １０ and Z ２０ The other is, 【Chemistry 25】 The compound according to any one of claims 6 to 18, wherein

27. Z １０ and Z ２０ each of which independently 【Chemistry 26】 The compound according to any one of claims 6 to 18, wherein

28. Z １０ but 【Chemistry 27】 If ２２ is H; and Z ２０ but 【Chemistry 28】 If ２４ is H, The compound according to any one of claims 6 to 18.

29. Z １０ and Z ２０ On the other hand, 【Chemistry 29】 and Z １０ and Z ２０ The other is, 【Transformation 30】 The compound according to any one of claims 6 to 18, wherein

30. X １ and X ２ 30. The compound of claim 29, wherein each is O.

31. R ２２ is not H;R ２４ is not H; and R ２３ and R ２５ 31. The compound of claim 29 or 30, wherein at least one of is not H.

32. Z １０ and Z ２０ On the other hand, 【Chemistry 31】 and Z １０ and Z ２０ The other is, 【Chemistry 32】 The compound according to any one of claims 6 to 18, wherein

33. 33. The compound of claim 32, wherein s is an integer from 1 to 3.

34. formula: 【Transformation 33】 The compound according to any one of claims 6 to 18, having the formula:

35. R ２２ and R ２３ are each independently an unsubstituted C ５ ~C ８ alkyl; or R ２３ is H and R ２２ is unsubstituted C １０ ~C １４ alkyl); R ２４ and R ２５ are each independently an unsubstituted C ５ ~C ８ alkyl; or R ２５ is H and R ２４ is unsubstituted C １０ ~C １４ is alkyl; Here, R ２３ and R ２５ are both not H, 35. The compound of claim 34.

36. formula: 【Transformation 34】 The compound according to any one of claims 6 to 18, having the formula:

37. X １ and X ２ and each is O.

38. R ２２ is not H;R ２４ is not H; and R ２３ and R ２５ 38. The compound of claim 36 or 37, wherein at least one of is not H.

39. formula: 【Chemistry 35】 The compound according to any one of claims 6 to 18, having the formula:

40. formula: 【Transformation 36】 The compound according to any one of claims 6 to 18, having the formula:

41. R ２３ is H.

42. The following structure: 【Chemistry 37】 【Transformation 38】 【Chemistry 39】 【Chemistry 40】 【Chemistry 41】 【Chemistry 42】 and During the ceremony, R = 【Chemistry 43】 and each R ２７ are independently H, C １ ~C １５ Alkyl, C ２ ~C ８ Alkenyl, or C ２ ~C ８ is alkynyl, The compound of claim 6.

43. Formula (AL-GI): 【Chemistry 44】 or a salt thereof, During the ceremony: R Ｎ is a substituted or unsubstituted C １ ~C ６ Alkyl or C ３ ~C ８ is cycloalkyl; R １ , R １’ , R ２ , R ２’ , R ３ , R ３’ , R ４ , and R ４’ Each of the following may be independently selected for each occurrence: H, branched C １ ~C ３ Alkyl, unbranched C １ ~C ３ Alkyl, branched C ２ ~C ３ Alkenyl or unbranched C ２ ~C ３ alkenyl; R １０ , R １１ , R １２ , and R １３ Each of the is independently H or a substituted or unsubstituted branched C １ ~C １５ Alkyl or unbranched C １ ~C １５ alkyl; １０ and R １１ At least one of is not H and R １２ and R １３ at least one of is not H; Z １ and Z ２ each of which independently 【Chemistry 45】 and X １ and X ２ Each of is independently O, S, or N(R ２１ ) and R ２０ is a substituted or unsubstituted branched C １ ~C １５ Alkyl or unbranched C １ ~C １５ is alkyl; R ２１ is H, substituted or unsubstituted C １ ~C ５ Alkyl, or substituted or unsubstituted C ３ ~C ８ is cycloalkyl; s is an integer from 1 to 4; n １ , n ２ , n ３ , and n ４ is independently an integer from 0 to 15, where n １＋ n ２ is in the range of 1 to 15, and n ３＋ n ４ is in the range of 1 to 15; Here, R Ｎ is substituted with hydroxy １ ~C ６ When it is alkyl, it is either (i) or (ii) below: (i) Z １ and Z ２ At least one of 【Chemistry 46】 or (ii) n １ and ２ The sum of ３ and ４ is not equal to the sum of At least one of the following applies: A compound or a salt thereof.

44. R Ｎ is C １ ~C ６ Alkyl, C ３ ~C ８ Cycloalkyl, -(CH ２ ) ｖ Q, -(CH ２ ) ｖ N(R”)Q, -C(Q)(R) ２ , or -(CH ２ ) ｖ C (Q) (R) ２ and Each Q is independently -OR", -SR", C ３ ~C ８ Cycloalkyl, C ３ ~C ８ Cycloalkenyl, heterocyclyl, heterocycloalkenyl, aryl, heteroaryl, —O(CH ２ ) ｖ N(R”) ２ , -C(O)OR", -OC(O)R, -C(R') ３ , -CN, -C(O)N(R") ２ , -N(R")C(O)R, -N(R")S(O) ２ R, -N(R”)C(O)N(R”) ２ , -N(R”)C(S)N(R”) ２ , -N(R")R ａ , -O(CH ２ ) ｖ OR", -N(R")C(=NR ｂ ) N (R") ２ , -N(R”)C(=CHR ｂ ) N (R") ２ , -OC(O)N(R”) ２ , -N(R")C(O)OR", -N(OR")C(O)R, -N(OR")S(O) ２ R, -N(OR")C(O)OR", -N(OR")C(O)N(R") ２ , -N(OR”)C(S)N(R”) ２ , -N(OR”)C(=NR ｂ ) N (R") ２ , -N(OR”)C(=CHR ｂ ) N (R") ２ , -C(=NR ｂ ) N (R") ２ , -C(=NR ｂ )R, or —C(O)N(R″)OR″; Each R is independently H, C １ ~C ３ Alkyl, C ２ ~C ３ alkenyl, amino, monoalkylamino, or dialkylamino; each R' is independently H, F, Cl, Br, or I; Each R" is independently H, C １ ~C ３ Alkyl, or C ２ ~C ３ alkenyl; Each R ａ are independently H or C ３ ~C ８ is cycloalkyl; Each R ｂ are independently H, CN, NO ２ , C １ ~C ６ Alkyl, —OR, —S(O) ２ R, -S(O) ２ N(R”) ２ , C ２ ~C ６ Alkenyl, C ３ ~C ８ cycloalkyl, or heterocyclyl; v is an integer from 1 to 6; Here, each of the alkyl group, cycloalkyl group, cycloalkenyl group, heterocyclyl group, heterocycloalkenyl group, aryl group, and heteroaryl group is oxo (=O), OH, amino, monoalkylamino, dialkylamino, and C １ ~C ３ optionally substituted with one or more substituents selected from the group consisting of alkyl; 44. The compound of claim 43.

45. R Ｎ is unsubstituted C １～４ Alkyl, -(CH ２ ) ｖ N(R")Q, or -(CH ２ ) ｖ Q; Q is -OH, -SH, -NHC(S)N(R") ２ , -NHC(O)N(R”) ２ , -N(R")C(O)R, -N(R")S(O) ２ R, -N(R")R ａ , -NHC(=NR ｂ ) N (R") ２ , -NHC (=CHR ｂ ) N (R") ２ , -OC(O)N(R”) ２ , —N(R″)C(O)OR″, heterocyclyl, or heteroaryl; v is 2, 3, 4, or 5; 45. The compound of claim 44.

46. R Ｎ is -(CH ２ ) ｖ 46. ​​The compound of claim 45, wherein n is 0, 1, 2, 3, or 4;

47. R Ｎ is -CH ２ CH ２ 46. ​​The compound of claim 45, wherein:

48. R Ｎ is -(CH ２ ) ｖ Q or -(CH ２ ) ｖ 46. ​​The compound of claim 45, wherein N(R")Q, wherein Q is heterocyclyl, heterocycloalkenyl, aryl, or heteroaryl optionally substituted with one or more substituents.

49. R Ｎ is -(CH ２ ) ｖ N(R")S(O) ２ 46. ​​The compound of claim 45, wherein R

50. R Ｎ teeth, 【Chemistry 47】 wherein each R ｃ are independently H or C １ ~C ３ 45. The compound of claim 44, wherein s is alkyl and s is an integer from 1 to 4.

51. R Ｎ teeth, 【Chemistry 48】 45. The compound of claim 44, wherein:

52. n １ +n ２ is in the range of 1 to 10, and n ３ +n ４ is in the range of 1 to 10.

53. n １ +n ２ is in the range of 2 to 7, and n ３ +n ４ is in the range of 2 to 7, and n １ +n ２ and n ３ +n ４ and are the same.

54. n １ +n ２ is in the range of 2 to 7, and n ３ +n ４ is in the range of 2 to 7, and n １ +n ２ and n ３ +n ４ is different.

55. Z １ and Z ２ each of which independently 【Chemistry 49】 The compound according to any one of claims 43 to 54, wherein

56. Z １ and Z ２ On the other hand, [Transformation 50] and Z １ and Z ２ The other is, 【Chemistry 51】 The compound according to any one of claims 43 to 54, wherein

57. Z １ and Z ２ each of which independently 【Chemistry 52】 The compound according to any one of claims 43 to 54, wherein

58. Z ２ but 【Chemistry 53】 If １１ is H; and Z １ but 【Chemistry 54】 If １３ 58. The compound of claim 56 or 57, wherein is H.

59. Z １ and Z ２ On the other hand, 【Transformation 55】 and Z １ and Z ２ The other is, 【Transformation 56】 The compound according to any one of claims 43 to 54, wherein

60. Z １ and Z ２ Each of the is independently the same or different 【Chemistry 57】 The compound according to any one of claims 43 to 54, wherein

61. Z １ and Z ２ On the other hand, 【Chemistry 58】 and Z １ and Z ２ The other is, 【Chemistry 59】 The compound according to any one of claims 43 to 54, wherein

62. Z １ and Z ２ Each of the is independently the same or different 【Transformation 60】 The compound according to any one of claims 43 to 54, wherein

63. formula: 【Chemistry 61】 The compound according to any one of claims 43 to 54, having the formula:

64. R １０ and R １１ are each independently an unsubstituted C ５ ~C ８ alkyl; or R １１ is H and R １０ is unsubstituted C １０ ~C １４ is alkyl; R １２ and R １３ are each independently an unsubstituted C ５ ~C ８ alkyl; or R １３ is H and R １２ is unsubstituted C １０ ~C １４ is alkyl; Here, R １１ and R １３ are both not H, 64. The compound of claim 63.

65. n １ +n ２ is an integer from 2 to 4, and n ３ +n ４ is an integer from 5 to 7; or n １ +n ２ is an integer from 5 to 7, and n ３ +n ４ is an integer from 2 to 4.

66. formula: 【Transformation 62】 The compound according to any one of claims 43 to 54, having the formula:

67. formula: 【Transformation 63】 The compound according to any one of claims 43 to 54, having the formula:

68. R １０ and R １１ are each independently an unsubstituted C ５ ~C ８ alkyl; or R １１ is H and R １０ is unsubstituted C ７ ~C １１ is alkyl; R １２ and R １３ are each independently an unsubstituted C ５ ~C ８ alkyl; or R １３ is H and R １２ is unsubstituted C ７ ~C １１ is alkyl; Here, R １１ and R １３ are both not H, 68. A compound according to claim 66 or 67.

69. X １ and X ２ Each of the groups is independently O or N(R ２１ ) and R ２１ is H or C １ ~C ３ 68. The compound of claim 66 or 67, which is alkyl.

70. n １ +n ２ is an integer from 4 to 7, and n ３ +n ４ is an integer from 6 to 7; or n １ +n ２ is an integer from 6 to 7, and n ３ +n ４ is an integer from 4 to 7. The compound according to claim 66 or 67.

71. formula: 【Chemistry 64】 The compound according to any one of claims 43 to 54, having the formula:

72. R １０ is unsubstituted C ７ ~C １１ is alkyl; R １２ and R １３ are each independently an unsubstituted C ５ ~C ８ alkyl; or R １３ is H and R １２ is unsubstituted C ７ ~C １１ is alkyl; Each R ２１ is H; Each R ２０ are independently unsubstituted C ２ ~C ９ is alkyl, 72. The compound of claim 71.

73. n １ +n ２ and n ３ +n ４ 72. The compound of claim 71, wherein each of is independently an integer from 5 to 7.

74. 74. A lipid-based carrier comprising a compound according to any one of claims 1 to 73, wherein the lipid-based carrier is a lipid nanoparticle.

75. 75. The lipid-based carrier of claim 74, further comprising a second lipid.

76. 76. The lipid-based carrier of claim 75, wherein the second lipid is a cationic lipid, an anionic lipid, an ionic lipid, or a zwitterionic lipid.

77. 75. The lipid-based carrier of claim 74, further comprising a PEGylated lipid, a sterol, a phospholipid, and / or a neutral lipid.

78. The lipid component of the lipid-based carrier is about 25 to 100 mol % of said compound; about 0-50 mol % phospholipids, about 0-50 mol % sterols, and Approximately 0-10 mol% PEGylated lipid 78. The lipid-based carrier of claim 77, comprising:

79. The lipid component of the lipid-based carrier may be about 30 to 60 mol % of said compound; about 0-30 mol % phospholipids, about 15-50 mol % sterols, and Approximately 0-10 mol% PEGylated lipid 79. The lipid-based carrier of claim 78, comprising:

80. 80. The lipid-based carrier of any one of claims 74 to 79, wherein the lipid nanoparticle further comprises a therapeutic agent.

81. 81. The lipid-based carrier of claim 80, wherein the therapeutic agent is a nucleic acid molecule.

82. 82. The lipid-based carrier of claim 81, wherein the nucleic acid molecule is a nucleic acid selected from the group consisting of a plasmid, an immunostimulatory oligonucleotide, an antisense oligonucleotide, an antagomir, an aptamer, a deoxyribozyme (DNAzyme), and a ribozyme.

83. 82. The lipid-based carrier of claim 81, wherein the nucleic acid molecule is DNA or RNA.

84. 84. The lipid-based carrier of claim 83, wherein the DNA is linear DNA, circular DNA, single-stranded DNA, or double-stranded DNA.

85. 84. The lipid-based carrier of claim 83, wherein the RNA is selected from the group consisting of mRNA, miRNA, siRNA or siRNA precursor, RNA aptamer, linear RNA, circular RNA, single-stranded RNA, double-stranded RNA, tRNA, microRNA (miRNA) or miRNA precursor, Dicer substrate small interfering RNA (dsiRNA), short hairpin RNA (shRNA), asymmetric interfering RNA (aiRNA), guide RNA (gRNA), lncRNA, ncRNA, sncRNA, rRNA, snRNA, piRNA, snoRNA, snRNA, scaRNA, exRNA, scaRNA, Y RNA, and hnRNA.

86. 84. The lipid-based carrier of claim 83, wherein the RNA is mRNA.

87. 87. The lipid-based carrier of any one of claims 81 to 86, wherein the nucleic acid molecule comprises one or more nucleic acid analogs selected from the group consisting of phosphoramide, phosphorothioate, phosphorodithioate, O-methyl phosphoramidate, morpholino, locked nucleic acid (LNA), glycerol nucleic acid (GNA), threose nucleic acid (TNA), and peptide nucleic acid (PNA).

88. 81. The lipid-based carrier of claim 80, wherein the therapeutic agent is a protein or a small molecule drug.

89. 80. The lipid-based carrier of any one of claims 74 to 79, wherein the lipid nanoparticle comprises an antigen.

90. 90. The lipid-based carrier of claim 89, wherein the antigen is a protein or a nucleic acid.

91. 91. The lipid-based carrier of claim 90, wherein the antigen is a protein.

92. 91. The lipid-based carrier of claim 90, wherein the antigen is a nucleic acid.

93. 80. The lipid-based carrier of any one of claims 74 to 79, wherein the lipid nanoparticle comprises an mRNA molecule comprising a nucleotide sequence encoding an antigen.

94. 89. A method of delivering a therapeutic agent to a subject, comprising administering to said subject the lipid-based carrier of any one of claims 80-88.

95. 94. A method of vaccinating a subject in need thereof, comprising administering to said subject an effective amount of the lipid-based carrier of any one of claims 89 to 93.

96. 94. A pharmaceutical composition comprising the lipid-based carrier of any one of claims 74 to 93 and a pharmaceutically acceptable excipient.

Images